sentences: Growth-Rate Dependent And Nutrient-Specific Gene Expression Resource Allocation In Fission Yeast Cellular resources are limited and their relative allocation to gene expression programmes determines physiological states and global properties such as the growth rate. Quantitative studies using various growth conditions have singled out growth rate as a major physiological variable explaining relative protein abundances. Here, we used the simple eukaryote Schizosaccharomyces pombe to determine the importance of growth rate in explaining relative changes in protein and mRNA levels during growth on a series of non-limiting nitrogen sources. Although half of fission yeast genes were significantly correlated with the growth rate, this came alongside wide-spread nutrient-specific regulation. Proteome and transcriptome often showed coordinated regulation but with notable exceptions, such as metabolic enzymes. Genes positively correlated with growth rate participated in every level of protein production with the notable exception of RNA polymerase II, whereas those negatively correlated mainly belonged to the environmental stress response programme. Critically, metabolic enzymes, which represent [~]55-70% of the proteome by mass, showed mainly condition-specific regulation. Specifically, many enzymes involved in glycolysis and NAD-dependent metabolism as well as the fermentative and respiratory pathways were condition-dependent and not consistently correlated with growth. In summary, we provide a rich account of resource allocation to gene expression in a simple eukaryote, advancing our basic understanding of the interplay between growth-rate dependent and nutrient-specific gene expression.

sentences: Generating minimum set of gRNA to cover multiple targets in multiple genomes with MINORg MINORg is an offline gRNA design tool that generates the smallest possible combination of gRNA capable of covering all desired targets in multiple non-reference genomes. As interest in pangenomic research grows, so does the workload required for large screens in multiple individuals. MINORg aims to lessen this workload by capitalising on sequence homology to favour multi-target gRNA while simultaneously screening multiple genetic backgrounds in order to generate reusable gRNA panels. We demonstrated the practical application of MINORg by knocking out a 11 homologous genes tandemly arrayed in a multigene cluster in two Arabidopsis thaliana lineages using three gRNA output by MINORg. Source code is freely available at https://github.com/rlrq/MINORg.

sentences: Sepsis-induced brain state instability Sepsis-associated brain dysfunction (SABD) is a frequent severe complication of sepsis and the systemic inflammatory response syndrome. It is associated with high mortality and a majority of survivors suffer long-term neurological consequences. Here, we studied the effects of SABD on oscillatory brain states in an acute rat model of sepsis induced by high dose LPS (10 mg/kg). To focus on intrinsically generated brain state dynamics, we used a urethane model that spares oscillatory activity in REM- and NREM-like sleep states. Soon after the intraperitoneal LPS injection we observed a robust fragmentation of both oscillatory states resulting in a three-fold increase in the number of state transitions that lasted for several hours, although the overall time spent in either state did not change. Analysis of power spectra showed opposing shifts in low frequency oscillations (1-9 Hz) that resulted in increased similarity between both states in 2-D state space. The described spectral characteristics of sepsis-induced EEG state instability might point to a mechanism underlying severe sleep fragmentation as described both in sepsis patients and in SABD animal models.

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Test-retest reproducibility of in vivo magnetization transfer ratio and saturation index in mice at 9.4 Tesla BackgroundMagnetization transfer saturation (MTsat) imaging was developed to reduce T1 dependence and improve specificity to myelin compared to the widely used MT ratio (MTR), while maintaining a feasible scan time. Knowledge of MTsat reproducibility is necessary to apply MTsat in preclinical neuroimaging.

PurposeTo assess the test-retest reproducibility of MTR and MTsat in the mouse brain at 9.4 T and calculate sample sizes required to detect various effect sizes.

Study TypeProspective

Animal ModelC57Bl/6 Mouse Model (6 females and 6 males, aged 12 - 14 weeks)

Field Strength/SequenceMagnetization Transfer Imaging at 9.4 T

AssessmentAll mice were scanned at two timepoints (5 days apart). MTR and MTsat maps were analyzed using mean region-of-interest (ROI), and whole brain voxel-wise analysis.

Statistical TestsBland-Altman plots assessed biases between test and retest measurements. Test-retest reproducibility was evaluated via between and within-subject coefficients of variation (CV). Sample sizes required were calculated (at a 95 % significance level and power of 80 %), given various minimum detectable effect sizes, using both between and within-subject approaches.

ResultsBland-Altman plots showed negligible biases between test and retest sessions. ROI-based and voxel-wise CVs revealed high reproducibility for both MTR (ROI: CVs < 8 %) and MTsat (ROI: CVs < 10 %). With a sample size of 6, changes on the order of 15% can be detected in MTR and MTsat, both between and within subjects, while smaller changes (6 - 8 %) require sample sizes of 10 - 15 for MTR, and 15 - 20 for MTsat.

Data ConclusionMTsat exhibits comparable reproducibility to MTR, while providing sensitivity to myelin with less T1 dependence than MTR. Our findings suggest both MTR and MTsat can detect moderate changes, common in pathologies, with feasible preclinical sample sizes.

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Stage dependent differential influence of metabolic and structural networks on memory across Alzheimer's disease continuum BackgroundLarge-scale neuronal network breakdown underlies memory impairment in Alzheimers disease (AD). However, the differential trajectories of the relationships between network organization and memory across pathology and cognitive stages in AD remain elusive. We determined whether and how the influences of individual-level structural and metabolic covariance network integrity on memory varied with amyloid pathology across clinical stages without assuming a constant relationship.

Methods708 participants from the Alzheimers Disease Neuroimaging Initiative were studied. Individual-level structural and metabolic covariance scores in higher-level cognitive and hippocampal networks were derived from magnetic resonance imaging and [18F]fluorodeoxyglucose positron emission tomography using seed-based partial least square analyses. The non-linear associations between network scores and memory across cognitive stages in each pathology group were examined using sparse varying coefficient modelling.

ResultsWe showed that the associations of memory with structural and metabolic networks in the hippocampal and default mode regions exhibited pathology-dependent differential trajectories across cognitive stages using sparse varying coefficient modelling. In amyloid pathology group, there was an early influence of hippocampal structural network deterioration on memory impairment in the preclinical stage, and a biphasic influence of the angular gyrus-seeded default mode network metabolism on memory in both preclinical and dementia stages. In non-amyloid pathology groups, in contrast, the trajectory of the hippocampus-memory association was opposite and weaker overall, while no metabolism covariance networks were related to memory. Key findings were replicated in a larger cohort of 1280 participants.

ConclusionsOur findings highlight potential windows of early intervention targeting network breakdown at the preclinical AD stage.

sentences: Uncertainty Quantification in Variable Selection for Genetic Fine-Mapping using Bayesian Neural Networks In this paper, we propose a new approach for variable selection using a collection of Bayesian neural networks with a focus on quantifying uncertainty over which variables are selected. Motivated by fine-mapping applications in statistical genetics, we refer to our framework as an "ensemble of single-effect neural networks" (ESNN) which generalizes the "sum of single-effects" regression framework by both accounting for nonlinear structure in genotypic data (e.g., dominance effects) and having the capability to model discrete phenotypes (e.g., case-control studies). Through extensive simulations, we demonstrate our methods ability to produce calibrated posterior summaries such as credible sets and posterior inclusion probabilities, particularly for traits with genetic architectures that have significant proportions of non-additive variation driven by correlated variants. Lastly, we use real data to demonstrate that the ESNN framework improves upon the state-of-the-art for identifying true effect variables underlying various complex traits.

sentences: mmSIM: An open toolbox for accessible structured illumination microscopy Since the first practical super-resolution structured illumination fluorescence microscopes (SIM) were demonstrated more than two decades ago the method has become increasingly popular for a wide range of bioimaging applications. The high cost and relative inflexibility of commercial systems, coupled with the conceptual simplicity of the approach and the desire to exploit and customise existing hardware, have led to the development of a large number of home-built systems. Several detailed hardware designs are available in the scientific literature, complemented by open-source software tools for SIM image validation and reconstruction. However, there remains a lack of simple open-source software to control these systems and manage the synchronization between hardware components, which is critical for effective SIM imaging. This article describes a new suite of software tools based on the popular Micro-Manager package, which enable the keen microscopist to develop and run a SIM system. We use the software to control two custom-built, high-speed, spatial light modulator-based SIM systems, evaluating their performance by imaging a range of fluorescent samples. By simplifying the process of SIM hardware development, we aim to support wider adoption of the technique.

sentences: The intrinsic spatiotemporal organization of the human brain - A multi-dimensional functional network atlas The human brain is a complex, integrative and segregative network that exhibits dynamic fluctuations in activity across space and time. A canonical set of large-scale networks has been historically identified from resting-state fMRI (rs-fMRI), including the default mode, visual, somatomotor, salience, attention, and executive control. However, the methods used in identification of these networks have relied on assumptions that may inadvertently constrain their properties and consequently our understanding of the human connectome. Here we define a brain "network" as a functional component that jointly describes its spatial distribution and temporal dynamics, where neither domain suffers from unrealistic constraints. Using our recently developed BrainSync algorithm and the Nadam-Accelerated SCAlable and Robust (NASCAR) tensor decomposition, we identified twenty-three brain networks using rs-fMRI data from a large group of healthy subjects acquired by the Human Connectome Project. These networks are spatially overlapped, temporally correlated, and highly reproducible across two independent groups and sessions. We show that these networks can be clustered into six distinct functional categories and naturally form a representative functional network atlas for a healthy population. Using this atlas, we demonstrate that individuals with attention-deficit/hyperactivity disorder display disproportionate brain activity increases, relative to neurotypical subjects, in visual, auditory, and somatomotor networks concurrent with decreases in the default mode and higher-order cognitive networks. Thus, this work not only yields a highly reproducible set of spatiotemporally overlapped functional brain networks, but also provides convergent evidence that individual differences in these networks can be used to explain individual differences in neurocognitive functioning.

sentences: Genomics analysis of hexanoic acid exposure in Drosophila species Drosophila sechellia is a dietary specialist endemic to the Seychelles islands that has evolved to consume the fruit of Morinda citrifolia. When ripe, the fruit of M. citrifolia contains octanoic acid and hexanoic acid, two medium chain fatty acid volatiles that deter and are toxic to generalist insects. D. sechellia has evolved resistance to these volatiles allowing it to feed almost exclusively on this host plant. The genetic basis of octanoic acid resistance has been the focus of multiple recent studies, but the mechanisms that govern hexanoic acid resistance in D. sechellia remain unknown. To understand how D. sechellia has evolved to specialize on M. citrifolia fruit and avoid the toxic effects of hexanoic acid, we exposed adult D. sechellia, D. melanogaster and D. simulans to hexanoic acid and performed RNA sequencing comparing their transcriptional responses to identify D. sechellia specific responses. Our analysis identified many more genes responding transcriptionally to hexanoic acid in the susceptible generalist species than in the specialist D. sechellia. Interrogation of the sets of differentially expressed genes showed that generalists regulated the expression of many genes involved in metabolism and detoxification whereas the specialist primarily downregulated genes involved in the innate immunity. Using these data we have identified interesting candidate genes that may be critically important in aspects of adaptation to their food source that contains high concentrations of HA. Understanding how gene expression evolves during dietary specialization is crucial for our understanding of how ecological communities are built and how evolution shapes trophic interactions.

sentences: CAR/Nr1i3 directs T cell adaptation to bile acids in the small intestine Bile acids (BAs) are lipid emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, BAs can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain BA homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high BA concentrations in the small intestine lamina propria (siLP). We previously reported that CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1/ABCB1 in the siLP to prevent BA toxicity and suppress Crohns disease-like small bowel inflammation4. Here, we identify the nuclear xenobiotic receptor, constitutive androstane receptor (CAR/NR1I3), as a regulator of MDR1 expression in T cells, and safeguard against BA toxicity and inflammation in the small intestine. CAR was activated and induced large-scale transcriptional reprograming in Teff cells infiltrating the siLP, but not the colon. CAR induced expression of detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine, Il10. Accordingly, CAR-deficiency in T cells exacerbated, whereas pharmacologic CAR activation suppressed, BA-driven ileitis in T cell-reconstituted Rag-/- mice. These data suggest that CAR acts locally in small intestinal T cells to detoxify BAs and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohns disease, and provides evidence of lymphocyte sub-specialization within the small intestine.

sentences: Developmental single-cell transcriptomics of hypothalamic POMC progenitors reveal the genetic trajectories of multiple neuropeptidergic phenotypes Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential to regulate food intake and energy balance. However, the ontogenetic transcriptional programs that specify the identity and functioning of these neurons are poorly understood. Here, we use scRNAseq to define the transcriptomes characterizing POMC progenitors in the developing hypothalamus and TRAP-seq to analyze the subsequent translatomes of mature POMC neurons. Our data show that Pomc-expressing neurons originate from multiple developmental pathways expressing distinct combinations of transcription factors. The predominant cluster, featured by high levels of Pomc and Prdm12 transcripts represents the precursor of canonical arcuate POMC neurons. Additional clusters of progenitors expressing lower levels of Pomc mature into different neuronal phenotypes characterized by distinct combinations of transcription factors, neuropeptides, processing enzymes, cell surface and nuclear receptors. We conclude that the genetic programs specifying the identity and differentiation of arcuate POMC neurons are diverse and generate a heterogeneous repertoire of neuronal phenotypes early in development that continue to evolve postnatally.

sentences: Polycomb Group Ring Finger Protein 6 suppresses Myc-induced lymphomagenesis Max is an obligate dimerization partner for the Myc transcription factors and for several repressors, such as Mnt, Mxd1-4 and Mga, collectively thought to antagonize Myc function in transcription and oncogenesis. Mga, in particular, is part of the variant Polycomb group repressive complex PRC1.6. Here, we show that ablation of the distinct PRC1.6 subunit Pcgf6 - but not Mga - accelerates Myc-induced lymphomagenesis in E{micro}-myc transgenic mice. Unexpectedly, however, Pcgf6 loss shows no significant impact on transcriptional profiles, in neither pre-tumoral B-cells, nor lymphomas. Altogether, these data unravel an unforeseen, Mga- and PRC1.6-independent tumor suppressor activity of Pcgf6.

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Transcriptome and DNA methylome dynamics reveal differential characteristics of inflorescence development between two ecotypes in Panicum hallii The morphological diversity of the inflorescence determines flower and seed production, which is critical for plant adaptation and fitness. Cytosine methylation is an epigenetic mark that contributes to gene expression regulation during inflorescence development. Panicum hallii is a wild perennial grass in the subfamily Panicoideae that has been developed as a model to study perennial grass biology and adaptive evolution. Highly divergent inflorescences have evolved between the two major ecotypes in P. hallii, the upland ecotype with compact inflorescence and large seed and the lowland ecotype with an open inflorescence and small seed. Here we performed a comparative transcriptome and DNA methylome analysis across different stages of inflorescence between these two divergent ecotypes of P. hallii. Global transcriptome analysis identified differentially expressed genes (DEGs) involved in panicle divergence, stage-specific expression, and co-expression modules underlying inflorescence development. Comparing DNA methylome profiles revealed a remarkable level of differential DNA methylation associated with the evolution of the P. hallii inflorescence. We found that most differentially methylated regions (DMRs) occurred within the flanking regulatory regions of genes, especially the promoter. Integrative analysis of DEGs and DMRs characterized the global features of DMR-associated DEGs in the divergence of P. hallii inflorescence, which includes homologs of important inflorescence and seed developmental genes that have been previously identified in domesticated crops. Evolutionary analysis measured by Ka/Ks ratio suggested that most DMR-associated DEGs are under relatively strong purifying selection. This study provides insights into the transcriptome and epigenetic landscape of inflorescence divergence in P. hallii and a novel genomic resource for perennial grass biology.

One sentence summaryA comparative transcriptome and DNA methylome analysis of inflorescence between upland and lowland ecotypes reveal gene expression and DNA methylation variation underlying inflorescence divergence in Panicum hallii.

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Searching for universal model of amyloid signaling motifs using probabilistic context-free grammars BackgroundAmyloid signaling motifs are a class of protein motifs which share basic structural and functional features despite lack of apparent sequence homology. They are hard to detect in large sequence databases either with the alignment-based profile methods (due to short length and diversity) or with generic amyloid- and prion-finding tools (due to insufficient discriminative power). We propose to address the challenge with a machine learning grammatical model capable of generalizing over diverse collections of unaligned yet related motifs.

ResultsFirst, we introduce and test improvements to our probabilistic context-free grammar framework for protein sequences that allow for inferring more sophisticated models achieving high sensitivity at low false positive rates. Then, we infer universal grammars for a collection of recently identified bacterial amyloid signaling motifs and demonstrate that the method is capable of generalizing by successfully searching for related motifs in fungi. The results are compared to available alternative methods. Finally, we conduct spectroscopy analyses of selected peptides to verify their structural and functional relationship.

ConclusionsWhile the profile HMMs remain the method of choice for modeling homologous sets of sequences, PCFGs seem more suitable for building meta-family descriptors and extrapolating beyond the seed sample.

sentences: Multi-tier mechanics control stromal adaptations in swelling lymph nodes Lymph nodes (LNs) comprise two main structural elements: Fibroblastic reticular cells (FRCs) that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. While LNs are fairly stable in size during homeostatic conditions, immunological challenge causes more than 10-fold increase in size within only a few days. How a solid organ can accommodate such extreme volumetric changes is poorly understood. Here, we characterize the biomechanics of LN swelling on the cellular and organ scale. We identify lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing FRCs and their associated conduits to stretch. After an initial phase of relaxation, FRCs sense the resulting strain via cell matrix adhesions, which coordinates local growth and remodeling of the stromal network. While the expanded FRC network adopts its typical configuration, a massive fibrotic reaction of the organ capsule sets in and counters further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multi-tier fashion.

sentences: Insight into Canary Island pine physiology provided by stable isotope patterns of water and plant tissues along an altitudinal gradient The Canary Islands, an archipelago east of Moroccos Atlantic coast, present steep altitudinal gradients covering various climatic zones from hot deserts to subalpine Mediterranean, passing through fog-influenced cloud forests. Unlike the majority of the Canarian flora, Pinus canariensis grow along most of these gradients, allowing the study of plant functioning in contrasting ecosystems. Here we assess the water sources (precipitation, fog) of P. canariensis and its physiological behavior in its different natural environments. We analyzed carbon and oxygen isotope ratios of water and organics from atmosphere, soil and different plant organs and tissues (including 10-year annual time series of tree-ring cellulose) of six sites from 480 to 1990 m asl on the Canary island La Palma. We found a decreasing {delta}18O trend in source water that was overridden by an increasing {delta}18O trend in needle water, leaf assimilates and tree-ring cellulose with increasing altitude, suggesting site-specific tree physiological responses to relative humidity. Fog-influenced and fog-free sites showed similar {delta}13C values, suggesting photosynthetic activity to be limited by stomatal closure and irradiance at certain periods. Besides, we observed an 18O-depletion (fog-free and timberline sites) and 13C-depletion (fog-influenced and fog-free sites) in latewood compared to earlywood caused by seasonal differences in: (i) water uptake (i.e. deeper ground water during summer drought, fog water frequency and interception) and (ii) meteorological conditions (stem radial growth and latewood {delta}18O correlated with winter precipitation). In addition, we found evidence for foliar water uptake and strong isotopic gradients along the pine needle axis in water and assimilates. These gradients are likely the reason for an unexpected underestimation of pine needle water {delta}18O when applying standard leaf water {delta}18O models. Our results indicate that soil water availability and air humidity conditions are the main drivers of the physiological behavior of pine along the Canary Islands altitudinal gradients.

sentences: Place Cells in the Claustrum Remap Under NMDA Receptor Control Place cells are cells exhibiting location-dependent responses; they have mostly been studied in the hippocampus. Place cells have also been reported in the rat claustrum, an underexplored paracortical region with extensive corto-cortical connectivity. It has been hypothesised that claustral neuronal responses are anchored to cortical visual inputs. We show rat claustral place cells remap when visual inputs are eliminated from the environment and that this remapping is NMDA-receptor-dependent. Eliminating visual input enhances delta-band oscillatory activity in the claustrum, without affecting simultaneously-recorded visual cortical activity. We conclude that, like the hippocampus, claustral place field remapping might be mediated by NMDA receptor activity, and is modulated by visual cortical inputs. (106 words)

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Pectin remodeling belongs to a homeostatic system and triggers transcriptomic and hormonal modulations O_LIHere, we focused on the biological modifications arisen from a strong and transient variation of the pectin methylesterification status during the seed-to-seedling transition.
C_LIO_LIA reverse genetic approach was used to trigger specific reduction of pectin de-methylesterification during the seed maturation stage and the related physiological effects were assessed using a combination of biochemical, transcriptomic and microscopic analyses.
C_LIO_LIArabidopsis PME36 is required to implement the characteristic pattern of de-methylesterified pectin in the mature seed. While this pattern is strongly impaired in pme36-1 and pme36-2 mature seed, no phenotypical effect is observed in the knockout mutant during seed germination. By analyzing hormone homeostasis and gene expression regulation, we show a strong and dynamic physiological disorder in the mutant, which reveals the existence of a complex compensatory mechanism overcoming the defect in pectin de-methylesterification.
C_LIO_LIOur results reveal that pectin methylesterification status acts as upstream modulator involved in an undescribed homeostatic system in which pectin remodeling, hormone signaling and transcriptomic regulations interact to ensure the maintenance of a normal seed-to-seedling developmental program.
C_LI

sentences: Functional universality in slow-growing microbial communities arises from thermodynamic constraints The dynamics of microbial communities is incredibly complex, determined by competition for metabolic substrates and cross-feeding of byproducts. Species in the community grow by harvesting energy from chemical reactions that transform substrates to products. In many anoxic environments, these reactions are close to thermodynamic equilibrium and growth is slow. To understand the community structure in these energy-limited environments, we developed a microbial community consumer-resource model incorporating energetic and thermodynamic constraints on an interconnected metabolic network. The central ingredient of the model is product inhibition, meaning that microbial growth may be limited not only by depletion of metabolic substrates but also by accumulation of products. We demonstrate that these additional constraints on microbial growth cause a convergence in the structure and function of the community metabolic network--independent of species composition and biochemical details--providing a possible explanation for convergence of community function despite taxonomic variation observed in many natural and industrial environments. Furthermore, we discovered that the structure of community metabolic network is governed by the thermodynamic principle of maximum heat dissipation. Overall, the work demonstrates how universal thermodynamic principles may constrain community metabolism and explain observed functional convergence in microbial communities.

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Systematic phenotyping and characterization of the 3xTg-AD mouse model of Alzheimer's Disease Animal models of disease are valuable resources for investigating pathogenic mechanisms and potential therapeutic interventions. However, for complex disorders such as Alzheimers disease (AD), the generation and availability of innumerous distinct animal models present unique challenges to AD researchers and hinder the success of useful therapies. Here, we conducted an in-depth analysis of the 3xTg-AD mouse model of AD across its lifespan to better inform the field of the various pathologies that appear at specific ages, and comment on drift that has occurred in the development of pathology in this line since its development 20 years ago. This modern characterization of the 3xTg-AD model includes an assessment of impairments in behavior, cognition, and long-term potentiation followed by quantification of amyloid beta (A{beta}) plaque burden and neurofibrillary tau tangles, biochemical levels of A{beta} and tau protein, and neuropathological markers such as gliosis and accumulation of dystrophic neurites. We also present a novel comparison of the 3xTg-AD model with the 5xFAD model using the same deep-phenotyping characterization pipeline. The results from these analyses are freely available via the AD Knowledge Portal (https://admodelexplorer.synapse.org). Our work demonstrates the utility of a characterization pipeline that generates robust and standardized information relevant to investigating and comparing disease etiologies of current and future models of AD.

Contribution to the Field StatementAlzheimers Disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory impairments and affects more than 30 million individuals worldwide. Using animal models of AD, researchers have elucidated disease progression and hallmark pathologies that may underpin the memory impairments seen in patients. However, therapeutic targets have failed to translate successfully from animal studies to human clinical trials, prompting a reassessment of the development, use, and interpretation of data acquired using the innumerous AD animal models available to researchers. To address these shortcomings, we have developed a robust and reproducible modern characterization of pathologies within current and future animal models of AD to better assess distinct pathologies that arise at specific brain regions and ages of different models. Using the popular 3xTg-AD mouse, we demonstrate the utility of these deep-phenotyping analyses and highlight the drift that affected development of pathologies in this line over the past two decades. Utilizing this same systematic characterization, we also perform a direct comparison with 5xFAD mice, another popular animal model of AD. The robust and standardized data generated from these systematic deep-phenotyping analyses are available for broad use by the AD research community to assess, compare, and determine appropriate animal models of AD.

sentences: Gene expression signatures underlying inbreeding depression as revealed by whole-transcriptome analysis of selectively bred strains of the Pacific Oyster (Crassostrea gigas) Exploring molecular mechanisms of inbreeding depression is significant for the conservation and sustainable use of the rare breed with a specific trait of high production value. In this work, we conducted whole-transcriptome analysis of two selectively bred Pacific oyster strains with one inbred strain showing significant growth depression. A total of 7980, 2677, and 28 differentially expressed protein-coding genes (DEGs), lncRNAs (DELs), and miRNAs (DEMs) were identified, respectively. The majority of DEGs and predicted target genes of DELs and DEMs were significantly enriched in biological process including immune response, cell proliferation, and apoptosis pathways. A set of genes with specific expression patterns as revealed by cluster profiling and enrichment analysis were identified, which may underlie inbreeding depression of the inbred strain. Furthermore, a competing endogenous RNA (ceRNA) network was constructed by integrative analysis of DEGs, DELs, and DEMs, supporting that ncRNAs, as regulators of gene expression, could be critical modulators in multiple subsystems involved in inbreeding depression.

sentences: Pervasive Under-Dominance in Gene Expression as Unifying Principle of Biomass Heterosis in Arabidopsis Heterosis, the generally superior performance in hybrids compared to their inbred parents, is one of the most enigmatic biological phenomena. Many different explanations have been put forward for heterosis, which begs the question whether common principles underpinning it do exist at all. We performed a systematic transcriptomic study in Arabidopsis thaliana involving 141 random crosses, to search for the general principles, if any, that heterotic hybrids share. Consistent additive expression in F1 hybrids was observed for only about 300 genes enriched for roles in stress response and cell death. Regulatory rare-allele burden affects the expression level of these genes but does not correlate with heterosis. Non-additive gene expression in F1 hybrids is much more common, with the vast majority of genes (over 90%) being expressed below parental average. These include genes that are quantitatively correlated with biomass accumulation in both parents and F1 hybrids, as well as genes strongly associated with heterosis. Unlike in the additive genes, regulatory rare allele burden in this non-additive gene set is strongly correlated with growth heterosis, even though it does not covary with the expression level of these genes. Together, our study suggests that while additive complementation is an intrinsic property of F1 hybrids, the major driver of growth in hybrids derives from the quantitative nature of non-additive gene expression, especially under-dominance and thus lower expression in hybrids than predicted from the parents.

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Hybrid Gaussian Process Models for continuous time series in bolus fed-batch cultures Hybrid modeling, meaning the integration of data-driven and knowledge-based methods, is quickly gaining popularity among many research fields, including bioprocess engineering and development. Recently, the data-driven part of hybrid methods have been largely extended with machine learning algorithms (e.g., artificial neural network, support vector regression), while the mechanistic part is typically using differential equations to describe the dynamics of the process based on its current state. In this work we present an alternative hybrid model formulation that merges the advantages of Gaussian Process State Space Models and the numerical approximation of differential equation systems through full discretization.

The use of Gaussian Process Models to describe complex bioprocesses in batch, fed-batch, has been reported in several applications. Nevertheless, handling the dynamics of the states of the system, known to have a continuous time-dependent evolution governed by implicit dynamics, has proven to be a major challenge. Discretization of the process on the sampling steps is a source of several complications, as are: 1) not being able to handle multi-rate date sets, 2) the step-size of the derivative approximation is defined by the sampling frequency, and 3) a high sensitivity to sampling and addition errors. We present a coupling of polynomial regression with Gaussian Process Models as representation of the right-hand side of the ordinary differential equation system and demonstrate the advantages in a typical fed-batch cultivation for monoclonal antibody production.

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DeepFlu: a deep learning approach for forecasting symptomatic influenza A infection based on pre-exposure gene expression Background and ObjectiveNot everyone gets sick after an exposure to influenza A viruses (IAV). Although KLRD1 has been identified as a potential biomarker for influenza susceptibility, it remains unclear whether forecasting symptomatic flu infection based on pre-exposure host gene expression might be possible.

MethodTo examine this hypothesis, we developed DeepFlu using the state-of-the-art deep learning approach on the human gene expression data infected with IAV subtype H1N1 or H3N2 viruses to forecast who would catch the flu prior to an exposure to IAV.

ResultsThe results indicated that such forecast is possible and, in other words, gene expression could reflect the strength of host immunity. In the leave-one-person-out cross-validation, DeepFlu based on deep neural network outperformed the models using convolutional neural network, random forest, or support vector machine, achieving 70.0% accuracy, 0.787 AUROC, and 0.758 AUPR for H1N1 and 73.8% accuracy, 0.847 AUROC, and 0.901 AUPR for H3N2. In the external validation, DeepFlu also reached 71.4% accuracy, 0.700 AUROC, and 0.723 AUPR for H1N1 and 73.5% accuracy, 0.732 AUROC, and 0.749 AUPR for H3N2, surpassing the KLRD1 biomarker. In addition, DeepFlu which was trained only by pre-exposure data worked the best than by other time spans and mixed training data of H1N1 and H3N2 did not necessarily enhance prediction. DeepFlu is available at https://github.com/ntou-compbio/DeepFlu.

ConclusionsDeepFlu is a prognostic tool that can moderately recognize individuals susceptible to the flu and may help prevent the spread of IAV.

sentences: APC-mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness Certain copy number alterations (CNAs) are strongly associated with particular cancer types. However, the mechanisms that underlie selection of specific CNAs remain unknown. Here, we identified functional relationships between recurrent CNAs in colorectal cancers (CRCs) and adenomatous polyposis coli (APC) mutations. Quantitative phenotyping of mitotic spindles highlighted APC functions at centrosomes where APC positively regulated Aurora A kinase (AURKA). Upon APC inactivation, elevated {beta}-catenin levels blocked AURKA activation, which caused chromosome instability and suppressed proliferation, resulting in the generation and selection of AURKA-activating CNAs. Arm-level amplification of chromosomes that contained AURKA and AURKA activator genes was observed in APC-mutant CRCs, early stage mouse tumours, and cells in culture, which was concomitant with an increase in growth potential. Our findings demonstrate a mechanism that restores tumour cell fitness through compensatory chromosome alterations to overcome adverse effects of prior mutations, which may affect the course of cancer type-specific CNA formation.

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Generation and characterization of Ccdc28b mutant mice links the Bardet-Biedl associated gene with social behavioral phenotypes CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). After confirming the depletion of Ccdc28b we performed a phenotypic characterization showing that Ccdc28b mut animals present a mild phenotype: i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors suggestive of autism spectrum disorder (ASD). This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Importantly however, our data suggests a possible causal link between CCDC28B and ASD-like phenotypes that exceeds the context of BBS: filtering for rare deleterious variants, we found CCDC28B mutations in eight probands from the Simmons Simplex Collection cohort. Furthermore, a phenotypic analysis showed that CCDC28B mutation carriers present lower BMI and mild communication defects compared to a randomly selected sample of SSC probands. Thus, our results suggest that mutations in CCDC28B lead to mild autism-like features in mice and humans. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and ASD-like phenotypes, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of ASD.

AUTHOR SUMMARYBardet-Biedl syndrome (BBS) is caused by mutations in any of 21 genes known to date. In some families, BBS can be inherited as an oligogenic trait whereby mutations in more than one BBS gene collaborate in the presentation of the syndrome. In addition, CCDC28B was identified as a modifier of BBS, associated with a more severe presentation of the syndrome. Different mechanisms, all relying on functional redundancy, have been proposed to explain this genetic interaction and the characterization of different BBS proteins supported this possibility as they were shown to play roles in the same cellular organelle, the primary cilium.

Similarly, CCDC28B also participates in cilia biology regulating the length of the organelle: knockdown of CCDC28B in cells results in cilia shortening and depletion in zebrafish also results in early embryonic phenotypes characteristic of other cilia mutants. Here, we sought to generate a mouse Ccdc28b mutant to determine whether it would be sufficient to cause a ciliopathy phenotype, and to generate a reagent critical to further dissect its modifying role in the context of BBS. Overall, Ccdc28b mutant mice presented a mild phenotype, a finding fully compatible with a modifier rather than a causal BBS gene. In addition, we found that Ccdc28b mutants showed a clear autism-like behavior, and autism is indeed a feature of several BBS patients. Importantly, we identified multiple individuals with autism from the Simmos Simplex Collection to carry disruptive mutations in CCDC28B suggesting that this gene is causally associated with autism independently of BBS.

sentences: HCN1 channels mediate mu opioid receptor long-term depression at insular cortex inputs to the dorsal striatum Mu opioid receptors (MORs) are expressed in the dorsal striatum, a brain region that mediates goal-directed (via the dorsomedial striatum), and habitual (via the dorsolateral striatum, DLS) behaviors. Our previous work indicates that glutamate transmission is depressed when MORs are activated in the dorsal striatum, inducing MOR-mediated long-term synaptic depression (MOR-LTD) or short-term depression (MOR-STD), depending on the input. In the DLS, MOR-LTD is produced by MORs on anterior insular cortex (AIC) inputs and MOR-STD occurs at thalamic inputs, suggesting input-specific MOR plasticity mechanisms. Here, we evaluated the mechanisms of induction of MOR-LTD and MOR-STD in the DLS using pharmacology and optogenetics combined with patch clamp electrophysiology. We found that cAMP/PKA signaling and protein synthesis are necessary for MOR-LTD expression, similar to previous studies of endocannabinoid-mediated LTD (eCB-LTD) in DLS. However, MOR-LTD does not require mTOR signaling as eCB-LTD does. MOR-STD does not utilize these same mechanisms. We characterized the role of presynaptic HCN1 channels in MOR-LTD induction as HCN1 channels expressed in AIC are necessary for MOR-LTD expression in the DLS. These results suggest a mechanism in which MOR activation needs HCN1 to induce MOR-LTD, suggesting a new target for pharmacological modulation of synaptic plasticity, providing new opportunities to develop novel drugs to treat alcohol and opioid use disorders.

sentences: Genetic code degeneracy is established by the decoding center of the ribosome The degeneracy of the genetic code confers a wide array of properties to coding sequences. Yet, its origin is still unclear. A structural analysis has shown that the stability of the Watson-Crick base pair at the second position of the anticodon-codon interaction is a critical parameter controlling the extent of non-specific pairings accepted at the third position by the ribosome, a flexibility at the root of degeneracy. Based on recent cryo-EM analyses, the present work shows that residue A1493 of the decoding center provides a significant contribution to the stability of this base pair, revealing that the ribosome is directly involved in the establishment of degeneracy. Building on existing evolutionary models, we show the evidence that the early appearance of A1493 and A1492 established the basis of degeneracy when an elementary kinetic scheme of translation was prevailing. Logical considerations on the expansion of this kinetic scheme indicate that the acquisition of the peptidyl transferase center was the next major evolutionary step, while the induced-fit mechanism, that enables a sharp selection of the tRNAs, necessarily arose later when G530 was acquired by the decoding center.

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Sample-wise unsupervised deconvolution of complex tissues MotivationComplex biological tissues are often a heterogeneous mixture of several molecularly distinct cell or tissue subtypes. Both subtype compositions and expressions in individual samples can vary across different biological states or conditions. Computational deconvolution aims to dissect patterns of bulk gene expression data into subtype compositions and subtype-specific expressions. Typically, existing deconvolution methods can only estimate averaged subtype-specific expressions in a population, while detecting differential expressions or co-expression networks in particular subtypes requires unique subtype expression estimates in individual samples. Different from population-level deconvolution, however, individual-level deconvolution is mathematically an underdetermined problem because there are more variables than observations.

ResultsWe report a sample-wise Convex Analysis of Mixtures (swCAM) method that can estimate subtype proportions and subtype-specific expressions in individual samples from bulk tissue transcriptomes. We extend our previous CAM framework to include a new term accounting for between-sample variations and formulate swCAM as a nuclear-norm and{ell} 2,1-norm regularized matrix factorization problem. We determine hyperparameter values using a cross-validation scheme with random entry exclusion and obtain a swCAM solution using an efficient alternating direction method of multipliers. The swCAM is implemented in open-source R scripts. Experimental results on realistic simulation data show that swCAM can accurately estimate subtype-specific expressions in individual samples and successfully extract co-expression networks in particular subtypes that are otherwise unobtainable using bulk expression data. Application of swCAM to bulk-tissue data of 320 samples from bipolar disorder patients and controls identified changes in cell proportions, expression and coexpression modules in patient neurons. Mitochondria related genes showed significant changes suggesting an important role of energy dysregulation in bipolar disorder.

Availability and implementationThe R Scripts of swCAM is freely available at https://github.com/Lululuella/swCAM. A users guide and a vignette are provided.

Contactyuewang@vt.edu

Supplementary informationSupplementary data are available at Bioinformatics online.

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The transcription factor EGR2 is indispensable for tissue-specific imprinting of alveolar macrophages in health and tissue repair Alveolar macrophages are the most abundant macrophages in the healthy lung where they play key roles in homeostasis and immune surveillance against air-borne pathogens. Tissue-specific differentiation and survival of alveolar macrophages relies on niche-derived factors, such as colony stimulating factor 2 (CSF-2) and transforming growth factor beta (TGF-{beta}). However, the nature of the downstream molecular pathways that regulate the identity and function of alveolar macrophages and their response to injury remains poorly understood. Here, we identify that the transcriptional factor EGR2 is an evolutionarily conserved feature of lung alveolar macrophages and show that cell-intrinsic EGR2 is indispensable for the tissue-specific identity of alveolar macrophages. Mechanistically, we show that EGR2 is driven by TGF-{beta} and CSF-2 in a PPAR-{gamma}-dependent manner to control alveolar macrophage differentiation. Functionally, EGR2 was dispensable for lipid handling, but crucial for the effective elimination of the respiratory pathogen Streptococcus pneumoniae. Finally, we show that EGR2 is required for repopulation of the alveolar niche following sterile, bleomycin-induced lung injury and demonstrate that EGR2-dependent, monocyte-derived alveolar macrophages are vital for effective tissue repair following injury. Collectively, we demonstrate that EGR2 is an indispensable component of the transcriptional network controlling the identity and function of alveolar macrophages in health and disease.

One Sentence SummaryEGR2 controls alveolar macrophage function in health and disease

sentences: Plasma exosomal miRNA analysis of Alzheimer's disease reveals the dysfunction of a neural network Exosomal microRNA (miRNA) is an emerging source for biomarkers of Alzheimers disease (AD). Here, we profiled miRNA expression in AD, mild cognitive impairment (MCI), and controls. The assessment and validation of differentially expressed miRNA represented their potential to be novel biomarkers for AD and MCI. We conducted 13 co-expression networks and a miRNA network module linked to neural function emerged as the most significantly associated with AD diagnosis. The conservation analysis revealed the M1 was highly preserved in controls but dysfunction in AD and MCI. The module pattern between MCI and NC was similar, but significantly differed from AD, suggesting that the neural network regulated by miRNA changed during the mild cognitive stage, and the total miRNA expression altered in AD stage. Additionally, 24 out of 26 M1 hub-miRNAs were derived from brain tissue, and 15 had been reported as AD biomarkers. We consequently proposed the other 11 miRNAs could play important roles in AD. Our study highlights that co-expression network analysis can provide a new path for finding novel biomarkers.

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Hippocampal cells multiplex positive and negative engrams The hippocampus is involved in processing a variety of mnemonic computations specifically the spatiotemporal components and emotional dimensions of contextual memory.1-3 Recent studies have demonstrated vast structural and functional heterogeneity along the dorsal-ventral axis1, 5 of the hippocampus, and while much is known about how the dorsal hippocampus processes spatial- temporal content, much less is known about whether or not the ventral hippocampus (vHPC) partitions positive and negative experiences into distinct sets of cells.4-9 Here, we combine transgenic and all-virus based activity-dependent tagging strategies to visualize multiple valence- specific engrams in the vHPC and demonstrate two partially segregated cell populations and projections that respond to positive and negative experiences. Next, using an RNA sequencing approach, we find that vHPC positive and negative engram cells display distinct transcriptional programs compared to a neutral engram population. Additionally, while optogenetic manipulation of tagged cell bodies in vHPC is not sufficient to drive preference or avoidance, stimulation of tagged vHPC terminals projecting to the amygdala and nucleus accumbens (NAc), but not the prefrontal cortex (PFC), drives preference and avoidance. These terminals can also undergo a "switch" or "reset" in their capacity to drive either, thereby demonstrating their flexible contributions to behavior. We conclude that the vHPC contains genetically, cellularly, and behaviorally distinct populations of cells processing positive and negative memory engrams. Together, our findings provide a novel means by which to visualize multiple engrams within the same brain and point to their unique genetic signatures as reference maps for the future development of new therapeutic strategies.

One sentence summaryThe hippocampus contains neurons that correspond to positive and negative engrams, which are segregated by their molecular, cellular, and projection-specific features

sentences: Sustained TNF signaling is required for the synaptic and behavioral response to acute stress Acute stress triggers plasticity of forebrain synapses as well as behavioral changes. Here we reveal that Tumor Necrosis Factor  (TNF) is a required downstream mediator of the stress response in mice, necessary for stress-induced synaptic potentiation in the ventral hippocampus and for an increase in anxiety-like behaviour. Acute stress is sufficient to activate microglia, triggering the long-term release TNF. Critically, on-going TNF signaling in the ventral hippocampus is necessary to sustain both the stress-induced synaptic and behavioral changes, as these could be reversed hours after induction by antagonizing TNF signaling. This demonstrates that TNF maintains the synaptic and behavioral stress response in vivo, making TNF a potential novel therapeutic target for stress disorders.

sentences: Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation There is an urgent need to limit and stop the worldwide coronavirus disease 2019 (COVID-19) pandemic via quick development of efficient and safe vaccination methods. Plasmid DNA vaccines are one of the most remarkable vaccines that can be developed in a short term. pVAX1-SARS-CoV2-co, which is a plasmid DNA vaccine, was designed to express severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. The produced antibodies lead to Immunoreactions against S protein, anti-receptor-binding-domain, and neutralizing action of pVAX1-SARS-CoV2-co, as confirmed in a previous study. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine, a pyro-drive jet injector (PJI) was employed. PJI is an injection device that can adjust the injection pressure depending on various target tissues. Intradermally-adjusted PJI demonstrated that pVAX1-SARS-CoV2-co vaccine injection caused a strong production of anti-S protein antibodies, triggered immunoreactions and neutralizing actions against SARS-CoV-2. Moreover, a high dose of pVAX1-SARS-CoV2-co intradermal injection via PJI did not cause any serious disorders in the rat model. Finally, virus infection challenge in mice, confirmed that intradermally immunized (via PJI) mice were potently protected from COVID-19 infection. Thus, pVAX1-SARS-CoV2-co intradermal injection via PJI is a safe and promising vaccination method to overcome the COVID-19 pandemic.

sentences: Mathematical Models of Retinitis Pigmentosa: The Trophic Factor Hypothesis Retinitis pigmentosa (RP) is the term used to denote a group of inherited retinal-degenerative conditions that cause progressive sight loss. Individuals with this condition lose their light-sensitive photoreceptor cells, known as rods and cones, over a period of years to decades; degeneration starting in the retinal periphery, and spreading peripherally and centrally over time. RP is a rod-cone dystrophy, meaning that rod health and function are affected earlier and more severely than that of cones. Rods degenerate due to an underlying mutation, whereas the reasons for cone degeneration are unknown. A number of mechanisms have been proposed to explain secondary cone loss and the spatio-temporal patterns of retinal degeneration in RP. One of the most promising is the trophic factor hypothesis, which suggests that rods produce a factor necessary for cone survival, such that, when rods degenerate, cone degeneration follows. In this paper we formulate and analyse mathematical models of RP under the trophic factor hypothesis. These models are constructed as systems of reaction-diffusion partial differential equations in one spatial dimension, and are solved and analysed using a combination of numerical and analytical methods. We predict the conditions under which cones will degenerate following the loss of a patch of rods from the retina, the critical trophic factor treatment rate required to prevent cone degeneration following rod loss and the spatio-temporal patterns of cone loss that would result if the trophic factor mechanism alone were responsible for retinal degeneration.

sentences: Brain network dynamics codify heterogeneity in seizure propagation Dynamic functional brain connectivity facilitates adaptive cognition and behavior. Abnormal alterations within such connectivity could result in disrupted functions observed across various neurological conditions. As one of the most common neurological disorders, epilepsy is defined by the seemingly random occurrence of spontaneous seizures. A central but unresolved question concerns the mechanisms by which extraordinarily diverse dynamics of seizures emerge. Here, we apply a graph-theoretical approach to assess dynamic reconfigurations in the functional brain connectivity before, during, and after seizures that display heterogeneous propagation patterns despite sharing similar origins. We demonstrate unique reconfigurations in globally-defined network properties preceding seizure onset that predict propagation patterns of impending seizures, and in locally-defined network properties that differentiate post-onset dynamics. These results characterize quantitative network features underlying the heterogeneity of seizure dynamics and the accompanying clinical manifestations. Decoding these network properties could improve personalized preventative treatment strategies for epilepsy as well as other neurological disorders.

sentences: Single cell RNA-seq identifies inflammation-induced loss of CFTR-expressing airway ionocytes in non-eosinophilic asthma Asthma is the most common chronic airways disease worldwide and the severe treatment resistant subtype of asthma is responsible for the majority of disease burden. Asthma is heterogeneous in nature and can be classified according to airway infiltrates as eosinophilic or non-eosinophilic (sometimes referred to as Type 2 low), which is further divided into paucigranulocytic (low levels of granulocytes), or neutrophilic asthma characterized by elevated neutrophils, and mixed Type 1 and Type 17 cytokines in airway tissue, sputum, and bronchoalveolar lavage. Severe non-eosinophilic asthma currently has fewer effective treatment options and many of these patients fail to qualify for newer biologic monoclonal therapies. The cystic fibrosis transmembrane conductance regulator (CFTR) is a key protein whose function is dysregulated in multiple respiratory diseases including cystic fibrosis and chronic obstructive pulmonary disease (COPD) and has proven a valuable therapeutic target. Using human bronchial epithelial cells (hBECs) isolated differentiated at air-liquid interface we demonstrated a reduced function of the CFTR in non-eosinophilic asthma. Characterization of the cell and molecular differences in airway epithelial cells between severe asthma subtypes using single cell RNA-sequencing (scRNAseq) revealed that airway epithelial cells from non-eosinophilic asthma, and in particular neutrophilic asthma patients, fail to differentiate into CFTR-expressing ionocytes compared with eosinophilic asthma or healthy donors. We identified a novel ionocyte transcriptional signature, which was present in both bronchial and tracheal airway epithelial samples indicating conserved anatomical gene regulation. Using protein markers and immunofluorescent quantification loss of ionocytes was confirmed in non-eosinophilic asthma hBECs. Similarly, ioncytes were also diminished in the airways of a murine model of neutrophilic-dominant but not eosinophilic allergen asthma models. Furthermore, treatment of hBECs from healthy donors with a neutrophilic asthma-like inflammatory cytokine mixture, but not IL-13, led to loss of ionocytes primarily due to IFN-{gamma}. Inflammation-induced loss of CFTR-expressing ionocytes in airway cells from non-eosinophilic asthma may represent a key feature of disease pathogenesis and a novel drug target for this difficult-to-treat disease.

sentences: Viruses of Asgard archaea Asgardarchaeota encode many eukaryotic signature proteins and are widely considered to represent the closest archaeal relatives of eukaryotes. Whether similarities between Asgard archaea and eukaryotes extend to their viromes remains unknown. Here we present 20 metagenome-assembled genomes of Asgardarchaeota from deep-sea sediments of the basin off the Shimokita Peninsula, Japan. By combining CRISPR spacer search of metagenomic sequences with phylogenomic analysis, we identify three family-level groups of viruses associated with Asgard archaea. The first group, Verdandiviruses, includes tailed viruses of the realm Duplodnaviria, the second one, Skuldviruses, consists of viruses with predicted icosahedral capsids that belong to the realm Varidnaviria, and the third group, Wyrdviruses, is related to spindle-shaped viruses previously identified in other archaea. More than 90% of the proteins encoded by these putative viruses of Asgard archaea show no sequence similarity to proteins encoded by other known viruses. Nevertheless, all three proposed families consist of viruses typical of prokaryotes, providing no indication of a specific evolutionary relationship between viruses infecting Asgard archaea and eukaryotes. Verdandiviruses and skuldviruses are likely to be lytic, whereas wyrdviruses, similar to all other known spindle-shaped viruses, probably establish chronic infection and are released without host cell lysis. All three groups of viruses were identified in sediment samples from distinct geographical locations and are expected to play important roles in controlling the Asgard archaea populations in deep-sea ecosystems.

sentences: Bio-inspired neural networks implement different recurrent visual processing strategies than task-trained ones do Behavioral studies suggest that recurrence in the visual system is important for processing degraded stimuli. There are two broad anatomical forms this recurrence can take, lateral or feedback, each with different assumed functions. Here we add four different kinds of recurrence--two of each anatomical form--to a feedforward convolutional neural network and find all forms capable of increasing the ability of the network to classify noisy digit images. Specifically, we take inspiration from findings in biology by adding predictive feedback and lateral surround suppression. To compare these forms of recurrence to anatomically-matched counterparts we also train feedback and lateral connections directly to classify degraded images. Counter-intuitively, we find that the anatomy of the recurrence is not related to its function: both forms of task-trained recurrence change neural activity and behavior similarly to each other and differently from their bio-inspired anatomical counterparts. By using several analysis tools frequently applied to neural data, we identified the distinct strategies used by the predictive versus task-trained networks. Specifically, predictive feedback de-noises the representation of noisy images at the first layer of the network and decreases its dimensionality, leading to an expected increase in classification performance. Surprisingly, in the task-trained networks, representations are not de-noised over time at the first layer (in fact, they become  noiser and dimensionality increases) yet these dynamics do lead to de-noising at later layers. The analyses used here can be applied to real neural recordings to identify the strategies at play in the brain. Our analysis of an fMRI dataset weakly supports the predictive feedback model but points to a need for higher-resolution cross-regional data to understand recurrent visual processing..

sentences: Modelling time evolution of in blood alcohol concentration A theoretical model based on mass conservation is proposed to predict blood alcohol concentration (BAC) over time. The proposed model takes as input the body parameters, the data timing for each drink, as well as his alcoholic strength and ingested amount. Three test cases with variations are presented and discussed. The results given are obtained by solving numerically the equation proposed. The solution predicts a well located alcohol peak and a reasonable time of alcohol body cleaning. This model represents a first order approach to a complex problem. Similar scenarios, like, for instance, drug concentrations can be modelled by using the same concept. The proposed model has been implemented on easy-to-use program COGORZA, which source code is available online at https://github.com/tonibois/Cogorza.

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Common signatures of differential microRNA expression in Parkinson's and Alzheimer's disease brains BackgroundDysregulation of microRNA (miRNA)-mediated gene expression has been implicated in the pathogenesis and course of many neurodegenerative diseases including Parkinsons disease (PD). However, the functionally relevant miRNAs remain largely unknown. Previous meta-analyses on differential miRNA expression data in post-mortem PD brains have highlighted several miRNAs showing consistent and statistically significant effects. However, these meta-analyses were based on exceedingly small sample sizes.

MethodsIn this study, we quantified the expression of the four most compelling PD candidate miRNAs from these meta-analyses in the superior temporal gyrus (STG) of one of the largest case-control post-mortem brain datasets available (261 samples), thereby quadruplicating previously investigated sample sizes. Furthermore, we probed for common differential miRNA expression signatures with Alzheimers disease (AD) by also analyzing these miRNAs in post-mortem STG of 190 AD patients and controls and by testing six top AD miRNAs in the PD brains.

ResultsOf all ten analyzed miRNAs, PD candidate miRNA homo sapiens (hsa-) miR-132-3p showed evidence for differential expression in both PD (p=4.89E-06) and AD (p=3.20E-24), and AD miRNAs hsa-miR-132-5p (p=4.52E-06) and hsa-miR-129-5p (p=0.0379) showed evidence for differential expression in PD. Combining these novel data with previously published data substantially improved the statistical support (=3.85E-03 using Bonferroni correction) of the corresponding meta-analyses clearly and compellingly implicating these miRNAs in both PD and AD. Furthermore, hsa-miR-132-3p/-5p (but not hsa-miR-129-5p) showed association with neuropathological Braak PD staging (p=3.51E-03/p=0.0117), suggesting that these miRNAs may play a role in -synuclein aggregation beyond the early disease phase.

ConclusionsOur study represents the largest independent assessment of recently highlighted candidate brain miRNAs in PD and AD post-mortem brain samples, to date. Our results implicate hsa-miR-132-3p/-5p and hsa-miR-129-5p to be differentially expressed in both PD and AD brains, potentially pinpointing shared pathogenic mechanisms across these neurodegenerative diseases.

sentences: Loss-of-function variants in the schizophrenia risk gene SETD1A alter neuronal network activity in human neurons through cAMP/PKA pathway Heterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, were shown to cause a novel neurodevelopmental syndrome and increase the risk for schizophrenia. We generated excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/-) using CRISPR/Cas9. Our data show that SETD1A haploinsufficiency resulted in morphologically increased dendritic complexity and functionally increased bursting activity. This network phenotype was primarily driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling revealed perturbations in gene sets associated with glutamatergic synaptic function. At the molecular level, we identified specific changes in the cAMP/PKA pathway pointing toward a hyperactive cAMP pathway in SETD1A+/- neurons. Finally, by pharmacologically targeting the cAMP pathway we were able to rescue the network deficits in SETD1A+/- cultures. Our results demonstrate a link between SETD1A and the cAMP-dependent pathway in human neurons.

sentences: ERp44 is Required for Endocardial Cushion Development by Regulating VEGFA Secretion in Myocardium Rationale: Endocardial cushions are precursors of the valvoseptal complex that separates the four heart chambers and control blood flow through the heart. Abnormalities in endocardial cushion development lead to atrioventricular septal defects (AVSDs), which affect 1 in 2,100 live births. Several genes have been implicated in the development of endocardial cushions. Specifically, endoplasmic reticulum-resident protein 44 (ERp44) has been found to play a role in the early secretory pathway, but its function in heart development has not been well studied. Objective: The goal of this study was to investigate the role of ERp44 in heart development in mice. Approach and Results: Using conventional and tissue-specific knockout mouse models, we demonstrated that ERp44 plays a specific role in heart development. ERp44 knockout (KO) mice were smaller in size, and most mice died during early postnatal life. KO hearts exhibited the typical phenotypes of congenital heart diseases, such as abnormal heart shapes as well as severe septal and valvular defects. Similar phenotypes were found in cTnt-cre+/-; Erp44fl/fl mice, which indicated that myocardial ERp44 principally controls endocardial cushion formation. Further studies demonstrated that the deletion of ERp44 significantly decreased the proliferation of cushion cells and impaired the endocardial-mesenchymal transition (EndMT), which was followed by endocardial cushion dysplasia. Finally, we found that ERp44 directly bound to VEGFA and controlled its release. Conclusions: ERp44 contributes to the development of the endocardial cushion by affecting the EndMT of cushion cells by regulating VEGFA release in myocardial cells.

sentences: Unrestrained growth of correctly oriented microtubules instructs axonal microtubule orientation In many eukaryotic cells, directed molecular transport occurs along microtubules. Within neuronal axons, transport over vast distances particularly relies on uniformly oriented microtubules, whose +-ends point towards the distal axon tip (+end out). However, axonal microtubules initially have mixed orientations, and how they orient during development is not yet fully understood. Using live imaging of primary Drosophila melanogaster neurons and physical modelling, we found that +end out microtubules are less likely to undergo catastrophe near the advancing axon tip, leading to their persistent long-term growth. In contrast, oppositely oriented microtubules remain short. Using chemical and physical perturbations of microtubule growth and genetic perturbations of the anti -catastrophe factor p150, which was enriched in the distal axon tip, we confirmed that the enhanced growth of +end out microtubules is critical for achieving uniform microtubule orientation. Computer simulations of axon development mimicking the enhanced +end out microtubule growth identified here along with previously proposed mechanisms correctly predicted the long-term evolution of axonal microtubule orientation as found in our experiments, highlighting the importance of the reduced catastrophe rate of +end out microtubules near the advancing axon tip in establishing uniform microtubule polarity. Our study thus leads to a holistic explanation of how axonal microtubules orient uniformly, a prerequisite for efficient long-range transport essential for neuronal functioning.

sentences: Modulation of RNA polymerase processivity affects double-strand break repair in the presence of a DNA-end binding protein Homologous recombination is the predominant double-strand DNA break repair pathway in Escherichia coli. A recent report of non-homologous end joining involving the Ku-like Gam protein of bacteriophage Mu (MuGam) has sparked interest in understanding the functions of DNA end-binding proteins in bacteria. MuGam binds to DNA ends, but how it interferes with DNA repair or transcription in live bacteria remains elusive. In E. coli, RNA polymerase secondary channel interactors, such as the Gre factors, play a role in coordinating transcription with DNA replication and break repair. Here we show that MuGam inhibits break repair by slowing down DNA resection and impeding recombination in living cells. Loss of GreA restores break repair in the presence of MuGam by allowing for increased DNA resection due to a potential release of MuGam from the DNA. Using MuGam as a DNA break sensor, we found that breaks are generated when translation is inhibited, more so in the presence of GreA, supporting the model where transcription-translation uncoupling increases transcription/replication collisions. Significantly, this work reveals that modulation of RNA polymerase can impact DNA break repair in presence of a Ku-like protein.

sentences: The importance of environmentally-acquired bacterial symbionts for the squash bug (Anasa tristis), a significant agricultural pest Most insects maintain associations with microbes that shape their ecology and evolution. Such symbioses have important applied implications when the associated insects are pests or vectors of disease. The squash bug, Anasa tristis (Coreoidea: Coreidae), is a significant pest of human agriculture in its own right and also causes damage to crops due to its capacity to transmit a bacterial plant pathogen. Here, we demonstrate that complete understanding of these insects requires consideration of their association with bacterial symbionts in the family Burkholderiaceae. Isolation and sequencing of bacteria housed in midgut crypts in these insects indicates that these bacteria are consistent and dominant members of the crypt-associated bacterial communities. These symbionts are closely related to Caballeronia spp. associated other true bugs in the superfamiles Lygaeoidea and Coreoidea. Fitness assays with representative Burkholderiaceae strains indicate that the association can significantly increase survival and decrease development time, though strains do vary in the benefits that they confer to their hosts, with Caballeronia spp. providing the greatest benefit. Experiments designed to assess transmission mode indicate that unlike many other beneficial insect symbionts, the bacteria are not acquired from parents before or after hatching but are instead acquired from the environment after molting to a later development stage. The bacteria do, however, have the capacity to escape adults to be transmitted to later generations, leaving the possibility for a combination of indirect vertical and horizontal transmission.

sentences: Pathogenic mutations in the chromokinesin KIF22 disrupt anaphase chromosome segregation The chromokinesin KIF22 generates forces that contribute to mitotic chromosome congression and alignment. Mutations in the 2 helix of the motor domain of KIF22 have been identified in patients with abnormal skeletal development, and we report the identification of a patient with a novel mutation in the KIF22 tail. We demonstrate that pathogenic mutations do not result in a loss of KIF22s functions in early mitosis. Instead, mutations disrupt chromosome segregation in anaphase, resulting in reduced proliferation, abnormal daughter cell nuclear morphology, and, in a subset of cells, cytokinesis failure. This phenotype could be explained by a failure of KIF22 to inactivate in anaphase. Consistent with this model, constitutive activation of the motor via a known site of phosphoregulation in the tail phenocopied the effects of pathogenic mutations. These results suggest the motor domain 2 helix may be an important site for regulation of KIF22 activity at the metaphase to anaphase transition. In support of this conclusion, mimicking phosphorylation of 2 helix residue T158 also prevents inactivation of KIF22 in anaphase. These findings demonstrate the importance of both the head and tail of the motor in regulating the activity of KIF22 and offer insight into the cellular consequences of preventing KIF22 inactivation and disrupting force balance in anaphase.

sentences: FLI1 and FRA1 transcription factors drive the transcriptional regulatory networks characterizing muscle invasive bladder cancer Bladder cancer is mostly present in the form of urothelium carcinoma, causing over 150.000 deaths each year. Its histopathological classification as muscle invasive (MIBC) and non-muscle invasive (NMIBC) is the most prominent aspect, affecting the prognosis and progression of this disease. In this study, we defined the active regulatory landscape of MIBC and NMIBC cell lines using H3K27ac-seq and used an integrative data approach to combine our findings with existing data. Our analysis revealed FRA1 and FLI1 as the two critical transcription factors differentially regulating MIBC regulatory landscape. Importantly, we show that FRA1 and FLI1 regulate the genes involved in epithelial cell migration and cell junction organization. Knock-down of FRA1 and FLI1 in MIBC revealed the downregulation of several EMT-related genes such as MAP4K4 and FLOT1. Further, ChIP-SICAP performed for FRA1 and FLI1 enabled us to infer chromatin binding partners of these two transcription factors and link this information with their target genes, providing a comprehensive regulatory circuit for the genes implicated in invasive ability of the bladder cancer cells. Finally, for the first time we show that knock-down of FRA1 and FRA1-FLI1 double knock-down results in significant reduction of invasion capacity of MIBC cells towards muscle microenvironment using IC-CHIP assays. Our results collectively highlight the role of these two transcription factors in invasive characteristics of bladder cancer in selection and design of targeted options for treatment of MIBC.

sentences: Obesity affects mitochondrial metabolism and proliferative potential of equine endometrial progenitor cells. The study aimed to investigate the influence of obesity on cellular features of equine endometrial progenitor cells (Eca EPCs), including viability, proliferation capacity, mitochondrial metabolism, and oxidative homeostasis. Eca EPCs derived from non-obese (Non-OB) and obese (OB) mares were characterized by cellular phenotype and multipotency. Obesity-induced changes in the activity of Eca EPCs include the decline of their proliferative activity, clonogenic potential, mitochondrial metabolism and enhanced oxidative stress. Eca EPCs isolated from obese mares were characterized by an increased occurrence of early apoptosis, loss of mitochondrial dynamics, and senescence-associated phenotype. Attenuated metabolism of Eca EPCs OB was related to increased expression of pro-apoptotic markers (CASP9, BAX, P53, P21), enhanced expression of OPN, PI3K and AKT, simultaneously with decreased signaling stabilizing cellular homeostasis (including mitofusin, SIRT1, FOXP3). Obesity alters functional features and the self-renewal potential of endometrial progenitor cells. The impaired cytophysiology of progenitor cells from obese endometrium predict lower regenerative capacity if used as autologous transplants.

sentences: Evolutionary rates of testes-expressed genes differ between monogamous and promiscuous Peromyscus species Reproductive proteins, including those expressed in the testes, are among the fastest evolving proteins across the tree of life. Sexual selection on traits involved in sperm competition is thought to be a primary driver of testes gene evolution and is expected to differ between promiscuous and monogamous species due to intense competition between males to fertilize females in promiscuous lineages and lack thereof in monogamous ones. Here, we employ the rodent genus Peromyscus as a model to explore differences in evolutionary rates between testes-expressed genes in monogamous and promiscuous species. We find candidate genes that may be associated with increased sperm production in promiscuous species and gene ontology categories that show patterns of molecular convergence associated with phenotypic convergence in independently evolved monogamous species. Overall, our results highlight the possible molecular consequences of differences in mating system, likely due to differences in selective pressures.

sentences: Comparative miRNA Transcriptomics of Mouse and Macaque Reveals MYOC is An Inhibitor for C. neoformans Invasion into Brain Cryptococcal meningoencephalitis is an emerging infection shifted from primarily ART-naive to being ART-experienced HIV/AIDS patients, COVID-19 patients and also in immune competent individuals, mainly caused by the human opportunistic pathogen Cryptococcus neoformans, yet mechanisms of the brain or CNS dissemination remain to elucidate, which is the deadest process for the disease. Meanwhile, illustrations of clinically relevant responses in cryptococcosis were limited, as the low availabilities of clinical samples. In this study, macaque and mouse infection models were employed and miRNA-mRNA transcriptomes were performed and combined, which revealed cytoskeleton, a major feather in HIV/AIDS patients, was a centric pathway regulated in both two infection models. Notably, assays of clinical immune cells confirmed an enhanced "Trojan Horse" in HIV/AIDS patients, which can be shut down by cytoskeleton inhibitors. Furthermore, we identified a novel enhancer for macrophage "Trojan Horse", myocilin, and an enhanced fungal burden was achieved in brains of MYOC transgenic mice. Taking together, this study reveals fundamental roles of cytoskeleton and MYOC in blocking fungal CNS dissemination, which not only helps to understand the high prevalence of cryptococcal meningitis in HIV/AIDS, but also facilitates the development of novel drugs for therapies of meningoencephalitis caused by C. neoformans and other pathogenic microorganisms.

sentences: A novel method for producing functionalized vesicles that efficiently deliver oligonucleotides in vitro in cancer cells and in vivo in mice Nano-based delivery systems have enhanced our ability to administer and target drugs and macromolecules to their targets. Oligonucleotide drugs have great therapeutic potential but often have off-target effects and stability issues. Therefore, they are often encapsulated in vesicles with targeting ligands such as antibodies (Ab) to deliver their cargo. Herein, we describe a novel, scalable and straightforward approach to producing functionalized vesicles called the "Functionalized Lipid Insertion Method," which differs from the older "Detergent-Dialysis Method." The latter method required excess detergent and extensive dialysis over many hours to produce the functionalized vesicles. With our method, only the functionalized lipid is detergent-solubilized during the engineering of the vesicle. The approach reduces the dialysis time, keeps the vesicles intact while orienting the targeting moieties of the functionalized lipid toward the outside of the vesicle. Pilot in vitro and in vivo experiments was performed to show the feasibility of our method. Dynamic light scattering (DLS) experiments suggested that the original vesicular structure was relatively unperturbed, and the functionalized lipid was inserted externally. Our approach efficiently delivered oligonucleotides and affected the function of liver cancer HepG2 cells. Furthermore, functionalized vesicles achieved targeted delivery of oligonucleotides in mice without inducing a significant innate immune response. The industrial and therapeutic significance and implications of functionalized vesicles produced by our method are also discussed. Additional experiments and analyses are recommended to bring out the full potential of this molecular delivery technology.

sentences: A computationally efficient clustering linear combination approach to jointly analyze multiple phenotypes for GWAS There has been an increasing interest in joint analysis of multiple phenotypes in genome-wide association studies (GWAS) because jointly analyzing multiple phenotypes may increase statistical power to detect genetic variants associated with complex diseases or traits. Recently, many statistical methods have been developed for joint analysis of multiple phenotypes in genetic association studies, including the Clustering Linear Combination (CLC) method. The CLC method works particularly well with phenotypes that have natural groupings, but due to the unknown number of clusters for a given data, the final test statistic of CLC method is the minimum p-value among all p-values of the CLC test statistics obtained from each possible number of clusters. Therefore, a simulation procedure must be used to evaluate the p-value of the final test statistic. This makes the CLC method computationally demanding. We develop a new method called computationally efficient CLC (ceCLC) to test the association between multiple phenotypes and a genetic variant. Instead of using the minimum p-value as the test statistic in the CLC method, ceCLC uses the Cauchy combination test to combine all p-values of the CLC test statistics obtained from each possible number of clusters. The test statistic of ceCLC approximately follows a standard Cauchy distribution, so the p-value can be obtained from the cumulative density function without the need for the simulation procedure. Through extensive simulation studies and application on the COPDGene data, the results demonstrate that the type I error rates of ceCLC are effectively controlled in different simulation settings and ceCLC either outperforms all other methods or has statistical power that is very close to the most powerful method with which it has been compared.

sentences: Bayesian model comparison for rare variant association studies Whole genome sequencing studies applied to large populations or biobanks with extensive phenotyping raise new analytic challenges. The need to consider many variants at a locus or group of genes simultaneously and the potential to study many correlated phenotypes with shared genetic architecture provide opportunities for discovery and inference that are not addressed by the traditional one variant, one phenotype association study. Here, we introduce a Bayesian model comparison approach that we refer to as MRP (Multiple Rare-variants and Phenotypes) for rare-variant association studies that considers correlation, scale, and direction of genetic effects across a group of genetic variants, phenotypes, and studies. The approach requires only summary statistic data. To demonstrate the efficacy of MRP, we apply our method to exome sequencing data (N = 184,698) across 2,019 traits from the UK Biobank, aggregating signals in genes. MRP demonstrates an ability to recover previously-verified signals such as associations between PCSK9 and LDL cholesterol levels. We additionally find MRP effective in conducting meta-analyses in exome data. Notable non-biomarker findings include associations between MC1R and red hair color and skin color, IL17RA and monocyte count, IQGAP2 and mean platelet volume, and JAK2 and platelet count and crit (mass). Finally, we apply MRP in a multi-phenotype setting; after clustering the 35 biomarker phenotypes based on genetic correlation estimates into four clusters, we find that joint analysis of these phenotypes results in substantial power gains for gene-trait associations, such as in TNFRSF13B in one of the clusters containing diabetes and lipid-related traits. Overall, we show that the MRP model comparison approach is able to improve upon useful features from widely-used meta-analysis approaches for rare variant association analyses and prioritize protective modifiers of disease risk.

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Maximizing the Anti-tumor Potential of Immune Checkpoint Blockade through Modulation of Myeloid-specific CXCL16 and STAT1 Signaling Sensitivity to immune checkpoint blockades (ICB) depends on the overall balance of immunogenic and immunosuppressive signals in the tumor immune microenvironment (TIME). Chemotherapy as an immunostimulatory strategy showed potential in improving ICBs clinical efficacy. Yet, evolution of highly plastic tumor-associated myeloid cells hinders ICBs potential to reach its full therapeutic potential. In this study, we leveraged single-cell transcriptomic and trajectory analyses to delineate TIME dynamics after chemotherapy priming. We found that metronomic chemotherapy (MCT) treatment led to an accelerated T cell exhaustion through CXCL16-mediated recruitment of peripheral immature myeloid cells and expansion of STAT1-driven PD-L1 expressing myeloid cells. Inhibiting STAT1 signaling in MCT-primed breast cancer relieved T cell exhaustion and significantly enhanced the efficacy of anti-PD-1 ICB treatment. Our study leveraged single-cell analyses to dissect the dynamics of breast cancer TIME and provides a pre-clinical rationale to translate the anti-STAT1 plus anti-PD-1 combinatorial immunotherapy regimen to maximize ICBs efficacy.

Manuscript SummarySingle-cell analyses on low dose chemotherapy primed breast tumor-associated immune cells demonstrates a parallel coexistence of immunogenic and immunosuppressive myeloid cell subsets. Modulating STAT1 signaling in the tumor microenvironment fine-tunes immunogenic and immunosuppressive balance and maximizes the anti-PD-1 immunotherapy efficacy in chemotherapy-primed breast cancer.

sentences: ProtView: A versatile tool for in silico protease evaluation and selection in a proteomic and proteogenomic context Tools have been created to generate in silico proteome digests with different protease enzymes and provide useful information for selecting optimal digest schemes for specific needs. This can save on time and resources and generate insights on the observable proteome. However, there remains a need for a tool that evaluates digest schemes beyond protein and amino acid coverages in the proteomic domain. Here, we present ProtView, a versatile in-silico protease/protease combination and digest evaluation workflow that maps in silico digested peptides to both protein and genome references, so that the potential observable sections of the proteome, transcriptome and genome can be identified. This supports the identification and quantification of the proteomic evidence of transcriptional, co-transcriptional, post-transcriptional and translational regulations. Benchmarking against biological data comparing multiple proteases shows that ProtView can correctly estimate the relative performances among the digest schemes. ProtView provides this information in a way that is easy to interpret, allowing for digest schemes to be evaluated before carrying out an analysis, in a broader context to optimize proteomic and proteogenomic experiments. ProtView is available at https://github.com/SSPuliasis/ProtView.

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Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue resulting in junction specific features. ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue specific pre-hydrogel digests. ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities which were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissuespecific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. ECM conditioned media had only a minor effect on upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, the extracellular matrix protein type XXII collagen had similar expression patterns as paxillin, with smaller effect sizes. Using tissue specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscletendon junctions to study the expression of MTJ specific proteins.

Impact StatementThe muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, it is overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction specific features and markers that should be included in tissue models of the muscletendon unit, while gaining a deeper understanding of the natural junction. This research aims to inform future methods to engineer a more relevant multi-tissue platform to study the muscletendon unit.

sentences: Performance of RNA purification kits and blood collection tubes in the Extracellular RNA Quality Control (exRNAQC) study The use of blood-based extracellular RNA (exRNA) as clinical biomarker requires the implementation of a validated procedure for sample collection, processing and profiling. So far, no study has systematically addressed the pre-analytical variables affecting transcriptome analysis of exRNAs. In the exRNAQC study, we evaluated 10 blood collection tubes, 3 time points between blood draw and downstream processing, and 8 RNA purification methods using the supplier-specified minimum and maximum biofluid input volumes. The impact of these pre-analytics is assessed by deep transcriptome profiling of both small and messenger RNA from healthy donors plasma or serum. Experiments are conducted in triplicate (for a total of 276 transcriptomes) using 189 synthetic spike-in RNAs as processing controls. When comparing blood tubes, so-called blood preservation tubes do not stabilize RNA very well, as is reflected by increasing RNA concentration and number of detected genes over time, and by compromised reproducibility. We also document large differences in RNA purification kit performance in terms of sensitivity, reproducibility, and observed transcriptome complexity. Our results are summarized in 11 performance metrics that enable an informed selection of the most optimal sample processing workflow for your own experiments. In conclusion, we put forward robust quality control metrics for exRNA quantification methods with validated standard operating procedures (SOPs) for processing, representing paramount groundwork for future exRNA-based precision medicine applications.

sentences: Archaic introgression and variation in pharmacogenes and implications for local adaptation Modern humans carry Neanderthal and Denisovan (archaic) genome elements which may have been adaptive. These effects may be particularly evident in pharmacogenes - genes responsible for the processing of exogenous substances such as food, pollutants, and medications. However, the health implications and contribution of archaic ancestry in pharmacogenes of modern humans remains understudied. We characterize eleven key cytochrome P450 (CYP450) genes involved in drug metabolizing reactions in three Neanderthal and one Denisovan individuals and examine archaic introgression in modern human populations. We infer the metabolizing efficiency of these eleven genes in archaic individuals and show important genetic differences relative to modern human variants. We identify archaic-specific SNVs in each CYP450 gene, including some that are potentially damaging, which may result in altered metabolism in modern human people carrying these variants. We highlight four genes which display interesting patterns of archaic variation: CYP2B6 - we find a large number of unique variants in the Vindija Neanderthal, some of which are shared with a small subset of African modern humans; CYP2C9 - containing multiple variants that are shared between Europeans and Neanderthals; CYP2A6*12 - a variant defined by a hybridization event that was found in humans and Neanderthals, suggesting the recombination event predates both species; and CYP2J2 - in which we hypothesize a Neanderthal variant was re-introduced in non-African populations by archaic admixture. The genetic variation identified in archaic individuals suggests that selective pressures from the environment may be involved in the evolution of CYP450 genes.

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Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner BackgroundPannexin 3 (PANX3), is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggests it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high fat diet (HFD).

MethodsC57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 - 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression.

ResultsIn females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight, and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. Additionally, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, in KO mice, multiple inflammatory genes were significantly differently expressed in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype compared to WT mice. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs.

ConclusionPANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

sentences: Functional and Genetic Analysis of Viral Receptor ACE2 Orthologs Reveals a Broad Potential Host Range of SARS-CoV-2 The pandemic of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major global health threat. Epidemiological studies suggest that bats are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. It has been shown that human, palm civet, pig and bat ACE2 can support virus entry, while the murine ortholog cannot. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. We found that ACE2 is expressed in a wide range of species, with especially high conservation in mammals. By analyzing amino acid residues of ACE2 critical for virus entry, based on structure of SARS-CoV spike protein interaction with human, bat, palm civet, pig and ferret ACE2, we identified approximately eighty ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 representative ACE2 orthologs among eighty orthologs for functional analysis and it showed that 44 of these mammalian ACE2 orthologs, including those of domestic animals, pets, livestock, and animals commonly found in zoos and aquaria, could bind SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. In summary, our study demonstrates that ACE2 from a remarkably broad range of species can facilitate SARS-CoV-2 entry. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.

sentences: Single-trial-based Temporal Principal Component Analysis on Extracting Event-related Potentials of Interest for an Individual Subject Temporal principal component analysis (t-PCA) has been widely used to extract event-related potentials (ERPs) at the group level of multiple subjects ERP data. The t-PCA is, however, poorly employed to isolate ERPs from single-trial data of an individual subject. Additionally, the effects of varied trial numbers on the yields of t-PCA have not been systematically examined. To fill both gaps, in an emotional experiment (22 subjects), we use t-PCA and Promax rotation to extract interesting P2/N2 from single-trial data of each subject with consecutive increasingly trial numbers (from 10 to 42) and all trials, respectively. Besides, time-domain analysis and other two group t-PCA strategies (trial-averaged and single-trial) are also employed to isolate ERPs of interest from all subjects. The results indicate that the proposed technique produces the internal consistent measure of N2 from few trials (i.e., 19) as from all trials compared with the other three approaches (more than 30 trials). As for P2, all approaches yield internal-subject consistent effect after approximately 33 trials are included in the average, but Cronbachs alpha values for the proposed technique are higher than the other two group PCA strategies over varied trials. Combined, the yields provide evidence that the proposed approach may efficiently temporally filter the data to extract more reliable and stable ERPs for an individual subject.

sentences: Illuminating links between cis-regulators and trans-acting variants in the human prefrontal cortex Psychiatric disorders exact immense human and economic tolls in societies globally. Underlying many of these disorders is a complex repertoire of genomic variants that influence the expression of genes involved in pathways and processes in the brain. Identifying such variants and their associated brain functions is thus essential for understanding the molecular underpinnings of psychiatric disorders. Genome-wide association studies (GWASes) have provided many variants associated with these disorders; however, our knowledge of the precise biological mechanisms by which these contribute to disease remains limited. In connection with this, expression quantitative trait loci (eQTLs) have provided useful information linking variants to genes and functions. However, most eQTL studies on human brain have focused exclusively on cis-eQTLs. A complete understanding of disease etiology should also include trans-regulatory mechanisms. Thus, we conduct one of the first genome-wide surveys of trans-eQTLs in the dorsolateral prefrontal cortex (DLPFC) by leveraging the large datasets from the PsychENCODE consortium. We identified [~]80,000 trans-eQTLs. We found that a significant number of these overlap with cis-eQTLs, thereby implicating cis-mediators as key players in trans-acting regulation. We show, furthermore, that trans-regulatory mechanisms provide novel insights into psychiatric disease. Particularly, colocalization analysis between trans-eQTLs and schizophrenia (SCZ) GWAS loci identified 90 novel SCZ risk genes and 23 GWAS loci previously uncharacterized by cis-eQTLs. Moreover, these 90 genes tend to be more central in transcriptome-wide co-expression networks and more susceptible to rare variants than SCZ-risk genes associated by cis-variation.

sentences: Locally synchronized ciliary domains and tissue-scale cilia alignment underlie global metachronal wave patterns Motile cilia are hair-like cell extensions present in multiple organs of the body. How cilia coordinate their regular beat in multiciliated epithelia to move fluids remains insufficiently understood, particularly due to lack of rigorous quantification. We combine here experiments, novel analysis tools, and theory to address this knowledge gap. We investigate collective dynamics of cilia in the zebrafish nose, due to its conserved properties with other ciliated tissues and its superior accessibility for non-invasive imaging. We revealed that cilia are synchronized only locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium. Despite the fact that synchronization is local only, we observed global patterns of traveling metachronal waves across the multiciliated epithelium. Intriguingly, these global wave direction patterns are conserved across individual fish, but different for left and right nose, unveiling a chiral asymmetry of metachronal coordination. To understand the implications of synchronization for fluid pumping, we used a computational model of a regular array of cilia. We found that local metachronal synchronization prevents steric collisions and improves fluid pumping in dense cilia carpets, but hardly affects the direction of fluid flow. In conclusion, we show that local synchronization together with tissue-scale cilia alignment are sufficient to generate metachronal wave patterns in multiciliated epithelia, which enhance their physiological function of fluid pumping.

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Altered hippocampal transcriptome dynamics following sleep deprivation Widespread sleep deprivation is a continuing public health problem in the United States and worldwide affecting adolescents and adults. Acute sleep deprivation results in decrements in spatial memory and cognitive impairments. The hippocampus is vulnerable to acute sleep deprivation with changes in gene expression, cell signaling, and protein synthesis. Sleep deprivation also has long lasting effects on memory and performance that persist after recovery sleep, as seen in behavioral studies from invertebrates to humans. Although previous research has shown that acute sleep deprivation impacts gene expression, the extent to which sleep deprivation affects gene regulation remains unknown. Using an unbiased deep RNA sequencing approach, we investigated the effects of acute sleep deprivation on gene expression in the hippocampus. We identified 1,146 genes that were significantly dysregulated following sleep deprivation with 507 genes upregulated and 639 genes downregulated, including protein coding genes and long non-coding RNAs not previously identified as impacted by sleep deprivation. Notably, genes significantly upregulated after sleep deprivation were associated with RNA splicing and the nucleus. In contrast, downregulated genes were associated with cell adhesion, dendritic localization, the synapse, and postsynaptic membrane. These results clearly demonstrate that sleep deprivation differentially regulates gene expression on multiple transcriptomic levels to impact hippocampal function.

Graphical Abstract

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sentences: Mitochondrial fusion regulates proliferation and differentiation in the type II neuroblast lineage in Drosophila Optimal mitochondrial function determined by mitochondrial dynamics, morphology and activity is coupled to stem cell differentiation and organism development. However, the mechanisms of interaction of signaling pathways with mitochondrial morphology and activity are not completely understood. We assessed the role of mitochondrial fusion and fission in differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depletion of mitochondrial inner membrane fusion protein Opa1 and mitochondrial outer membrane protein Marf in the Drosophila type II neuroblast lineage led to mitochondrial fragmentation and loss of activity. Opa1 and Marf depletion did not affect the numbers and polarity of type II neuroblasts but led to a decrease in proliferation and differentiation of cells in the lineage. On the contrary, loss of mitochondrial fission protein Drp1 led to mitochondrial fusion but did not show defects in proliferation and differentiation. Depletion of Drp1 along with Opa1 or Marf also led to mitochondrial fusion and suppressed fragmentation, loss of mitochondrial activity, proliferation and differentiation in the type II NB lineage. We found that Notch signaling depletion via the canonical pathway showed mitochondrial fragmentation and loss of differentiation similar to Opa1 mutants. An increase in Notch signaling required mitochondrial fusion for NB proliferation. Further, Drp1 mutants in combination with Notch depletion showed mitochondrial fusion and drove differentiation in the lineage suggesting that fused mitochondria can influence Notch signaling driven differentiation in the type II NB lineage. Our results implicate a crosstalk between Notch signalling, mitochondrial activity and mitochondrial fusion as an essential step in type II NB differentiation.

sentences: Critical interactions for SARS-CoV-2 spike protein binding to ACE2 identified by machine learning Both SARS-CoV and SARS-CoV-2 bind to the human ACE2 receptor. Based on high-resolution structures, the two viruses bind in practically identical conformations, although several residues of the receptor-binding domain (RBD) differ between them. Here we have used molecular dynamics (MD) simulations, machine learning (ML), and free energy perturbation (FEP) calculations to elucidate the differences in RBD binding by the two viruses. Although only subtle differences were observed from the initial MD simulations of the two RBD-ACE2 complexes, ML identified the individual residues with the most distinctive ACE2 interactions, many of which have been highlighted in previous experimental studies. FEP calculations quantified the corresponding differences in binding free energies to ACE2, and examination of MD trajectories provided structural explanations for these differences. Lastly, the energetics of emerging SARS-CoV-2 mutations were studied, showing that the affinity of the RBD for ACE2 is increased by N501Y and E484K mutations but is slightly decreased by K417N.

sentences: Inhibiting adult neurogenesis differentially affects spatial learning in females and males Adult hippocampal neurogenesis has been implicated in the spatial processing functions of the hippocampus but ablating neurogenesis does not consistently lead to behavioral deficits in spatial tasks. Parallel studies have shown that adult-born neurons also regulate behavioral responses to stressful and aversive stimuli. We therefore hypothesized that spatial functions of adult-born neurons may be more prominent under conditions of stress, and may differ between males and females given established sex differences in stress responding. To test this we trained intact and neurogenesis-deficient rats in the spatial water maze at temperatures that vary in their degree of aversiveness. At standard temperatures (25{degrees}C) ablating neurogenesis did not alter learning and memory in either sex, consistent with prior work. However, in cold water (16{degrees}C), ablating neurogenesis had divergent sex-dependent effects: relative to intact rats, male neurogenesis-deficient rats were slower to escape and female neurogenesis-deficient rats were faster. Neurogenesis promoted temperature-related changes in search strategy in females, but it promoted search strategy stability in males. Females displayed greater recruitment of the dorsal hippocampus than males, particularly at 16{degrees}C. However, blocking neurogenesis did not alter activity-dependent immediate-early gene expression in either sex. Finally, morphological analyses of retrovirally-labelled neurons revealed greater experience-dependent plasticity in new neurons in males. Neurons had comparable morphology in untrained rats but 16{degrees}C training increased spine density, and 25{degrees}C training caused shrinkage of mossy fiber presynaptic terminals, specifically in males. Collectively, these findings indicate that neurogenesis functions in memory are prominent under conditions of stress, they provide the first evidence for sex differences in the behavioral function of newborn neurons, and they suggest possibly distinct roles for neurogenesis in cognition and mental health in males and females.

sentences: Theta oscillations in frontal cortex differentially modulate accuracy and speed in flexible stimulus- and action-based learning Flexible reward learning relies on frontal cortex, with substantial evidence indicating the anterior cingulate cortex (ACC) and orbitofrontal cortex (OFC) play important roles. Recent studies in both rat and macaque suggest theta oscillations (5-10 Hz) may be a spectral signature that coordinates this learning. However, causal network-level interactions between ACC and OFC in flexible learning remain unclear. We investigated the learning of stimulus-outcome (S-O) and action-outcome (A-O) associations using a combination of simultaneous in-vivo electrophysiology in dorsal ACC and ventral OFC, partnered with bilateral inhibitory DREADDs in ACC. In freely-behaving male and female rats and using a within-subject design, we examined accuracy and speed of response across distinct and precisely-defined trial epochs including correct choice, incorrect choice, and reward collection. We report significant modulation of accuracy by theta power in both ACC and OFC. Both ACC and OFC theta oscillations consistently signaled accuracy in the initial discrimination phase, whereas it was OFC theta alone in the reversal phases of both S-O and A-O learning. Theta power in either region was not correlated with deliberation speed. Indeed, theta modulation of accuracy was dissociable from its involvement in speed of response which was affected by ACC inhibition, most prominently in A-O learning. Results are discussed with reference to the nonhuman primate literature, where there is by contrast more reported specialization of OFC and ACC in flexible learning of stimuli and actions. The present results also point to a specific role of OFC theta in signaling a reversal of either kind.

sentences: Using ultraconserved elements to reconstruct the termite tree of life The phylogenetic history of termites has been investigated using mitochondrial genomes and transcriptomes. However, both sets of markers have specific limitations. Mitochondrial genomes represent a single genetic marker likely to yield phylogenetic trees presenting incongruences with species trees, and transcriptomes can only be obtained from well-preserved samples. In contrast, ultraconserved elements (UCEs) include a great many independent markers that can be retrieved from poorly preserved samples. Here, we designed termite-specific baits targeting 50,616 UCE loci. We tested our UCE bait set on 42 samples of termites and three samples of Cryptocercus, for which we generated low-coverage highly-fragmented genome assemblies and successfully extracted in silico between 3,426 to 42,860 non-duplicated UCEs per sample. Our maximum likelihood phylogenetic tree, reconstructed using the 5,934 UCE loci retrieved from upward of 75% of samples, was congruent with transcriptome-based phylogenies, demonstrating that our UCE bait set is reliable and phylogenetically informative. Combined with non-destructive DNA extraction protocols, our UCE bait set provides the tool needed to carry out a global taxonomic revision of termites based on poorly preserved specimens such as old museum samples. The Termite UCE database is maintained at: https://github.com/oist/TER-UCE-DB/.

sentences: Dorsal striatum coding for the timely execution of action sequences The automatic initiation of actions can be highly functional. But occasionally these actions cannot be withheld and are released at inappropriate times, impulsively. Striatal activity has been shown to participate in the timing of action sequence initiation and it has been linked to impulsivity. Using a self- initiated task, we trained adult rats to withhold a rewarded action sequence until a waiting time interval has elapsed. By analyzing neuronal activity we show that the striatal response preceding the initiation of the learned sequence is strongly modulated by the time subjects wait before eliciting the sequence. Interestingly, the modulation is steeper in adolescent rats, which show a strong prevalence of impulsive responses compared to adults. We hypothesize this anticipatory striatal activity reflects the animals subjective reward expectation, based on the elapsed waiting time, while its steeper waiting modulation in adolescence reflects age-related differences in temporal discounting, internal urgency states or explore- exploit balance.

sentences: The dorsal hippocampus is required for the formation of long-term duration memories in rats Interval timing--the perception of durations mainly in seconds or minutes--is a ubiquitous behavior in organisms. Animal studies have suggested that the hippocampus plays an essential role in duration memory; however, the memory processes involved are unclear. To clarify the role of the dorsal hippocampus in the acquisition of long-term duration memories, we adapted the "time-shift paradigm" to a peak-interval procedure. After a sufficient number of training with an initial target duration (20 s), the rats underwent "shift sessions" with a new target duration (40 s) under a muscimol (0.5 g per side) infusion into the bilateral dorsal hippocampus. The memory of the new target duration was then tested in drug-free "probe sessions," including trials in which no lever presses were reinforced. In the probe sessions, the mean response rate distribution of the muscimol group was located leftward to the control group, but these two response rate distributions were superimposed on the standardized time axis, suggesting a scalar property. In the session-by-session analysis, the mean peak time (an index of timing accuracy) of the muscimol group was lower than that of the control group in the probe sessions, but not in the shift sessions. These findings suggest that the dorsal hippocampus is required for the formation of long-term duration memories within the range of interval timing.

sentences: A burst of regulatory and protein innovation at the origin of placental mammals drove the emergence of placenta and underpins divergent early pregnancy strategies in modern mammals. Summary paragraphThe origin of live birth in mammals [~]148 million years ago was a dramatic shift in reproductive strategy, yet the molecular changes that established mammal viviparity are largely unknown. Although progesterone receptor signalling predates the origin of mammals andis highly conserved in, and critical for, successful mammal pregnancy, it alone cannot explain the origin and subsequent diversity of implantation strategies throughout the placental mammal radiation. MiRNAs are known to be flexible and dynamic regulators with a well established role in the pathophysiology of mammal placenta. We propose that a dynamic core microRNA (miRNA) network originated early in placental mammal evolution, responds to conserved mammal pregnancy cues (e.g. progesterone), and facilitates species-specific responses. Here we identify 13 miRNAs that arose at the origin of placental mammals and were subsequently retained in all descendent lineages. The expression of these 13 miRNAs in response to early pregnancy molecules is regulated in a species-specific manner in endometrial epithelia of species with extremes of implantation strategies. Furthermore, these 13 miRNAs preferentially target 84 proteins under positive selective pressure on ancestral eutherian. Discovery of this core "live-birth" toolkit and specifically adapted proteins helps explain the origin and evolution of the placenta in mammals.

sentences: Global patterns and rates of habitat transitions across the eukaryotic tree of life The successful colonisation of new habitats has played a fundamental role during the evolution of life. Salinity is one of the strongest barriers for organisms to cross, which has resulted in the evolution of distinct marine and terrestrial (including both freshwater and soil) communities. Although microbes represent by far the vast majority of eukaryote diversity, the role of the salt barrier in shaping the diversity across the eukaryotic tree is poorly known. Traditional views suggest rare and ancient marine-terrestrial transitions, but this view is being challenged by the discovery of several recently transitioned lineages. Here, we investigate habitat evolution across the tree of eukaryotes using a unique set of taxon-rich environmental phylogenies inferred from a combination of long-read and short-read metabarcoding data spanning the ribosomal DNA operon. Our results show that overall marine and terrestrial microbial communities are phylogenetically distinct, but transitions have occurred in both directions in almost all major eukaryotic lineages, with at least 350 transition events detected. Some groups have experienced relatively high rates of transitions, most notably fungi for which crossing the salt barrier has most likely been an important aspect of their successful diversification. At the deepest phylogenetic levels, ancestral habitat reconstruction analyses suggest that eukaryotes may have first evolved in non-saline habitats, and that the two largest known eukaryotic assemblages (TSAR and Amorphea) arose in different habitats. Overall, our findings indicate that crossing the salt barrier has played an important role in eukaryotic evolution by providing new ecological niches to fill.

sentences: Allogeneic CAR-invariant Natural Killer T Cells Exert Potent Antitumor Effects Through Host CD8 T cell Cross-Priming The development of allogeneic chimeric antigen receptor (CAR) T cell therapies for off-the-shelf use is a major goal yet faces two main immunological challenges, namely the risk of graft-versus-host-disease (GvHD) induction by the transferred cells and the rejection by the host immune system limiting their persistence. We demonstrate that allogeneic CAR-engineered invariant natural killer T (iNKT) cells, a cell population without GvHD-induction potential that displays immunomodulatory properties, exerted potent direct and indirect antitumor activity in murine models of B-cell lymphoma when administered across major MHC-barriers. In addition to their known direct cytotoxic effect, allogeneic CAR iNKT cells induced tumor-specific antitumor immunity through host CD8 T cell cross-priming, resulting in a potent antitumor effect lasting longer than the physical persistence of the allogeneic cells. The utilization of off-the-shelf allogeneic CAR iNKT cells could meet significant unmet needs in the clinic.

sentences: Genome characteristic of Bordetella parapertussis isolated from Iran Pertussis also known as whooping cough is a respiratory infection in humans particularly in infants and usually caused by Bordetella pertussis. However, Bordetella parapertussis can also cause a similar clinical syndrome. During 2012 to 2015, from nasal swabs sent from different provinces to the pertussis reference laboratory of Pasture Institute of Iran for pertussis confirmation, seven B. parapertussis isolates were identified by bacterial culture, biochemical tests, and the presence of IS1001 insertion in the genome by real-time PCR. Furthermore, the expression of pertactin (Prn) as one the major virulence factor for bacterial adhesion was investigated using western blot. Moreover, the genomic characteristic of one recently collected isolate, IRBP134, from a seven-month infant was investigated using Illumina NextSeq sequencing protocol. The results revealed the genome with G+C content 65% and genome size 4.7 Mbp. A total of 81 single nucleotide polymorphisms (SNPs) and 13 short insertion and deletions were found in the genome compared to the B. parapertussis 12822 as a reference genome showing ongoing evolutionary changes in our isolate. A phylogeny relationship of IRBP134 was also investigated using global B. parapertussis available genomes.

sentences: Stress relaxation amplitude of hydrogels determines migration, proliferation, and morphology of cells in 3-D The viscoelastic behavior of hydrogel matrices sensitively influences the cell behavior in 3D culture and biofabricated tissue model systems. Previous reports have demonstrated that cells tend to adhere, spread, migrate and proliferate better in hydrogels with pronounced stress relaxation. However, it is currently unknown if cells respond more sensitively to the amplitude of stress relaxation, or to the relaxation time constant. To test this, we compare the behavior of fibroblasts cultured for up to 10 days in alginate and oxidized alginate hydrogels with similar Youngs moduli but diverging stress relaxation behavior. We find that fibroblasts elongate, migrate and proliferate better in hydrogels that display a higher stress relaxation amplitude. By contrast, the cells response to the relaxation time constant was less pronounced and less consistent. Together, these data suggest that it is foremost the stress relaxation amplitude of the matrix that determines the ability of cells to locally penetrate and remodel the matrix, which subsequently leads to better spreading, faster migration, and higher cell proliferation. We conclude that the stress relaxation amplitude is a central design parameter for optimizing cell behavior in 3-D hydrogels.

sentences: Analyzing SARS CoV-2 Patient Data Using Quantum Supervised Machine Learning The novel coronavirus disease 2019 (COVID-19) has created a serious threat to global health. We developed a new quantum machine learning (QML) assisted diagnostic method that can provide an accurate diagnosis to aid decision processes of medical providers. One of the key elements in our method was to implement the quantum variational method to efficiently classify data, taking crucial multiple correlations among the features into account. We established and fine-tuned this quantum classifier by using a group of data drawn from publicly available COVID-19 cases. We have shown that QML is capable of processing patient information efficiently and accurately for the diagnosis of COVID-19.

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Category-specific fMRI Correlates of Picture Naming: a study with Arabs and Filipinos Cross cultural neuroimaging work has demonstrated differences in neural correlates of some cognitive processes between individuals from different cultures, often comparing American and Chinese subjects. In contrast, a limited number of studies examined Arab and/or Filipino participants. This fMRI study aimed to demonstrate neural activations during animal and tool picture naming by 18 healthy Arabs and 18 healthy Filipino participants. In animal naming contrasted with tool naming, Arabs preferentially activated regions in the right lateral occipital and fusiform cortices, whereas Filipinos recruited bilateral visual areas. Cross-group comparisons of animal naming revealed that Arabs recruited right visual areas more than Filipinos, who in turn recruited the cerebellum more than Arabs. In tool naming, Arabs preferentially activated a predominantly left frontoparietal network, whereas no regions were identified in Filipinos, and no differences in activation between groups were found. Using a low-demand picture-naming task, this study revealed category-specific neural activations during picture naming by Arabs and Filipinos, as well as between-group differences in animal naming. The results suggest that Arabs and Filipinos may have culture-specific differences in processing animate and inanimate pictures, and caution against generalizing findings from the more commonly studied populations, especially in verbal tasks such as picture naming.

HIGHLIGHTS{blacksquare} The neural correlates of animal and tool picture naming in Arabs and Filipinos are category specific.
{blacksquare}Animal naming by Arabs tended to preferentially activate the nondominant ventral visual stream.
{blacksquare}Animal naming by Filipinos activated bilateral visual areas, and the cerebellum.
{blacksquare}Tool naming by Arabs activated dominant frontoparietal areas related to praxis.
{blacksquare}Results suggest that Arabs and Filipinos have culture-specific differences in visual processing.

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A unified physiological framework of transitions between seizures, sustained ictal activity, and depolarization block at the single neuron level The majority of seizures recorded in humans and experimental animal models can be described by a generic phenomenological mathematical model, The Epileptor. In this model, seizure-like events (SLEs) are driven by a slow variable and occur via saddle node (SN) and homoclinic bifurcations at seizure onset and offset, respectively. Here we investigated SLEs at the single cell level using a biophysically relevant neuron model including a slow/fast system of four equations. The two equations for the slow subsystem describe ion concentration variations and the two equations of the fast subsystem delineate the electrophysiological activities of the neuron. Using extracellular K+ as a slow variable, we report that SLEs with SN/homoclinic bifurcations can readily occur at the single cell level when extracellular K+ reaches a critical value. In patients and experimental models, seizures can also evolve into sustained ictal activity (SIA) and, depolarization block (DB), activities which are also parts of the dynamic repertoire of the Epileptor. Increasing extracellular concentration of K+ in the model to values found during experimental status epilepticus and DB, we show that SIA and DB can also occur at the single cell level. Thus, seizures, SIA and DB, which have been first identified as network events, can exist in a unified framework of a biophysical model at the single neuron level and exhibit similar dynamics as observed in the Epileptor.

Author SummaryEpilepsy is a neurological disorder characterized by the occurrence of seizures. Seizures have been characterized in patients in experimental models at both macroscopic and microscopic scales using electrophysiological recordings. Experimental works allowed the establishment of a detailed taxonomy of seizures, which can be described by mathematical models. We can distinguish two main types of models. Phenomenological (generic) models have few parameters and variables and permit detailed dynamical studies often capturing a majority of activities observed in experimental conditions. But they also have abstract parameters, making biological interpretation difficult. Biophysical models, on the other hand, use a large number of variables and parameters due to the complexity of the biological systems they represent. Because of the multiplicity of solutions, it is difficult to extract general dynamical rules. In the present work, we integrate both approaches and reduce a detailed biophysical model to sufficiently low-dimensional equations, and thus maintaining the advantages of a generic model. We propose, at the single cell level, a unified framework of different pathological activities that are seizures, depolarization block, and sustained ictal activity.

sentences: Single-Cell Proteomics Study of Immune Cell Diversity by Quantitating 465 Proteins The identification and characterization of T cell subpopulations is critical to reveal cell development throughout life and immune responses to environmental factors. Next-generation sequencing technologies have dramatically advanced the single-cell genomics and transcriptomics for T cell classification. However, gene expression is often not correlated with protein expression, and immunotyping is mostly accepted in the protein format. Current single-cell proteomics technologies are either limited in multiplex capacity or not sensitive enough to detect the critical functional proteins. Herein we present a cyclic multiplex in situ tagging (Cyclic MIST) technology to simultaneously measure 465 proteins, a scale of >10 times than similar technologies, in single cells. Such a high multiplexity is achieved by reiterative staining of the single cells coupled with a MIST array for detection. This technology has been thoroughly validated through comparison with flow cytometry and fluorescence immunostaining techniques. Both THP1 and CD4+ T cells are analyzed by the Cyclic MIST technology, and over 300 surface markers have been profiled to classify the subpopulations. This represents the most comprehensive mapping of the diversity of immune cells at the protein level. With additional information from intracellular proteins of the same single cells, our technology can potentially facilitate mechanistic studies of immune responses, particularly cytokine storm that results in sepsis.

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The fish pathogen Aliivibrio salmonicida LFI1238 can degrade and metabolize chitin despite major gene loss in the chitinolytic pathway The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that Al. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose ((GlcNAc)2), the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate compared to the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both - and {beta}-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity compared to other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on {beta}-chitin, whereas the LPMO gene-deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of Al. salmonicida LFI1238 growth on {beta}-chitin showed expression of all three chitinolytic enzymes, and intriguingly also three of the disrupted chitinases. In conclusion, our results show that Al. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability.

IMPORTANCEThe ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida, the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study we demonstrate that Al. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.

sentences: In-silico Structural and Molecular Docking-Based Drug Discovery Against Viral Protein (VP40) of Marburg Virus: A Causative Agent of MAVD The Marburg virus (MARV) is reported to induce extreme hemorrhagic fever (MHF) with a high degree of infectivity and lethality in both human and non-human primates. An appropriate vaccination for this viruss treatment is not yet usable, and thus needs intensive attempts on multiple scales. In this study, we employed the Computer-Aided Drug Design (CADD) based approach to identify the drug-like compounds inhibiting the replication of the Viral protein (VP40) of MARV. Our database search using an online database "PubChem" retrieved [~]3000 compounds structure-based similarity. Lipinskis rule was applied to evaluate further the drug-like compounds, followed by molecular docking-based screening, and the selection of screening ligand complex with VP40 based on S-score (lower than reference Favipiravir inhibitor) and root-mean-square-deviation (RMSD) value (probably less than 2) using AutoDock 4.2. Resultantly, [~]100 compounds were identified having strong interaction with VP40 of MARV. After evaluating their binding energy using the AutoDock 4.2 software, four compounds (CID-67534452, CID-72201087, CID-123273976, CID-153708661) were identified that showed strongest binding energy with VP40 of MARV and strong inhibition effect than the Favipiravir. Robust binding energy, useful ADMET parameters and drug-likeness suggest that these candidates "CID-67534452, CID-72201087, CID-123273976, CID-153708661" have tremendous potential to stop the replication of MARV, hence might lead to the cure of MAVD.

sentences: Estimates of genetic load suggest frequent purging of deleterious alleles in small populations It is commonly thought that declining populations will experience negative genetic consequences as a result of increased inbreeding. Here we estimated the average deleteriousness of derived alleles in a range of mammals and found that species with historically small population size and low genetic diversity often have lower genetic load than species with large population sizes. This is likely the result of genetic purging - the more efficient removal of partially deleterious recessive alleles from inbred populations. Our findings suggest that genetic purging occurs over long evolutionary time frames, and therefore rapid population declines are likely to dis-proportionally increase mutational load in species with high diversity, as they carry many deleterious alleles that can reach fixation before genetic purging can remove them.

sentences: Mutations in two SARS-CoV-2 variants of concern reflect two distinct strategies of antibody escape Understanding the factors that contribute to antibody escape of SARS-CoV-2 and its variants is key for the development of drugs and vaccines that provide broad protection against a variety of virus variants. Using microfluidic diffusional sizing, we determined the dissociation constant (KD) for the interaction between receptor binding domains (RBDs) of SARS-CoV-2 in its original version (WT) as well as alpha and beta variants with the host-cell receptor angiotensin converting enzyme 2 (ACE2). For RBD-alpha, the ACE2-binding affinity was increased by a factor of ten when compared with RBD-WT, while ACE2-binding of RBD-beta was largely unaffected. However, when challenged with a neutralizing antibody that binds to both RBD-WT and RBD-alpha with low nanomolar KD values, RBD-beta displayed no binding, suggesting a substantial epitope change. In SARS-CoV-2 convalescent sera, RBD-binding antibodies showed low nanomolar affinities to both wild-type and variant RBD proteins--strikingly, the concentration of antibodies binding to RBD-beta was half that of RBD-WT and RBD-alpha, again indicating considerable epitope changes in the beta variant. Our data therefore suggests that one factor contributing to the higher transmissibility and antibody evasion of SARS-CoV-2 alpha and beta is a larger fraction of viruses that can form a complex with ACE2. However, the two variants employ different mechanisms to achieve this goal. While SARS-CoV-2 alpha RBD binds with greater affinity to ACE2 and is thus more difficult to displace from the receptor by neutralizing antibodies, RBD-beta is less accessible to antibodies due to epitope changes which increases the chances of ACE2-binding and infection.

sentences: Age differences in the functional architecture of the human brain The intrinsic functional organization of the brain changes into older adulthood. Age differences are observed at multiple spatial scales, from global reductions in modularity and segregation of distributed brain systems, to network-specific patterns of dedifferentiation. Whether dedifferentiation reflects an inevitable, global shift in brain function with age, circumscribed, experience dependent changes, or both, is uncertain. We employed a multi-method strategy to interrogate dedifferentiation at multiple spatial scales. Multi-echo (ME) resting-state fMRI was collected in younger (n=181) and older (n=120) healthy adults. Cortical parcellation sensitive to individual variation was implemented for precision functional mapping of each participant, while preserving group-level parcel and network labels. ME-fMRI processing and gradient mapping identified global and macroscale network differences. Multivariate functional connectivity methods tested for microscale, edge-level differences. Older adults had lower BOLD signal dimensionality, consistent with global network dedifferentiation. Gradients were largely age-invariant. Edge-level analyses revealed discrete, network-specific dedifferentiation patterns in older adults. Visual and somatosensory regions were more integrated within the functional connectome; default and frontoparietal control network regions showed greater connectivity; and the dorsal attention network was more integrated with heteromodal regions. These findings highlight the importance of multi-scale, multi-method approaches to characterize the architecture of functional brain aging.

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scDALI: Modelling allelic heterogeneity of DNA accessibility in single-cells reveals context-specific genetic regulation While the functional impact of genetic variation can vary across cell types and states, capturing this diversity remains challenging. Current studies, using bulk sequencing, ignore much of this heterogeneity, reducing discovery and explanatory power. Single-cell approaches combined with F1 genetic designs provide a new opportunity to address this problem, however suitable computational methods to model these complex relationships are lacking.

Here, we developed scDALI, an analysis framework that integrates single-cell chromatin accessibility for unbiased cell state identification with allelic quantifications to assay genetic effects. scDALI builds on Gaussian process regression and can differentiate between homogeneous (pervasive) allelic imbalances and cell state-specific regulation. As a proof-of-principle, we applied scDALI to whole Drosophila embryos from F1 crosses, profiling sciATAC-seq at three embryonic stages. Even in these very complex samples, scDALI discovered hundreds of peaks with heterogeneous allelic imbalance, having effects in specific lineages and/or developmental stages. Our study provides a general strategy to identify the cellular context of allelic imbalance, a crucial step in linking genetic traits to cellular phenotypes.

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Unravelling the Physiological Correlates of Mental Workload Variations in Tracking and Collision Prediction Tasks: Implications for Air Traffic Controllers ObjectiveWe have designed tracking and collision prediction tasks to elucidate the differences in the physiological response to the workload variations in basic ATC tasks to untangle the impact of workload variations experienced by operators working in a complex ATC environment.

BackgroundEven though several factors influence the complexity of ATC tasks, keeping track of the aircraft and preventing collision are the most crucial.

MethodsPhysiological measures, such as electroencephalogram (EEG), eye activity, and heart rate variability (HRV) data, were recorded from 24 participants performing tracking and collision prediction tasks with three levels of difficulty.

ResultsThe neurometrics of workload variations in the tracking and collision prediction tasks were markedly distinct, indicating that neurometrics can provide insights on the type of mental workload. The pupil size, number of blinks and HRV metric, root mean square of successive difference (RMSSD), varied significantly with the mental workload in both these tasks in a similar manner.

ConclusionOur findings indicate that variations in task load are sensitively reflected in physiological signals, such as EEG, eye activity and HRV, in these basic ATC-related tasks.

ApplicationThese findings have applicability to the design of future mental workload adaptive systems that integrate neurometrics in deciding not just  when but also  what to adapt. Our study provides compelling evidence in the viability of developing intelligent closed-loop mental workload adaptive systems that ensure efficiency and safety in ATC and beyond.

PrecisThis article identifies the physiological correlates of mental workload variation in basic ATC tasks. The findings assert that neurometrics can provide more information on the task that contributes to the workload, which can aid in the design of intelligent mental workload adaptive system.

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Moving Beyond the 'CAP' of the Iceberg: Intrinsic Connectivity Networks in fMRI are Continuously Engaging and Overlapping Resting-state functional magnetic resonance imaging is currently the mainstay of functional neuroimaging and has allowed researchers to identify intrinsic connectivity networks (aka functional networks) at different spatial scales. However, little is known about the temporal profiles of these networks and whether it is best to model them as continuous phenomena in both space and time or, rather, as a set of temporally discrete events. Both categories have been supported by series of studies with promising findings. However, a critical question is whether focusing only on time points presumed to contain isolated neural events and disregarding the rest of the data is missing important information, potentially leading to misleading conclusions. In this work, we argue that brain networks identified within the spontaneous blood oxygenation level-dependent (BOLD) signal are not limited to temporally sparse burst moments and that these event present time points (EPTs) contain valuable but incomplete information about the underlying functional patterns.

We focus on the default mode and show evidence that is consistent with its continuous presence in the BOLD signal, including during the event absent time points (EATs), i.e., time points that exhibit minimum activity and are the least likely to contain an event. Moreover, our findings suggest that EPTs may not contain all the available information about their corresponding networks. We observe distinct default mode connectivity patterns obtained from all time points (AllTPs), EPTs, and EATs. We show evidence of robust relationships with schizophrenia symptoms that are both common and unique to each of the sets of time points (AllTPs, EPTs, EATs), likely related to transient patterns of connectivity. Together, these findings indicate the importance of leveraging the full temporal data in functional studies, including those using event-detection approaches.

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Systematic benchmarking of all-in-one microbial SNP calling pipelines Clinical and public health microbiology is increasingly utilising whole genome sequencing (WGS) technology and this has lead to the development of a myriad of analysis tools and bioinformatics pipelines. Single nucleotide polymorphism (SNP) analysis is an approach used for strain characterisation and determining isolate relatedness. However, in order to ensure the development of robust methodologies suitable for clinical application of this technology, accurate, reproducible, traceable and benchmarked analysis pipelines are necessary. To date, the approach to benchmarking of these has been largely ad-hoc with new pipelines benchmarked on their own datasets with limited comparisons to previously published pipelines.

In this study, Snpdragon, a fast and accurate SNP calling pipeline is introduced. Written in Nextflow, Snpdragon is capable of handling small to very large and incrementally growing datasets. Snpdragon is benchmarked using previously published datasets against six other all-in-one microbial SNP calling pipelines, Lyveset, Lyveset2, Snippy, SPANDx, BactSNP and Nesoni. The effect of dataset choice on performance measures is demonstrated to highlight some of the issues associated with the current available benchmarking approaches.

The establishment of an agreed upon gold-standard benchmarking process for microbial variant analysis is becoming increasingly important to aid in its robust application, improve transparency of pipeline performance under different settings and direct future improvements and development.

Snpdragon is available at https://github.com/FordeGenomics/SNPdragon.

Impact statementWhole-genome sequencing has become increasingly popular in infectious disease diagnostics and surveillance. The resolution provided by single nucleotide polymorphism (SNP) analyses provides the highest level of insight into strain characteristics and relatedness. Numerous approaches to SNP analysis have been developed but with no established gold-standard benchmarking approach, choice of bioinformatics pipeline tends to come down to laboratory or researcher preference. To support the clinical application of this technology, accurate, transparent, auditable, reproducible and benchmarked pipelines are necessary. Therefore, Snpdragon has been developed in Nextflow to allow transparency, auditability and reproducibility and has been benchmarked against six other all-in-one pipelines using a number of previously published benchmarking datasets. The variability of performance measures across different datasets is shown and illustrates the need for a robust, fair and uniform approach to benchmarking.

Data SummaryO_LIPreviously sequenced reads for Escherichia coli O25b:H4-ST131 strain EC958 are available in BioProject PRJNA362676. BioSample accession numbers for the three benchmarking isolates are:
O_LIEC958: SAMN06245884
C_LIO_LIMS6573: SAMN06245879
C_LIO_LIMS6574: SAMN06245880
C_LI
C_LIO_LIAccession numbers for reference genomes against the E. coli O25b:H4-ST131 strain EC958 benchmark are detailed in table 2.
C_LIO_LISimulated benchmarking data previously described by Yoshimura et al. is available at http://platanus.bio.titech.ac.jp/bactsnp (1).
C_LIO_LISimulated datasets previously described by Bush et al. is available at http://dx.doi.org/10.5287/bodleian:AmNXrjYN8 (2).
C_LIO_LIReal sequencing benchmarking datasets previously described by Bush et al. are available at http://dx.doi.org/10.5287/bodleian:nrmv8k5r8 (2).
C_LI

O_TBL View this table:
org.highwire.dtl.DTLVardef@3cb198org.highwire.dtl.DTLVardef@af9421org.highwire.dtl.DTLVardef@1261244org.highwire.dtl.DTLVardef@793b42org.highwire.dtl.DTLVardef@1217387_HPS_FORMAT_FIGEXP  M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONReference genomes used in the EC958 benchmarking dataset and the percent identity against the three included samples.

C_TABLECAPTION C_TBL

sentences: A network approach to genetic circuit designs As genetic circuits become more sophisticated, the size and complexity of data about their designs increases. This data captured goes beyond monolithic genetic sequences and towards circuit modularity and functional details, which are beneficial for analyzing circuit performance and establishing design automation techniques. However, the accessibility, visualisation and usability of design data (and metadata) have received relatively little attention to date. Here, we present a method to turn circuit designs into networks and showcase its potential to enhance the utility of design data. Since networks are dynamic structures, initial graphs can be interactively shaped into sub-networks of relevant information based on requirements such as abstraction, hierarchy and protein interactions. Additionally, several visual changes can be applied, such as colouring or clustering nodes based on types (e.g., genes or promoters), resulting in easier comprehension from a user perspective. This approach allows circuit designs to be coupled to other networks, such as metabolic pathways or implementation protocols captured in graph-like formats. Therefore, we advocate using networks to structure, access and improve synthetic biology information.

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Grp78  alleviates sodium iodate-induced retinal cell injury in vivo and in vitro ObjectiveGlucose-regulated protein 78 (Grp78) has been regarded as a main member of the endoplasmic reticulum proteins, Grp78 could protect cells from apoptosis under stress conditions. However, whether Grp78 could protect retinal pigment epithelium (RPE) cells from oxidative injury and then protect retinas from morphological changes and functional abnormalities remain undetermined. Here, we try to explore the effect of Grp78 on retinal cell injury induced by sodium iodate in vivo and in vitro.

MethodsTo investigate whether Grp78 has a protective effect on RPE injury in vitro, human retinal pigment epithelium (ARPE-19) cells were treated with sodium iodate. The cell proliferation, morphology, apoptosis and ROS production assays were detected. In vivo, We established sodium iodate-induced retinal injury model in mice by intravenous injection of sodium iodate into tail vein. After that, we examined the morphology and function of retina in mice by fundus photography, OCT and ERG. Finally, we removed the retina of mice for histological examination.

ResultsGrp78 significantly inhibited sodium iodate-induced reactive oxygen species (ROS), and decreased apoptosis of RPE in vitro. Furthermore, Grp78 significantly decreased the apoptosis of retinal cells in vivo, resulting in the inhibition of morphological changes of retina, and improving the function of retina. The underlying mechanisms included inhibited caspase3 and Nos, and increased expression of Bcl2, thereby protecting RPE from SI-induced ROS and apoptosis.

ConclusionGrp78 could reduce the injury of retinal cells induced by sodium iodate in vitro and in vivo. These findings suggested Grp78 may become a new therapeutic target for retinal injury in clinical practice.

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Plant Metabolic Network: A multi-species resource of plant metabolic information Plant metabolism is a pillar of our ecosystem, food security, and economy. To understand and engineer plant metabolism, we first need a comprehensive and accurate annotation of all metabolic information across plant species. As a step towards this goal, we previously created the Plant Metabolic Network (PMN), an online resource of curated and computationally predicted information about the enzymes, compounds, reactions, and pathways that make up plant metabolism. Here we report PMN 15, which contains genome-scale metabolic pathway databases of 126 algal and plant genomes, ranging from model organisms to crops to medicinal plants, and new tools for analyzing and viewing metabolism information across species and integrating omics data in a metabolic context. We systematically evaluated the quality of the databases, which revealed that our semi-automated validation pipeline dramatically improves the quality. We then compared the metabolic content across the 126 organisms using multiple correspondence analysis and found that Brassicaceae, Poaceae, and Chlorophyta appeared as metabolically distinct groups. To demonstrate the utility of this resource, we used recently published sorghum transcriptomics data to discover previously unreported trends of metabolism underlying drought tolerance. We also used single-cell transcriptomics data from the Arabidopsis root to infer cell-type specific metabolic pathways. This work shows the continued growth and refinement of the PMN resource and demonstrates its wide-ranging utility in integrating metabolism with other areas of plant biology.

One-sentence SummaryThe Plant Metabolic Network is a collection of databases containing experimentally-supported and predicted information about plant metabolism spanning many species.

sentences: Scalability of large neural network simulations via activity tracking with time asynchrony and procedural connectivity We present a new algorithm to efficiently simulate random models of large neural networks satisfying the property of time asynchrony. The model parameters (average firing rate, number of neurons, synaptic connection probability, and postsynaptic duration) are of the order of magnitude of a small mammalian brain, or of human brain areas. Through the use of activity tracking and procedural connectivity (dynamical regeneration of synapses), both computational and memory complexities of this algorithm are proved to be theoretically linear with the number of neurons. These results are experimentally validated by sequential simulations of millions of neurons and billions of synapses running in few minutes using a single thread of an equivalent desktop computer.

sentences: Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore's adaptive sequencing Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopores adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective  human host depletion resulted in a 1.70 fold ({+/-} 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.

sentences: Csf1r-GCaMP5 Reporter Mice Reveal Immune Cell Communication in Vitro and in Vivo Interconnected cells are responsible for emergent functions ranging from cognition in the brain to cyclic contraction in the heart. In electrically excitable cells, methods for studying cell communication are highly advanced, but in non-excitable cells, generalized methods for studying cell communication are less mature. Immune cells have generally been classified as non-excitable cells with diverse pathophysiologic roles that span every tissue in the body, yet little is known about their interconnectedness because assays are destructive and have low temporal resolution. In this work, we hypothesize that non-excitable immune cells are functionally interconnected in previously unrecognized cell communication networks. To test the hypothesis, we created a hematopoietic calcium reporter mouse (Csf1r-Cre x GCaMP5) and non-destructively quantified the spatiotemporal dynamics of intracellular calcium in vitro and in vivo. In vitro, bone marrow derived macrophages calcium reporters reveal that fatal immune stimulatory DNA-sensing induces rapid intercellular communication to neighboring cells. In vivo, using intravital microscopy through a dorsal window chamber in the context of MC38-H2B-mCherry tumors, Csf1r-GCaMP5 reporters exhibit spatiotemporal dynamics consistent with cell communication. We present a theoretical framework and analysis pipeline for identifying spatiotemporal locations of "excess synchrony" of calcium spiking as a means of inferring previously unrecognized cell communication events. Together, these methods provide a toolkit for investigating known and as-yet-undiscovered cell communication events in vitro and in vivo.

sentences: Ancestral diversity improves discovery and fine-mapping of genetic loci for anthropometric traits - the Hispanic/Latino Anthropometry Consortium Hispanic/Latinos have been underrepresented in genome-wide association studies (GWAS) for anthropometric traits despite notable anthropometric variability with ancestry proportions, and a high burden of growth stunting and overweight/obesity in Hispanic/Latino populations. This address this knowledge gap, we analyzed densely-imputed genetic data in a sample of Hispanic/Latino adults, to identify and fine-map common genetic variants associated with body mass index (BMI), height, and BMI-adjusted waist-to-hip ratio (WHRadjBMI). We conducted a GWAS of 18 studies/consortia as part of the Hispanic/Latino Anthropometry (HISLA) Consortium (Stage 1, n=59,769) and validated our findings in 9 additional studies (HISLA Stage 2, n=9,336). We conducted a trans-ethnic GWAS with summary statistics from HISLA Stage 1 and existing consortia of European and African ancestries. In our HISLA Stage 1+2 analyses, we discovered one novel BMI locus, as well two novel BMI signals and another novel height signal, each within established anthropometric loci. In our trans-ethnic meta- analysis, we identified three additional novel BMI loci, one novel height locus, and one novel WHRadjBMI locus. We also identified three secondary signals for BMI, 28 for height, and two for WHRadjBMI. We replicated >60 established anthropometric loci in Hispanic/Latino populations at genome-wide significance--representing up to 30% of previously-reported index SNP anthropometric associations. Trans-ethnic meta-analysis of the three ancestries showed a small-to-moderate impact of uncorrected population stratification on the resulting effect size estimates. Our novel findings demonstrate that future studies may also benefit from leveraging differences in linkage disequilibrium patterns to discover novel loci and additional signals with less residual population stratification.

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Lipophorin receptors regulate mushroom bodies development and participate in learning, memory, and sleep in flies BackgroundDrosophila melanogaster Lipophorin Receptors (LpRs), LpR1 and LpR2, mediate lipid uptake. The orthologs of these receptors in vertebrates, ApoER2 and VLDL-R, bind Reelin, a glycoprotein not present in flies. These receptors are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain. It is currently unknown whether LpRs play similar roles in the Drosophila brain.

ResultsWe report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the Mushroom Bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurites. This effect depends on LpRs and Dab, the Drosophila ortholog of the reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin.

ConclusionsThese findings demonstrate that LpRs contribute to MB development and function, supporting the existence of LpR-dependent signaling in Drosophila.

sentences: Differences in IgG antibody responses following BNT162b2 and mRNA-1273 Vaccines Studies examining antibody responses by vaccine brand are lacking and may be informative for optimizing vaccine selection, dosage, and regimens. The purpose of this study is to assess IgG antibody responses following immunization with BNT162b2 (30 g S protein) and mRNA-1273 (100 g S protein) vaccines. A cohort of clinicians at a non-for-profit organization is being assessed clinically and serologically following immunization with BNT162b2 or mRNA-1273. IgG responses were measured at the Remington Laboratory by an IgG against the SARS-CoV-2 spike protein-receptor binding domain. Mixed-effect linear (MEL) regression modeling was used to examine whether the SARS-CoV-2 IgG level differed by vaccine brand, dosage, or days since vaccination. Among 532 SARS-CoV-2 seronegative participants, 530 (99.6%) seroconverted with either vaccine. After adjustments for age and gender MEL regression modeling revealed that the average IgG increased after the second dose compared to the first dose (p<0.001). Overall, titers peaked at week six for both vaccines. Titers were significantly higher for mRNA-1273 vaccine on days 14-20 (p < 0.05), 42-48 (p < 0.01), 70-76 (p < 0.05), 77-83 (p < 0.05), and higher for BNT162b2 vaccine on days 28-34 (p < 0.001). In two participants taking immunosuppressive drugs SARS-CoV-2 IgG remained negative. mRNA-1273 elicited both earlier and higher IgG antibody responses than BNT162b2, possibly due to the higher S-protein delivery. Prospective clinical and serological follow-up of defined cohorts such as this may prove useful in determining antibody protection and whether differences in antibody kinetics between the vaccines have manufacturing relevance and clinical significance.

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Residual errors in visuomotor adaptation persist despite extended motor preparation periods A consistent finding in sensorimotor adaptation is a persistent undershoot of full compensation, such that performance asymptotes with residual errors greater than seen at baseline. This behaviour has been attributed to limiting factors within the implicit adaptation system, which reaches a sub-optimal equilibrium between trial-by-trial learning and forgetting. However, recent research has suggested that allowing longer motor planning periods prior to movement eliminates these residual errors. The additional planning time allows required cognitive processes to be completed before movement onset, thus increasing accuracy. Here we looked to extend these findings by investigating the relationship between increased motor preparation time and the size of imposed visuomotor rotation (30{degrees}, 45{degrees} or 60{degrees}), with regards to the final asymptotic level of adaptation. We found that restricting preparation time to 0.35 seconds impaired adaptation for moderate and larger rotations, resulting in larger residual errors compared to groups with additional preparation time. However, we found that even extended preparation time failed to eliminate persistent errors, regardless of magnitude of cursor rotation. Thus, the asymptote of adaptation was significantly less than the degree of imposed rotation, for all experimental groups. Additionally, there was a positive relationship between asymptotic error and implicit retention. These data suggest that a prolonged motor preparation period is insufficient to reliably achieve complete adaptation and therefore our results provide support for the proposal that limitations within the implicit learning system contributes to asymptotic adaptation levels.

New & NoteworthyResidual errors in sensorimotor adaptation are commonly attributed to an equilibrium between trial-by-trial learning and forgetting. Recent research suggested that allowing sufficient time for mental rotation eliminates these errors. In a number of experimental conditions, we show that while restricted motor preparation time does limit adaptation - consistent with mental rotation - extending preparation time fails to eliminate the residual errors in motor adaptation.

sentences: SPICEMIX: Integrative single-cell spatial modeling of cell identity Spatial transcriptomics technologies promise to reveal spatial relationships of cell-type composition in complex tissues. However, the development of computational methods that can utilize the unique properties of spatial transcriptome data to unveil cell identities remains a challenge. Here, we introduce SO_SCPLOWPICEC_SCPLOWMO_SCPLOWIXC_SCPLOW, a new interpretable method based on probabilistic, latent variable modeling for effective joint analysis of spatial information and gene expression from spatial transcriptome data. Both simulation and real data evaluations demonstrate that SO_SCPLOWPICEC_SCPLOWMO_SCPLOWIXC_SCPLOW markedly improves upon the inference of cell types and their spatial patterns compared with existing approaches. By applying to spatial transcriptome data of brain regions in human and mouse acquired by seqFISH+, STARmap, and Visium, we show that SO_SCPLOWPICEC_SCPLOWMO_SCPLOWIXC_SCPLOW can enhance the inference of complex cell identities, reveal interpretable spatial metagenes, and uncover differentiation trajectories. SO_SCPLOWPICEC_SCPLOWMO_SCPLOWIXC_SCPLOW is a generalizable framework for analyzing spatial transcriptome data to provide critical insights into the cell type composition and spatial organization of cells in complex tissues.

sentences: The cellular architecture of memory modules in Drosophila supports stochastic input integration The ability to associate neutral stimuli with valence information and to store these associations as memories forms the basis for decision making. To determine the underlying computational principles, we build a realistic computational model of a central decision module within the Drosophila mushroom body (MB), the flys center for learning and memory. Our model combines the electron microscopy-based architecture of one MB output neuron (MBON-3), the synaptic connectivity of its 948 presynaptic Kenyon cells (KCs), and its in vivo membrane properties obtained from patch-clamp recordings. We show that this neuron is electrotonically compact and that synaptic input corresponding to simulated odor input robustly drives its spiking behavior. Therefore, sparse innervation by KCs can efficiently control and modulate MBON activity in response to learning with minimal requirements on the specificity of synaptic localization. This architecture allows efficient storage of large numbers of memories using the flexible stochastic connectivity of the circuit.

sentences: Extremely reduced supergroup F Wolbachia: transition to obligate insect symbionts Wolbachia are widely distributed symbionts among invertebrates that manifest by a broad spectrum of lifestyles from parasitism to mutualism. Wolbachia Supergroup F is considered a particularly interesting group which gave rise to symbionts of both arthropods and nematodes, and some of its members are obligate mutualists. Further investigations on evolutionary transitions in symbiosis have been hampered by a lack of genomic data for Supergroup F members. In this study, we present genomic data for five new supergroup F Wolbachia strains associated with four chewing lice species. These new strains in different evolutionary stages show genomic characteristics well-illustrating the evolutionary trajectory which symbiotic bacteria experience during their transition to mutualism. Three of the strains have not yet progressed with the transition, the other two show typical signs of ongoing gene deactivation and removal (genome size, coding density, low number of pseudogenes). Particularly, wMeur1, a symbiont fixed in all Menacanthus eurysternus populations across four continents, possesses a highly reduced genome of 733,850 bp with a horizontally acquired capacity for pantothenate synthesis. Comparing with other strains showed wMeur1 genome as the smallest currently known among all Wolbachia and the first example of Wolbachia which has completed genomic streamlining known from the gammaproteobacterial obligate symbionts.

sentences: The reintroduction of a dung-beetle associated primate increases the recruitment success of large seeds The biased loss of large and medium frugivores alters seed dispersal and plant regeneration. Species reintroductions have been proposed as a strategy to reverse the consequences of species loss. However, the effects of reintroductions on ecological processes are seldom accessed, which hinders the comprehension of reintroductions potential to reestablish functioning ecosystems. In this study, we investigate the effect of howler monkey (Alouatta guariba) reintroduction on the plant regeneration of Tijuca National Park (TNP), a defaunated Atlantic Forest fragment. Howlers are folivore-frugivore primates, whose large clumped defecations attract dung beetles, which provide secondary dispersal by burying seeds present in the howlers feces. Thus, we expect that the fate of seeds dispersed by howlers will differ from those dispersed by other frugivores present in the Park. We followed the fate of seeds between 3 and 14mm in diameter in three steps of the seed dispersal loop, each one consisting of a different experiment. First, we estimated secondary seed dispersal and burial depth probabilities according to the frugivores defecation pattern; then, predation probability in different burial depths and defecation patterns; and, finally, recruitment probability in different burial depths. Considering the final result of the three experiments, the howlers reintroduction affected positively the regeneration of large seeds. The fate of 3mm seeds was little affected because they were seldom preyed upon at shallower depths anyway and could not recruit when deeply buried. On the other hand, seeds larger than 3mm reached the seedling stage more frequently when dispersed by howlers than when dispersed by other animals present in the Park. Thus, howler monkey reintroduction in defaunated areas, in addition to smaller frugivores, whose defecation patterns are less attractive for dung beetles, improves the regeneration of large seeds. We hope that this study will stimulate new howler reintroductions in defaunated areas.

sentences: Sex Differences in Pubertal Circadian and Ultradian Rhythmic Development Under Naturalistic Conditions Biological rhythms in core body temperature (CBT) provide informative markers of adolescent development under controlled laboratory conditions. However, it is unknown if these markers are preserved under more variable naturalistic conditions, and if CBT may therefore prove useful in a real-world setting. To evaluate this possibility, we examined fecal steroid concentrations and CBT rhythms from pre-adolescence (p26) through early adulthood (p76) in intact male and female rats under natural light and climate at the University of California, Berkeley Field Station. Despite greater environmental variability, CBT markers of pubertal onset and its rhythmic progression were comparable to those previously reported in laboratory conditions in female rats and extend actigraphy-based findings in males. Specifically, sex differences emerged in circadian rhythm (CR) power and temperature amplitude prior to pubertal onset and persisted into early adulthood, with females exhibiting elevated CBT and decreased CR power compared to males. Within-day (ultradian rhythm; UR) patterns also exhibited a pronounced sex difference associated with estrous cyclicity. Pubertal onset, defined by vaginal opening, preputial separation, and sex steroid concentrations, occurred later than previously reported under lab conditions for both sexes. Vaginal opening and increased fecal estradiol concentrations were closely tied to the commencement of 4-day oscillations in CBT and UR power in female rats. By contrast, preputial separation and the first rise in testosterone concentration were not associated with adolescent changes to CBT rhythms in male rats. Together, males and females exhibited unique temporal patterning of CBT and sex steroids across pubertal development, with tractable associations between hormonal concentrations, external development, and temporal structure in females. The preservation of these features outside the laboratory supports CBT as a strong candidate for translational pubertal monitoring under naturalistic conditions in females.

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A selective glucocorticoid receptor modulator attenuates lung inflammation and improves alveolarization in a neonatal rat model of bronchopulmonary dysplasia. BackgroundBronchopulmonary dysplasia (BPD) is a major problem for extremely preterm infants. Glucocorticoids effectively treat BPD; however, they have many side effects. Compound A (Cpd A) is a nonsteroidal Selective Glucocorticoid Receptor Modulator (SEGRM) that acts as a glucocorticoid receptor ligand without inducing the expression of glucocorticoid-response element-driven genes. Cpd A reportedly has anti-inflammatory properties with fewer side effects than glucocorticoids.

MethodsUsing a bleomycin (Bleo)-induced BPD model, we evaluated the therapeutic effects of Cpd A. 0-day-old Sprague-Dawley rats were administered Bleo for 10 days and treated with dexamethasone (Dex) or Cpd A from day 0 to 13. We evaluated lung pathology by histology and the mRNA levels of interleukin (IL)-1{beta}, transforming growth factor (TGF)-{beta}1 and chemokines, CXCL1 and CCL2.

ResultsBleo-treated mice had lungs with impaired alveolarization. Dex and Cpd A treatments improved the alveolar structure, attenuating the lung injury. Bleo-exposed lungs had increased inflammatory cells recruitment and inflammatory mediator mRNA levels. Cpd A treatment reduced inflammatory cells infiltration and CXCL1, CCL2 and TGF-{beta}1 expression.

ConclusionCpd A improved lung inflammation and alveolar maturation arrest, and restored histological and biochemical changes in a model of BPD. SEGRMs, including Cpd A, are promising candidates for the therapy of BPD.

Impact Statement{circ} What is the key message of your article?
Compound A decreased lung inflammation and improved lung morphometric changes in Bleomycin-exposed lungs.
{circ}What does it add to the existing literature?
Compound A has anti-inflammatory effects in an experimental model of BPD.
{circ}What is the impact?
SEGRMs, including Cpd A, may be promising candidates for the therapy of BPD.

sentences: When Vibrios Take Flight: A Meta-analysis of Pathogenic Vibrios Species in Wild and Domestic Birds Of the over 100 species in the genus Vibrio, approximately twelve are associated with clinical disease, such as cholera and vibriosis. Crucially, eleven of those twelve--V. alginolyticus, V. cholerae, V. cincinnatiensis, V. hollinsae, e.g., Grimontia hollinsae, V. furnissii, V. mimicus, V. parahaemolyticus, V. vulnificus, V. harveyi, V. scophthalmi and V. metschnikovi--have also been isolated from birds. Since 1965, pathogenic Vibrio species have been consistently isolated from aquatic and ground-foraging bird species, which has implications for public health, as well as the One Health paradigm defined as an ecology-inspired, integrative framework for the study of health and disease, inclusive of environmental, human, and animal health. In this meta-analysis, we identified 76 studies from the primary literature which report on or examine birds as hosts for pathogenic Vibrio species. We found that the burden of disease in birds was most commonly associated with V. cholerae, followed by V. metschnikovi and V. parahaemolyticus. Meta-analysis wide prevalences of the Vibrio pathogens varied from 19% for V. parahaemolyticus to 1% for V. mimicus. Wild and domestic birds were both affected, which may have implications for conservation, as well as agriculturally associated avian species. As pathogenic Vibrios become more abundant throughout the world as a result of warming estuaries and oceans, susceptible avian species should be continually monitored as potential reservoirs for these pathogens.

sentences: Integrated single-cell sequencing reveals principles of epigenetic regulation of human gastrulation and germ cell development in a 3D organoid model The emergence of different cell types and the role of the epigenome in regulating transcription is a key yet understudied event during human gastrulation. Investigating these questions remain infeasible due to the lack of availability of embryos at these stages of development. Further, human gastrulation is marked by dynamic changes in cell states that are difficult to isolate at high purity, thereby making it challenging to map how epigenetic reprogramming impacts gene expression and cellular phenotypes. To overcome these limitations, we describe scMAT-seq, a high-throughput one-pot single-cell multiomics technology to simultaneously quantify DNA methylation, DNA accessibility and the transcriptome from the same cell. Applying scMAT-seq to 3D human gastruloids, we characterized the epigenetic landscape of major cell types corresponding to the germ layers and primordial germ cell-like cells (hPGCLC). As the identity of the progenitors that give rise to human PGCLCs remain unclear, we used this system to discover that the progenitors emerge from epiblast cells and show transient characteristics of both amniotic- and mesoderm-like cells, before getting specified towards hPGCLCs. Finally, as cells differentiate along different lineages during gastrulation, we surprisingly find that while changes in DNA accessibility are tightly correlated to both upregulated and downregulated genes, reorganization of gene body DNA methylation is strongly related to only genes that get downregulated, with genes that turn on displaying a lineage trajectory-dependent correlation with DNA methylation. Collectively, these results demonstrate that scMAT-seq is a high-throughput and sensitive approach to elucidate epigenetic regulation of gene expression in complex systems such as human gastrulation that are marked by rapidly transitioning cell states.

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Mechanosensitivity of nucleocytoplasmic transport Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Here we show that nuclear forces differentially control passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than facilitated diffusion. Due to this differential effect, force leads to the translocation into or out of the nucleus of cargoes within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism, and engineered exogenously by introducing appropriate nuclear localization signals. Our work sets a novel framework to understand mechanically induced signalling, with potential general applicability across signalling pathways and pathophysiological scenarios.

One sentence summaryForce application to the nucleus leads to nuclear accumulation of proteins by differentially affecting passive versus facilitated nucleocytoplasmic transport.

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Fatty acid metabolic reprogramming promotes C. elegans development Acetylcholine signaling has been reported to play essential roles in animal metabolic regulation and disease affected by diets. However, the underlying mechanisms that how diets regulate animal physiology and health are not well understood. Here we found that the acetylcholine receptor gene eat-2 was expressed in most of the pharyngeal muscles, which is in accordance to our previous report that EAT-2 received synaptic signals not only from pharyngeal MC neurons. The expression of fatty acid synthesis genes was significantly increased in both eat-2 and tmc-1 fast-growth mutants on CeMM food environment, compared to the wild-type. Excitingly, dietary fatty acids such as 15-methyl-hexadecanoic acid (C17ISO), palmitic acid (PA, C16:0) and stearic acid (SA, C18:0) supplementation, significantly accelerated wild-type worm development on CeMM, indicating that the fatty acid synthesis reprogramming is an essential strategy for C. elegans to regulate its development and growth on CeMM diet. Furthermore, we found that fatty acid elongase gene elo-6 knock-out significantly attenuated eat-2 mutant fast growth, while overexpression of elo-6 could rescue the eat-2; elo-6 double mutant slow development, which suggested that elo-6 played a major role in the above metabolic remodeling. Taken together, our report indicates that diets regulate neuromuscular circuit and modulate C. elegans development via fatty acid metabolic reprogramming. As most of the key genes and metabolites found in this study are conserved in both invertebrate and vertebrate animals, we believed that our results might provide essential clues to the molecular mechanisms underlying interactions among animal nutrition sensation, metabolism reprogramming and developmental regulation.

Significance StatementDiets and nutritional composition affect animal development and human health, however the underlying mechanisms remain elusive. We demonstrate that the acetylcholine receptor gene eat-2 is expressed in most of pharyngeal muscles, and the expression of fatty acid synthesis genes is significantly increased in both eat-2 and tmc-1 fast-growth mutants on the synthetic chemical defined CeMM food environment. Dietary supplementation of several fatty acids significantly speed up animal development. Furthermore, we demonstrate that the fatty acid elongase gene elo-6 knock-out attenuates eat-2 mutant fast growth, and overexpression of wild-type elo-6 promotes the eat-2; elo-6 double mutant slow development. Our findings describe that acetylcholine signaling coordinate nutrition sensation and developmental regulation through fatty acid metabolic remodeling.

sentences: Life history adaptations to fluctuating environments: Combined effects of demographic buffering and lability of demographic parameters Demographic buffering and lability have been identified as adaptive strategies to optimise fitness in a fluctuating environment. These are not mutually exclusive, however we lack efficient methods to measure their relative importance for a given life history. Here, we decompose the stochastic growth rate (fitness) into components arising from nonlinear responses and variance-covariance of demographic parameters to an environmental driver, which allows studying joint effects of buffering and lability. We apply this decomposition for 154 animal matrix population models under different scenarios, to explore how these main fitness components vary across life histories. Faster-living species appear more responsive to environmental fluctuations, either positively or negatively. They have the highest potential for strong adaptive demographic lability, while demographic buffering is a main strategy in slow-living species. Our decomposition provides a comprehensive framework to study how organisms adapt to variability through buffering and lability, and to predict species responses to climate change.

sentences: Electrophysiological Indicators of Sleep-associated Memory Consolidation in 5- to 6-Year-Old Children In adults, the synchronised interplay of sleep spindles (SP) and slow oscillations (SO) supports memory consolidation. Given tremendous developmental changes in SP and SO morphology, it remains elusive whether across childhood the same mechanisms as identified in adults are functional. Based on topography and frequency, we characterise slow and fast SPs and their temporal coupling to SOs in 24 pre-school children. Further, we ask whether slow and fast SPs and their modulation during SOs are associated with behavioural indicators of declarative memory consolidation as suggested by the literature on adults. Employing an individually tailored approach, we reliably identify an inherent, development-specific fast centro-parietal SP type, nested in the adult-like slow SP frequency range, along with a dominant slow frontal SP type. Further, we provide evidence that the modulation of fast centro-parietal SPs during SOs is already present in pre-school children. However, the temporal coordination between fast centro-parietal SPs and SOs is weaker and less precise than expected from research on adults. While we do not find evidence for a critical contribution of SP-SO coupling for memory consolidation, crucially, slow frontal and fast centro-parietal SPs are each differentially related to sleep-associated consolidation of items of varying quality. While a higher number of slow frontal SPs is associated with stronger maintenance of medium-quality memories, a higher number of fast centro-parietal SPs is linked to a greater gain of low-quality items. Our results demonstrate two functionally relevant inherent SP types in pre-school children although SP-SO coupling is not yet fully mature.

sentences: Genome-wide association study of open fieldbehavior in outbred heterogeneous stock ratsidentifies multiple loci implicated inpsychiatric disorders Many personality traits are influenced by genetic factors. Rodents models provide an efficient system for analyzing genetic contribution to these traits. Using 1,246 adolescent heterogeneous stock (HS) male and female rats, we conducted a genome-wide association study (GWAS) of behaviors measured in an open field, including locomotion, novel object interaction, and social interaction. We identified 30 genome-wide significant quantitative trait loci (QTL). Using multiple criteria, including the presence of high impact genomic variants and co-localization of cis-eQTL, we identified 13 candidate genes (Adarb2, Ankrd26, Cacna1c, Clock, Crhr1, Ctu2, Cyp26b1, Eva1a, Fam114a1, Kcnj9, Mlf2, Rab27b, Sec11a) for these traits. Most of these genes have been implicated by human GWAS of various psychiatric traits. For example, Cacna1c, a gene known to be critical for social behavior in rodents and implicated in human schizophrenia and bipolar disorder, is a candidate gene for distance to the social zone. In addition, the QTL region for total distance to the novel object zone, on Chr1 at 144 Mb, is syntenic to a hotspot on human Chr15 (82.5-90.8 Mb) that contains 14 genes associated with psychiatric or substance abuse traits. Although some of the genes identified by this study appear to replicate findings from prior human GWAS, others likely represent novel findings that can be the catalyst for future molecular and genetic insights into human psychiatric diseases. Together, these findings provide strong support for the use of the HS population to study psychiatric disorders.

sentences: Impaired glutamate reuptake induces synaptic mistuning in rat hippocampal slices, that can be counteracted by ketamine Mistuning of synaptic transmission has been proposed to underlie many psychiatric disorders, with decreased reuptake of the excitatory neurotransmitter glutamate as one contributing factor. Synaptic tuning occurs through several diverging and converging forms of plasticity. By recording evoked field postsynaptic potentials in the CA1 area in hippocampal slices, we found that inhibiting glutamate transporters using DL-TBOA causes retuning of synaptic transmission, resulting in a new steady state with reduced synaptic strength and a lower threshold for inducing long-term synaptic potentiation (LTP). Moreover, we also found reduced threshold for LTP in a rat model of depression that has decreased levels of glutamate transporters. Most importantly, we found that the antidepressant ketamine counteracts the effects of increased glutamate on the various steps involved in synaptic retuning. We therefore propose that ketamines mechanism of action as an antidepressant is to restore adequate synaptic tuning.

sentences: Multistep loading of PCNA onto DNA by RFC The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded/double-stranded DNA junctions harboring a recessed 3 end (3 ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATP{gamma}S and 3 ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and indicating that PCNA ring-closure is not mechanistically coupled to ATP-hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.

sentences: A computational study on the optimization of transcranial temporal interfering stimulation with high-definition electrode using unsupervised neural network Transcranial temporal interfering stimulation (tTIS) can focally stimulate deep parts of the brain, which are related to specific functions, by using beats at two high AC frequencies that do not affect the human brain. However, it has limitations in terms of calculation time and precision for optimization because of its complexity and non-linearity. We aimed to propose a method using an unsupervised neural network (USNN) for tTIS to optimize quickly the interfering current value of high-definition electrodes, which can finely stimulate the deep part of the brain, and analyze the performance and characteristics of tTIS. A computational study was conducted using 16 realistic head models. This method generated the strongest stimulation on the target, even when targeting deep areas or multi-target stimulation. The tTIS was robust with target depth compared with transcranial alternating current stimulation, and mis-stimulation could be reduced compared with the case of using two-pair inferential stimulation. Optimization of a target could be performed in 3 min. By proposing the USNN for tTIS, we showed that the electrode currents of tTIS can be optimized quickly and accurately, and the possibility of stimulating the deep part of the brain precisely with transcranial electrical stimulation was confirmed.

sentences: Causal inference using deep-learning variable selection identifies and incorporates direct and indirect causalities in complex biological systems In complex diseases, causal structure learning across biological variables is critical to identify modifiable triggers or potential therapeutic agents. A limitation of existing causal learning methods is that they cannot identify indirect causal relations, those that would interact through latent mediating variables. We developed the first computational method that identifies both direct and indirect causalities, causal inference using deep-learning variable-selection (causalDeepVASE). To accurately identify indirect causalities and incorporate them with direct causalities, causalDeepVASE develops a deep neural network approach and extends a flexible causal inference method. In simulated and biological data of various contexts, causalDeepVASE outperforms existing methods in identifying expected or validated causal relations. Further, causalDeepVASE facilitates a systematic understanding of complex diseases. For example, causalDeepVASE uniquely identified a possible causal relation between IFN{gamma} and creatinine suggested in a polymicrobial sepsis model. In future biomedical studies, causalDeepVASE can facilitate the identification of driver genes and therapeutic agents.

sentences: Coupling between cerebrovascular oscillations and CSF flow fluctuation during wakefulness: An fMRI study Cerebrospinal fluid (CSF) plays an important role in the clearance of metabolic waste products from the brain, yet the driving forces of CSF movement are not fully understood. It is commonly believed that CSF movement is facilitated by blood vessel wall movements (i.e., hemodynamic oscillations) in the brain. A coherent pattern of low frequency hemodynamic oscillations and CSF movement was recently found during non-rapid eye movement (NREM) sleep via functional MRI. However, questions remain regarding 1) the explanation of coupling between hemodynamic oscillations and CSF movement from fMRI signals; 2) the existence of the coupling during wakefulness; 3) the direction of CSF movement. In this resting state fMRI study, we proposed a mechanical model to explain the coupling between hemodynamics and CSF movement through the lens of fMRI. We found that the observed delays between hemodynamics and CSF movement match those predicted by the model. Moreover, by conducting separate fMRI scans of the brain and neck, we confirmed the low frequency CSF movement at the fourth ventricle is bidirectional. Our finding also demonstrates that CSF movement is facilitated by hemodynamic oscillations mainly in the low frequency range, even when the individual is awake.

sentences: Plasmin-mediated cleavage of EphA4 at central amygdala inhibitory synapses controls anxiety Severe stress can trigger complex behavioural changes such as high anxiety (1). Inhibitory GABA-ergic interneurons in the lateral division of the central amygdala (CEl) control anxiety through feedforward inhibition of their target cells in the medial division (CEm) (2, 3). In particular, PKC{delta}-positive (PKC{delta}+) interneurons in CEl are critical elements of the neuronal circuitry of fear and anxiety (3-5), but the molecular mechanisms they employ are poorly understood. Here, we show that, during stress, GABA-ergic synapses of amygdala PKC{delta}+ interneurons are regulated by a serine protease plasmin. On stress, plasmin cleaves the extracellular portion of the tyrosine kinase receptor EphA4 triggering its dissociation from gephyrin, a postsynaptic GABA-receptor anchoring protein. Dynamic EphA4/gephyrin interaction leads to modification of dendritic spine morphology and synaptic GABA-receptor expression profile. Consistent with the critical role for the plasmin/EphA4/gephyrin signalling axis in anxiogenesis, viral delivery of plasmin-resistant (prEphA4) form of EphA4 into the central amygdala prevents the development of stress-induced anxiety in mice, while the delivery of plasmin-truncated EphA4 (tEphA4) dramatically enhances this effect. Thus, our studies identify a novel, critical molecular cascade regulating GABA-ergic signalling in the central amygdala synapses that allows bidirectional switching of animal behaviour from high to low anxiety states.

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Advancing admixture graph estimation via maximum likelihood network orientation MotivationAdmixture, the interbreeding between previously distinct populations, is a pervasive force in evolution. The evolutionary history of populations in the presence of admixture can be modeled by augmenting phylogenetic trees with additional nodes that represent admixture events. While enabling a more faithful representation of evolutionary history, admixture graphs present formidable inferential challenges, and there is an increasing need for methods that are accurate, fully automated, and computationally efficient. One key challenge arises from the size of the space of admixture graphs. Given that exhaustively evaluating all admixture graphs can be prohibitively expensive, heuristics have been developed to enable efficient search over this space. One heuristic, implemented in the popular method TreeMix, consists of adding edges to a starting tree while optimizing a suitable objective function.

ResultsHere, we present a demographic model (with one admixed population incident to a leaf) where TreeMix and any other starting-tree-based maximum likelihood heuristic using its likelihood function is guaranteed to get stuck in a local optimum and return an incorrect network topology. To address this issue, we propose a new search strategy that we term maximum likelihood network orientation (MLNO). We augment TreeMix with an exhaustive search for a MLNO, referring to this approach as OrientA-Graph. In evaluations including previously published admixture graphs, OrientAGraph outperformed TreeMix on 4/8 models (there are no differences in the other cases). Overall, OrientAGraph found graphs with higher likelihood scores and topological accuracy while remaining computationally efficient. Lastly, our study reveals several directions for improving maximum likelihood admixture graph estimation.

AvailabilityOrientAGraph is available under the GNU General Public License v3.0 on Github (https://github.com/sriramlab/OrientAGraph).

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Motor dysfunction in Drosophila melanogaster as a biomarker for developmental neurotoxicity Humans interact with numerous chemical compounds with direct health implications, with several able to induce developmental neurotoxicity (DNT), which bear developmental, behavioral, and cognitive consequences from a young age. Current guidelines for DNT testing are notably costly, time consuming, and unsuitable for testing large numbers of chemicals. Therefore, there is a need for adequate alternatives to conventional animal testing for neurotoxicity and DNT. Here we show that detailed kinematic analysis can provide a strong indicator for DNT, using known (chlorpyrifos, CPS) or putative ({beta}-N-methylamino-L-alanine, BMAA) neurotoxic compounds. We exposed Drosophila melanogaster to these compounds during development and evaluated for common general toxicity -- notably developmental survival and pupal positioning, together with the FlyWalker system, a detailed adult kinematics evaluation method.

At concentrations that do not induce general toxicity, the solvent DMSO had a significant effect on kinematic parameters. Nonetheless, CPS not only induced developmental lethality but also significantly impaired coordination in comparison to DMSO, altering 16 motor parameters, validating the usefulness of our kinematic approach.

Interestingly, BMAA, although not lethal during development, induced a dose-dependent motor decay, targeting most parameters in young adult animals, phenotypically resembling normally aged, non-exposed flies. This effect was subsequently attenuated during ageing, indicating an adaptive response. Furthermore, BMAA induced an abnormal terminal differentiation of leg motor neurons, without inducing degeneration, underpinning the observed altered mobility phenotype. Overall, our results support our kinematic approach as a novel, highly sensitive and reliable tool to assess potential DNT of chemical compounds.

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Discovery of archaeal Fusexins homologous to eukaryotic HAP2/GCS1 gamete fusion proteins Sexual reproduction consists of genome reduction by meiosis and subsequent gamete fusion. Presence of meiotic genes in prokaryotes suggests that DNA repair mechanisms evolved toward meiotic recombination; however, fusogenic proteins resembling those found in eukaryotes were not identified in prokaryotes. Here, we identify archaeal proteins that are homologs of fusexins, a superfamily of fusogens that mediate eukaryotic gamete and somatic cell fusion, as well as virus entry. The crystal structure of a trimeric archaeal Fusexin1 reveals novel features such as a six-helix bundle and an additional globular domain. Ectopically expressed Fusexin1 can fuse mammalian cells, and this process involves the additional domain and a conserved fusion loop. Archaeal fusexin genes exist within integrated mobile elements, potentially linking ancient archaeal gene exchanges and eukaryotic sex.

One-Sentence SummaryCell membrane fusion proteins of viruses and eukaryotes are also present in archaea.

sentences: Influence of age on functional memory T cell diversity Memory T cells exhibit considerable diversity that determines their ability to be protective and their durability. Here, we examined whether changes in T cell heterogeneity contribute to the age-associated failure of immune memory. By screening for age-dependent T cell surface markers, we have identified CD4 and CD8 memory T cell subsets that are unrelated to previously defined subsets of central and effector memory cells. Memory T cells expressing the ecto-5-nucleotidase CD73 constitute a functionally distinct subset of memory T cells that declines with age. They exhibit many features favorable for immune protection, including longevity and polyfunctionality. They have a low turnover, but are poised to display effector functions and to develop into cells resembling tissue-resident memory T cells (TRM). Upstream regulators of differential chromatin accessibility and transcriptomes include transcription factors that are characteristic for conferring these superior memory features as well as facilitating CD73 expression. CD73 is not just a surrogate marker of these regulatory networks but is directly involved in T cell survival and TRM differentiation Interventions preventing the decline of this T cell subset or increasing CD73 expression have the potential to improve immune memory in older adults.

sentences: Model biomolecular condensates have heterogeneous structure quantitatively dependent on the interaction profile of their constituent macromolecules Biomolecular condensates play numerous roles in cells by selectively concentrating client proteins while excluding others. These functions are likely to be sensitive to the spatial organization of the scaffold proteins forming the condensate. We use coarse-grained molecular simulations to show that model intrinsically-disordered proteins phase separate into a heterogeneous, structured fluid characterized by a well-defined length scale. The proteins are modelled as semi-flexible polymers with punctate, multifunctional binding sites in good solvent conditions. Their dense phase is highly solvated with a spatial structure that is more sensitive to the separation of the binding sites than their affinity. We introduce graph theoretic measures to show that the proteins are heterogeneously distributed throughout the dense phase, an effect that increases with increasing binding site number, and exhibit multi-timescale dynamics. The simulations predict that the structure of the dense phase is modulated by the location and affinity of binding sites distant from the termini of the proteins, while sites near the termini more strongly affect its phase behaviour. The relations uncovered between the arrangement of weak interaction sites on disordered proteins and the material properties of their dense phase can be experimentally tested to give insight into the biophysical properties and rational design of biomolecular condensates.

sentences: The universal language network: A cross-linguistic investigation spanning 45 languages and 12 language families. To understand the architecture of human language, it is critical to examine diverse languages; yet most cognitive neuroscience research has focused on a handful of primarily Indo-European languages. Here, we report an investigation of the fronto-temporo-parietal language network across 45 languages and establish the robustness to cross-linguistic variation of its topography and key functional properties, including left-lateralization, strong functional integration among its brain regions, and functional selectivity for language processing.

sentences: Serotonergic neuron ribosomes regulate the neuroendocrine control of Drosophila development. The regulation of ribosome function is a conserved mechanism of growth control. While studies in single cell systems have defined how ribosomes contribute to cell growth, the mechanisms that link ribosome function to organismal growth are less clear. Here we explore this issue using Drosophila Minutes, a class of heterozygous mutants for ribosomal proteins (Rps). These animals exhibit a delay in larval development caused by decreased production of the steroid hormone ecdysone, the main regulator of larval maturation. We found that this developmental delay is not caused by decreases in either global ribosome numbers or translation rates. Instead, we show that they are due in part to loss of Rp function specifically in a subset of serotonin (5-HT) neurons that innervate the prothoracic gland to control ecdysone production. We found that these 5-HT neurons have defective secretion in Minute animals, and that overexpression of synaptic vesicle proteins in 5-HTergic cells can partially reverse the Minute developmental delay. These results identify a cell-specific role for ribosomal function in the neuroendocrine control of animal growth and development.

sentences: Condensates in RNA Repeat Sequences are Heterogeneously Organized and Exhibit Reptation-like Dynamics Although it is known that RNA undergoes liquid-liquid phase separation (LLPS), the interplay between the molecular driving forces and the emergent features of the condensates, such as their morphologies and dynamical properties, is not well understood. We introduce a coarse-grained model to simulate phase separation of trinucleotide repeat RNAs, which are implicated in neurological disorders such as Huntington disease and amyotrophic lateral sclerosis. After establishing that the simulations reproduce key experimental findings (length and concentration dependence of the phase transition in (CAG)n repeats), we show that once recruited inside the liquid droplets, the monomers transition from hairpin-like structures to extended states. Interactions between the monomers in the condensates result in the formation of an intricate and dense intermolecular network, which severely restrains the fluctuations and mobilities of the RNAs inside large droplets. In the largest densely packed high viscosity droplets, the mobility of RNA chains is best characterized by reptation, reminiscent of the dynamics in polymer melts. Our work provides a microscopic framework for understanding liquid-liquid phase separation in RNA, which is not easily discernible in current experiments.

sentences: Personality, Subjective Well-Being, and the Serotonin 1a Receptor Gene in Common Marmosets (Callithrix jacchus) Studies of personality traits in common marmosets (Callithrix jacchus) indicate that there are five or six constructs--sociability, dominance, neuroticism, openness, and two related to conscientiousness--that define personality in common marmosets. The present study attempted to determine whether our earlier study of laboratory-housed individuals only yielded three domains--Dominance, Sociability, and Neuroticism--because of a low amount of between-subjects variance. We therefore increased our sample size from 77 to 128. In addition, we ascertained the reliability and validity of ratings and whether polymorphisms related to the serotonin 1a receptor were associated with personality. We found Sociability, Dominance, and Negative Affect factors that resembled three domains found in previous studies, including ours. We also found an Openness and Impulsiveness factor, the latter of which bore some resemblance to Conscientiousness, and two higher-order factors, Pro-sociality and Boldness. Further analyses could not exclude the possibility that Pro-sociality and Boldness represented a higher-level of personality organization. Correlations between personality factors and well-being were consistent with the definitions of the factors. There were no significant associations between personality and genotype. These results are consistent with the possibility that common marmoset personality structure varies as a function of rearing or housing variables that have not yet been investigated systematically.

sentences: Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.

sentences: SARS-CoV-2 Encodes a PPxY Late Domain Motif Known to Enhance Budding and Spread in Enveloped RNA Viruses The current COVID-19 (Coronavirus Disease-2019) pandemic is affecting the health and/or socioeconomic welfare of almost everyone in the world. Finding vaccines and therapeutics is therefore urgent, but elucidation of the molecular mechanisms that allow some viruses to cross host species boundaries, becoming a threat to human health, must also be given close attention. Here, analysis of all proteins of SARS-CoV-2 revealed a unique PPxY Late (L) domain motif, 25PPAY28, in a spike (S) protein inside a predicted hot disordered loop subject to phosphorylation and binding. PPxY motifs in enveloped RNA viruses are known to recruit Nedd4 E3 ubiquitin ligases and ultimately the ESCRT complex to enhance virus budding and release, resulting in higher viral loads, hence facilitating new infections. Interestingly, proteins of SARS-CoV-1 do not feature PPxY motifs, which could explain why SARS-CoV-2 is more contagious than SARS-CoV-1. Should an experimental assessment of this hypothesis show that the PPxY motif plays the same role in SARS-CoV-2 as it does in other enveloped RNA viruses, this motif will become a promising target for the development of novel host-oriented antiviral therapeutics for preventing S proteins from recruiting Nedd4 E3 ubiquitin ligase partners.

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Differential Requirement of DICER1 Activity during Development of Mitral and Tricuspid Valves Mitral and tricuspid valves are essential for unidirectional blood flow in the heart. They are derived from similar cell sources, and yet congenital dysplasia affecting both valves is clinically rare, suggesting the presence of differential regulatory mechanisms underlying their development. We specifically inactivated Dicer1 in the endocardium during cardiogenesis, and unexpectedly found that Dicer1-deletion caused congenital mitral valve stenosis and regurgitation, while it had no impact on other valves. We showed that hyperplastic mitral valves were caused by abnormal condensation and extracellular matrix (ECM) remodeling. Our single-cell RNA Sequencing analysis revealed impaired maturation of mesenchymal cells and abnormal expression of ECM genes in mutant mitral valves. Furthermore, expression of a set of miRNAs that target ECM genes was significantly lower in tricuspid valves compared to mitral valves, consistent with the idea that the miRNAs are differentially required for mitral and tricuspid valve development. Our study thus reveals miRNA-mediated gene regulation as a novel molecular mechanism that differentially regulates mitral and tricuspid valve development, thereby enhancing our understanding of the non-association of inborn mitral and tricuspid dysplasia observed clinically.

Summary statementAlthough mitral and tricuspid heart valves are both derived from same cell sources in the atrioventricular region through similar initiation and remodeling processes, congenital dysplasia simultaneously affecting both valves is clinically uncommon. We show that DICER1 activity is only essential for mitral valve development whereas it is dispensable for other valves. Our study thus reveals miRNA-mediated gene regulation as a novel molecular mechanism that is differentially required to regulate mitral and tricuspid valve development, thereby enhancing our understanding of the non-association of inborn mitral and tricuspid dysplasia observed clinically.

sentences: MinION nanopore sequencing provides similar methylation estimates to Sanger bisulfite sequencing in the TRPA1 promoter region Bisulfite sequencing has long been considered the gold standard for measurement of DNA methylation at single CpG resolution. In the meantime, several new approaches have been developed, which are regarded as less error-prone. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter obtained from our previous studies. For this purpose, we compared methylation rates obtained via direct bisulfite sequencing and nanopore sequencing. Thus, we were able to confirm our previous findings to a large extent.

sentences: Hierarchical Computational Modeling and Dynamic Network Analysis of Allosteric Regulation in the SARS-CoV-2 Spike Omicron Trimer Structures: Omicron Mutations Cooperate to Allosterically Control Balance of Protein Stability and Conformational Adaptability Structural and computational studies of the Omicron spike protein in various functional states and complexes provided important insights into molecular mechanisms underlying binding, high transmissibility, and escaping immune defense. However, the regulatory roles and functional coordination of the Omicron mutations are poorly understood and often ignored in the proposed mechanisms. In this work, we explored the hypothesis that the SARS-CoV-2 spike protein can function as a robust allosterically regulated machinery in which Omicron mutational sites are dynamically coupled and form a central engine of the allosteric network that regulates the balance between conformational plasticity, protein stability, and functional adaptability. In this study, we employed coarse-grained dynamics simulations of multiple full-length SARS-CoV-2 spike Omicron trimers structures in the closed and open states with the local energetic frustration analysis and collective dynamics mapping to understand the determinants and key hotspots driving the balance of protein stability and conformational adaptability. We have found that the Omicron mutational sites at the inter-protomer regions form regulatory clusters that control functional transitions between the closed and open states. Through perturbation-based modeling of allosteric interaction networks and diffusion analysis of communications in the closed and open spike states, we quantify the allosterically regulated activation mechanism and uncover specific regulatory roles of the Omicron mutations. The network modeling demonstrated that Omicron mutations form the inter-protomer electrostatic bridges that connect local stable communities and function as allosteric switches of signal transmission. The results of this study are consistent with the experiments, revealing distinct and yet complementary role of the Omicron mutational sites as a network of hotspots that enable allosteric modulation of structural stability and conformational changes which are central for spike activation and virus transmissibility.

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Use of a graph neural network to the weighted gene co-expression network analysis of Korean native cattle In the general framework of the weighted gene co-expression network analysis (WGCNA), a hierarchical clustering algorithm is commonly used to module definition. However, hierarchical clustering depends strongly on the topological overlap measure. In other words, this algorithm may assign two genes with low topological overlap to different modules even though their expression patterns are similar. Here, a novel gene module clustering algorithm for WGCNA is proposed. We develop a gene module clustering network (gmcNet), which simultaneously addresses single-level expression and topological overlap measure. The proposed gmcNet includes a "co-expression pattern recognizer" (CEPR) and "module classifier". The CEPR incorporates expression features of single genes into the topological features of co-expressed ones. Given this CEPR-embedded feature, the module classifier computes module assignment probabilities. We validated gmcNet performance using 4,976 genes from 20 native Korean cattle. We observed that the CEPR generates more robust features than single-level expression or topological overlap measure. Given the CEPR-embedded feature, gmcNet achieved the best performance in terms of modularity (0.261) and the differentially expressed signal (27.739) compared with other clustering methods tested. Furthermore, gmcNet detected some interesting biological functionalities for carcass weight, backfat thickness, intramuscular fat, and beef tenderness of Korean native cattle. Therefore, gmcNet is a useful framework for WGCNA module clustering.

Author summaryA graph neural network is a good alternative algorithm for WGCNA module clustering. Even though the graph-based learning methods have been widely applied in bioinformatics, most studies on WGCNA did not use graph neural network for module clustering. In addition, existing methods depend on topological overlap measure of gene pairs. This can degrade similarity of expression not only between modules, but also within module. On the other hand, the proposed gmcNet, which works similar to message-passing operation of graph neural network, simultaneously addresses single-level expression and topological overlap measure. We observed the higher performance of gmcNet comparing to existing methods for WGCNA module clustering. To adopt gmcNet as clustering algorithm of WGCNA, it remains future research issues to add noise filtering and optimal k search on gmcNet. This further research will extend our proposed method to be a useful module clustering algorithm in WGCNA. Furthermore, our findings will be of interest to computational biologists since the studies using graph neural networks to WGCNA are still rare.

sentences: Squamation and scale morphology at the root of jawed vertebrates Placoderms, the most basal jawed vertebrates, are crucial to understanding how the characters of crown gnathostomes comprising Chondrichthyes and Osteichthyes evolved from their stem relatives. Despite the growing knowledge on the anatomy and diversity of placoderms over the past decade, the dermal scales of placoderms are predominantly known from isolated material, either morphologically or histologically, resulting in their squamation being poorly understood. Here we provide a comprehensive description of the squamation and scale morphology of a primitive taxon of Antiarcha (a clade at the root of jawed vertebrates), Parayunnanolepis xitunensis, based on the virtual restoration of an articulated specimen by using X-ray computed tomography. Thirteen morphotypes of scales are classified to exhibit how the morphology changes with their position on the body in primitive antiarchs, based on which nine areas of the post-thoracic body are distinguished to show their scale variations in the dorsal, flank, ventral, and caudal lobe regions. In this study, the histological structure of yunnanolepidoid scales was described for the first time based on disarticulated scales from the type locality and horizon of P. xitunensis, demonstrating that yunnanolepidoid scales are remarkably different from the dermal plates of antiarchs including yunnanolepidoids in the absence of developed middle layer. Together, our study reveals that the high regionalization of squamation and the bipartite histological structure of scale might be plesiomorphic for antiarchs, and jawed vertebrates in general.

sentences: Septins and a formin have distinct functions in anaphase chiral cortical rotation in the C. eleganszygote. Many cells and tissues exhibit chirality that stems from the chirality of proteins and polymers. In the C. elegans zygote actomyosin contractility drives chiral rotation of the entire cortex circumferentially around the division plane during anaphase. How contractility is translated to cell-scale chirality, and what dictates handedness, are unknown. Septins are candidate contributors to cell-scale chirality because they anchor and crosslink the actomyosin cytoskeleton. We report that septins are required for anaphase cortical rotation. In contrast, the formin CYK-1, which we found to be enriched in the posterior in early anaphase, is not required for cortical rotation, but contributes to its chirality. Simultaneous loss of septin and CYK-1 function led to abnormal and often reversed cortical rotation. Our results suggest that anaphase contractility leads to chiral rotation by releasing torsional stress generated during formin-based polymerization, which is polarized along the cell anterior-posterior axis, and which accumulates due to actomyosin network connectivity. Our findings shed light on the molecular and physical bases for cellular chirality in the C. elegans zygote. We also identify conditions in which chiral rotation fails but animals are developmentally viable, opening avenues for future work on the relationship between early embryonic cellular chirality and animal body plan.

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Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval pigmentation in Bombyx mori The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body color became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori x B. mandarina F1 hybrid individuals. Knockout of the B. mandarina allele of apt-like in F1 embryos results in depigmented patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.

Brief abstractBombyx mori and its wild progenitor are an important model for the study of phenotypic evolution associated with domestication. As proof-of-principle, we used CRISPR/Cas9 to generate targeted knockouts of two pigmentation-related genes. By generating a homozygous knockout of yellow-y in B. mandarina, we confirmed this gene"s role in pigment formation. Further, by generating allele-specific knockouts of apontic-like (apt-like) in B. mori x B. mandarina F1 hybrids, we establish that evolution of apt-like contributed to reduced pigmentation during B. mori domestication.

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sentences: Coping with oxidative stress in extreme environments: the distinctive roles played by Acinetobacter sp. Ver3 superoxide dismutases Acinetobacter sp. Ver3 is a polyextremophilic strain characterized by a high tolerance to radiation and pro-oxidants. The Ver3 genome comprises the sodB and sodC genes encoding an iron (AV3SodB) and a copper/zinc superoxide dismutase (AV3SodC), respectively; however, the specific role(s) of these genes has remained elusive. We show that the expression of sodB remained unaltered in different oxidative stress conditions whereas sodC was up-regulated in the presence of blue light. Besides, we studied the changes in the in vitro activity of each SOD enzyme in response to diverse agents and solved the crystal structure of AV3SodB at 1.34 [A], one of the highest resolutions achieved for a SOD. Cell fractionation studies interestingly revealed that AV3SodB is located in the cytosol whereas AV3SodC is also found in the periplasm. Consistently, a bioinformatic analysis of the genomes of 53 Acinetobacter species pointed out the presence of at least one SOD type in each compartment, suggesting that these enzymes are separately required to cope with oxidative stress. Surprisingly, AV3SodC was found in an active state also in outer membrane vesicles, probably exerting a protective role. Overall, our multidisciplinary approach highlights the relevance of SOD enzymes when Acinetobacter spp. are confronted with oxidizing agents.

sentences: EGFR-MAPK adaptor proteins mediate the epithelial response to Candida albicans via the cytolytic peptide toxin, candidalysin Candida albicans (C. albicans) is a dimorphic human fungal pathogen that can cause severe oropharyngeal candidiasis (OPC, oral thrush) in susceptible hosts. During invasive infection, C. albicans hyphae invade oral epithelial cells (OECs) and secrete candidalysin, a pore-forming cytolytic peptide that is required for fungal pathogenesis at mucosal surfaces. Candidalysin induces cell damage and activates multiple MAPK-based innate signaling events that collectively drive the production of downstream inflammatory mediators. The activities of candidalysin are also dependent on the epidermal growth factor receptor (EGFR), but how these signals are integrated is undefined. Here, we identified five essential adaptor proteins as key mediators of the epithelial response to C. albicans infection on cultured OECs, including growth factor receptor bound protein 2 (Grb2), Grb2-associated-binding protein 1 (Gab1), Src homology and collagen (Shc), SH2 containing protein tyrosine phosphatase-2 (Shp2) and casitas B-lineage lymphoma (c-Cbl). All these signaling effectors were inducibly phosphorylated in response to C. albicans, in a candidalysin-dependent mechanism but additionally required EGFR phosphorylation, matrix metalloproteinases (MMPs) and cellular calcium flux. Of these, Gab1, Grb2 and Shp2 were the dominant drivers of ERK1/2 signaling and production of downstream cytokines. Together, these results identify the key adaptor proteins that drive EGFR signaling mechanisms, which determine oral epithelial responses to C. albicans.

sentences: A Focused Small-Molecule Screen Identifies PHA-680626 as an Amphosteric Inhibitor Disrupting the Interaction between Aurora-A and N-Myc Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcriptional factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation, by directly binding to a highly conserved N-Myc region, i.e. Myc Box I. As a result, elevated levels of N-Myc, which are required for the growth of MYCN amplified cells, are observed. During the last years, it has been demonstrated that the ATP competitive inhibitors of AURKA CD532, MLN8054 and Alisertib also cause essential conformational changes in the structure of the activation loop of the kinase that prevent N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complex with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results, identifying PHA-680626 as an amphosteric inhibitor both in vitro and MYCN overexpressing cell lines, expand the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex, and confirm that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.

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The peripheral T cell population is associated with pneumonia severity in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus 2 The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that began in December 2019. Lymphopenia is a common feature in severe cases of COVID-19; however, the role of T cell responses during infection is unclear. Here, we inoculated six cynomolgus monkeys, divided into two groups according to the CD3+ T cell population in peripheral blood, with two clinical isolates of SARS-CoV-2: one of East Asian lineage and one of European lineage. After initial infection with the isolate of East Asian lineage, all three monkeys in the CD3+ low group showed clinical symptoms, including loss of appetite, lethargy, and transient severe anemia with/without short-term fever, within 14 days post-infection (p.i.). By contrast, all three monkeys in the CD3+ high group showed mild clinical symptoms such as mild fever and loss of appetite within 4 days p.i. and then recovered. After a second inoculation with the isolate of European lineage, three of four animals in both groups showed mild clinical symptoms but recovered quickly. Hematological, immunological, and serological tests suggested that the CD3+ high and low groups mounted different immune responses during the initial and second infection stages. In both groups, anti-viral and innate immune responses were activated during the early phase of infection and re-infection. However, in the CD3+ low group, inflammatory responses, such as increased production of monocytes and neutrophils, were stronger than those in the CD3+ high group, leading to more severe immunopathology and failure to eliminate the virus. Taken together, the data suggest that the peripheral T lymphocyte population is associated with pneumonia severity in cynomolgus monkeys experimentally infected with SARS-CoV-2.

Author summarySARS-CoV-2 infection causes an illness with clinical manifestations that vary from asymptomatic or mild to severe; examples include severe pneumonia and acute respiratory distress syndrome. Lymphopenia, which is common in severe COVID-19 cases, is characterized by markedly reduced numbers of CD4+ T cells, CD8+ T cells, B cells, and natural killer cells. Here, we showed that cynomolgus monkeys selected according to the T cell populations in peripheral blood have different outcomes after experimental infection with SARS-CoV-2. These findings will increase our understanding of disease pathogenesis and may facilitate the development of animal models for vaccine evaluation.

sentences: Liver enhancer signature and regulatory network of non-alcoholic steatohepatitis resistance in humans with obesity The molecular underpinnings of non-alcoholic steatohepatitis (NASH) development in patients are poorly understood. Active enhancer landscapes are known to determine cell states and behaviors. Super-enhancers, in particular, have helped reveal key disease drivers in several cancer types; however, they remain unexplored in human NASH. To define the enhancer signature of NASH-prone (NP) and NASH-resistant (NR) phenotypes in humans with obesity, we performed chromatin run-on sequencing (ChRO-seq) analysis on liver biopsies of individuals with obesity who were stratified into either NP or NR. We first demonstrated that NP and NR groups exhibit distinct active enhancer signatures. The subsequent identification of NP- and NR-specific super-enhancers revealed the specific genes that are likely the most critical for each of the phenotypes, including HES1 for NP and GATM for NR. Integrative analysis with results from genome-wide association studies of NAFLD and related traits identified disease/trait-loci specific to NP or NR enhancers. Further analysis of the ChRO-seq data pointed to critical roles for serine/glycine metabolism in NASH resistance, which was corroborate by profiling of circulating amino acids in the same patients. Overall, the distinct enhancer signatures of human NP and NR phenotypes revealed key genes, pathways, and transcription factor networks that promote NASH development.

sentences: Biohybrid Microswimmers Against Bacterial Infections Biohybrid microswimmers exploit the natural abilities of motile microorganisms e.g. in releasing cargo on-demand with high spatial and temporal control. However, using such engineered swarms to deliver antibiotics addressing bacterial infections has not yet been realized. In the present study, a design strategy for biohybrid microswimmers is reported, which features the covalent attachment of antibiotics to the motile green algae Chlamydomonas reinhardtii via a photo-cleavable linker. The surface engineering of the algae does not rely on genetic manipulations, proceeds with high efficiency, does not impair the viability or phototactic ability of microalgae, and allows for caging of the antibiotic on the surface for subsequent release via external stimuli. Two different antibiotic classes have been separately utilized, which result in activity against both gram-positive and gram-negative strains. Guiding the biohybrid microswimmers by an external beacon, and on-demand delivery of the drugs by light with high spatial and temporal control, allowed for strong inhibition of bacterial growth in vivo. This efficient strategy could potentially allow for the selective treatment of bacterial infections by engineered algal microrobots with high precision in space and time. Overall, this work presents an operationally simple production of biohybrid microswimmers loaded with antibiotic cargo to combat bacterial infections precisely delivered in three-dimensional space.

sentences: Linkage disequilibrium and population structure in a core collection of Brassica napus (L.) Estimation of genetic diversity in rapeseed/canola is important for sustainable breeding program to provide an option for the development of new breeding lines. The objective of this study was to elucidate the patterns of genetic diversity within and among different structural groups, and measure the extent of linkage disequilibrium (LD) of 383 globally distributed rapeseed/canola germplasm using 8,502 single nucleotide polymorphism (SNP) markers. The germplasm accessions were divided into five subpopulations (P1 to P5) with obvious geographic and growth habit-related patterns. All subpopulations showed moderate genetic diversity (average H = 0.22 and I = 0.34). The pairwise Fst comparison revealed a great degree of divergence (Fst > 0.24) between most of the combinations. The rutabaga type showed highest divergence with spring and winter types. Higher divergence was also found between winter and spring types. Overall, mean linkage disequilibrium was 0.03 and it decayed to its half maximum within < 45 kb distance for whole genome. The LD decay was slower in C genome (< 93 kb), relative to the A genome (< 21 kb) which was confirmed by availability of larger haplotype blocks in C genome than A genome. To maximize genetic gain, hybridization between rutabaga and other types are potentially the best option. Hybridization between spring and winter, semi-winter type is also helpful to maximize the diversity in subsequent populations. Low genetic differentiation between both spring type subpopulations (P4 and P3) will accelerate favorable allele accumulation for specific traits in elite lines. The Neighbor-Joining tree and kinship matrix will assist to identify distantly related genotypes from subpopulations to utilize in hybridization. The low levels of LD and population structure make the core collection an important resource for association mapping efforts to identify genes useful in crop improvement as well as for selection of parents for hybrid breeding.

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Subfunctionalization of paralog transcription factors contributes to regulation of alkaloid pathway branch choice in Catharanthus roseus Catharanthus roseus produces a diverse range of specialized metabolites of the monoterpenoid indole alkaloid (MIA) class in a heavily branched pathway. Recent great progress in identification of MIA biosynthesis genes revealed that the different pathway branch genes are expressed in a highly cell type- and organ-specific and stress-dependent manner. This implies a complex control by specific transcription factors (TFs), only partly revealed today. We generated and mined a comprehensive compendium of publicly available C. roseus transcriptome data for MIA pathway branch-specific TFs. Functional analysis was performed through extensive comparative gene expression analysis and profiling of over 40 MIA metabolites in the C. roseus flower petal expression system. We identified additional members of the known BIS and ORCA regulators. Further detailed study of the ORCA TFs suggests subfunctionalization of ORCA paralogs in terms of target gene-specific regulation and synergistic activity with the central jasmonate response regulator MYC2. Moreover, we identified specific amino acid residues within the ORCA DNA-binding domains that contribute to the differential regulation of some MIA pathway branches. Our results advance our understanding of TF paralog specificity for which, despite the common occurrence of closely related paralogs in many species, comparative studies are scarce.

SIGNIFICANCE STATEMENTA gene discovery program for regulators of monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus advances our understanding of paralog specificity and subfunctionalization of the renowned class of ORCA transcription factors, particularly in terms of target gene-specificity and synergistic activity with other jasmonate-responsive transcription factors.

sentences: SCON - A Short Conditional intrON for conditional knockout with one-step zygote injection The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables rapid generation of conditional alleles via one-step zygote injection. SCON has conditional intronic function in various vertebrate species and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.

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Copper signaling promotes proteostasis and animal development via allosteric activation of ubiquitin E2D conjugases Nutrient copper supply is critical for cell growth and differentiation, and its disturbance is associated with major pathologies including cancer and neurodegeneration. Although increasing copper bioavailability in late Precambrian facilitated emergence of novel cuproproteins, their intricate regulation by this essential trace element remains largely cryptic. We found that subtle rises in cellular copper strikingly increase polyubiquitination and accelerate protein degradation within 30 minutes in numerous mammalian cell lines. We track this surprising observation to allostery induced in the UBE2D ubiquitin conjugase clade through a conserved CXXXC sub-femtomolar-affinity Cu+ binding motif. Thus, physiologic fluctuation in cytoplasmic Cu+ is coupled to the prompt degradation of UBE2D protein targets, including p53. In Drosophila harboring a larval-lethal knockdown of the nearly identical fly orthologue UbcD1, complementation with human UBE2D2 restored near-normal development, but mutation of its CXXXC Cu+ binding motif profoundly disrupted organogenesis. Nutrient Cu+ emerges as a trophic allosteric modulator of UBE2D activity through a structural motif whose evolution coincides with animal multicellularity.

One Sentence SummaryModulation of nutrient copper impacts protein turnover and animal morphogenesis through conserved allostery of ubiquitin E2D conjugases.

HilightsO_LINutrient copper supply is critical for cell growth and differentiation
C_LIO_LIThe E2D clade of ubiquitin conjugases contains a sub-femtomolar-affinity Cu+ binding motif
C_LIO_LIAllosteric activation by Cu+ markedly accelerates protein polyubiquitination
C_LIO_LIThis sensor couples physiologic fluctuations in cytoplasmic Cu+ with the degradation rate of E2D targets, including p53
C_LIO_LIThis metazoan signaling mechanism is critical for drosophila morphogenesis
C_LI

In BriefConserved allostery of ubiquitin E2D conjugases links nutrient copper signaling to protein degradation and animal morphogenesis.

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sentences: NyuWa Genome Resource: Deep Whole Genome Sequencing Based Chinese Population Variation Profile and Reference Panel The lack of Chinese population specific haplotype reference panel and whole genome sequencing resources has greatly hindered the genetics studies in the worlds largest population. Here we presented the NyuWa genome resource based on deep (26.2X) sequencing of 2,999 Chinese individuals, and constructed NyuWa reference panel of 5,804 haplotypes and 19.3M variants, which is the first publicly available Chinese population specific reference panel with thousands of samples. Compared with other panels, NyuWa reference panel reduces the Han Chinese imputation error rate by the range of 30% to 51%. Population structure and imputation simulation tests supported the applicability of one integrated reference panel for both northern and southern Chinese. In addition, a total of 22,504 loss-of-function variants in coding and noncoding genes were identified, including 11,493 novel variants. These results highlight the value of NyuWa genome resource to facilitate genetics research in Chinese and Asian populations.

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Rhythm of The Night (and Day): Predictive metabolic modeling of circadian growth in Chlamydomonas Algal cells experience strong circadian rhythms under diurnal light, with regular changes in both biomass composition and transcriptomic environment. However, most metabolic models - critical tools for bioengineering organisms - assume a steady state. The conflict between these assumptions and the reality of the cellular environment make such models inappropriate for algal cells, creating a significant obstacle in engineering cells that are viable under natural light. By transforming a set of discreet transcriptomic measurements from synchronized Chlamydomonas cells grown in a 12/12 diel light regime (1) into continuous curves, we produced a complete representation of the cells transcriptome that can be interrogated at any arbitrary timepoint. We clustered these curves, in order to find genes that were expressed in similar patterns, and then also used it to build a metabolic model that can accumulate and catabolize different biomass components over the course of a day. This model predicts qualitative phenotypical outcomes for the sta6 mutant, including excess lipid accumulation (2) and a failure to thrive when grown diurnally in minimal media (3), representing a qualitative prediction of phenotype from genotype even under dynamic conditions. We also extended this approach to simulate all single-knockout mutants with genes represented in the model and identified potential targets for rational engineering efforts.

SIGNIFICANCE STATEMENTWe have developed the first transient metabolic model for diurnal growth of algae based on experimental data and capable of predicting phenotype from genotype. This model enables us to evaluate the impact of genetic and environmental changes on the growth, biomass composition and intracellular fluxes of the model green alga, Chlamydomonas reinhardtii. The availability of this model will enable faster and more efficient design of cells for production of fuels, chemicals and pharmaceuticals.

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A joint distribution framework to improve presence-only species distribution models by exploiting opportunistic surveys {whitebullet} AimWe propose a Bayesian framework for modelling species distributions using presence-only biodiversity occurrences obtained from historical opportunistic surveys.

 {whitebullet}LocationGlobal applicability with two case studies in south-east Mexico.

 {whitebullet}MethodsThe framework defines a bivariate spatial process separable into ecological and sampling effort processes that jointly generate occurrence observations of biodiversity records. Presence-only data are conceived as incomplete observations where some presences have been filtered out. A choosing principle is used to separate out presences, missing data and absences relative to the species of interest and the sampling observations. The framework provides three modelling alternatives for accounting the spatial autocorrelation structure: independent latent variables (model I); common latent spatial random effect (model II); and correlated latent spatial random effects (model III).

The framework was compared against the Maximum Entropy (MaxEnt) algorithm in two case studies: one for the prediction of pines (Class: Pinopsida), using botanical records as sampling observations and another for the prediction of Flycatchers (Family: Tyranidae), using bird sightings as sampling records.

 {whitebullet} [a]ResultsIn both case studies, at least one of the proposed models achieved higher predictive accuracy than MaxEnt. The model with correlated spatial effects fit best when the sampling effort was informative, while the one with a shared spatial effect was more suitable in cases with high proportion of non sampled sites.

 {whitebullet}Main ConclusionsOur approach provides a flexible framework for presence-only SDMs aided by a sampling effort process informed by the accumulated observations of independent and heterogeneous surveys. For the two case studies, the framework provided a model with a higher predictive accuracy than an optimised version MaxEnt.

sentences: Nuclease proteins CifA and CifB promote spermatid DNA damage associated with symbiont-induced cytoplasmic incompatibility The worldwide endosymbiosis between arthropods and Wolbachia bacteria is an archetype for reproductive parasitism. This parasitic strategy rapidly increases the proportion of symbiont-transmitting mothers, and the most common form, cytoplasmic incompatibility (CI), impacts insect evolution and arboviral control strategies. During CI, sperms from symbiotic males kill embryos of aposymbiotic females via two nuclear-targeting proteins, CifA and CifB, that alter sperm chromatin organization in Drosophila melanogaster. Here we hypothesize that Cif proteins metabolize nucleic acids of developing sperm to initiate genome integrity changes. Using in vitro and in situ transgenic, mutant, enzymatic, and cytochemical assays, we show that CifA is a previously-unrecognized DNase and RNase, and CifB is a DNase. Notably, in vitro nuclease activity translates to in situ spermatid DNA damage at the canoe stage of spermiogenesis. Evolution-guided mutations ablate Cif enzymatic activity. Nucleic acid metabolism by Cif enzymes expands a fundamental understanding of the mechanism of symbiont-mediated reproductive parasitism.

sentences: Improving long-read consensus sequencing accuracy with deep learning The PacBio HiFi sequencing technology combines less accurate, multi-read passes from the same molecule (subreads) to yield consensus sequencing reads that are both long (averaging 10-25 kb) and highly accurate. However, these reads can retain residual sequencing error, predominantly insertions or deletions at homopolymeric regions. Here, we train deep learning models to polish HiFi reads by recognizing and correcting sequencing errors. We show that our models are effective at reducing these errors by 25-40% in HiFi reads from human as well as E. coli genomes.

sentences: Enhancing Sampling of Water Rearrangements on Ligand Binding: A Comparison of Techniques 1Water often plays a key role in protein structure, molecular recognition, and mediating protein-ligand interactions. Thus, free energy calculations must adequately sample water motions, which often proves challenging in typical MD simulation timescales. Thus, the accuracy of methods relying on MD simulations ends up limited by slow water sampling. Particularly, as a ligand is removed or modified, bulk water may not have time to fill or rearrange in the binding site. In this work, we focus on several molecular dynamics (MD) simulation-based methods attempting to help rehydrate buried water sites: BLUES, using nonequilibrium candidate Monte Carlo (NCMC); grand, using grand canonical Monte Carlo (GCMC); and normal MD. We assess the accuracy and efficiency of these methods in rehydrating target water sites. We selected a range of systems with varying numbers of waters in the binding site, as well as those where water occupancy is coupled to the identity or binding mode of the ligand. We analyzed rehydration of buried water sites in binding pockets using both clustering of trajectories and direct analysis of electron density maps. Our results suggest both BLUES and grand enhance water sampling relative to normal MD and grand is more robust than BLUES, but also that water sampling remains a major challenge for all of the methods tested. The lessons we learned for these methods and systems are discussed.

sentences: Integrative single cell and spatial transcriptomic analysis reveal reciprocal microglia - plasma cell crosstalk in the mouse brain during chronic Trypanosoma brucei infection Human African trypanosomiasis, or sleeping sickness, is caused by the protozoan parasite Trypanosoma brucei and induces profound reactivity of glial cells and neuroinflammation when the parasites colonise the central nervous system. However, the transcriptional and functional responses of the brain to chronic T. brucei infection remain poorly understood. By integrating single cell and spatial transcriptomics of the mouse brain, we identified that glial responses triggered by infection are readily detected in the proximity to the circumventricular organs, including the lateral and 3rd ventricle. This coincides with the spatial localisation of both slender and stumpy forms of T. brucei. Furthermore, in silico predictions and functional validations led us to identify a previously unknown crosstalk between homeostatic Cx3cr1+ microglia and Cd138+ plasma cells mediated by IL-10 and B cell activating factor (BAFF) signalling. This study provides important insights and resources to improve understanding of the molecular and cellular responses in the brain during infection with African trypanosomes.

sentences: Environment-imposed constraints can make Brownian walkers efficient searchers Pathogen-specific CD8 T cells face the problem of finding rare cells that present their cognate antigen either in the lymph node or infected tissue. While quantitative details of T cell movement strategies in some tissues such lymph nodes or skin have been relatively well characterized, we still lack quantitative understanding of T cell movement in many other important tissues such as the spleen, lung, liver, and gut. Furthermore, how environmental constraints influence movement patterns of T cells in tissues remains incompletely characterized. Liver is one of the major organs in mice and humans and is the target site for many pathogens such as hepatitis B and C viruses in humans and Plasmodium parasites in multiple mammalian species. We developed a protocol to generate stable numbers of liver-located CD8 T cells, used intravital microscopy to record movement patterns of CD8 T cells in livers of live mice, and analyzed these and previously published data using well-established statistical and computational methods. We show that in most of our experiments Plasmodium-specific liver-localized CD8 T cells perform correlated random walks characterized by transiently superdiffusive displacement with persistence times of 10-15 min that exceed those observed for T cells in lymph nodes. Liver-localized CD8 T cells typically crawl on the lumenal side of liver sinusoids (i.e., are in the blood); simulating T cell movement in digital structures derived from the liver sinusoids illustrates that liver structure alone is sufficient to explain the relatively long superdiffusive displacement of T cells. In experiments when CD8 T cells in the liver poorly attach to the sinusoids (e.g., one week post immunization with radiation-attenuated Plasmodium sporozoites) T cells also undergo Levy flights - large displacements occurring due to cell deattaching from the endothelium, floating with the blood flow, and re-attaching at another location. Our analysis, thus, provides quantitative details of movement patterns of liver-localized CD8 T cells and illustrates how structural and physiological details of the tissue may impact T cell movement patterns.

sentences: Hydrodynamics of spike proteins dictate a transport-affinity competition for SARS-CoV-2 and other enveloped viruses Many viruses, such as SARS-CoV-2 or Influenza, possess spike-decorated envelopes. Depending on the virus type, a large variability is present in spikes number, morphology and reactivity, which remains generally unexplained. Since viruses transmissibility depend on features beyond their genetic sequence, new tools are required to discern the effects of spikes functionality, interaction, and morphology. Here, we postulate the relevance of hydrodynamic interactions in the viral infectivity of enveloped viruses and propose micro-rheological characterization as a platform for viruses differentiation. To understand how the spikes affect virion mobility and infectivity, we investigate the diffusivity of spike-decorate structures using mesoscopic-hydrodynamic simulations. Furthermore, we explored the interplay between affinity and passive viral transport. Our results revealed that the diffusional mechanism of SARS-CoV-2 is strongly influenced by the size and distribution of its spikes. We propose and validate a universal mechanism to explain the link between optimal virion structure and maximal infectivity for many virus families.

sentences: SARS-CoV-2 Delta breakthrough infections in vaccinated patients The continuous emergence of SARS-CoV-2 variants with increased transmission and immune evasion has caused breakthrough infections in vaccinated population. It is important to determine the threshold of neutralizing antibody titers that permit breakthrough infections. Here we tested the neutralization titers of vaccinated patients who contracted Delta variant. All 75 patients with Delta breakthrough infections exhibited neutralization titers (NT50) of less than 70. Among the breakthrough patients, 76%, 18.7%, and 5.3% of them had the NT50 ranges of <20, 20-50, and 50-69, respectively. These clinical laboratory results have implications in vaccine strategy and public health policy.

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Caveolae mediated endocytosis of VLDL particles in macrophages requires NPC1 and STARD3 for further lysosomal processing Macrophages accumulate triglycerides under certain pathological conditions such as atherosclerosis. Triglycerides are carried in the bloodstream as part of very low-density lipoproteins (VLDL) and chylomicrons. How macrophages take up and process VLDL-lipids is not very well known. Here, using VLDL-sized triglyceride-rich emulsion particles, we aimed to study the mechanism by which VLDL-triglycerides are taken up, processed, and stored in macrophages. Our results show that macrophage uptake of emulsion particles mimicking VLDL (VLDLm) is dependent on lipoproteins lipase (LPL) and requires the lipoprotein-binding C-terminal domain of LPL but not the catalytic N-terminal domain. Subsequent internalization of VLDLm-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by triglyceride hydrolysis catalyzed by lysosomal acid lipase. Transfer of lysosomal fatty acids to the ER for subsequent storage as triglycerides is mediated by Stard3, whereas NPC1 was found to promote the extracellular efflux of fatty acids from lysosomes. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete part of the internalized triglycerides as fatty acids.

SummaryTriglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a pro-inflammatory effect on macrophage. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. We show that uptake by macrophages of VLDL-like particles is dependent on the enzyme lipoproteins lipase via its C-terminal domain. Subsequent internalization of VLDL-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by hydrolysis by lysosomal acid lipase. Transfer of lysosomal fatty acids to the ER for lipid storage is mediated by Stard3, while NPC1 promotes the extracellular efflux of fatty acids. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete internalized triglycerides as fatty acids.

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sentences: Machine Learning Identification of Capsid Mutations to Improve AAV Production Fitness The adeno-associated virus (AAV) holds great potential for gene therapy efforts by providing a viable vector. However, current efforts are constrained by a lack of AAV variants that exhibit specific tropisms or immunogenicity and a lack of sustainable industrial projection. Departing from experimental approaches to addressing these issues, we built a model to predict residue mutations to improve AAV production fitness. Our model leverages the evolutionary paradigm and microenvironment characteristics by analyzing structural AAV data without needing domain knowledge or experimental fitness data for AAV as inputs. When testing our model's predictions for AAV2 residue mutations, we found a threefold increase in the percent of mutations yielding variants with better production fitness than wild type compared to random mutations, achieving a p-value of 7.46x10-12. Given these results, our machine learning approach of using structural data to approximate fitness data has the potential to accelerate AAV development.

sentences: Evolutionary genetic integration of behavioural and endocrine components of the stress response The vertebrate stress response comprises a suite of behavioural and physiological traits that must be functionally integrated to ensure organisms cope adaptively with acute stressors. Natural selection should favour functional integration, leading to a prediction of genetic integration of these traits. Despite the implications of such genetic integration for our understanding of human and animal health, as well as evolutionary responses to natural and anthropogenic stressors, formal quantitative genetic tests of this prediction are lacking. Here we demonstrate that acute stress response components in Trinidadian guppies are both heritable and integrated on the major axis of genetic covariation. This integration could either facilitate or constrain evolutionary responses to selection, depending upon the alignment of selection with this axis. Such integration also suggests artificial selection on the genetically correlated behavioural responses to stress could offer a viable non-invasive route to the improvement of health and welfare in captive animal populations.

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Drug-driven reclassification of multiple tumour subtypes reveals intrinsic molecular concordance of therapy across histologically disparate cancers Cancers that are histologically defined as the same type of cancer often need a distinct therapy based on underlying heterogeneity; likewise, histologically disparate cancers can require similar treatment approaches due to intrinsic similarities. A comprehensive analysis integrated with drug response data and genomic alterations, particularly to reveal therapeutic concordance mechanisms across histologically disparate tumour subtypes, has not yet been fully exploited.

In this study, we used pharmacogenomic profiling data provided from the Cancer Genome Project (CGP) in a systematic in silico investigation of the pharmacological subtypes of cancers and the intrinsic concordance of molecular mechanisms leading to similar therapeutic responses across histologically disparate tumour subtypes. We further developed a novel approach to redefine cell-to-cell similarity and drug-to-drug similarity from the therapeutic concordance, providing a new point of view to study cancer heterogeneity.

Our study identified that histologically different tumours, such as malignant melanoma and colorectal adenocarcinoma, could belong to the same pharmacological subtype regarding drug sensitivity to MEK inhibitors, which was determined by their genomic alterations, high occurrence of BRAF or KRAS mutations. Therapeutic concordance for chemotherapy drugs was identified across histologically disparate hematological tumors mainly due to the extraordinary activation of the cell cycle in blood cancers. A subcluster of SCLC had a more similar profile with hematological tumors, and was associated with the malignant phenotype, with a higher level of MYC expression. We developed a website to store and visualize the pharmacological subtypes of drugs, as well as their connected genomic and expression alterations.

sentences: Stratification Of Chemotherapy-Treated Stage III Colorectal Cancer Patients Using Multiplexed Imaging And Single Cell Analysis Of T Cell Populations Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Predictive biomarkers for identification of patients with increased risk for disease recurrence are currently lacking, with progress in biomarker discovery hindered by the diseases inherent heterogeneity. The immune profile of colorectal tumors has previously been found to have prognostic value. The aims of this study were to evaluate immune signatures in the tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single cell analysis technology (Cell DIVE). Tissue microarrays (TMAs) with up to three 1mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin chemotherapy. Single sections underwent multilplexed immunofluorescence with Cy3- and Cy5-conjugated antibodies for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase and S6). We applied a probabilistic multi-class, multi-label classification algorithm based on multi-parametric models to build statistical models of protein expression to classify immune cells. Expert annotations of immune cell markers were made on a range of images, and Support Vector Machines (SVM) were used to derive a statistical model for cell classification. Images were also manually scored independently by a Pathologist as  high,  moderate or  low, for stromal and total immune cell content. Excellent agreement was found between manual and total automated scores (p<0.0001). Higher levels of multi-marker classified regulatory T cells (CD3+CD4+FOXP3+PD1-) were significantly associated with disease-free survival (DFS) and overall-survival (OS) (p=0.049 and 0.032), compared to FOXP3 alone. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cells with greater potential for predicting patient outcomes.

sentences: Automated slice-specific z-shimming for fMRI of the human spinal cord Functional magnetic resonance imaging (fMRI) of the human spinal cord faces many challenges, such as signal loss due to local magnetic field inhomogeneities. This issue can be addressed with slice-specific z-shimming, which compensates for the dephasing effect of the inhomogeneities using a slice-specific gradient pulse. Here, we aim to address outstanding issues regarding this technique by evaluating its effects on several aspects that are directly relevant for spinal fMRI and by developing two automated procedures in order to improve upon the time-consuming and subjective nature of manual selection of z-shims: one procedure finds the z-shim that maximizes signal intensity in each slice of an EPI reference-scan and the other finds the through-slice field inhomogeneity for each EPI-slice in field map data and calculates the required compensation gradient moment. We demonstrate that the beneficial effects of z-shimming are apparent across different echo times, hold true for both the dorsal and ventral horn, and are also apparent in the temporal signal-to-noise ratio (tSNR) of EPI time-series data. Both of our automated approaches were faster than the manual approach, lead to significant improvements in gray matter tSNR compared to no z-shimming and resulted in beneficial effects that were stable across time. While the field-map-based approach performed slightly worse than the manual approach, the EPI-based approach performed as well as the manual one and was furthermore validated on an external corticospinal data-set (N>100). Together, automated z-shimming may improve the data quality of future spinal fMRI studies and lead to increased reproducibility in longitudinal studies.

sentences: Coregistration of heading to visual cues in retrosplenial cortex Spatial cognition depends on an accurate representation of orientation within an environment. Head direction cells in distributed brain regions receive a range of sensory inputs, but visual input is particularly important for aligning their responses to environmental landmarks. To investigate how population-level heading responses are aligned to visual input, we recorded from retrosplenial cortex (RSC) of head-fixed mice in a moving environment using two-photon calcium imaging. We show that RSC neurons are tuned to the animals relative orientation in the environment, even in the absence of head movement. Next, we found that RSC receives functionally distinct projections from visual and thalamic areas, and contains several functional classes of neurons. While some functional classes mirror RSC inputs, a newly discovered class coregisters visual and thalamic signals. Finally, decoding analyses reveal unique contributions to heading from each class. Our results suggest an RSC circuit for anchoring heading representations to environmental visual landmarks.

sentences: Vision-dependent and -independent molecular maturation of mouse retinal ganglion cells The development and connectivity of retinal ganglion cells (RGCs), the retinas sole output neurons, are patterned by activity-independent transcriptional programs and activity-dependent remodeling. To inventory the molecular correlates of these influences, we applied high-throughput single-cell RNA sequencing (scRNA-seq) to mouse RGCs at six embryonic and postnatal ages. We identified temporally regulated modules of genes that correlate with, and likely regulate, multiple phases of RGC development, ranging from differentiation and axon guidance to synaptic recognition and refinement. Some of these genes are expressed broadly while others, including key transcription factors and recognition molecules, are selectively expressed by one or a few of the 45 transcriptomically distinct types defined previously in adult mice. Next, we used these results as a foundation to analyze the transcriptomes of RGCs in mice lacking visual experience due to dark rearing from birth or to mutations that ablate either bipolar or photoreceptor cells. 98.5% of visually deprived (VD) RGCs could be unequivocally assigned to a single RGC type based on their transcriptional profiles, demonstrating that visual activity is dispensable for acquisition and maintenance of RGC type identity. However, visual deprivation significantly reduced the transcriptomic distinctions among RGC types, implying that activity is required for complete RGC maturation or maintenance. Consistent with this notion, transcriptomic alternations in VD RGCs significantly overlapped with gene modules found in developing RGCs. Our results provide a resource for mechanistic analyses of RGC differentiation and maturation, and for investigating the role of activity in these processes.

sentences: Differential effects of two catalytic mutations on full-length PRDM9 and its isolated PR/SET domain reveal a case of pseudo-modularity PRDM9 is a DNA-binding histone methyltransferase that designates and activates recombination hotspots in mammals by locally trimethylating lysines 4 and 36 of histone H3. In mice, we recently reported two independently produced point mutations at the same residue, glu360pro (Prdm9EP) and glu360lys (Prdm9EK), which severely reduce its H3K4 and H3K36 methyltransferase activities in vivo. Prdm9EP is slightly less hypomorphic than Prdm9EK, but both mutations reduce both the number and amplitude of PRDM9-dependent H3K4me3 and H3K36me3 peaks in spermatocytes. While both mutations cause infertility with complete meiotic arrest in males, Prdm9EP, but not Prdm9EK, is compatible with some female fertility. When we tested the effects of these mutations in vitro, both Prdm9EP and Prdm9EK abolished H3K4 and H3K36 methyltransferase activity in full-length PRDM9. However, in the isolated PRDM9 PR/SET domain, these mutations selectively compromised H3K36 methyltransferase activity, while leaving H3K4 methyltransferase activity intact. The difference in these effects on the PR/SET domain versus the full-length protein show that PRDM9 is not an intrinsically modular enzyme; its catalytic domain is influenced by its tertiary structure and possibly by its interactions with DNA and other proteins in vivo. These two informative mutations illuminate the enzymatic chemistry of PRDM9, and potentially of PR/SET domains in general, reveal the minimal threshold of PRDM9-dependent catalytic activity for female fertility, and potentially have some practical utility for genetic mapping and genomics.

sentences: Patterns of item nonresponse behavior to survey questionnaires are systematic and have a genetic basis Response to survey questionnaires is vital for social and behavioral research, and most analyses assume full and accurate response by survey participants. However, nonresponse is common and impedes proper interpretation and generalizability of results. We examined item nonresponse behavior across 109 questionnaire items from the UK Biobank (UKB) (N=360,628). Phenotypic factor scores for two participant-selected nonresponse answers, "Prefer not to answer" (PNA) and "I dont know" (IDK), each predicted participant nonresponse in follow-up surveys, controlling for education and self-reported general health. We performed genome-wide association studies on these factors and identified 39 genome-wide significant loci, and further validated these effects with polygenic scores in an independent study (N=3,414), gaining information that we could not have had from phenotypic data alone. PNA and IDK were highly genetically correlated with one another and with education, health, and income, although unique genetic effects were also observed for both PNA and IDK. We discuss how these effects may bias studies of traits correlated with nonresponse and how genetic analyses can further enhance our understanding of nonresponse behaviors in survey research, for instance by helping to correct for nonresponse bias.

sentences: Chromosomal rearrangements but no change of genes and transposable elements repertoires in an invasive forest-pathogenic fungus AO_SCPLOWBSTRACTC_SCPLOWChromosomal rearrangements have been largely described among eukaryotes, and may have important consequences on evolution of species. High genome plasticity has been often reported in Fungi, which may explain their apparent ability to quickly adapt to new environments. Cryphonectria parasitica, causing the Chestnut blight disease, is an invasive fungal pathogen species associated with several recent host shifts during its successive introductions from Asia to North America and Europe. Previous cytological karyotyping and genomic studies suggested several chromosomal rearrangements which remains to be described in detail for this species. A serious limitation for valid genome comparisons is the access to robust genome assemblies that usually contain genomic regions of low complexity. We present a new de novo whole-genome assembly obtained from a new method of DNA extraction and long-reads sequencing Nanopore technology obtained from a Japanese isolate sampled in the native area of the species. The comparison with a recently published reference genome revealed stable gene and transposable elements (TEs) repertoires. We also showed that the C. parasitica genome is lowly compartmentalized, with a poor association between TEs and genes, such as those potentially involved in host interactions (i.e., genes coding for small secreted proteins or for secondary metabolites). This genome comparison, however, detected several large chromosomal rearrangements that may have important consequences in gene regulations and sexual mating in this invasive species. This study opens the way for more comparisons of high-quality assembled genomes, and questions the role of structural variations in the invasive success of this fungal pathogen species.

sentences: Endogenous μ-opioid receptor system modulates sex drive in human males The endogenous mu-opioid receptor (MOR) system modulates a multitude of social and reward-related functions, and exogenous opiates also influence sex drive in humans and animals. Sex drive shows substantial variation across humans, and it is possible that individual differences in MOR availability underlie interindividual of variation in human sex drive. Here we measured healthy male subjects (n=52) brains MOR availability with positron emission tomography (PET) using an agonist radioligand, [11C]carfentanil, that has high affinity for MORs. Sex drive was measured using self-reports of engaging in sexual behaviour (sex with partner and masturbating). Bayesian hierarchical regression analysis revealed that sex drive was positively associated with MOR availability in cortical and subcortical areas, notably in caudate nucleus, hippocampus, and cingulate cortices. These results were replicated in full-volume GLM analysis. These widespread effects are in line with high spatial autocorrelation in MOR expression in human brain. Complementary voxel-based morphometry analysis (n=108) provided limited evidence for association between sex drive and cortical density in the midcingulate cortex. We conclude that endogenous MOR tone is associated with individual differences in sex drive in human males.

sentences: Retinal OFF ganglion cells allow detection of quantal shadows at starlight Perception of light in darkness requires no more than a handful of photons and this remarkable behavioral performance can be directly linked to a particular retinal circuit - the retinal ON pathway. However, the neural limits to shadow detection in very dim light have remained unresolved. Here, we unravel the neural mechanisms that determine the sensitivity of mice to the dimmest light decrements by measuring signals from the most sensitive ON and OFF retinal ganglion cell types and by correlating their signals with visually guided behavior. We show that mice can detect shadows when only a few photon absorptions are missing among thousands of rods. Behavioral detection of "quantal" shadows relies only on the retinal OFF pathway and is limited by noise and losses of single-photon signals in retinal processing. Thus, in the dim-light regime, light increments and decrements are encoded separately via the ON and OFF retinal pathways, respectively.

sentences: A Genomic Perspective on the Evolutionary Diversification of Turtles To examine phylogenetic heterogeneity in turtle evolution, we collected thousands of high-confidence single-copy orthologs from 19 genome assemblies representative of extant turtle diversity and estimated a phylogeny with multispecies coalescent and concatenated partitioned methods. We also collected next-generation sequences from 26 turtle species and assembled millions of biallelic markers to reconstruct phylogenies based on annotated regions from the western painted turtle (Chrysemys picta bellii) genome (coding regions, introns, untranslated regions, intergenic, and others). We then measured gene tree-species tree discordance, as well as gene and site heterogeneity at each node in the inferred trees, and tested for temporal patterns in phylogenomic conflict across turtle evolution. We found strong and consistent support for all bifurcations in the inferred turtle species phylogenies. However, a number of genes, sites, and genomic features supported alternate relationships between turtle taxa. Our results suggest that gene tree-species tree discordance in these datasets is likely driven by population-level processes such as incomplete lineage sorting. We found very little effect of substitutional saturation on species tree topologies, and no clear phylogenetic patterns in codon usage bias and compositional heterogeneity. There was no correlation between gene and site concordance, node age, and DNA substitution rate across most annotated genomic regions. Our study demonstrates that heterogeneity is to be expected even in well resolved clades such as turtles, and that future phylogenomic studies should aim to sample as much of the genome as possible in order to obtain accurate phylogenies for assessing conservation priorities in turtles.

sentences: Unraveling three-dimensional chromatin structural dynamics during spermatogonial differentiation Spermatogonial stem cells (SSCs) are able to undergo self-renewal and differentiation. Unlike the self-renewal that replenishes the SSC and progenitor pool, the differentiation is an irreversible process committed to meiosis. While the preparations for meiotic events in differentiating spermatogonia (Di-SG) are likely to be accompanied by alterations in chromatin structure, the three-dimensional (3D) chromatin architectural difference between SSCs and Di-SG, and the higher-order chromatin dynamics during spermatogonial differentiation, have not been systematically investigated. Here, we performed in situ high-throughput chromosome conformation capture (Hi-C), RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses on porcine undifferentiated spermatogonia (Un-SG, which consist of SSCs and progenitors) and Di-SG. By integrating and analyzing these data, we identified that Di-SG exhibited increased disorder but weakened compartmentalization and topologically associating domains (TADs) in comparison with Un-SG, suggesting that diminished higher-order chromatin architecture in meiotic cells, as shown by recent reports, is preprogramed in Di-SG. Our data also revealed that A/B compartments and TADs were related to dynamic gene expression during spermatogonial differentiation. We further unraveled the contribution of promoter-enhancer interactions (PEIs) to pre-meiotic transcriptional regulation, which has not been accomplished in previous studies due to limited cell input and resolution. Together, our study uncovered the 3D chromatin structure of SSCs/progenitors and Di-SG, as well as the interplay between higher-order chromatin architecture and dynamic gene expression during spermatogonial differentiation, providing novel insights into the mechanisms for SSC self-renewal and differentiation and having implications for diagnosis and treatment of male sub-/infertility.

sentences: A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in the regulation of aging and metabolism. Targeted sequencing identified a SIRT6 allele containing two linked substitutions (N308K/A313S) as enriched in Ashkenazi Jewish (AJ) centenarians as compared to AJ control individuals. Characterization of this SIRT6 (centSIRT6) allele demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double strand break repair, and more robust cancer cell killing compared to the wild type. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

sentences: Altered low frequency brain rhythms precede changes in gamma power during tauopathy Alzheimers disease and other dementias are associated with disruptions of electrophysiological brain activity, including low frequency and gamma rhythms. Many of these dementias are also associated with the malfunction of the membrane associated protein tau. Tauopathy disrupts neuronal function and the stability of synapses and is a key driver of neurodegeneration. Here we ask how brain rhythms are affected by tauopathy, at different stages of its progression. We performed local field potential recordings from visual cortex of rTg4510 and control animals at early stages of neurodegeneration (5 months) and at a more advanced stage where pathology is evident (8 months). We measured brain activity in the presence or absence of external visual stimulation, and while monitoring pupil diameter and locomotion to establish animal behavioural states. At 5 months, before substantial pathology, we found an increase in low frequency rhythms during resting state in tauopathic animals. This was because tauopathic animals entered intermittent periods of increased neural synchronisation, where activity across a wide band of low frequencies was strongly correlated. At 8 months, when the degeneration was more advanced, the increased synchronisation and low frequency power was accompanied by a reduction in power in the gamma range, with diverse effects across different components of the gamma rhythm. Our results indicate that slower rhythms are impaired earlier than gamma rhythms in tauopathy, suggesting that electrophysiological measurements can indicate both the presence and progression of tauopathic degeneration.

sentences: Hyperspectral Counting of Multiplexed Nanoparticle Emitters in Single Cells and Organelles Nanomaterials are the subject of a range of biomedical, commercial, and environmental investigations involving measurements in living cells and tissues. Accurate quantification of nanomaterials, at the tissue, cell, and organelle levels, is often difficult, however, in part due to their inhomogeneity. Here, we propose a method that uses the diverse optical properties of a nanomaterial preparation in order to improve quantification at the single-cell and organelle level. We developed  hyperspectral counting, which employs diffraction-limited imaging via hyperspectral microscopy of a diverse set of nanomaterial emitters, to estimate nanomaterial counts in live cells and sub-cellular structures. A mathematical model was developed, and Monte Carlo simulations were employed, to improve the accuracy of these estimates, enabling quantification with single-cell and single-endosome resolution. We applied this nanometrology technique to identify an upper-limit of the rate of uptake into cells - approximately 3,000 particles endocytosed within 30 minutes. In contrast, conventional ROI counting results in a 230% undercount. The method identified significant heterogeneity and a broad non-Gaussian distribution of carbon nanotube uptake within cells. For example, while a particular cell contained an average of 1 nanotube per endosome, the heterogenous distribution resulted in over 7 nanotubes localizing within some endosomes, substantially changing the accounting of subcellular nanoparticle concentration distributions. This work presents a method to quantify cellular and subcellular concentrations of a heterogeneous carbon nanotube reference material, with implications for nanotoxicology, drug/gene delivery, and nanosensor fields.

sentences: A thermodynamic model for water activity and redox potential in evolution and development Reactions involving water and oxygen are basic features of geological and biological processes. To understand how life interacts with its environment requires monitoring interactions with H2O and O2 at timescales relevant to not only organismal growth but also billions of years of geobiological evolution. Here, chemical transformations intrinsic to evolution and development were characterized by analyzing data from recent phylostratigraphic and proteomic studies. This two-stage analysis involves obtaining chemical metrics (carbon oxidation state and stoichiometric hydration state) from the elemental compositions of proteins followed by modeling the relative stabilities of target proteins against a proteomic background to infer thermodynamic parameters (oxygen fugacity, water activity, and virtual redox potential (Eh)). The main results of this study are a rise in carbon oxidation state of proteins spanning the time of the Great Oxidation Event, a rise in virtual redox potential that coincides with the likely emergence of aerobic metabolism, a rise in carbon oxidation state of proteins inferred from the transcriptome in late stages of Bacillus subtilis biofilm growth, and a drop in stoichiometric hydration state of the fruit fly developmental proteome at the same time as a drop in organismal water content. Stoichiometric hydration state also decreases for proteins with more recent gene ages and through stages of biofilm development, leading to predictions of higher hydration potentials at earlier time points. By building chemical representations of protein evolution and developmental proteomes, exciting new types of geobiochemical proxies can be developed.

sentences: Influence of sensory modality and control dynamics on human path integration Path integration is a sensorimotor computation that can be used to infer latent dynamical states by integrating self-motion cues. We studied the influence of sensory observation (visual/vestibular) and latent control dynamics (velocity/acceleration) on human path integration using a novel motion-cueing algorithm. Sensory modality and control dynamics were both varied randomly across trials, as participants controlled a joystick to steer to a memorized target location in virtual reality. Visual and vestibular steering cues allowed comparable accuracies only when participants controlled their acceleration, suggesting that vestibular signals, on their own, fail to support accurate path integration in the absence of sustained acceleration. Nevertheless, performance in all conditions reflected a failure to fully adapt to changes in the underlying control dynamics, a result that was well explained by a bias in the dynamics estimation. This work demonstrates how an incorrect internal model of control dynamics affects navigation in volatile environments in spite of continuous sensory feedback.

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Shade-induced WRKY transcription factors restrict root growth during the shade avoidance response Shade-intolerant plants rapidly elongate their stems, branches, and leaf stalks to compete with their neighboring vegetation to maximize sunlight capture for photosynthesis. This rapid growth adaptation, known as the shade avoidance response (SAR), comes at a cost; reduced biomass, crop yield, and root growth. Significant progress has been made on the mechanistic understanding of hypocotyl elongation during SAR; however, the molecular account of how root growth is repressed is not well understood. Here, we explore the mechanisms by which low red:far-red induced SAR restrict the primary and lateral root (LR) growth. By analyzing whole-genome transcriptome, we identified a core set of shade-induced genes in the roots of Arabidopsis and tomato seedlings grown in the shade. Abiotic and biotic stressors also induce many of these shade-induced genes and are predominantly regulated by the WRKY transcription factors. Correspondingly, a majority of the WRKYs were also among the shade-induced genes. Functional analysis using transgenics of these shade-induced WRKYs revealed their role is essentially to restrict primary root and LR growth in the shade, and captivatingly, they did not affect hypocotyl elongation. Similarly, we also show that ethylene hormone signaling is necessary to limit root growth in the shade. Our study proposes that during SAR, shade-induced WRKY26, 45, and 75, and ethylene reprogram gene expression in the root to restrict its growth and development. The reduced growth of root organs helps the plant divert its critical resources to the elongating organs in the shoot to ensure competitiveness under limiting photosynthetic radiation.

One sentence summaryShade represses root growth by inducing WRKY transcription factors.

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Diet outweighs genetics in shaping gut microbiomes in Asian honeybee BackgroundThe gut microbiome is a crucial element that facilitates a hosts adaptation to a changing environment. Host-specificity often coincides with distinctions in gut microbes, suggesting a co-evolution of the holobionts. However, it is unclear how gut microbiota shared by a common host ancestor would co-diversify with hosts and eventually become distinct among sister hosts. In this context, understanding the evolutionary pathway of gut microbiomes of the same host species could provide insight on how holobionts co-adapt along environmental gradients. Specifically, we ask which factor, nature or nurture, i.e., genetics or diets, contributes more to the shaping of gut microbiome, along with host diversification and range expansion.

ResultsWe compared and analyzed the gut microbiomes of 99 Asian honeybees, Apis cerana, from genetically diverged populations covering 13 provinces across China. Bacterial composition varied significantly across populations at phylotype, sequence-discrete population (SDP), and strain levels, but with extensive overlaps, indicating the diversity of microbial community among A. cerana populations is driven by nestedness. Taken together, genetics exhibited tangential impacts, while pollen diets were significantly correlated with both the composition and function of gut microbiome. Core bacteria, Gilliamella and Lactobacillus Firm-5, showed antagonistic turnovers and contributed to the enrichment in carbohydrate transport and metabolism. By feeding and inoculation bioassays, we confirmed that the variations in pollen polysaccharide composition contributed to the trade-off of these core bacteria.

ConclusionsProgressive change, i.e., nestedness, is the foundation of gut microbiome evolution in the Asian honeybee. Such a transition during the co-diversification of gut microbiomes is shaped primarily by environmental factors, diets in general, pollen polysaccharide in particular.

sentences: Structural basis for ligand recognition and G protein-coupling promiscuity of the cholecystokinin A receptor Cholecystokinin A receptor (CCKAR) belongs to family A G protein-coupled receptors (GPCRs) and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with sulfated ligand and to couple with divergent G protein subtypes, including Gs, Gi, and Gq. However, the basis for G protein coupling promiscuity and ligand recognition by CCKAR remain unknown. Here we present three cryo-electron microscopy (cryo-EM) structures of sulfated CCK-8 activated CCKAR in complex with Gs, Gi, and Gq heterotrimers, respectively. In these three structures, CCKAR presents a similar conformation, whereas conformational differences in "wavy hook" of G subunits and ICL3 of the receptor serve as determinants in G protein coupling selectivity. These structures together with mutagenesis data provide the framework for understanding the G protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8.

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Genomic variation across a clinical Cryptococcus population linked to disease outcome Cryptococcus neoformans is the causative agent of cryptococcosis, a disease with poor patient outcomes, accounting for approximately 180,000 deaths each year. Patient outcomes may be impacted by the underlying genetics of the infecting isolate, however, our current understanding of how genetic diversity contributes to clinical outcomes is limited. Here, we leverage clinical, in vitro growth and genomic data for 284 C. neoformans isolates to identify clinically relevant pathogen variants within a population of clinical isolates from patients with HIV-associated cryptococcosis in Malawi. Through a genome-wide association study (GWAS) approach, we identify variants associated with fungal burden and growth rate. We also find both small and large-scale variation, including aneuploidy, associated with alternate growth phenotypes, which may impact the course of infection. Genes impacted by these variants are involved in transcriptional regulation, signal transduction, glycolysis, sugar transport, and glycosylation. When combined with clinical data, we show that growth within the CNS is reliant upon glycolysis in an animal model, and likely impacts patient mortality, as CNS burden modulates patient outcome. Additionally, we find genes with roles in sugar transport are under selection in the majority of these clinical isolates. Further, we demonstrate that two hypothetical proteins identified by GWAS impact virulence in animal models. Our approach illustrates links between genetic variation and clinically relevant phenotypes, shedding light on survival mechanisms within the CNS and pathways involved in this persistence.

ImportanceInfection outcomes for cryptococcosis, most commonly caused by C. neoformans, are influenced by host immune responses, as well as host and pathogen genetics. Infecting yeast isolates are genetically diverse, however, we lack a deep understanding of how this diversity impacts patient outcomes. To better understand both clinical isolate diversity and how diversity contributes to infection outcome, we utilize a large collection of clinical C. neoformans samples, isolated from patients enrolled in a clinical trial across 3 hospitals in Malawi. By combining whole-genome sequence data, clinical data, and in vitro growth data, we utilize genome-wide association approaches to examine the genetic basis of virulence. Genes with significant associations show virulence phenotypes in both murine and rabbit models, demonstrating that our approach successfully identifies links between genetic variation and biologically significant phenotypes.

sentences: DecoPath: A web application for decoding pathway enrichment analysis The past two decades have brought a steady growth of pathway databases and pathway enrichment methods. However, the advent of pathway data has not been accompanied by an improvement with regards to interoperability across databases, thus, hampering the use of pathway knowledge from multiple databases for pathway enrichment analyses. While integrative databases have attempted to address this issue by collating pathway knowledge from multiple resources, these approaches do not account for redundant information across them. On the other hand, the majority of studies that employ pathway enrichment analyses still rely upon a single database, though the use of another resource could yield differing results, which is similarly the case when different pathway enrichment methods are employed. These shortcomings call for approaches that investigate the differences and agreements across databases and enrichment methods as their selection in the experimental design of a pathway analysis can be a crucial first step in ensuring the results of such an analysis are meaningful. Here we present DecoPath, a web application to assist in the interpretation of the results of pathway enrichment analysis. DecoPath provides an ecosystem to run pathway enrichment analysis or directly upload results and facilitate the interpretation of these results with custom visualizations that highlight the consensus and/or discrepancies at the pathway- and gene-levels. DecoPath is available at https://decopath.scai.fraunhofer.de and its source code and documentation can be found on GitHub at https://github.com/DecoPath/DecoPath.

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The nature of intraspecific genome size variation in taxonomically complex eyebrights O_LIGenome size (GS) is a key trait related to morphology, life history, and evolvability. Although GS is, by definition, affected by presence/absence variants (PAVs), which are ubiquitous in population sequencing studies, GS is often treated as an intrinsic property of a species. Here, we studied intra- and interspecific GS variation in taxonomically complex British eyebrights (Euphrasia).
C_LIO_LIWe generated GS data for 192 individuals of diploid and tetraploid Euphrasia and analysed GS variation in relation to ploidy, taxonomy, population affiliation, and geography. We further compared the genomic repeat content of 30 samples.
C_LIO_LIWe found considerable genuine intraspecific GS variation, and observed isolation-by-distance for GS in outcrossing diploids. Tetraploid Euphrasia showed contrasting patterns, with GS increasing with latitude in outcrossing Euphrasia arctica, but little GS variation in the highly selfing Euphrasia micrantha. Interspecific differences in GS genomic repeat percentages were small.
C_LIO_LIWe show the utility of treating GS as the outcome of polygenic variation. Like other types of genetic variation, such as single nucleotide polymorphisms, GS variation may be increased through hybridisation and population subdivision. In addition to selection on associated traits, GS is predicted to be affected indirectly by selection due to pleiotropy of the underlying PAVs.
C_LI

sentences: The HIV-1 capsid core is an opportunistic nuclear import receptor The movement of viruses and other large macromolecular cargo through nuclear pore complexes (NPCs) is poorly understood. The human immunodeficiency virus type 1 (HIV-1) provides an attractive model to interrogate this process due to the genetic and cell biological assays to score virus nuclear entry in living cells. Although initial studies of HIV-1 infection of nondividing cells focused on karyophilic virion proteins, subsequent work revealed the viral capsid (CA), the chief structural component of the pre-integration complex (PIC), to be a critical determinant in nuclear transport1. In support of this model, HIV-1 interactions with NPCs can be altered through CA mutation2, which makes direct contact with nucleoporins (Nups)3-5. Here we identify Nup35, Nup153, and POM121 to coordinately support HIV-1 nuclear entry. For Nup35 and POM121, this dependence was strongly dependent cyclophilin A (CypA) interaction with CA. Mutation of CA or removal of soluble host factors changed the interaction with the NPC. Collectively, these findings implicate the HIV-1 CA hexameric lattice that encapsulates the viral genome as a macromolecular nuclear transport receptor (NTR) that exploits soluble host factors to modulate NPC requirements during nuclear invasion.

sentences: Distinct profiles of spatio-temporal brain dynamics along symptoms dimensions in autism Autism Spectrum Disorders are accompanied by atypical brain activity and impairments in brain connectivity. In particular, dynamic functional connectivity approaches highlighted aberrant brain fluctuations at rest in individuals with autism compared to a group composed of typically developed individuals, matched in age and gender. However, the characterization of these variations remains unclear. Here, we quantified the spatio-temporal network dynamics using two novel dynamic group-based measures, namely system diversity and spatio-temporal diversity. Using the public database ABIDE 1, we explored the differences between individuals with autism and typically developed individuals. Our results show evidence that individuals with autism have atypical connectivity patterns over time characterized by a lower integration of heterogeneous cognitive processes and unstable functional activity, except for the default mode network presenting its own specific dynamic pattern. Within the autism group, we find this pattern of results to be stronger in more severely affected patients with a predominance of symptoms in the social affect domain. However, patients with prominently restricted and repetitive behaviours demonstrate a more conservative profile of brain dynamics characterized by a lower spatio-temporal diversity of the default mode network.

sentences: Investigation of Chordwise Functionally Graded Flexural Rigidity in Flapping Wings Using a Two-Dimensional Pitch-Plunge Model Insect wings are heterogeneous structures, with flexural rigidity varying one to two orders of magnitude over the wing surface. This heterogeneity influences the deformation the flapping wing experiences during flight. However, it is not well understood how this flexural rigidity gradient affects wing performance. Here, we develop a simplified 2D model of a flapping wing as a pitching, plunging airfoil using the assumed mode method and unsteady vortex lattice method to model the structural and fluid dynamics, respectively. We conduct parameter studies to explore how variable flexural rigidity affects mean lift production, power consumption and the forces required to flap the wing. We find that there is an optimal flexural rigidity distribution that maximizes lift production; this distribution generally corresponds to a 3:1 ratio between the wings flapping and natural frequencies, though the ratio is sensitive to flapping kinematics. For hovering flight, the optimized flexible wing produces 20% more lift and requires 15% less power compared to a rigid wing but needs 20% higher forces to flap. Even when flapping kinematics deviate from those observed during hover, the flexible wing outperforms the rigid wing in terms of aerodynamic force generation and power across a wide range of flexural rigidity gradients. Peak force requirements and power consumption are inversely proportional with respect to flexural rigidity gradient, which may present a trade-off between insect muscle size and energy storage requirements. The model developed in this work can be used to efficiently investigate other spatially variant morphological or material wing features moving forward.

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Raman microspectroscopy imaging analysis of extracellular vesicles (EVs) biogenesis by filamentous fungus Penicilium chrysogenum Mechanism of production of extracellular vesicles (EVs) and their molecular contents are of great interest owing to their diverse roles in biological systems and are far from being completely understood. Even though, cellular cargo release mediated by EVs have been demonstrated in several cases, their role in secondary metabolite production and release remains elusive. In this study we investigate this aspect in detail using Raman micro-spectroscopic imaging. We provide considerable evidence to suggest that the release of antibiotic penicillin by filamentous fungus Penicillium chrysogenum involves EVs. Morphological modifications of the fungal body during biogenesis, changes in cell composition at the locus of biogenesis, and major molecular contents of the released EVs are also revealed in this study.

ImportanceExtracellular vesicles (EVs) play a key role in cellular communications. EVs role in functioning of fungi are relatively less explored. Here we show selective enrichment of chemical contents at certain locations of mycelium of P. chrysogenum forming protruding regions. Secondary metabolite penicillin is excessively localized in them. We provide evidence to show that EVs are released from these protrusions. Raman imaging has been applied for molecular profiling of the mycelium and for characterizing chemical contents of the EVs. Our study suggests a possible general role of EVs in the release of antibiotics from the producing organisms.

sentences: Single cell sequencing reveals microglia induced angiogenesis by specific subsets of endothelial cells following spinal cord injury Spinal cord injury (SCI) results in dynamic alterations of the microenvironment at the lesion site, which inevitably leads to neuron degeneration and functional deficits. The prominent deterioration of the milieu, derived from the destruction of spinal vascular system, not only activates innate immunity but also makes cells in the lesion lose nutrient supports. Limited endogenous angiogenesis happens after SCI, but the cell events at the lesion site underpinning this process have not been delineated so far. Here, we conducted single-cell RNA sequencing (scRNA-seq) of tissues in the spinal lesion at different time points after rat SCI. After performing clustering and cell-type identification, we focused on the vascular endothelial cells (ECs), which play a pivot role in angiogenesis, and drew a comprehensive cellular and molecular atlas for endogenous angiogenesis after SCI. We found that microglia and macrophage promote endogenous angiogenesis by regulating EC subsets through SPP1 and IGF1 signal pathways. Our results indicated that immune cells promotes angiogenesis by the regulation of specific cell subsets of vascular ECs, which provides new clues for the development of interventions for SCI.

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Cerebral biodistribution and real-time particle tracking in vivo of ultrabright fluorescent PLGA nano-carriers Poly(lactic-co-glycolic acid) (PLGA)-based drug formulations are approved for the use in humans, however, the potential of PLGA to design nanoparticles (NPs) and target the central nervous system remains to be exploited.

The aim of the current study was design PLGA NPs, loading them with bulky fluorophores thereby increasing single particle fluorescence to a level visible by in vivo microscopy, and investigate their brain biodistribution. We developed, highly fluorescent 70 nm PLGA NPs significantly brighter then quantum dots enabling their visualization by intravital real-time 2-photon microscopy. We found that PLGA NPs coated with pluronic F-68 (PF-68) had a substantially longer plasma half-life than uncoated NPs and were taken up by cerebro-vascular endothelial cells. High resolution confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1 hour after systemic injection and were more readily taken up by endothelial cells in peripheral organs.

The current data suggest that PF-68 coated PLGA NPs are taken up by mouse cerebral and peripheral endothelial cells in vivo. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers.

sentences: Chromatin dynamics are constrained by loops and driven by the INO80 nucleosome remodeler The chromosomes - DNA polymers and their binding proteins - are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of these polymers to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse-model polymer simulations. We first investigated the model that the activity of two loop extrusion factors, cohesin and condensin, drives chromatin dynamics. Surprisingly, deactivating cohesin or condensin increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. We also find that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taken together we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops.

sentences: Viability, task switching, and fall avoidance of the simplest dynamic walker Humans display great versatility when performing goal-directed tasks while walking. However, the extent to which such versatility helps with fall avoidance remains unclear. We recently demonstrated a functional connection between the motor regulation needed to achieve task goals (e.g. maintaining walking speed) and a simple walkers ability to reject large disturbances. Here, for the same model, we identify the viability kernel--the state space region in which the walker can step forever via at least one sequence of push-off inputs per state. We further find that only a few basins of attraction of the speed-regulated walkers steady-state gaits can fully cover the viability kernel. This highlights a potentially important role of task-level motor regulation in fall avoidance. Therefore, we posit an adaptive hierarchical control/regulation strategy that switches between different task-level regulators to avoid falls. Our hierarchical task switching controller only requires a target value of the regulated observable--a  task switch--at each walking step, each chosen from a small, predetermined collection. Because humans have typically already learned to perform such tasks during nominal walking conditions, this suggests that the  information cost of biologically implementing such controllers for the nervous system, including cognitive demands in humans, could be relatively low.

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Molecular architecture of the autoinhibited kinesin-1 lambda particle Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the alpha-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1, in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drive kinesin-1 activation.

Summary statementIntegration of computational structure prediction with electron microscopy reveals the coiled-coil architecture of the autoinhibited compact conformer of the microtubule motor, kinesin-1.

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Neurodevelopmental oscillatory basis of speech processing in noise Humans extraordinary ability to understand speech in noise relies on multiple processes that develop with age. Using magnetoencephalography (MEG), we characterize the underlying neuromaturational basis by quantifying how cortical oscillations in 144 participants (aged 5 to 27 years) track phrasal and syllabic structures in connected speech mixed with different types of noise. While the extraction of prosodic cues from clear speech was stable during development, its maintenance in a multi-talker background matured rapidly up to age 9 and was associated with speech comprehension. Furthermore, while the extraction of subtler information provided by syllables matured at age 9, its maintenance in noisy backgrounds progressively matured until adulthood. Altogether, these results highlight distinct behaviorally relevant maturational trajectories for the neuronal signatures of speech perception. In accordance with grain-size proposals, neuromaturational milestones are reached increasingly late for linguistic units of decreasing size, with further delays incurred by noise.

TeaserThe neural signature of speech processing in silence and noise features multiple behaviorally relevant developmental milestones

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The interplay between developmental stage and environment determines the adaptive effect of a natural transposable element insertion Establishing causal links between adaptive mutations and their ecologically relevant phenotypes is key to understanding adaptation, a central goal in evolutionary biology. Although progress has been made, the number of causal adaptive mutations identified so far is still limited as gene by gene, and gene by environment interactions, among others, complicates linking genetic variation with its fitness-related effects. Transposable elements, often ignored in the quest for the genetic basis of adaptive evolution, are known to be a genome-wide source of regulatory elements across organisms that at times can lead to adaptive phenotypes. In this work, we combine gene expression, in vivo reporter assays, CRISPR/Cas9 genome editing and survival experiments to characterize in detail the molecular and phenotypic consequences of a natural Drosophila melanogaster transposable element insertion: the roo solo-LTR FBti0019985. This transposable element provides an alternative promoter to the transcription factor Lime, involved in cold- and immune-stress responses, and acts as an enhancer in response to immune-stress. We found that the effect of FBti0019985 on Lime expression depends on the interplay between the developmental stage and the environmental conditions. We further establish a causal link between the presence of FBti0019985 and increased survival to cold- and immune-stress. Our results exemplify how several developmental stages and environmental conditions need to be considered to characterize the molecular and functional effects of a genetic variant, and add to the growing body of evidence that transposable elements can induce complex mutations with ecologically relevant effects.

SIGNIFICANT STATEMENTTransposable elements (TEs) are repetitive DNA sequences with the ability to move along the genome. TEs are a genome-wide source of regulatory elements capable of regulating the expression of nearby genes. TEs can act as enhancers in specific tissues or developmental stages, and can also regulate the expression of nearby genes depending on the environmental context. In this work, we characterize a natural TE insertion whose effects on the expression of its nearby gene depends on the interplay between the developmental stage and the environmental conditions. A precise deletion of this TE insertion in natural populations using the CRISPR/Cas9 editing tool further proves that the insertion is the causal mutation leading to increased survival to cold- and immune-stress.

sentences: Manganese modulates metabolic activity and redox homeostasis in translationally-blocked Lactococcus cremoris, impacting metabolic persistence, cell-culturability, and flavor formation. Manganese (Mn) is an essential trace element that is supplemented in microbial media with varying benefits across species and growth conditions. We found that growth of Lactococcus cremoris was unaffected by manganese omission from the growth medium. The main proteome adaptation to manganese omission involved increased manganese transporter production (up to 2000-fold), while the remaining 10 significant proteome changes were between 1.4 and 4 fold. Further investigation in translationally-blocked (TB), non-growing cells showed that Mn supplementation (20 M) led to approximately 1.5X faster acidification compared to Mn-free conditions. However, this faster acidification stagnated within 24 hours, likely due to draining of intracellular NADH that coincides with substantial loss of culturability. Conversely, without manganese, non-growing cells persisted to acidify for weeks, albeit at a reduced rate, but maintaining redox balance and culturability. Strikingly, despite being unculturable, -keto acid-derived aldehydes continued to accumulate in cells incubated in the presence of manganese, whereas without manganese cells predominantly formed the corresponding alcohols. This is most likely reflecting NADH availability for the alcohol dehydrogenase-catalyzed conversion. Overall, manganese influences the lactococcal acidification rate, and flavor formation capacity in a redox dependent manner. These are important industrial traits especially during cheese ripening, where cells are in a non-growing, often unculturable state.

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Synthetic metabolic pathways for conversion of CO2 into secreted short- to medium-chain hydrocarbons using cyanobacteria The objective of this study was to implement direct sunlight-driven conversion of CO2 into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to medium-chain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO2 into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of bioreaction ingredients it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the bioreaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8-C10 preferring thioestersaes. When UndB instead was combined with a C12-specific  UcFatB1 thioesterase, this resulted in ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Optimization of the RBS, promoter and gene order in these synthetic operons resulted in 1-alkene bioproductivity of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO2 into secreted and ready-to-use hydrocarbon fuel was accomplished and optimal results were obtained with UndB and a C12 chain-length specific thioesterase.

HighlightsO_LIMultiple repression of endogenous aldehyde reductases/dehydrogenases by CRISPRi enabled propane biosynthesis
C_LIO_LIBiosynthesis of short-medium chain hydrocarbons (C7-C11) in a cyanobacterium was demonstrated for the first time
C_LIO_LIThe final enzymes of the hydrocarbon pathways influenced both productivity and product profile
C_LIO_LIAll volatile products were naturally secreted and accumulated outside of the cell
C_LI

sentences: Development and validation of a cellular host response test as an early diagnostic for sepsis Sepsis must be diagnosed quickly to avoid morbidity and mortality. However, the clinical manifestations of sepsis are highly variable and emergency department (ED) clinicians often must make rapid, impactful decisions before laboratory results are known. We previously developed a technique that allows the measurement of the biophysical properties of white blood cells as they are stretched through a microfluidic channel. In this study we describe and validate the resultant output as a model and score - the IntelliSep Index (ISI) - that aids in the diagnosis of sepsis in patients with suspected or confirmed infection from a single blood draw performed at the time of ED presentation. By applying this technique to a high acuity cohort with a 23.5% sepsis incidence (n=307), we defined specific metrics - the aspect ratio and visco-elastic inertial response - that are more sensitive than cell size or cell count in predicting disease severity. The final model was trained and cross-validated on the high acuity cohort, and the performance and generalizability of the model was evaluated on a separate low acuity cohort with a 6.4% sepsis incidence (n=94) and healthy donors (n=72). For easier clinical interpretation, the ISI is divided into three interpretation bands of Green, Yellow, and Red that correspond to increasing disease severity. The ISI agreed with the diagnosis established by retrospective physician adjudication, and accurately identified subjects with severe illness as measured by SOFA, APACHE-II, hospital-free days, and intensive care unit admission. Measured using routinely collected blood samples, with a short run-time and no requirement for patient or laboratory information, the ISI is well suited to aid ED clinicians in rapidly diagnosing sepsis.

sentences: The sperm protein SPACA4 is required for efficient fertilization in mice Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious mammalian homologs. We have recently identified the Ly6/uPAR protein Bouncer as a new, essential fertilization factor in zebrafish (Herberg et al., 2018). Here, we show that Bouncers homolog in mammals, SPACA4, is also required for efficient fertilization in mice. In contrast to fish, where Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely sub-fertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

sentences: Limiting the loss of terrestrial ecosystems to safeguard nature for biodiversity and humanity Humanity is on a pathway of unsustainable loss of the natural systems upon which we, and all life, rely. To date, global efforts to achieve internationally-agreed goals to reduce carbon emissions, halt biodiversity loss, and retain essential ecosystem services, have been poorly integrated. However, these different goals all rely on preserving natural ecosystems. Here, we show how to unify these goals by empirically deriving spatially-explicit, quantitative area-based targets for the retention of natural terrestrial ecosystems. We found that at least 67 million km2 of Earths natural terrestrial ecosystems (~79% of the area remaining) require retention - via a combination of strict protection but more prominently through sustainably managed land use regimes complemented by restoration actions - to contribute to biodiversity, climate, soil and freshwater objectives under four United Nations Resolutions. This equates to retaining natural ecosystems across ~50% of the total terrestrial (excluding Antarctica) surface of Earth. Our results show where retention efforts could be focussed to contribute to multiple goals simultaneously. The retention targets concept that we present explicitly recognises that such management can and should co-occur alongside and be driven by the people who live in and rely on places where natural and semi-natural ecosystems remain on Earth.

sentences: Understanding Hybrid Lipid-Polymer Gene Delivery Nanoparticle Assembly with Fluorescence Cross Correlation Spectroscopy Hybrid lipid-polymer-nucleic acid nanoparticles (LPNPs) provide a range of delivery strategies for nonviral gene therapy. However, due to several pairwise interactions between their components, these formulations are difficult to predict and characterize. Here, we employed, for the first time, a novel methodology based on fluorescence cross correlation spectroscopy (FCCS) to directly quantify the extent of association between polycation-DNA cores (polyplexes) and cationic liposome shells. This leads to a rapid and easy evaluation of LPNP formation. As a result, we were able to unveil two critical insights for predicting LPNP assembly. Firstly, the interaction between the polycation (polylysine) and DNA is robust, with the polycation not being displaced by liposomes. Hence, the polyplex cores and liposome shells have to be oppositely charged to associate. Secondly, and most importantly, the liposome:poliplex number ratio ({rho}N), as opposed to the more common charge ratio, is a critical parameter to predict stable LPNP formation. We find that{rho} N[≥]1 is required to ensure that every polyplex is enveloped by a liposome, avoiding the coexistence of oppositely charged species, thus inhibiting aggregation. Since the chosen LPNP components are common, these observations should be applicable to many LPNP systems. Furthermore, this FCCS-based methodology is relevant to other self-assembly composite materials, for applications beyond gene delivery.

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Evolutionary analysis of the LORELEI gene family in angiosperms reveals regulatory subfunctionalization A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and regulate growth, reproduction, immunity, and stress responses in Arabidopsis. Genes encoding these proteins are members of multi-gene families in most angiosperms and could generate thousands of signaling complex variants. However, the link(s) between expansion of these gene families and the functional diversification of this critical signaling complex as well as the evolutionary factors underlying the maintenance of gene duplicates remain unknown. Here, we investigated LLG gene family evolution, function, and expression in angiosperms. We found that LLGs in monocots and eudicots are descendants of a duplication early in angiosperm evolution and that both ancient and recent LLG duplicates are retained. Complementation and expression analysis showed that expression divergence of LLGs (regulatory subfunctionalization), rather than functional divergence, explains the retention of paralogs in Brassicales. All but one extant monocot and eudicot species examined maintained an LLG copy with preferential expression in male reproductive tissues, with the other duplicate copies showed highest levels of expression in female or vegetative tissues. Interestingly, the single LLG copy in Amborella (sister to all other angiosperms) is expressed vastly higher in male compared to female reproductive or vegetative tissues. Reconstruction of expression evolution showed that the highest inferred expression levels for the single copy ancestral angiosperm LLG was in male reproductive tissues. We propose that expression divergence played an important role in maintenance of LLG duplicates in angiosperms.

One Sentence SummaryExpression divergence played an important role in maintenance of two sub-groups of LLG duplicates in angiosperms

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Preferred Conformations in the Intrinsically Disordered Region of Human CPEB3, a Functional Amyloid Linked to Memory Consolidation While implicated in neurodegenerative diseases, amyloids are also essential to some physiological processes, including memory consolidation by neuronal-specific isoforms of the Cytoplasmic Polyadenylation Element Binding (CPEB) protein family. CPEB mediates memory persistence by the formation of self-sustaining amyloid assemblies through its intrinsically disordered region (IDR). Here, we characterize the atomic level conformation and ps-ns dynamics of the 426-residue IDR of human CPEB3 (hCPEB3), which has been associated with episodic memory in humans, by NMR spectroscopy. We found that the first 29 residues: M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical+disordered motif. Residues 86-93: P83QQPPPP93, and 166-175: P166PPPAPAPQP175 form polyproline II (PPII) helices. While the (VG)5 repeat motif is completely disordered, residues 200-250 adopt three partially populated -helices. Residues 345-355, which comprise the nuclear localization signal (NLS), form a modestly populated -helix and border a phosphoTyr which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids. Besides revealing some aspects of the molecular basis of memory, these findings could aid the future development of treatments for post-traumatic stress disorder.

Areas: Biophysics, Structural Biology, Biochemistry & Neurosciences.

sentences: Integrating genomics and multivariate evolutionary quantitative genetics: A case study of multivariate constraints on sexual selection in Drosophila serrata In evolutionary quantitative genetics, the genetic variance-covariance matrix, G, and the vector of directional selection gradients, {beta}, are key parameters for predicting multivariate selection responses and genetic constraints. Historically, investigations of G and {beta} have not overlapped with those dissecting the genetic basis of quantitative traits. Thus, it remains unknown whether these parameters reflect pleiotropic effects at individual loci. Here, we integrate multivariate GWAS with G and {beta} estimation in a well-studied system of multivariate constraint; sexual selection on male cuticular hydrocarbons (CHCs) in Drosophila serrata. In a panel of wild-derived resequenced lines, we augment genome-based REML, (GREML) to estimate G alongside multivariate SNP effects, detecting 532 significant associations from 1,652,276 SNPs. Constraint was evident, with {beta} lying in a direction of G with low evolvability. Interestingly, minor frequency alleles typically increased male CHC-attractiveness suggesting opposing natural selection on {beta}. SNP effects were significantly misaligned with the major eigenvector of G, gmax, but well aligned to the second and third eigenvectors g2 and g3. We discuss potential factors leading to these varied results including multivariate stabilising selection and mutational bias. Our framework may be useful as researchers increasingly access genomic methods to study multivariate selection responses in wild populations.

sentences: Adaptive cognitive maps for curved surfaces in the 3D world Terrains in a 3D world can be undulating. Yet, most prior research has exclusively investigated spatial representations on a flat surface, leaving a 2D cognitive map as the dominant model in the field. Here, we investigated whether humans represent a curved surface by building a dimension-reduced flattened 2D map or a full 3D map. Participants learned the location of objects positioned on a flat and curved surface in a virtual environment by driving on the concave side of the surface (Experiment 1), driving and looking vertically (Experiment 2), or flying (Experiment 3). Subsequently, they were asked to retrieve either the path distance or the 3D Euclidean distance between the objects. Path distance estimation was good overall, but we found a significant underestimation bias for the path distance on the curve, suggesting an influence of potential 3D shortcuts, even though participants were only driving on the surface. Euclidean distance estimation was better when participants were exposed more to the global 3D structure of the environment by looking and flying. These results suggest that the representation of the 2D manifold, embedded in a 3D world, is neither purely 2D nor 3D. Rather, it is flexible and dependent on the behavioral experience and demand.

sentences: Non-glycosylated Seipin to Cause a Motor Neuron Disease Induces ER stress and Apoptosis by Inactivating the ER Calcium Pump SERCA2b A causal relationship between endoplasmic reticulum (ER) stress and the development of neurodegenerative diseases remains controversial. Here, we focused on Seipinopathy, a dominant motor neuron disease, based on the finding that its causal gene product, Seipin, is a hairpin-like transmembrane protein in the ER. Gain-of-function mutations of Seipin produce non-glycosylated Seipin (ngSeipin), which was previously shown to induce ER stress and apoptosis at both cell and mouse levels albeit with no clarified mechanism. We found that aggregation-prone ngSeipin dominantly inactivated SERCA2b, the major calcium pump in the ER, and decreased the calcium concentration in the ER, leading to ER stress and apoptosis. This inactivation required oligomerization of ngSeipin and direct interaction of the ngSeipin C-terminus with SERCA2b, and was observed in Seipin-deficient human colorectal carcinoma-derived cells (HCT116) expressing ngSeipin at a level comparable with that in neuroblastoma cells (SH-SY5T). Our results thus provide a new direction to controversy noted above.

sentences: The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA The control of mRNA translation has been increasingly recognized as a key regulatory step for gene control but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf) genes. In the study, we focused on the Ogura CMS system in rapeseed and showed that the suppression to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific inhibition of the translation of the mitochondria-encoded CMS-causing mRNA orf138. We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.

sentences: Evolution of thermal physiology alters projected species distributions under climate change Species distribution models (SDMs) are widely used to predict range shifts but could be unreliable under climate change scenarios because they do not account for evolution. The thermal physiology of a species is a key determinant of range and thus incorporating thermal trait evolution into SDMs might be expected to alter projected ranges. We identified a genetic basis for physiological and behavioural traits that evolve in response to temperature change in natural populations of threespine stickleback (Gasterosteus aculeatus). Using these data, we created geographic range projections using a mechanistic niche area approach under two climate change scenarios. Under both scenarios, trait data was either static ( no evolution models), allowed to evolve at observed evolutionary rates ( evolution models), or allowed to evolve at a rate of evolution scaled by the trait variance that is explained by quantitative trait loci (QTL;  scaled evolution models). We show that incorporating these traits and their evolution substantially altered the projected ranges for a widespread panmictic marine population, with over 7-fold increases in area under climate change projections when traits are allowed to evolve. Evolution-informed SDMs should improve the precision of forecasting range dynamics under climate change, and aid in their application to management and the protection of biodiversity.

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Assessing biomass and primary production of microphytobenthos in depositional coastal systems using spectral information In depositional intertidal coastal systems, primary production is dominated by benthic microalgae (microphytobenthos) inhabiting the mudflats. This benthic productivity is supporting secondary production and supplying important services to humans including food provisioning. Increased frequencies of extreme events in weather (such as heatwaves, storm surges and cloudbursts) are expected to strongly impact the spatiotemporal dynamics of the microphytobenthos and subsequently their contribution to coastal food webs. Within north-western Europe, the years 2018 and 2019 were characterized by record-breaking summer temperatures and accompanying droughts. Field-calibrated satellite data (Sentinel 2) were used to quantify the seasonal dynamics of microphytobenthos biomass and production at an unprecedented spatial and temporal resolution during these years. We demonstrate that the Normalized Difference Vegetation Index (NDVI) should be used with caution in depositional coastal intertidal systems, because it may reflect import of remains of allochthonous pelagic productivity rather than local benthic biomass. We show that the reduction in summer biomass of the benthic microalgae cannot be explained by grazing but was most probably due to the high temperatures. The fivefold increase in salinity from January to September 2018, resulting from reduced river run-off during this exceptionally dry year, cannot have been without consequences for the vitality of the microphytobenthos community and its resistance to wind stress and cloud bursts. Comparison to historical information revealed that primary productivity of microphytobenthos may vary at least fivefold due to variations in environmental conditions. Therefore, ongoing changes in environmental conditions and especially extreme events because of climate change will not only lead to changes in spatiotemporal patterns of benthic primary production but also to changes in biodiversity of life under water and ecosystem services including food supply. Satellite MPB data allows for adequate choices in selecting coastal biodiversity conservation and coastal food supply.

HIGHLIGHTSO_LIExpected seasonality changes require large-scale and high-resolution coastal data
C_LIO_LINDVI of tidal flats reflects local benthic biomass and allochthonous phytoplankton
C_LIO_LIHigh summer temperatures reduced biomass and productivity of benthic microalgae
C_LIO_LILong-term data revealed a five-fold variation in MPB biomass and production
C_LIO_LISatellite MPB data allow for adequate conservation of coastal biodiversity
C_LI

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Physiological and molecular responses to combinatorial iron and phosphate deficiencies in hexaploid wheat seedlings Iron (Fe) and phosphorus (P) are the essential mineral nutrient for plant growth and development. However, the molecular interaction of the Fe and P pathways in crops remained largely obscure. In this study, we provide a comprehensive physiological and molecular analysis of hexaploid wheat response to single (Fe, P) and its combinatorial deficiencies. Our data showed that inhibition of the primary root growth occurs in response to Fe deficiency; however, growth was rescued when combinatorial deficiencies occurred. Analysis of RNAseq revealed that distinct molecular rearrangements during combined deficiencies with predominance for genes related to metabolic pathways and secondary metabolite biosynthesis primarily include genes for UDP-glycosyltransferase, cytochrome-P450s, and glutathione metabolism. Interestingly, the Fe-responsive cis-regulatory elements in the roots in Fe stress conditions were enriched compared to the combined stress. Our metabolome data also revealed the accumulation of distinct metabolites such as amino-isobutyric acid, arabinonic acid, and aconitic acid in the combined stress environment. Overall, these results are essential in developing new strategies to improve the resilience of crops in limited nutrients.

HIGHLIGHTSO_LIThis study was performed to understand the molecular changes occurring during the interaction of Phosphorus (P) and Iron (Fe) in hexaploid wheat roots.
C_LIO_LIP and Fe show cross-talk as Fe deficiency-induced phenotype that was restored by the withdrawal of P.
C_LIO_LIA total of 2780 differentially expressed genes were identified in the roots with the combined -Fe-P deficiencies with predominance for UDP-glycosyltransferases, cytochrome-450 and glutathione-S-transferases transcripts.
C_LIO_LIThe metabolomic changes identified the importance of amino-isobutyric acid, arabinonic acid and aconitic acid during dual deficiency
C_LIO_LIThis work provides a comprehensive insight to understand the molecular rearrangements occurring in wheat roots during Fe and P interaction.
C_LI

sentences: The Instability of Functional Connectomes Across the First Year of Life The uniqueness and stability of the adolescent and adult functional connectome has been demonstrated to be high (80-95% identification) using connectome-based identification (ID) or "fingerprinting". However, it is unclear to what extent individuals exhibit similar distinctiveness and stability in infancy, a developmental period of rapid and unparalleled brain development. In this study, we examined connectome-based ID rates within and across the first year of life using a longitudinal infant dataset at 1 month and 9 months of age. We also calculated the test-retest reliability of individual connections across the first year of life using the intraclass correlation coefficient (ICC). Overall, we found substantially lower infant ID rates than have been reported in adult and adolescent populations. Within-session ID rates were moderate and significant (ID = 48.94-70.83%). Between-session ID rates were very low and not significant, with task-to-task connectomes resulting in the highest between-session ID rate (ID = 26.6%). Similarly, average edge-level test-retest reliability was higher within-session than between-session (mean within-session ICC = 0.17, mean between-session ICC = 0.10). These findings suggest a lack of uniqueness and stability in functional connectomes across the first year of life consistent with the unparalleled changes in brain functional organization during this critical period.

sentences: Genetic association of TMPRSS2 rs2070788 polymorphism with COVID-19 Case Fatality Rate among Indian populations SARS-CoV2, the causative agent for COVID-19, an ongoing pandemic, engages the ACE2 receptor to enter the host cell through S protein priming by a serine protease, TMPRSS2. Variation in the TMPRSS2 gene may account for the difference in population disease susceptibility. The haplotype-based genetic sharing and structure of TMPRSS2 among global populations have not been studied so far. Therefore, in the present work, we used this approach with a focus on South Asia to study the haplotypes and their sharing among various populations worldwide. We have used next-generation sequencing data of 393 individuals and analysed the TMPRSS2 gene. Our analysis of genetic relatedness for this gene showed a closer affinity of South Asians with the West Eurasian populations therefore, host disease susceptibility and severity particularly in the context of TMPRSS2 will be more akin to West Eurasian instead of East Eurasian. This is in contrast to our prior study on ACE2 gene which shows South Asian haplotypes have a strong affinity towards West Eurasians. Thus ACE2 and TMPRSS2 have an antagonistic genetic relatedness among South Asians. We have also tested the SNPs frequencies of this gene among various Indian state populations with respect to the case fatality rate. Interestingly, we found a significant positive association between the rs2070788 SNP (G Allele) and the case fatality rate in India. It has been shown that the GG genotype of rs2070788 allele tends to have a higher expression of TMPRSS2 in the lung compared to the AG and AA genotypes, thus it might play a vital part in determining differential disease vulnerability. We trust that this information will be useful in underscoring the role of the TMPRSS2 variant in COVID-19 susceptibility and using it as a biomarker may help to predict populations at risk.

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On the sensitivity analysis of porous finite element models for cerebral perfusion estimation Computational physiological models are promising tools to enhance the design of clinical trials and to assist in decision making. Organ-scale haemodynamic models are gaining popularity to evaluate perfusion in a virtual environment both in healthy and diseased patients. Recently, the principles of verification, validation, and uncertainty quantification of such physiological models have been laid down to ensure safe applications of engineering software in the medical device industry. The present study sets out to establish guidelines for the usage of a three-dimensional steady state porous cerebral perfusion model of the human brain following principles detailed in the verification and validation (V&V 40) standard of the American Society of Mechanical Engineers. The model relies on the finite element method and has been developed specifically to estimate how brain perfusion is altered in ischaemic stroke patients before, during, and after treatments. Simulations are compared with exact analytical solutions and a thorough sensitivity analysis is presented covering every numerical and physiological model parameter.

The results suggest that such porous models can approximate blood pressure and perfusion distributions reliably even on a coarse grid with first order elements. On the other hand, higher order elements are essential to mitigate errors in volumetric blood flow rate estimation through cortical surface regions. Matching the volumetric flow rate corresponding to major cerebral arteries is identified as a validation milestone. It is found that inlet velocity boundary conditions are hard to obtain and that constant pressure inlet boundary conditions are feasible alternatives. A one-dimensional model is presented which can serve as a computationally inexpensive replacement of the three-dimensional brain model to ease parameter optimisation, sensitivity analyses and uncertainty quantification.

The findings of the present study can be generalised to organ-scale porous perfusion models. The results increase the applicability of computational tools regarding treatment development for stroke and other cerebrovascular conditions.

sentences: Structural Insights of SARS-CoV-2 Spike Protein from Delta and Omicron Variants Given the continuing heavy toll of the COVID-19 pandemic and the emergence of the Delta (B.1.617.2) and Omicron (B.1.1.529) variants, the WHO declared both as variants of concern (VOC). There are valid concerns that the latest Omicron variant might have increased infectivity and pathogenicity. In addition, the sheer number of S protein mutations in the Omicron variant raise concerns of potential immune evasion and resistance to therapeutics such as monoclonal antibodies. However, structural insights that underpin the potential increased pathogenicity are unknown. Here we adopted an artificial intelligence (AI)-based approach to predict the structural changes induced by mutations of the Delta and Omicron variants in the spike (S) protein using Alphafold. This was followed by docking the human angiotensin-converting enzyme 2 (ACE2) with the predicted S proteins for Wuhan-Hu-1, Delta, and Omicron variants. Our in-silico structural analysis indicates that S protein for Omicron variant has a higher binding affinity to ACE-2 receptor, compared to Wuhan-Hu-1 and Delta variants. In addition, the recognition sites of the receptor binding domains for Delta and Omicron variants showed lower electronegativity compared to Wuhan-Hu-1. Importantly, further molecular insights revealed significant changes induced at fusion protein (FP) site, which may mediate enhanced viral entry. These results represent the first computational analysis of structural changes associated with Omicron variant using Alphafold, Collectively, our results highlight potential structural basis for enhanced pathogenicity of the Omicron variant, however further validation using X-ray crystallography and cryo-EM are warranted.

sentences: Multiplexed fluorescence imaging of GPCR downstream signaling dynamics at the single-cell level. G-protein-coupled receptors (GPCRs) play an important role in sensing various extracellular stimuli, such as neurotransmitters, hormones, and tastants, and transducing the input information into the cell. While the human genome encodes more than 800 GPCR genes, only four G-proteins (Gs, Gi/o, Gq/11, and G12/13) are known to couple with GPCRs. It remains unclear how such divergent GPCR information is translated into the downstream G-protein signaling dynamics. To answer this question, we report a live-cell fluorescence imaging system for monitoring GPCR downstream signaling dynamics at the single-cell level. Genetically encoded biosensors for cAMP, Ca2+, RhoA, and ERK were selected as markers for GPCR downstream signaling, and were stably expressed in HeLa cells. GPCR was further transiently overexpressed in the cells. As a proof-of-concept, we visualized GPCR signaling dynamics of 5 dopamine receptors and 12 serotonin receptors, and found heterogeneity between GPCRs and between cells. Even when the same G proteins were known to be coupled, the patterns of dynamics in GPCR downstream signaling, including the signal strength and duration, were substantially distinct among GPCRs. These results suggest the importance of dynamical encoding in GPCR signaling.

sentences: Regulation of Sox8 through lncRNA Mrhl mediated chromatin looping in mouse spermatogonia Sox8 is a developmentally important transcription factor that plays an important role in sex maintenance and fertility of adult mice. In the B-type spermatogonial cells, Sox8 is regulated by the lncRNA Mrhl in a p68-dependant manner under the control of the Wnt signalling pathway. The downregulation of Mrhl leads to the meiotic commitment of the spermatogonial cells in a Sox8-dependant manner. While the molecular players involved in the regulation of transcription at the Sox8 promoter have been worked out, our current study points to the involvement of the architectural proteins CTCF and cohesin in mediating a chromatin loop that brings the Sox8 promoter in contact with a silencer element present within the gene body in the presence of lncRNA Mrhl concomitant with transcriptional repression. Further, lncRNA Mrhl interacts with the Sox8 locus through the formation of a DNA:DNA:RNA triplex which is necessary for the recruitment of PRC2 to the locus. The downregulation of lncRNA Mrhl results in the promoter-silencer loop giving way to a promoter-enhancer loop. This active transcription associated chromatin loop is mediated by YY1 and brings the promoter in contact with the enhancer present downstream of the gene.

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One gene - two proteins: The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria Muscarinic acetylcholine receptors are G protein-coupled receptors activated by the endogenous neurotransmitter acetylcholine. We show here that the carboxyl terminal fragment of the muscarinic M2 receptor, containing the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site. Radioligand binding, biochemical and molecular biology experiments, combined to advanced fluorescence microscopy reveal that the M2tail, whose expression is upregulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here it controls oxygen consumption, cell proliferation and the formation of ROS by reducing oxidative phosphorylation. Genetic editing of the key methionine where cap-independent translation occurs, reveals the physiological role of this process in influencing cell proliferation and oxygen consumption. The expression of the C-terminal of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably unrelated to the canonical signalling function of the GPCR at the plasma membrane.

TeaserA G protein-coupled receptor fragment localizing in the mitochondria unexpectedly links two hitherto unrelated aspects of cell biology.

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High photosynthesis rate in the selected wild rice is driven by leaf anatomy mediating high Rubisco activity and electron transport rate The importance of increasing photosynthetic efficiency for sustainable crop yield increases to feed the growing world population is well recognized. The natural genetic variation for leaf photosynthesis in crop plants is largely unexploited for increasing genetic yield potential. The genus Oryza, including cultivated rice and wild relatives, offers tremendous genetic variability to explore photosynthetic differences, and underlying biochemical, photochemical, and developmental bases. We quantified leaf photosynthesis and related physiological parameters for six cultivated and three wild rice genotypes, and identified photosynthetically efficient wild rice accessions. Fitting A/Ci curves and biochemical analyses showed that the leaf photosynthesis in cultivated rice varieties, IR64 and Nipponbare, was limited due to leaf nitrogen content, Rubisco activity, and electron transport rate compared to photosynthetically efficient accessions of wild rice Oryza australiensis and Oryza latifolia. The selected wild rice accessions with high leaf photosynthesis per unit area had striking anatomical features, such as larger mesophyll cells with more chloroplasts, fewer mesophyll cells between two consecutive veins, and higher mesophyll cell and chloroplast surface area exposed to intercellular space. Our results show the existence of desirable variations in Rubisco activity, electron transport rate, and leaf anatomical features in the rice system itself that could be targeted for increasing the photosynthetic efficiency of cultivated rice varieties.

HighlightDistinct leaf biochemical, photochemical, and developmental features contribute to efficient photosynthesis in the selected wild rice accessions that could potentially be exploited to increase rice leaf photosynthesis.

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Neuronal responses to focused ultrasound are gated by pre-stimulation brain rhythms BackgroundOwing to its high spatial resolution and penetration depth, transcranial focused ultrasound stimulation (tFUS) is one of the most promising approaches to non-invasive neuromodulation. Identifying the impact of the stimulation waveform and endogenous neural activity on neuromodulation outcome is critical to harnessing the potential of tFUS.

ObjectiveHere we tested a new form of tFUS where the amplitude of the ultrasonic waveform is modulated at a rate much slower than the operating frequency. Moreover, we sought to identify the relationship between pre-stimulation neural activity and the neuronal response to tFUS.

MethodsWe applied three minutes of amplitude modulated (AM) tFUS at 40 Hz to the rat hippocampus while recording local field potentials (LFP) and multi-unit activity (MUA)from the sonicated region. To assess the role of AM, we also tested continuous-wave (CW) stimulation.

ResultsAM tFUS reduced firing rate during and immediately after stimulation. On the other hand, CW tFUS produced an acute firing rate increase that was abolished after sonication. For both waveforms, firing rate changes were stronger in units exhibiting high baseline LFP power, particularly in the gamma band (30-250 Hz). The neuromodulatory effect was also influenced by the prevalence of sharp wave ripples (SWR) during the pre-stimulation period, with firing rates modulated by up to 33% at units showing frequent baseline SWR.

ConclusionOur findings suggest that AM and CW tFUS produce qualitatively different neuronal outcomes, and that baseline rhythms may effectively "gate" the response to tFUS.

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An exploratory analysis of 4844 withdrawn articles and their retraction notes. The objective of our study was to obtain an updated image of the dynamic of retractions and retraction notes, retraction reasons for questionable research and publication practices, countries producing retracted articles, and the scientific impact of retractions by studying 4844 PubMed indexed retracted articles published between 2009 and 2020 and their retraction notes.

RESULTSMistakes/inconsistent data account for 32% of total retractions, followed by images(22,5%), plagiarism(13,7%) and overlap(11,5%).

Thirty countries account for 94,79% of 4844 retractions. Top five are: China(32,78%), United States(18,84%), India(7,25%), Japan(4,37%) and Italy(3,75%).

The total citations number for all articles is 140810(Google Scholar), 96000(Dimensions).

Average exposure time(ET) is 28,89 months. Largest ET is for image retractions(49,3 months), lowest ET is for editorial errors(11,2 months).

The impact of retracted research is higher for Spain, Sweden, United Kingdom, United States, and other nine countries and lower for Pakistan, Turkey, Malaysia, and other six countries, including China.

CONCLUSIONSMistakes and data inconsistencies represent the main retraction reason; images and ethical issues show a growing trend, while plagiarism and overlap still represent a significant problem. There is a steady increase in QRP and QPP article withdrawals. Retraction of articles seems to be a technology-dependent process.

The number of citations of retracted articles shows a high impact of papers published by authors from certain countries. The number of retracted articles per country does not always accurately reflect the scientific impact of QRP/QPP articles.

The country distribution of retraction reasons shows structural problems in the organization and quality control of scientific research, which have different images depending on geographical location, economic development, and cultural model.

sentences: Drug-like small molecules that inhibit expression of the oncogenic microRNA-21 We report the discovery of a series of drug-like small molecules which bind specifically to the precursor of the oncogenic and pro-fibrotic microRNA-21 with mid-nanomolar affinity. These molecules are highly ligand-efficient (MW<330) and display specific biochemical and cellular activity by suppressing maturation of miR-21, thereby providing an avenue towards therapeutic intervention in multiple diseases where miR-21 is abnormally expressed. The small molecules target a local structure at the Dicer cleavage site and induce distinctive structural changes in the RNA which correlate with specific inhibition of miRNA processing. Structurally conservative single nucleotide substitutions eliminate the conformational change, which is not observed in other miRNA precursors. The most potent of these compounds reduces cellular proliferation and miR-21 levels in cancer cell lines without inhibiting kinases or classical receptors, while closely related compounds without this specific binding activity are inactive in cells.

sentences: SOYBEAN RESPONSE TO INITIAL STAGE OF Fusarium virguliforme INFECTION Fusarium virguliforme causes the Sudden Death Syndrome, an important disease in soybean crops. In this work, we investigated the defensive response mechanisms in soybean root, at cell wall level, during F. virguliforme infection using an in vitro culture system. We measured total root lignin content by the acetyl bromide method and estimated the in-situ lignin and suberin deposition by confocal microscopy on local and systemic root tissues, i.e. adjacent and distant to the pathogen entry site respectively. Moreover, the expression dynamics of phenylalanine ammonia lyase (PAL), shikimate/quinate hydroxycinnamoyltransferase (HCT) and cinnamyl alcohol dehydrogenase (CAD) was evaluated by real-time quantitative PCR. The results showed that, although the most significant increment of lignin deposition was observed in the epidermal cells of local tissues, F. virguliforme also induced lignin deposition changes in a sistemic fashion. In fact, inoculated plants presented a higher deposition of lignin in hypodermis and cortex than the control ones, independently of the distance from the inoculum source, while suberin deposition was higher in local zones. Moreover, the gene expression analysis showed an up-regulation of PAL, HCT and CAD genes after the inoculation with the pathogen, which correlates with the cell wall modifications observed in the local tissues. The results presented here suggest that the increase in lignin and suberin deposition during soybean root/F. virguliforme interaction is probably a strategy not only to stop the pathogen entrance, but to provide the plant more time to prepare its defences as well.

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Overcoming Tumor Heterogeneity by Ex Vivo Arming of T Cells Using Multiple Bispecific Antibodies PurposeTumoral heterogeneity is a hallmark of tumor evolution and cancer progression, being a longstanding challenge to targeted immunotherapy. Ex vivo armed T cells (EATs) using IgG-[L]- scFv bispecific antibodies (BsAbs) are potent tumor-specific cytotoxic effectors. To improve the anti-tumor efficacy of EATs against heterogeneous solid tumors, we explored multi-antigen targeting approaches.

MethodsEx vivo expanded T cells were armed with BsAbs built on the IgG-[L]-scFv platform, where an anti-CD3 (huOKT3) scFv was attached to the carboxyl end of both light chains of a tumor specific IgG. Multispecificity was created by combining monospecific EATs, combining BsAbs on the same T cell, or combining specificities on the same antibody. Three multi-antigens targeting EAT strategies were tested: (1) pooled EATs (simultaneous combination of monospecific EATs or alternate EATs (alternating combination of monospecific EATs), (2) dual-EATs or multi- EATs (T cells simultaneously armed with [≥] 2 BsAbs), and (3) TriAb-EATs [T cells armed with BsAb specific for two tumor targets besides CD3 (TriAb)]. The properties and efficiencies of these 3 strategies were evaluated by flow cytometry, in vitro cytotoxicity, cytokine release assays, and in vivo studies performed in BALB-Rag2-/-IL-2R-{gamma}c-KO (BRG) mice xenografted with cancer cell line (CDX) or patient-derived tumor (PDX).

ResultsMulti-EATs retained target antigen specificity and anti-tumor potency. Cytokine release with multi-EATs in the presence of tumor cells was substantially less than when multiple BsAbs were mixed with unarmed T cells. When tested against CDXs or PDXs, dual- or multi-EATs effectively suppressed tumor growth without clinical toxicities. Most importantly, dual- or multi- EATs were highly efficient in preventing clonal escape while mono- or TriAb- EATs were not as efficient.

ConclusionArming T cells with multiple BsAbs enabled multi-specific T cell immunotherapy which overcomes tumor heterogeneity without excessive cytokine release.

sentences: Dynamic nuclear lamina-chromatin interactions during G1 progression The chromatin associated with the nuclear lamina (NL) is referred to as Lamina-Associated Domains (LADs). While mapping of this feature has been done using various technologies, technical limitations exist for each of the methods. Here, we present an adaptation of the Tyramide-Signal Amplification sequencing (TSA-seq) protocol, which we call chromatin pull down-based TSA-seq (cTSA-seq), that can be used to map chromatin regions at or near the NL from as little as 50,000 cells without using carriers. The cTSA-seq mapped regions are composed of LADs and smaller chromatin regions that fall within the chromatin B-compartment known to be enriched for heterochromatin and be present at the nuclear periphery. As a proof of principle, we used cTSA-seq to map chromatin at or near the assembling NL as cells exit mitosis and progress through early and later G1. Consistent with previous reports, lamin-B1 based cTSA-seq revealed that regions toward the distal ends of chromosomes are near or at the reassembling NL during early G1. The cTSA-seq mapping and analyses revealed similarity between the early G1 chromatin and oncogene-induced senescent cell populations. The cTSA-seq reported here represents a useful method for analyzing chromatin at or near the NL from small numbers of cells.

sentences: Low pathogenic avian influenza virus infection retards colon microbiome diversification in two different chicken lines A commensal microbiome regulates and is in turn regulated by viruses during host infection which can influence virus infectivity. In this study, analysis of colon microbiome population changes following a low pathogenicity avian influenza virus (AIV) of the H9N2 subtype infection of two different chicken breeds was conducted. Using 16S rRNA sequencing and subsequent data analysis we found reduced microbiome alpha diversity in the acute period of AIV infection (day 2-3) in both Rhode Island Red and VALO chicken lines. From day 4 post infection a gradual increase in diversity of the colon microbiome was observed, but the diversity did not reach the same level as in uninfected chickens by day 10 post infection, suggesting that AIV infection retards the natural accumulation of colon microbiome diversity, which may further influence chicken health following recovery from infection. Beta diversity analysis indicated differences in diversity between the chicken lines during and following acute influenza infection suggesting the impact of host gut microflora dysbiosis following H9N2 influenza virus infection could differ for different breeds.

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Lysophosphatidylcholine acyltransferase 2 (LPCAT2) Regulates The Expression of Macrophage Phenotype Markers in RAW264.7 Cells. 1Macrophages are key antigen presenting cells that also secrete cytokines during inflammation. They can be polarised to M1 or M2 phenotypes. Molecules such as CD206 and inducible Nitric Oxide Synthase are considered macrophage phenotype markers because they are highly expressed in either M1 or M2 macrophages. LPCAT2 is a phospholipid modifying enzyme that influences inflammatory responses in macrophages. However, how LPCAT2 influences inflammation is not fully understood.

In this study, we have used genetic technology to study the influence of LPCAT2 on macrophage phenotype markers.

Our results show for the first time that overexpression of LPCAT2 promotes the expression of M1 macrophage phenotype markers, and attenuates the expression of M2 macrophage markers.

sentences: The widespread nature of Pack-TYPE transposons reveals their importance for plant genome evolution Pack-TYPE transposable elements (TEs) are a group of non-autonomous DNA transposons found in plants. These elements can efficiently capture and shuffle coding DNA across the host genome, accelerating the evolution of genes. Despite their relevance for plant genome plasticity, the detection and study of Pack-TYPE TEs are challenging due to the high similarity these elements have with genes. Here, we produced an automated annotation procedure designed to study Pack-TYPE elements and used it to successfully annotate and analyse more than 4000 new Pack-TYPE TEs in the rice and maize genomes. Our analysis indicates that Pack-TYPE TEs are an abundant and heterogeneous group of elements. We found that these elements are associated with all main superfamilies of Class II DNA transposons in plants and likely share a similar mechanism to capture new chromosomal DNA sequences. Furthermore, we report examples of the direct contribution of these TEs to coding genes, suggesting a generalised and extensive role of Pack-TYPE TEs in plant genome evolution.

sentences: Mirror Neurons are Modulated by Grip Force and Reward Expectation in the Sensorimotor Cortices (S1, M1, PMd, PMv) Mirror Neurons (MNs) respond similarly when primates make, or observe, grasping movements. Recent work indicates that reward expectation influences rostral M1 (rM1) during manual, observational, and Brain Machine Interface (BMI) reaching movements. Previous work showed MNs are modulated by subjective value. Here we expand on the above work utilizing two non-human primates (NHPs), one male Macaca Radiata (NHP S) and one female Macaca Mulatta (NHP P), that were trained to perform a cued reward level isometric grip-force task, where the NHPs had to apply visually cued grip-force to move and transport a virtual object. We found a population of (S1 area 1-2, rM1, PMd, PMv) units that significantly represented grip-force during manual and observational trials. We found the neural representation of visually cued force was similar during observational trials and manual trials for the same units, however, the representation was weaker during observational trials. Comparing changes in neural time lags between manual and observational tasks indicated that a subpopulation fit the standard MN definition of observational neural activity lagging the visual information. Neural activity in (S1 areas 1-2, rM1, PMd, PMv) significantly represented force and reward expectation. In summary, we present results indicating that sensorimotor cortices have MNs for visually cued force and value.

sentences: Fresh insights into Mediterranean biodiversity: Environmental DNA reveals spatio-temporal patterns of stream invertebrate communities on Sicily The Mediterranean region with its islands is among top biodiversity hotspots. It houses numerous freshwater taxa with a high rate of endemism, but is heavily impacted by anthropogenic pressures and global climate change. To conserve biodiversity, reliable data on species and genetic diversity are needed especially for the scarcely known insular freshwater ecosystems. Environmental DNA metabarcoding provide a straight-forward opportunity to assess aquatic biodiversity. Therefore, we conducted the first eDNA metabarcoding study in one stream catchment on Sicily. Specifically, we aimed to i) investigate spatial diversity patterns of macroinvertebrate communities, ii) assess seasonal changes, and iii) check if dispersal barriers can be identified. Water samples were taken at 27 different sites in two seasons and eDNA metabarcoding performed using the COI gene. In total, we detected 98 macroinvertebrate species, including 28 taxa potentially new to Sicily. Exact sequence variant (ESV) and species composition data showed that diversity differed between seasons with less taxa detected in winter. We also detected a dispersal barrier, which had a stronger effect in autumn. Our findings show that eDNA metabarcoding provides valuable information on Sicilian freshwater biodiversity. We therefore encourage its application for understudied regions to better understand the state and dynamics of freshwater biodiversity.

sentences: A hybrid boundary element-finite element method to solve the EEG forward problem This article presents a hybrid boundary element-finite element (BE-FE) method to solve the EEG forward problem and take advantages of both the boundary element method (BEM) and finite element method (FEM). Although realistic EEG forward problems with heterogeneous and anisotropic regions can be solved by FEM accurately, the FEM modeling of the brain with dipolar sources may lead to singularity. In contrast, the BEM can solve EEG forward problems with isotropic tissue regions and dipolar sources using a suitable integral formulation. This work utilizes both FEM and BEM strengths attained by dividing the regions into some homogeneous BE regions with sources and some heterogeneous and anisotropic FE regions. Furthermore, the BEM is applied for modeling the brain, including dipole sources and the FEM for other head layers. To validate the proposed method, inhomogeneous isotropic/anisotropic three- and four-layer spherical head models are studied. Moreover, a four-layer realistic head model is investigated. Results for six different dipole eccentricities and two different dipole orientations are computed using the BEM, FEM, and hybrid BE-FE method together with statistical analysis and the related error criteria are compared. The proposed method is a promising new approach for solving realistic EEG forward problems.

sentences: Neuronal morphology and physiology, functional brain networks and memory in temporal lobe epilepsy Temporal lobe epilepsy (TLE) patients are at risk of memory deficits, which have been linked to functional network disturbances, particularly of integration of the default mode network (DMN). However, the cellular substrates of functional network integration are unknown. We leverage a unique cross-scale dataset of therapy-resistant TLE patients, who underwent fMRI, MEG and/or neuropsychological testing before neurosurgery. fMRI and MEG underwent atlas-based connectivity analyses. Functional network centrality of the lateral middle temporal gyrus, part of the DMN, was used as a measure of local network integration. Subsequently, non-pathological cortical tissue from this region was used for single cell morphological and electrophysiological patch-clamp analysis, assessing integration in terms of total dendritic length and action potential rise speed. As could be hypothesized, greater network centrality related to better memory performance. Moreover, greater network centrality correlated with more integrative properties at the cellular level across patients. We conclude that individual differences in cognitively relevant functional network integration of a DMN region are mirrored by differences in cellular integrative properties of this region in TLE patients. These findings connect previously separate scales of investigation, increasing translational insight into focal pathology and large-scale network disturbances in TLE.

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Disrupted choline clearance and sustained acetylcholine release in vivo by a common choline transporter coding variant associated with poor attentional control in humans Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional control as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, in vivo, CHT-mediated choline transport, and ACh release. Relative to wild type (WT) mice, CHT-mediated clearance of choline in mice expressing one or two Val89 alleles was reduced by over 80% cortex and by over 50% in striatum. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10 min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. When challenged with a visual disruptor to reveal attentional control mechanisms, Val89 mice failed to adopt a more conservative response bias. Structural modeling revealed that Val89 may attenuate choline transport by altering conformational changes of CHT that support normal transport rates. Our findings support the view that diminished, sustained cholinergic signaling capacity underlies perturbed attentional performance in individuals expressing CHT Val89. The CHT Val89 mouse serves as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction.

Significance StatementAcetylcholine (ACh) signaling depends on the functional capacity of the neuronal choline transporter (CHT). Previous research demonstrated that humans expressing the common CHT coding variant Val89 exhibit attentional vulnerabilities and attenuated fronto-cortical activation during attention. Here, we find that mice engineered to express the Val89 variant exhibit reduced CHT-mediated choline clearance and a diminished capacity to sustain ACh release. Additionally, Val89 mice lack cognitive flexibility in response to an attentional challenge. These findings provide a mechanistic and cognitive framework for interpreting the attentional phenotype associated with the human Val89 variant and establish a model that permits a more invasive interrogation of CNS effects as well as the development of therapeutic strategies for those, including Val89 carriers, with presynaptic cholinergic perturbations.

sentences: Mapping the nuclear microenvironment of genes at agenome-wide scale The nuclear folding of chromosomes relative to nuclear bodies is an integral part of gene function. Here, we demonstrate that population-based modeling--from ensemble Hi-C data--can provide a detailed description of the nuclear microenvironment of genes and its role on gene function. We define the microenvironment by the subnuclear positions of genomic regions with respect to nuclear bodies, local chromatin compaction, and preferences in chromatin compartmentalization. These structural descriptors are determined in single cell models on a genome-wide scale, thereby revealing the structural variability between cells. We demonstrate that the structural microenvironment of a genomic region is linked to its functional potential in gene transcription, replication and chromatin compartmentalization. Some chromatin regions are distinguished by their strong preferences to a single microenvironment, due to associations to specific nuclear bodies in most cells. Other chromatin shows high structural variability, which is a strong indicator of functional heterogeneity. Moreover, we identify specialized nuclear microenvironments, which distinguish chromatin in different functional states and reveal a key role of nuclear speckles in chromosome organization. We demonstrate that our method produces highly predictive 3-dimensional genome structures, which accurately reproduce data from TSA-seq, DamID, GPSeq and super-resolution imaging. Thus, our method considerably expands the range of Hi-C data analysis and is widely applicable.

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Strobemers: an alternative to k-mers for sequence comparison K-mer-based methods are widely used in bioinformatics for various types of sequence comparison. However, a single mutation will mutate k consecutive k-mers and makes most k-mer based applications for sequence comparison sensitive to variable mutation rates. Many techniques have been studied to overcome this sensitivity, e.g., spaced k-mers and k-mer permutation techniques, but these techniques do not handle indels well. For indels, pairs or groups of small k-mers are commonly used, but these methods first produce k-mer matches, and only in a second step, a pairing or grouping of k-mers is performed. Such techniques produce many redundant k-mer matches due to the size of k.

Here, we propose strobemers as an alternative to k-mers for sequence comparison. Intuitively, strobemers consist of linked minimizers. We use simulated data to show that strobemers provide more evenly distributed sequence matches and are less sensitive to different mutation rates than k-mers and spaced k-mers. Strobemers also produce a higher match coverage across sequences. We further implement a proof-of-concept sequence matching tool StrobeMap, and use synthetic and biological Oxford Nanopore sequencing data to show the utility of using strobemers for sequence comparison in different contexts such as sequence clustering and alignment scenarios. A reference implementation of our tool StrobeMap together with code for analyses is available at https://github.com/ksahlin/strobemers.

sentences: Metformin Suppresses Monocyte Immunometabolic Activation by SARS-CoV-2 and Spike Protein Subunit 1 A hallmark of COVID-19 is a hyperinflammatory state that is associated with severity. Various anti-inflammatory therapeutics have shown mixed efficacy in treating COVID-19, and the mechanisms by which hyperinflammation occurs are not well understood. Previous research indicated that monocytes, a key innate immune cell, undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism could inhibit the inflammatory response in monocytes. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism that was associated with production of pro-inflammatory cytokines including interleukin-6 and tumor necrosis factor-. This response was dependent on hypoxia-inducible factor-1, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production in monocytes, and abrogated glycolytic and mitochondrial metabolism. Likewise, metformin pre-treatment blocked cytokine induction by SARS-CoV-2 strain WA1/2020 in direct infection experiments in monocytes. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by metformin, and metformin likewise suppresses inflammatory responses to live SARS-CoV-2. This has potential implications for the treatment of hyperinflammation during COVID-19.

sentences: Platform for Remote, Multifocal Brain Interventions Transcranial focused ultrasound brings precision healthcare to the human brain, delivering focal interventions to deep brain sites without the need for surgeries or systemic drug administrations. The approaches have included neuromodulation and localized delivery of drugs through an intact or temporarily opened blood brain barrier. Clinical translation of these approaches requires a system that can deliver ultrasound into specific brain targets on command. Here, we demonstrate a platform with these features, and validate its function in nonhuman primates. Specifically, we used the system to flexibly modulate two deep brain nuclei--the left and right lateral geniculate nucleus-during visual choice behavior. We found that ultrasound induced a behavioral bias whose polarity depended on the stimulation waveform. Moreover, repeated applications of ultrasound over 39 consecutive behavioral sessions produced no signs of harm to the stimulated circuits. The system can be be applied to diagnose and possibly treat disorders of brain function with a precision and flexibility previously unattainable.

sentences: The maintenance of polygenic sex determination depends on the dominance of fitness effects which are predictive of the role of sexual antagonism In species with polygenic sex determination, multiple male- and female-determining loci on different proto-sex chromosomes segregate as polymorphisms within populations. The extent to which these polymorphisms are at stable equilibria is not yet resolved. Previous work demonstrated that polygenic sex determination is most likely to be maintained as a stable polymorphism when the proto-sex chromosomes have opposite (sexually antagonistic) fitness effects in males and females. However, these models usually consider polygenic sex determination systems with only two proto-sex chromosomes, or they do not broadly consider the dominance of the alleles under selection. To address these shortcomings, I used forward population genetic simulations to identify selection pressures that can maintain polygenic sex determination under different dominance scenarios in a system with more than two proto-sex chromosomes (modeled after the house fly). I found that overdominant fitness effects of male-determining proto-Y chromosomes are more likely to maintain polygenic sex determination than dominant, recessive, or additive fitness effects. The overdominant fitness effects that maintain polygenic sex determination tend to have proto-Y chromosomes with sexually antagonstic effects (male-beneficial and female-detrimental). In contrast, dominant fitness effects that maintain polygenic sex determination tend to have sexually antagonistic multi-chromosomal genotypes, but the individual proto-sex chromosomes do not have sexually antagonstic effects. These results demonstrate that sexual antagonism can be an emergent property of the multi-chromosome genotype without individual sexually antagonistic chromosomes. My results further illustrate how the dominance of fitness effects has consequences for both the likelihood that polygenic sex determination will be maintained as well as the role sexually antagonistic selection is expected to play in maintaining the polymorphism.

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Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for [~]9-10 months after the primary 2-dose mRNA vaccine series, as well as for [~]3 months after a 3rd mRNA vaccine dose. Notably, antibody decay slowed significantly between 6- and 9-months post-primary vaccination, essentially stabilizing at the time of the 3rd dose. Antibody quality also continued to improve for at least 9 months after primary 2-dose vaccination. Spike- and RBD-specific memory B cells were stable through 9 months post-vaccination with no evidence of decline over time, and [~]40-50% of RBD-specific memory B cells were capable of simultaneously recognizing the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells induced by the first 2 doses of mRNA vaccine were boosted significantly by a 3rd dose and the magnitude of this boosting was similar to memory B cells specific for other variants. Pre-3rd dose memory B cell frequencies correlated with the increase in neutralizing antibody titers after the 3rd dose. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit reactivation of immunological memory and constrain further antibody boosting by mRNA vaccines. These data provide a deeper understanding of how the quantity and quality of antibody and memory B cell responses change over time and number of antigen exposures. These data also provide insight into potential immune dynamics following recall responses to additional vaccine doses or post-vaccination infections.

Graphical Summary

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Accelerated Predictive Healthcare Analytics with Pumas, A High Performance Pharmaceutical Modeling and Simulation Platform Pharmacometric modeling establishes causal quantitative relationships between administered dose, tissue exposures, desired and undesired effects and patients risk factors. These models are employed to de-risk drug development and guide precision medicine decisions. However, pharmacometric tools have not been designed to handle todays heterogeneous big data and complex models. We set out to design a platform that facilitates domain-specific modeling and its integration with modern analytics to foster innovation and readiness in healthcare.

Pumas demonstrates estimation methodologies with dramatic performance advances. New ODE solver algorithms, such as coeficient-optimized higher order integrators and new automatic stiffness detecting algorithms which are robust to frequent discontinuities, give rise to a median 4x performance improvement across a wide range of stiff and non-stiff systems seen in pharmacometric applications. These methods combine with JIT compiler techniques, such as statically-sized optimizations and discrete sensitivity analysis via forward-mode automatic differentiation, to further enhance the accuracy and performance of the solving and parameter estimation process. We demonstrate that when all of these techniques are combined with a validated clinical trial dosing mechanism and non-compartmental analysis (NCA) suite, real applications like NLME fitting see a median 81x acceleration while retaining the same accuracy. Meanwhile in areas with less prior software optimization, like optimal experimental design, we see orders of magnitude performance enhancements over competitors. Further, Pumas combines these technical advances with several workflows that are automated and designed to boost productivity of the day-to-day user activity. Together we show a fast pharmacometric modeling framework for next-generation precision analytics.

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Protein allocation and utilization in the versatile chemolithoautotroph Cupriavidus necator Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile  knallgas bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy.

HighlightsO_LIA large fraction of the C. necator proteome is not utilized and not essential
C_LIO_LIHighly utilized enzymes are more abundant and less variable
C_LIO_LIAutotrophy related enzymes are largely underutilized
C_LIO_LIRe-assimilation of CO2 via the CBB cycle is unlikely to provide a fitness benefit
C_LI

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Aspergillus fumigatus acetate utilisation impacts virulence traits and pathogenicity Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilisation of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolised via different pathways in A. fumigatus and that acetate utilisation is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilisation is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, to anti-fungal drugs and to human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilisation in A. fumigatus and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of anti-fungal strategies.

ImportanceAspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonisation and invasion. This work shows that A. fumigatus metabolises acetate via different pathways, a process that is dependent on the transcription factor FacB. Furthermore, the type and concentration of the extracellular available carbon source were determined to shape A. fumigatus virulence determinants such as secondary metabolite secretion and cell wall composition. Subsequently, interactions with immune cells are altered in a carbon source-specific manner. FacB is required for A. fumigatus in vivo virulence in both insect and mammalian models of invasive aspergillosis. This is the first report that characterises acetate utilisation in A. fumigatus and highlights the importance of available host-specific carbon sources in shaping virulence traits and potentially subsequent disease outcome.

sentences: Thermal Effects on Neurons During Stimulation of the Brain All artificial stimulation of the brain deposits thermal energy in the brain. This occurs through either Joule heating of the conductors carrying current through electrodes and magnetic coils, or through dissipation of energy in the conductive brain. Similarly, temperature affects all biological processes and chemical reactions. Although electrical interaction with brain tissue is inseparable from thermal effects when electrodes are used, magnetic induction enables us to separate Joule heating from induction effects by contrasting AC and DC driving of magnetic coils using the same energy deposition within the conductors. Since mammalian cortical neurons have no known sensitivity to static magnetic fields, and if there is no evidence of effect on spike timing to oscillating magnetic fields, we can presume that the induced electrical currents within the brain are below the molecular shot noise where any interaction with tissue is purely thermal. In this study, we examined a range of frequencies produced from micromagnetic coils operating below the molecular shot noise threshold for electrical interaction with single neurons. We found that small temperature increases and decreases of 1{degrees}C caused consistent transient suppression and excitation of neurons during temperature change. Numerical modeling of the biophysics demonstrated that the Na-K pump, and to a lesser extent the Nernst potential, could account for these transient effects. Such effects are dependent upon compartmental ion fluxes, and the rate of temperature change. A new bifurcation is described in the model dynamics that accounts for the transient suppression and excitation; in addition, we note the remarkable similarity of this bifurcations rate dependency with other thermal rate-dependent tipping points in planetary warming dynamics. Furthermore, bifurcations in the steady state dynamics leading to stable firing suppression are described for slightly higher temperatures. These experimental and theoretical findings demonstrate that stimulation of the brain must take into account small thermal effects that are ubiquitously present in electrical and magnetic stimulation. More sophisticated models of electrical current interaction with neurons combined with thermal effects will be required in order to more accurately enable model-based control of neuronal circuitry.

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Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement. The retinoic acid receptor-related orphan receptor {gamma} (ROR{gamma}) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of ROR{gamma} is far from complete. Like most nuclear receptors, ROR{gamma} must recruit coregulatory protein to enact the ROR{gamma} target gene program. To date, a majority of structural studies have been focused exclusively on the ROR{gamma} ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length ROR{gamma}:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that ROR{gamma} becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory proteins may be able to  sense the ligand- and DNA-bound status of ROR{gamma}. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which ROR{gamma} is bound. Overall, the efforts described herein will illuminate important aspects of full length ROR{gamma} and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes.

GRAPHICAL ABSTRACT

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DDN2.0: R and Python packages for differential dependency network analysis of biological systems Data-driven differential dependency network analysis identifies in a complex and often unknown overall molecular circuitry a network of differentially connected molecular entities (pairwise selective coupling or uncoupling depending on the specific phenotypes or experimental conditions) (Herrington, et al. 2018; Zhang, et al., 2009; Zhang and Wang, 2010; Zhang, et al., 2016). Such differential dependency networks are typically used to assist in the inference of potential key pathways. Based on our previously developed Differential Dependency Network (DDN) method, we report here the fully implemented R and Python software tool packages for public use. The DDN2.0 algorithm uses a fused Lasso model and block-wise coordinate descent to estimate both the common and differential edges of dependency networks. The identified DDN can help to provide plausible interpretation of data, gain new insight of disease biology, and generate novel hypotheses for further validation and investigations.

To address the imbalanced sample group problem, we propose a sample-size normalized formulation to correct systematic bias. To address high computational complexity, we propose four strategies to accelerate DDN2.0 learning. The experimental results show that new DDN2.0+ learning speed with combined four accelerating strategies is hundreds of times faster than that of DDN2.0 algorithm on medium-sized data (Fu, 2019). To detect intra-omics and inter-omics network rewiring, we propose multiDDN using a multi-layer signaling model to integrate multi-omics data. The simulation study shows that the multiDDN method can achieve higher accuracy of detecting network rewiring (Fu, 2019).

sentences: Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-care Platform Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of 7 wastewater samples from COVID-19 hotspots. RT{square}LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best surrogate marker for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be used as a surrogate marker for detection of SARS-CoV-2. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips.

sentences: Cell Stiffening Contributes to Complement-mediated Injury of Choroidal Endothelial Cells in Early AMD Age-related macular degeneration (AMD) is the leading cause of blindness in the aging population. Yet, no therapies exist for [~]85% of all AMD patients who have the dry form that is marked by degeneration of the retinal pigment epithelium (RPE) and underlying choroidal vasculature. As the choroidal vessels are crucial for RPE development and maintenance, understanding how they degenerate may lead to effective therapies for dry AMD. One likely causative factor for choroidal vascular loss is the cytolytic membrane attack complex (MAC) of the complement pathway that is abundant on choroidal vessels of humans with early dry AMD. To examine this possibility, we studied the effect of complement activation on choroidal endothelial cells (ECs) isolated from a rhesus monkey model of early AMD that, we report, exhibits MAC deposition and choriocapillaris endothelial loss similar to that seen in human early AMD. Treatment of choroidal ECs from AMD eyes with complement-competent normal human serum caused extensive actin cytoskeletal injury that was significantly less pronounced in choroidal ECs from young normal monkey eyes. We further show that ECs from AMD eyes are significantly stiffer than their younger counterparts and exhibit peripheral actin organization that is distinct from the longitudinal stress fibers in young ECs. Finally, these differences in complement susceptibility and mechanostructural properties were found to be regulated by the differential activity of small GTPases Rac and Rho because Rac inhibition in AMD cells led to simultaneous reduction in stiffness and complement susceptibility while Rho inhibition in young cells exacerbated complement injury. Thus, by identifying cell stiffness and cytoskeletal regulators Rac and Rho as important determinants of complement susceptibility, the current findings offer a new mechanistic insight into choroidal vascular loss in early AMD that warrants further investigation for assessment of translational potential.

sentences: Population structure limits parallel evolution Population genetic theory predicts that small effective population sizes (Ne) and restricted gene flow limit the potential for local adaptation. In particular, the probability of evolving similar phenotypes based on shared genetic mechanisms (i.e. parallel evolution), is expected to be reduced. We tested these predictions in a comparative genomic study of two ecologically similar and geographically co-distributed stickleback species (viz. Gasterosteus aculeatus and Pungitius pungitius). We found that P. pungitius harbours less genetic diversity and exhibits higher levels of genetic differentiation and isolation-by-distance than G. aculeatus. Conversely, G. aculeatus exhibits a stronger degree of genetic parallelism across freshwater populations than P. pungitius: 2996 vs. 379 SNPs located within 26 vs nine genomic regions show evidence of selection in multiple freshwater populations of G. aculeatus and P. pungitius, respectively. Most regions involved in parallel evolution in G. aculeatus showed increased levels of divergence, suggestive of selection on ancient haplotypes. In contrast, regions involved in freshwater adaptation in P. pungitius were younger, and often associated with reduced diversity. In accordance with theory, the results suggest that connectivity and genetic drift play crucial roles in determining the levels and geographic distribution of standing genetic variation, providing evidence that population subdivision limits local adaptation and therefore also the likelihood of parallel evolution.

sentences: Impact of 98 LRRK2 variants linked to Parkinson's Disease on kinase activity and microtubule binding Mutations enhancing the kinase activity of LRRK2 cause Parkinsons disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 98 LRRK2 variants linked to PD, including previously described pathogenic mutations, and identify 22 variants that robustly stimulate LRRK2 kinase activity. These include variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK (R767H), LRR (R1067Q, R1325Q)] as well as variants residing within the GTPase domain, predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORB (S1761R, L1795F)] and CORB:CORB dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer910/pSer935), consistent with the notion that the active kinase conformation blocks their phosphorylation. We find that the R1628P variant that has been linked to PD does not stimulate LRRK2 activity. We observe that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation at Thr73 compared to an in vitro kinase assay, as only a minority of activating variants enhance the activity of immunoprecipitated LRRK2, comprising the CORB (Y1699C, R1728H, S1761R) and kinase (G2019S, I2020T, T2031S) variants. Twelve variants including some that activated LRRK2, suppressed microtubule association in the presence of a Type 1 kinase inhibitor [ARM (M712V), LRR (R1320S), ROC (A1442P, K1468E, S1508R), CORA (A1589S), CORB (Y1699C, R1728H/L) and WD40 (R2143M, S2350I, G2385R)]. Our findings will aid in understanding the impact of these variants on LRRK2 kinase activation, stimulate work to reveal mechanisms by which variants impact biology, and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 clinical trials.

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An initial 'snapshot' of sensory information biases the likelihood and speed of subsequent changes of mind We often need to rapidly change our mind about perceptual decisions in order to account for new information and correct mistakes. One fundamental, unresolved question is whether information processed prior to a decision being made ( pre-decisional information) has any influence on the likelihood and speed with which that decision is reversed. We investigated this using a luminance discrimination task in which participants indicated which of two flickering greyscale squares was brightest. Following an initial decision, the stimuli briefly remained on screen, and participants could change their response. Using psychophysical reverse correlation, we examined how moment-to-moment fluctuations in stimulus luminance affected participants decisions. This revealed that the strength of even the very earliest (pre-decisional) evidence was associated with the likelihood and speed of later changes of mind. To account for this effect, we propose an extended diffusion model in which an initial  snapshot of sensory information biases ongoing evidence accumulation.

Author SummaryTo avoid harm in an ever-changing world we need to be able to rapidly change our minds about our decisions. For example, imagine being unable to overrule a decision to run across a street when you realise a speeding car is approaching. In this study, we examined the information processing dynamics which underlie perceptual judgements and changes of mind. By reverse correlating participants decisions with the moment-to-moment sensory evidence they received, we show that the very earliest information, processed prior to an initial decision being made, can have a lasting influence over the speed and likelihood of subsequent changes of mind. To account for this, we develop a model of perceptual decisions in which initial sensory evidence exerts a lasting bias over later evidence processing. When fit to participants behavioural responses alone, this model predicted their observed information usage patterns. This suggests that an initial  snapshot of sensory information may influence the ongoing dynamics of the perceptual decision process, thus influencing the speed and likelihood of decision reversals.

sentences: A unique class of Zn2+-binding PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile {beta}-Lactam antibiotics, particularly cephalosporins, are major risk factors for C. difficile infection (CDI), the most common hospital acquired infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), essential enzymes that assemble the bacterial cell wall. Little is known about the C. difficile PBPs, yet they play central roles in the growth, infection, and transmission of this pathogen. In this study we discover that PBP2, essential for vegetative growth, is the primary bactericidal target for {beta}-lactams in C. difficile. We further demonstrate PBP2 is insensitive to cephalosporin inhibition, revealing a key cause of the well-documented, but poorly understood, cephalosporin resistance in C. difficile. For the first time, we determine the crystal structures of C. difficile PBP2, which bears several highly unique features, including significant ligand-induced conformational changes and an active site Zn2+-binding motif that influences {beta}-lactam binding and protein stability. Remarkably, this motif is shared in two other C. difficile PBPs essential for sporulation, PBP3 and SpoVD. While these PBPs are present in a wide range of bacterial taxa, including species in extreme environments and the human gut, they are mostly found in anaerobes, typically Firmicutes. The widespread presence of this convergently evolved thiol-containing motif and its cognate Zn2+ suggests it may function as a redox-sensor to regulate cell wall synthesis for survival in adverse environments. Collectively, our findings address important etiological questions surrounding C. difficile, characterize new elements of PBP structure and function, and lay the groundwork for antibiotic development targeting both C. difficile growth and sporulation.

sentences: Activation of the Crtc2/Creb1 transcriptional network in skeletal muscle enhances weight loss during intermittent fasting The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc 2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, supported by maintenance of higher energy expenditure during the fast. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

sentences: Regulation of EGF-stimulated activation of the PI-3K/AKT pathway by exocyst-mediated exocytosis The phosphoinositide-3 kinase (PI-3K)/AKT cell survival pathway is an important pathway activated by EGFR signaling. Here we show, that in addition to previously described critical components of this pathway, i.e., the docking protein Gab1, the PI-3K/AKT pathway in epithelial cells is regulated by the exocyst complex, which is a vesicle tether that is essential for exocytosis. Using live-cell imaging, we demonstrate that PI(3,4,5)P3 levels fluctuate at the membrane on a minutes time scale and that these fluctuations are associated with local PI(3,4,5)P3 increases at sites where recycling vesicles undergo exocytic fusion. Supporting a role for exocytosis in PI(3,4,5)P3 generation, acute promotion of exocytosis by optogenetically driving exocyst-mediated vesicle tethering upregulates PI(3,4,5)P3 production and AKT activation. Conversely, acute inhibition of exocytosis using Endosidin2, a small-molecule inhibitor of the exocyst subunit Exo70, impairs PI(3,4,5)P3 production and AKT activation induced by EGF stimulation of epithelial cells. Moreover, prolonged inhibition of EGF signaling by EGFR tyrosine kinase inhibitors results in spontaneous reactivation of AKT without a concomitant relief of EGFR inhibition. However, this reactivation can be negated by acutely inhibiting the exocyst. These experiments demonstrate that exocyst-mediated exocytosis - by regulating PI(3,4,5)P3 levels at the plasma membrane - subserves activation of the PI-3K/AKT pathway by EGFR in epithelial cells.

sentences: Polymer skulls with integrated transparent electrode arrays for cortex-wide opto-electrophysiological recordings Electrophysiological and optical imaging provide complementary neural sensing capabilities - electrophysiological recordings have the highest temporal resolution, while optical imaging allows recording the activities of genetically defined populations at high spatial resolution. Combining these complementary, yet orthogonal modalities to perform simultaneous large-scale, multimodal sensing of neural activity across multiple brain regions would be very powerful. Here we show that transparent, inkjet-printed electrocorticography (ECoG) electrode arrays can be seamlessly integrated with morphologically conformant transparent polymer skulls for multimodal recordings across the cortex. These  eSee-Shells were implanted on transgenic mice expressing the Ca2+ indicator GCaMP6f in cortical excitatory cells and provided a robust opto-electrophysiological interface for over 100 days. eSee-Shells enable simultaneous mesoscale Ca2+ imaging and ECoG acquisition under anesthesia as well as in awake animals presented with sensory stimuli. eSee-Shells further show sufficient clarity and transparency to observe single-cell Ca2+ signals directly below the electrodes and interconnects. Simultaneous multimodal measurement of cortical dynamics reveals changes in both ECoG and Ca2+ signals that depend on the behavioral state.

sentences: The Petasites hybridus CO2-extract (Ze 339) blocks SARS-CoV-2 replication in vitro The coronavirus disease 2019 (COVID-19), caused by a novel coronavirus (SARS-CoV-2), has spread worldwide, affecting over 250 million people and resulting in over five million deaths. Antivirals that are effective are still limited. The antiviral activities of the Petasites hybdridus CO2-extract Ze 339 were previously reported. Thus, to assess the anti-SARS-CoV-2 activity of Ze 339 as well as isopetasin and neopetasin as major active compounds, a CPE- and plaque reduction assay in Vero E6 cells was used for viral output. Antiviral effects were tested using the original virus (Wuhan) and the Delta variant of SARS-CoV-2. The antiviral drug remdesivir was used as control. Pre-treatment with Ze 339 in SARS-CoV-2 infected Vero E6 cells with either virus variant significantly inhibited virus replication with IC50 values of 0.10 and 0.40 g/mL, repectively. The IC50 values obtained for isopetasin ranged between 0.37-0.88 M for both virus variants, that of remdesivir between 1.53-2.37 M. In conclusion, Ze 339 as well as the petasins potently inhibited SARS-Cov-2 replication in vitro of the Wuhan and Delta variants. Since time is of essence in finding effective treatments, clinical studies will have to demonstrate if Ze339 can become a therapeutic option to treat SARS-CoV-2 infections.

sentences: Decision value signals in the ventromedial prefrontal cortex andmotivational and hedonic symptoms across mood and psychotic disorders Deficits in motivation and pleasure are common across many psychiatric disorders, and manifest as symptoms of amotivation and anhedonia, which are prominent features of both mood and psychotic disorders. Here we provide evidence for a shared transdiagnostic mechanism underlying impairments in motivation and pleasure across major depression, bipolar disorder, and schizophrenia. We found that value signals in the ventromedial prefrontal cortex (vmPFC) during decision-making were dampened in individuals with greater motivational and hedonic deficits, regardless of the primary diagnosis. This relationship remained significant while controlling for diagnosis-specific symptoms of mood and psychosis, such as depression as well as positive and negative symptoms. Our results demonstrate that dysfunction in the vmPFC during value-based decision-making is specifically linked to motivational and hedonic impairments across various psychiatric conditions. These findings provide a quantitative neural target for the potential development of novel treatments for amotivation and anhedonia.

sentences: Uniformity of spheroid-on-chip by surface treatment of PDMS microfluidic platforms Spheroids have emerged as a more reliable model for drug screening when compared with 2D culture models. Microfluidic based biochips have many advantages over other 3D cell culture models for drug testing on spheroids, including precise control of the cellular microenvironment. The control of the cell adhesion to the surface is one of the most important challenges affecting the size and the geometry of the spheroids which could be controlled by appropriate surface engineering methods. We have studied the modification of the PDMS surface properties treated by applying different concentrations of the two anti-fouling coatings (BSA and Pluronic F-68). The desired treatment of PDMS surface effectively inhibits cell adhesion to the surface and promotes cells self-aggregations to form more uniform and healthy spheroids for a longer period of time. The microscopic observations with qualitative and quantitate data revealed that surface properties drastically affect the number of the spheroids formed on-chip and their geometry. We used human breast cancer cell line (MDA-MB-231-GFP) while the concentration of the chemical coatings and incubation time were adjusted. Proper repellent PDMS surfaces were provided with minimum cell attachment and facilitated spheroid formation when compared with non-treated PDMS. The results demonstrate fundamental and helpful patterns for microfluidic based cell culture applications to improve the quantity and quality of spheroid formation on-chip which are strongly manipulated by surface properties (i.e., morphology, roughness, wettability and etc.)

sentences: Volumetric Calcium Imaging of 1 Million Neurons Across Cortical Regions at Cellular Resolution Using Light Beads Microscopy Two-photon microscopy together with genetically encodable calcium indicators has emerged as a standard tool for high-resolution imaging of neuroactivity in scattering brain tissue. However, its various realizations have not overcome the inherent tradeoffs between speed and spatiotemporal sampling in a principled manner which would be necessary to enable, amongst other applications, mesoscale volumetric recording of neuroactivity at cellular resolution and speed compatible with resolving calcium transients. Here, we introduce Light Beads Microscopy (LBM), a scalable and spatiotemporally optimal acquisition approach limited only by fluorescence life-time, where a set of axially-separated and temporally-distinct foci record the entire axial imaging range near-simultaneously, enabling volumetric recording at 1.41 x 108 voxels per second. Using LBM, we demonstrate mesoscopic and volumetric imaging at multiple scales in the mouse cortex, including cellular resolution recordings within ~3x5x0.5 mm3 volumes containing >200,000 neurons at ~5 Hz, recording of populations of ~1 million neurons within ~5.4x6x0.5 mm3 volumes at ~2Hz as well as higher-speed (9.6 Hz) sub-cellular resolution volumetric recordings. LBM provides an unprecedented opportunity for discovering the neurocomputations underlying cortex-wide encoding and processing of information in the mammalian brain.

sentences: BitterMatch: Recommendation systems for matching molecules with bitter taste receptors Bitterness is an aversive cue elicited by thousands of chemically diverse compounds. Bitter taste may prevent consumption of foods and jeopardize drug compliance. The G protein-coupled receptors for bitter taste, TAS2Rs, have species-dependent number of subtypes and varying expression levels in extraoral tissues. Molecular recognition by TAS2R subtypes is physiologically important, and presents a challenging case study for ligand-receptor matchmaking. Inspired by hybrid recommendation systems, we developed a new set of similarity features, and created the BitterMatch algorithm that predicts associations of ligands to receptors with ~80% precision at ~50% recall. Associations for several compounds were tested in-vitro, resulting in 80% precision and 42% recall. The encouraging performance was achieved by including receptor properties and integrating experimentally determined ligand-receptor associations with chemical ligand-to-ligand similarities. BitterMatch can predict off-targets for bitter drugs, identify novel ligands and guide flavor design. Inclusion of neighbor-informed similarities improves as experimental data mounts, and provides a generalizable framework for molecule-biotarget matching.

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The effects of sequencing depth on the assembly of coding and noncoding transcripts in the human genome BackgroundInvestigating the functions and activities of genes requires proper annotation of the transcribed units. However, transcript assembly efforts have produced a surprisingly large variation in the number of transcripts, and especially so for noncoding transcripts. The heterogeneity of the assembled transcript sets might be partially explained by sequencing depth.

ResultsHere, we used real and simulated short-read sequencing data as well as long-read data to systematically investigate the impact of sequencing depths on the accuracy of assembled transcripts. We assembled and analyzed transcripts from 671 human short-read data sets and four long-read data sets. At the first level, there is a positive correlation between the number of reads and the number of recovered transcripts. However, the effect of the sequencing depth varied based on cell or tissue type, the type of read considered and the nature and expression levels of the transcripts. The detection of coding transcripts saturated rapidly for both short-read and long-reads, however, there was no sign of saturation for noncoding transcripts at any sequencing depth. Increasing long-read sequencing depth specifically benefited transcripts containing transposable elements. Finally, we show how single-cell RNA-seq can be guided by transcripts assembled from bulk long-read samples, and demonstrate that noncoding transcripts are expressed at similar levels to coding transcripts but are expressed in fewer cells.

ConclusionsThis study shows the impact of sequencing depth on transcript assembly. Sequencing read depth has a relatively minor impact on coding transcript assembly, but a major effect on the assembly of noncoding transcripts. This study highlights important factors to consider when deciding the sequencing read depths to be used for transcript assembly.

sentences: Three Reagents for in-Solution Enrichment of Ancient Human DNA at More than a Million SNPs In-solution enrichment for hundreds of thousands of single nucleotide polymorphisms (SNPs) has been the source of >70% of all genome-scale ancient human DNA data published to date. This approach has made it possible to generate data for one to two orders of magnitude lower cost than random shotgun sequencing, making it economical to study ancient samples with low proportions of human DNA, and increasing the rate of conversion of sampled remains into working data thereby facilitating ethical stewardship of human remains. So far, nearly all ancient DNA data obtained using in-solution enrichment has been generated using a set of bait sequences targeting about 1.24 million SNPs (the  1240k reagent). These sequences were published in 2015, but synthesis of the reagent has been cost-effective for only a few laboratories. In 2021, two companies made available reagents that target the same core set of SNPs along with supplementary content. Here, we test the properties of the three reagents on a common set of 27 ancient DNA libraries across a range of richness of DNA content and percentages of human molecules. All three reagents are highly effective at enriching many hundreds of thousands of SNPs. For all three reagents and a wide range of conditions, one round of enrichment produces data that is as useful as two rounds when tens of millions of sequences are read out as is typical for such experiments. In our testing, the "Twist Ancient DNA" reagent produces the highest coverages, greatest uniformity on targeted positions, and almost no bias toward enriching one allele more than another relative to shotgun sequencing. Allelic bias in 1240k enrichment has made it challenging to carry out joint analysis of these data with shotgun data, creating a situation where the ancient DNA community has been publishing two important bodes of data that cannot easily be co-analyzed by population genetic methods. To address this challenge, we introduce a subset of hundreds of thousands of SNPs for which 1240k data can be effectively co-analyzed with all other major data types.

sentences: The Nicotiana tabacum Mediator subunit MED25 regulates nicotine biosynthesis through interacting with the basic helix-loop-helix (bHLH) transcription factor NtMYC2s Nicotine is one of the most important secondary metabolites in tobacco, and its biosynthesis can be induced by topping and jasmonic acid treatment. NtMYC2s play pivotal roles in the regulation of nicotine. The mediator server as a bridge betwen the transcription factors and RNA polymerase in order to facilitates transcription and functions in plants. However, the role of mediator in the regulation of nicotine biosynthesis remains unknown. In this study, we firstly identify the NtMED25 through homologous analysis. NtMED25 interacts with NtMYC2s through the MD region. Interestingly, the nicotine content is decreased in the the knock-down transgenic lines of NtMED25, and the expression levels of two nicotine biosynthesis genes, NtQPT2 and NtPMT2, are also reduced when compared with that in the wild-type plants. Furthermore, NtMED25 enhances the binding of NtMYC2a/ NtMYC2b to the promoter of NtPMT2 and NtQPT2, and then facilitates the nicotine biosynthesis. Therefore, our study revealed the function of mediator in the regulation of nicotine, and provide the insight role on the transcriptional regulation of plant secondary metabolites.

sentences: Mapping Consistent, Reproducible, and Transcriptionally Relevant Connectome Hubs of the Human Brain Human brain connectomes include sets of densely connected regions, known as connectome hubs, which play a vital role in understanding global brain communication, cognitive processing, and brain disorders. However, the consistency and reproducibility of functional connectome hubs anatomical localization have not been established to date and the genetic signatures underlying robust connectome hubs remain unknown. Here, we conduct the first worldwide, harmonized meta-connectomic analysis by pooling resting-state functional MRI data of 5,212 healthy young adults across 61 independent cohorts. We identify highly consistent and reproducible functional connectome hubs in heteromodal and unimodal regions both across cohorts and across individuals. These connectome hubs show heterogeneous connectivity profiles and are critical for both intra- and inter-network communications. Using post-mortem gene expression data, we show that these connectome hubs have a spatiotemporally distinctive transcriptomic pattern dominated by genes involved in the neuropeptide signaling pathway, neurodevelopmental processes, and metabolic processes. These results highlight the robustness of macroscopic connectome hubs and their potential cellular and molecular underpinnings.

sentences: Plasma cell maintenance and antibody secretion are under the control of Sec22b-mediated regulation of organelle dynamics Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the SNARE Sec22b as a unique and critical regulator of plasma cell maintenance and function. In absence of Sec22b, plasma cells were barely detectable and serum antibody titres were dramatically reduced. Accordingly, Sec22b deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b is indispensable for efficient antibody secretion but also for plasma cell fitness through the regulation of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil a critical role for Sec22b-mediated regulation of plasma cell biology through the control of organelle dynamics.

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Nephrotoxicity of Calcineurin Inhibitors in kidney epithelial cells is independent of Calcineurin and NFAT signaling. BackgroundCalcineurin inhibitors (CNI) such as Cyclosporine A (CsA) and Tacrolimus (FK506) are commonly used after renal transplantation in order to suppress the immune system. In lymphoid cells, CsA acts via the Calcineurin-NFAT axis, whereas in non-lymphoid cells, such as kidney epithelial cells, CsA induces Calcineurin inhibitor toxicity (CNT). Up to date, it is unknown via which off-targets CsA induces CNT in kidney epithelial cells.

MethodsIn vitro experiments using a surrogate marker to measure CNT induction as well as in vivo studies with acute CNT, were used in order to elucidate the underlying molecular mechanism.

ResultsInhibition of the NFAT axis does not show any nephrotoxicity. However, inhibition of p38 and PI3K/Akt Kinases showed induction of nephrotoxicity.

ConclusionsThese findings show that CsA acts NFAT independent on kidney epithelial cells. Moreover, inhibition of certain protein kinases mimic CsA activity on kidney epithelial cells indicating that p38 and PI3K/Akt kinase pathways might be involved in CNT progression on kidney epithelial cells.

sentences: An interactome landscape of SARS-CoV-2 virus-human protein-protein interactions by protein sequence-based multi-label classifiers The new coronavirus species, SARS-CoV-2, caused an unprecedented global pandemic of COVID-19 disease since late December 2019. A comprehensive characterization of protein-protein interactions (PPIs) between SARS-CoV-2 and human cells is a key to understanding the infection and preventing the disease. Here we present a novel approach to predict virus-host PPIs by multi-label machine learning classifiers of random forests and XGBoost using amino acid composition profiles of virus and human proteins. Our models harness a large-scale database of Viruses.STRING with >80,000 virus-host PPIs along with evidence scores for multi-level evidence prediction, which is distinct from predicting binary interactions in previous studies. Our multi-label classifiers are based on 5 evidence levels binned from evidence scores. Our best model of XGBoost achieves 74% AUC and 68% accuracy on average in 10-fold cross validation. The most important amino acids are cysteine and histidine. In addition, our model predicts experimental PPIs with higher accuracy than text mining-based PPIs by 4% despite their smaller data size by more than 6-fold. We then predict evidence levels of [~]2,000 SARS-CoV-2 virus-human PPIs from public experimental proteomics data. Interactions with SARS-CoV-2 Nsp7b show high evidence. We also predict evidence levels of all pairwise PPIs of [~]550,000 between the SARS-CoV-2 and human proteomes to provide a draft virus-host interactome landscape for SARS-CoV-2 infection in humans in a comprehensive and unbiased way in silico. Most human proteins from 140 highest evidence predictions interact with SARS-CoV-2 Nsp7, Nsp1, and ORF14, with significant enrichment in the top 2 pathways of vascular smooth muscle contraction (CALD1, NPR2, CALML3) and Myc targets (CBX3, PES1). Our prediction also suggests that histone H2A components are targeted by multiple SARS-CoV-2 proteins.

sentences: Assessment of AI-based Protein Structure Prediction for the NLRP3 Target The recent success of AlphaFold and RoseTTAFold has demonstrated the values of AI methods in predicting highly accurate protein structures. Despite the advances, their roles in the context of small-molecule drug discovery need to be thoroughly explored. In this study, we evaluated the possibility whether the AI-based models can lead to reliable three-dimensional structures of protein-ligand complexes. The structure we predicted was NLRP3, a challenging protein target in terms of obtaining the three-dimensional model both experimentally and computationally. The conformation of the binding pockets generated by the AI models were carefully characterized and compared with the experimental structures.

sentences: Twitches emerge postnatally during quiet sleep in human infants and are synchronized with sleep spindles SO_SCPLOWUMMARYC_SCPLOWIn humans and other mammals, the stillness of sleep is punctuated by bursts of rapid eye movements (REMs) and myoclonic twitches of the limbs [1]. Contrary to the notion that twitches are mere by-products of dreams, sensory feedback arising from twitching limbs provides a rich and unique source of activation to the developing sensorimotor system [2]. In fact, it is partly because of the behavioral activation of REM sleep that this state is also called active sleep (AS), in contrast with the behavioral quiescence that gives quiet sleep (QS)--the second major stage of sleep--its name. In human infants, for which AS occupies eight or more hours of each day [3], limb twitching is one among several components that help to identify the state [4-7]; nonetheless, we know relatively little about the structure and functions of twitching across development. Recently, in sleeping infants over the first seven postnatal months [8], we documented a pronounced shift in the temporal expression of twitching beginning around three months of age that suggested a qualitative shift in how twitches are produced. Here, we combine behavioral assessments of twitching with high-density electroencephalography (EEG) and demonstrate that this shift reflects the developmental emergence of limb twitches during QS. Twitches during QS are not only unaccompanied by REMs, but they also occur synchronously with sleep spindles, a hallmark of QS. As QS-related twitching increases with age, sleep spindle rate also increases along the sensorimotor strip. The emerging synchrony between subcortically generated twitches and cortical oscillations suggests the development of functional connectivity among distant sensorimotor structures, with potential implications for detecting and explaining atypical developmental trajectories.

sentences: Quantitative mapping of dense microtubule arrays in mammalian neurons The neuronal microtubule cytoskeleton underlies the polarization and proper functioning of neurons, amongst others by providing tracks for motor proteins that drive intracellular transport. Different subsets of neuronal microtubules, varying in composition, stability and motor preference, are known to exist, but the high density of microtubules has so far precluded mapping their relative abundance and three-dimensional organization. Here we use different super-resolution techniques (STED, Expansion Microscopy) to explore the nanoscale organization of the neuronal microtubule network. This revealed that in dendrites stable, acetylated microtubules are enriched in the core of the dendritic shaft, while dynamic, tyrosinated microtubules enrich near the plasma membrane, thus forming a shell around the stable microtubules. Moreover, using a novel analysis pipeline we quantified the absolute number of acetylated and tyrosinated microtubules within dendrites and found that they account for 65-75% and [~]20-30% of all microtubules, respectively, leaving only few microtubules that do not fall in either category. Because these different microtubule subtypes facilitate different motor proteins, these novel insights help to understand the spatial regulation of intracellular transport.

sentences: Brief sensory deprivation triggers plasticity of dopamine-synthesising enzyme expression in genetically labelled olfactory bulb dopaminergic neurons In the glomerular layer of the olfactory bulb, local dopaminergic interneurons play a key role in regulating the flow of sensory information from nose to cortex. These dual dopamine- and GABA-releasing cells are capable of marked experience-dependent changes in the expression of neurotransmitter-synthesising enzymes, including tyrosine hydroxylase (TH). However, such plasticity has most commonly been studied in cell populations identified by their expression of the enzyme being studied, and after long periods of sensory deprivation. Here, instead, we used brief 1- or 3-day manipulations of olfactory experience in juvenile mice, coupled with a conditional genetic approach that labelled neurons contingent upon their expression of the dopamine transporter (DAT-tdTomato). This enabled us to evaluate the potential for rapid changes in neurotransmitter-synthesising enzyme expression in an independently identified neuronal population. Our labelling strategy showed good specificity for olfactory bulb dopaminergic neurons, whilst revealing a minority sub-population of non-dopaminergic DAT-tdTomato cells that expressed the calcium-binding protein calretinin. Crucially, the proportions of these neuronal subtypes were not affected by brief alterations in sensory experience. Short-term olfactory manipulations also produced no significant changes in immunofluorescence or whole-bulb mRNA for the GABA-synthesising enzyme GAD67/Gad1. However, in bulbar DAT-tdTomato neurons brief sensory deprivation was accompanied by a transient, small drop in immunofluorescence for the dopamine-synthesising enzyme dopa decarboxylase (DDC), and a sustained decrease for TH. Deprivation also produced a sustained decrease in whole-bulb Th mRNA. Careful characterisation of an independently identified, genetically labelled neuronal population therefore enabled us to uncover rapid experience-dependent changes in dopamine-synthesising enzyme expression.

sentences: Environment, rather than Hematodinium parasitization, determines collateral disease contraction in a crustacean host Host, pathogen, and environment are determinants of the disease triangle, the latter being a key driver of disease outcomes and persistence within a community. The dinoflagellate genus Hematodinium is detrimental to crustaceans globally - considered to suppress the innate defences of hosts, making them more susceptible to co-infections. Evidence supporting immune-suppression is largely anecdotal and sourced from diffuse accounts of compromised decapods. We used a population of shore crabs (Carcinus maenas), where Hematodinium sp. is endemic, to determine the extent of collateral infections across two distinct environments (open water, semi- closed dock). Using a multi-resource approach (PCR, histology, haematology, population genetics, eDNA), we identified 162 Hematodinium-positive crabs and size/sex-matched these to 162 Hematodinium-free crabs out of 1,191 analysed. Crabs were interrogated for additional disease-causing agents; haplosporidians, microsporidians, mikrocytids, Vibrio spp., fungi, Sacculina, trematodes, and haemolymph bacterial loads. We found no significant differences in occurrence, severity or composition of collateral infections between Hematodinium-positive and Hematodinium-free crabs at either site, but crucially, we recorded site-restricted blends of pathogens. We found no gross signs of host cell immune reactivity toward Hematodinium in the presence or absence of other pathogens. We contend Hematodinium sp. is an immune-evader rather than immune-suppressor, which suggests an evolutionary drive toward latency in this environmentally plastic host.

sentences: Thioesterase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a futile cycle that inhibits hepatic β-oxidation 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing {beta}-oxidation. In this study, the mechanism of {beta}-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete {beta}-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).

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Evolution and spread of SARS-CoV-2 likely to be affected by climate COVID-19 pandemic has been extensively studied by many researchers. However, it is still unclear why it was restricted to higher latitudes during the initial days and later cascaded in the tropics. Here, we analyzed 176 SARS-CoV-2 genomes across different latitudes and climate (Koppens climate) that provided insights about within species virus evolution and its relation to abiotic factors. Two genetically variant groups, named as G1 and G2 were identified, well defined by four mutations. The G1 group (ancestor), is mainly restricted to warm and moist, temperate climate (Koppens C climate) while its descendent G2 group surpasses the climatic restrictions of G1, initially cascading into neighboring cold climate (D) of higher latitudes and later into hot climate of the tropics (A). It appears that the gradation of temperate climate (Cfa-Cfb) to cold climate (Dfa-Dfb) drives the evolution of G1 into G2 variant group which later adapted to tropical climate (A) as well. It seems this virus followed inverse latitudinal gradient in the beginning due to its preference towards temperate (C) and cold climate (D). Our work elucidates virus evolutionary studies combined with climatic studies can provide crucial information about the pathogenesis and natural spreading pathways in such outbreaks which is hard to achieve through individual studies. Mutational insights gained may help design an efficacious vaccine.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/147074v3_ufig1.gif" ALT="Figure 1">
View larger version (28K):
org.highwire.dtl.DTLVardef@f39a3org.highwire.dtl.DTLVardef@1910598org.highwire.dtl.DTLVardef@6516corg.highwire.dtl.DTLVardef@cbb48c_HPS_FORMAT_FIGEXP  M_FIG C_FIG In BriefThe authors elucidate adaptation of SARS-CoV-2 to different climates by studying phylogenetics and the distribution of strains on Koppens climate map.

HighlightsO_LIPhylogenetic network divides SARS-CoV-2 strains into two variant groups, G1 and G2.
C_LIO_LIG1 strains is restricted to Koppens "temperate" climate (mainly Cfa-Cfb).
C_LIO_LIG2 strains has evolved from G1 to sustain in other climates mainly "humid-continental" (Dfa-Dfb) and "tropical-savannah" (Aw) climate.
C_LI

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The complex genetics of symbiotic extended phenotypes across environments in a model mutualism A goal of modern biology is to develop the genotype-phenotype (G[->]P) map, a predictive understanding of how genomic information generates trait variation that forms the basis of both natural and managed communities. As microbiome research advances, however, it has become clear that many of these traits are symbiotic extended phenotypes, being governed by genetic variation encoded not only by the hosts own genome, but also by the genomes of myriad cryptic symbionts. Building a reliable G[->]P map therefore requires accounting for the multitude of interacting genes and even genomes involved in symbiosis. Here we use naturally-occurring genetic variation in 191 strains of the model microbial symbiont Ensifer meliloti in four mapping experiments to study the genomic architecture of a key symbiotic extended phenotype - partner quality, or the fitness benefit conferred to a host by a particular symbiont genotype, within and across environmental contexts and host genotypes. We define three novel categories of loci in rhizobium genomes that must be accounted for if we want to build a reliable G[->]P map of partner quality; namely, 1) loci whose identities depend on the environment, 2) those that depend on the host genotype with which rhizobia interact, and 3) universal loci that are likely important in all or most environments.

IMPORTANCEGiven the rapid rise of research on how microbiomes can be harnessed to improve host health, understanding the contribution of microbial genetic variation to host phenotypic variation is pressing, and will better enable us to predict the evolution of (and select more precisely for) symbiotic extended phenotypes that impact host health. We uncover extensive context-dependency in both the identity and functions of symbiont loci that control host growth, which makes predicting the genes and pathways important for determining symbiotic outcomes under different conditions more challenging. Despite this context-dependency, we also resolve a core set of universal loci that are likely important in all or most environments, and thus, serve as excellent targets both for genetic engineering and future coevolutionary studies of symbiosis.

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A novel regulatory gene promotes novel cell fate by suppressing ancestral fate in the sea anemone Nematostella vectensis Cnidocytes ("stinging cells") are an unequivocally novel cell type used by cnidarians (corals, jellyfish, and their kin) to immobilize prey. Although they are known to share a common evolutionary origin with neurons, the developmental program that promoted the emergence of cnidocyte fate is not known. Using functional genomics in the sea anemone, Nematostella vectensis, we show that cnidocytes evolved by suppression of neural fate in a subset of neurons expressing RFamide. We further show that a single regulatory gene, a C2H2-type zinc finger transcription factor (ZNF845), coordinates both the gain of novel (cnidocyte-specific) traits and the inhibition of ancestral (neural) traits during cnidocyte development and that this gene arose by domain shuffling in the stem cnidarian. Thus, we uncover a mechanism by which a truly novel regulatory gene (ZNF845) promoted the origin of a truly novel cell type (cnidocyte) through duplication of an ancestral cell lineage (neuron) and inhibition of its ancestral identity (RFamide).

SignificanceIn this study, we demonstrate how new cell types can arise in animals through duplication of an ancestral (old) cell type followed by functional divergence of the new daughter cell. Specifically, we show that stinging cells in cnidarians (jellyfish and corals) evolved by duplication of an ancestral neuron followed by inhibition of the RFamide neuropeptide it once secreted. This is the first evidence that stinging cells evolved from a specific subtype of neurons and suggests some neurons may be easier to co-opt for novel functions than others.

sentences: A lateral protrusion latticework connects neuroepithelial cells and is regulated during neurogenesis Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple protrusive structures in neuroepithelial apical endfeet of the chick embryonic spinal cord, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape, and cytoskeleton of these lateral protrusions and distinguish these structures from cytonemes/filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1: mutant-WAVE1 misexpression attenuated protrusion and generated a round-ended apical endfoot morphology. However, this did not alter apico-basal cell polarity nor reduce tissue integrity. During normal neuronal delamination sub-apical protrusions were withdrawn, but mutant-WAVE1-induced precocious protrusion loss was insufficient to trigger neurogenesis. This study uncovers a new form of cell-cell contact within the developing neuroepithelium regulation of which prefigures neuronal delamination.

sentences: Utilizing occupancy-detection models with museum specimen data: promise and pitfalls Historical museum records provide potentially useful data for identifying drivers of change in species occupancy. However, because museum records are typically obtained via many collection methods, methodological developments are needed in order to enable robust inferences. Occupancy-detection models, a relatively new and powerful suite of methods, are a potentially promising avenue because they can account for changes in collection effort through space and time. Here we present a methodological road-map for using occupancy models to analyze historical museum records. We use simulated data-sets to identify how and when patterns in data and/or modelling decisions can bias inference. We focus primarily on the consequences of contrasting methodological approaches for dealing with species ranges and inferring species non-detections in both space and time. We find that not all data-sets are suitable for occupancy-detection analysis but, under the right conditions (namely, data-sets that span long durations and contain a high fraction of community-wide collections, or collection events that focus on communities of organisms), models can accurately estimate trends. Finally, we present a case-study on eastern North American odonates where we calculate long-term trends of occupancy by using our most robust workflow.

sentences: Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly by ensuring PCNA-dependent CAF-1 recycling The inability to protect stalled replication forks from nucleolytic degradation drives genome instability and is associated with chemosensitivity in BRCA-deficient tumors. An emerging hallmark of BRCA deficiency is the inability to suppress replication-associated single-stranded DNA (ssDNA) gaps. Here, we report that ssDNA gaps on the lagging strand interfere with the ASF1-CAF-1 pathway of nucleosome assembly, and drive fork degradation in BRCA-deficient cells. We show that CAF-1 function at replication forks is lost in BRCA-deficient cells, due to its sequestration at inactive replication factories during replication stress. This CAF-1 recycling defect is caused by the accumulation of Pol-dependent lagging strand gaps, which preclude PCNA unloading, causing sequestration of PCNA-CAF-1 complexes on chromatin. Importantly, correcting PCNA unloading defects in BRCA-deficient cells restores fork stability in a CAF-1-dependent manner. We also show that the activation of a HIRA-dependent compensatory histone deposition pathway restores fork stability to BRCA-deficient cells upon CAF-1 loss. We thus define nucleosome assembly as a critical determinant of BRCA-mediated fork stability. We further reveal lagging strand ssDNA gaps as drivers of fork degradation in BRCA-deficient cells, which operate by inhibiting PCNA unloading and CAF-1-dependent nucleosome assembly.

sentences: Deficiency in Lyst function leads to accumulation of secreted proteases and predisposition to mechanic stress-induced retinal detachment Chediak-Higashi syndrome, caused by mutations in the Lysosome Trafficking Regulator (Lyst) gene, is a recessive hypopigmentation disorder characterized by albinism, neuropathies, neurodegeneration, and defective immune responses, with enlargement of lysosomes and lysosome-related organelles. Although recent studies have suggested that Lyst mutations impair the regulation of sizes of lysosome and lysosome-related organelle, the underlying pathogenic mechanism of Chediak-Higashi syndrome is still unclear. Here we show striking evidence that deficiency in LYST protein function leads to accumulation of photoreceptor outer segment phagosomes in retinal pigment epithelial cells, and reduces adhesion between photoreceptor outer segment and retinal pigment epithelial cells in a mouse model of Chediak-Higashi syndrome. In addition, we observe elevated levels of cathepsins, matrix metallopeptidase (MMP) 3 and oxidative stress markers in the retinal pigment epithelium of Lyst mutants. Previous reports showed that impaired degradation of photoreceptor outer segment phagosomes causes elevated oxidative stress, which could consequently lead to increases of cysteine cathepsins and MMPs in the extracellular matrix. Taken together, we conclude that the loss of LYST function causes accumulation of phagosomes in the retinal pigment epithelium and elevation of several extracellular matrix-remodeling proteases through oxidative stress, which may, in turn, reduce retinal adhesion. Our work reveals previously unreported pathogenic events in the retinal pigment epithelium caused by Lyst deficiency, which may place Chediak-Higashi syndrome patients at increased risk for retinal detachment. The same pathogenic events may be conserved in other professional phagocytic cells, such as macrophages in the immune system, contributing to overall Chediak-Higashi syndrome pathology.

sentences: Characterizing and explaining impact of disease-associated mutations in proteins without known structures or structural homologues Mutations in human proteins lead to diseases. The structure of these proteins can help understand the mechanism of such diseases and develop therapeutics against them. With improved deep learning techniques such as RoseTTAFold and AlphaFold, we can predict the structure of proteins even in the absence of structural homologues. We modeled and extracted the domains from 553 disease-associated human proteins without known protein structures or close homologues in the Protein Databank (PDB). We noticed that the model quality was higher and the RMSD lower between AlphaFold and RoseTTAFold models for domains that could be assigned to CATH families as compared to those which could only be assigned to Pfam families of unknown structure or could not be assigned to either. We predicted ligand-binding sites, protein-protein interfaces, conserved residues in these predicted structures. We then explored whether the disease-associated missense mutations were in the proximity of these predicted functional sites, if they destabilized the protein structure based on ddG calculations or if they were predicted to be pathogenic. We could explain 80% of these disease-associated mutations based on proximity to functional sites, structural destabilization or pathogenicity. When compared to polymorphisms a larger percentage of disease associated missense mutations were buried, closer to predicted functional sites, predicted as destabilising and/or pathogenic. Usage of models from the two state-of-the-art techniques provide better confidence in our predictions, and we explain 93 additional mutations based on RoseTTAFold models which could not be explained based solely on AlphaFold models.

sentences: Myonuclear maturation dynamics in aged and adult regenerating mouse skeletal muscle Skeletal muscle cells are multinucleated syncytial cells arising from cell fusion, yet despite sharing a common cytoplasm individual myonuclei express distinct transcriptional programs. Whether individual myonuclei acquire heterogenous transcriptional states via differences in their progenitors, during differentiation, or once their anatomical position is acquired, is not known. We performed transcriptome and pseudotime analysis of single myogenic nuclei from uninjured and post-injury murine skeletal muscle to assess when myonuclear heterogeneity is acquired. Two distinct progenitors contribute to myonuclei, one a non-myogenic fibroblast subtype, and skeletal muscle stem cells the other. Both progenitors enter a single pseudotime trajectory that bifurcates as myonuclei mature into two branches segregated by myosin isoform expression and metabolic profiles, suggesting transcriptional heterogeneity is acquired as myonuclei mature. In aged skeletal muscle myogenic progenitor expansion is perturbed and nuclei from aged muscle display distinct pseudotemporal kinetics compared to nuclei from young mice. In aged mice, the inferred myogenic differentiation trajectory is delayed, altering the distribution of myogenic nuclei in pseudotime, suggesting that altered transcriptional dynamics in nuclei in aged mice may drive age-associated muscle deficits and bias myonuclei towards acquiring oxidative metabolic profiles.

sentences: Dystonia-specific mutations in THAP1 alter transcription of genes associated with neurodevelopment and myelin Dystonia is a neurologic disorder associated with an increasingly large number of variants in many genes, resulting in characteristic disturbances in volitional movement. Dissecting the relationships between these mutations and their functional outcomes is a critical step in understanding the key pathways that drive dystonia pathogenesis. Here we established a pipeline for characterizing an allelic series of dystonia-specific mutations in isogenic induced pluripotent stem cells (iPSCs). We used this strategy to investigate the molecular consequences of variation in THAP1, which encodes a transcription factor that has been linked to neural differentiation. Multiple pathogenic mutations that have been associated with dystonia cluster within distinct THAP1 functional domains and are predicted to alter its DNA binding properties and/or protein interactions differently, yet the relative impact of these varied changes on molecular signatures and neural deficits is unclear. To determine the effects of these mutations on THAP1 transcriptional activity, we engineered an allelic series of eight mutations in a common iPSC background and differentiated these lines into a panel of near-isogenic neural stem cells (n = 94 lines). Transcriptome profiling of these neural derivatives followed by joint analysis of the most robust individual signatures across mutations identified a convergent pattern of dysregulated genes functionally related to neurodevelopment, lysosomal lipid metabolism, and myelin. Based on these observations, we examined mice bearing Thap1-disruptive alleles and detected significant changes in myelin gene expression and reduction of myelin structural integrity relative to tissue from control mice. These results suggest that deficits in neurodevelopment and myelination are common consequences of dystonia-associated THAP1 mutations and highlight the potential role of neuron-glial interactions in the pathogenesis of dystonia.

sentences: Plague risk in the western United States over seven decades of environmental change After several pandemics over the last two millennia, the wildlife reservoirs of plague (Yersinia pestis) now persist around the world, including in the western United States. Routine surveillance in this region has generated comprehensive records of human cases and animal seroprevalence, creating a unique opportunity to test how plague reservoirs are responding to environmental change. Here, we develop a new method to detect the signal of climate change in infectious disease distributions, and test whether plague reservoirs and spillover risk have shifted since 1950. We find that plague foci are associated with high-elevation rodent communities, and soil biochemistry may play a key role in the geography of long-term persistence. In addition, we find that human cases are concentrated only in a small subset of endemic areas, and that spillover events are driven by higher rodent species richness (the amplification hypothesis) and climatic anomalies (the trophic cascade hypothesis). Using our detection model, we find that due to the changing climate, rodent communities at high elevations have become more conducive to the establishment of plague reservoirs--with suitability increasing up to 40% in some places--and that spillover risk to humans at mid-elevations has increased as well, although more gradually. These results highlight opportunities for deeper investigation of plague ecology, the value of integrative surveillance for infectious disease geography, and the need for further research into ongoing climate change impacts.

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A Genome-wide CRISPR Screen Identifies WDFY3 as a Novel Regulator of Macrophage Efferocytosis AbstractsPhagocytic clearance of dying cells, termed efferocytosis, must occur efficiently to maintain homeostasis and prevent disease. Yet, our understanding of this important biological process remains incomplete. To search for novel regulators of efferocytosis, we performed a FACS-based genome-wide CRISPR knockout screen in primary murine macrophages. We identified a novel role for WDFY3 in efferocytosis by macrophages. WDFY3 deficiency in macrophages specifically impaired uptake, not binding, of apoptotic cells due to defective actin depolymerization. We further revealed that WDFY3 directly interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Although the C-terminus of WDFY3 was sufficient to rescue impaired degradation, full-length WDFY3 is still required for regulating uptake. Finally, WDFY3 is required for efficient efferocytosis in vivo in mice and in primary human macrophages. The work expands our knowledge of the mechanisms of macrophage efferocytosis, and more broadly, provides a general strategy for genome-wide CRISPR screen to interrogate complex functional phenotypes in primary macrophages.

HighlightsO_LIFunctional readout for pooled genome-wide CRISPR screen in primary macrophages.
C_LIO_LIWDFY3 is discovered as a regulator of macrophage efferocytosis in vitro and in vivo.
C_LIO_LIWDFY3 deficiency led to impaired uptake, as opposed to binding, of apoptotic cells due to defective actin depolymerization.
C_LIO_LIWDFY3 directly interacts with GABARAP, facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components.
C_LIO_LIC-terminal WDFY3 is sufficient to regulate the degradation of engulfed apoptotic cells while full-length WDFY is required for regulating uptake.
C_LI

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Yeast galactokinase in closed conformation can switch between catalytic and signal transducer states. S.cerevisiae galactokinase (ScGal1p), in closed conformation catalyzes the phosphorylation of galactose to galactose 1-phopshate using ATP as the phosphate donor as well as allosterically activates the GAL genetic switch in response to galactose and ATP as ligands. How both kinase and signaling activities of ScGal1p are associated with closed conformation of the protein is not understood. Conformational sampling of ScGal1p indicated that this protein samples closed kinase and closed non-kinase conformers. Closed non-kinase conformers are catalytically incompetent to phosphorylate galactose and act as a bonafide signal transducer. It was observed that toggling of side chain of highly conserved K266 of ScGal1p between S171and catalytic base D217 is responsible for transitioning of ScGal1p between signal transducer and kinase states. Interestingly in ScGal3p, the paralog of ScGal1p, which has only signal transduction activity and lacks kinase activity, a H bond between a non-conserved Y433, and a highly conserved Y57, gets broken during MD simulation. The corresponding H-bond present in ScGal1p between residues Y441 and Y63 respectively, remains intact throughout the simulations of ScGal1p.Therefore, we predicted that K266 and Y441 have a role in bifunctionality of ScGal1p. To test the above predictions, we monitored the signaling and kinase activity of ScGal1K266Rp and ScGal1Y441Ap variants. Signaling activity increased in both ScGal1Y441Ap and ScGal1K266Rp variants as compared to ScGal1wtp, whereas the kinase activity increased in ScGal1Y441Ap, but decreased in ScGal1K266Rp Based on the above, we propose that K266 and Y441 are crucial for conferring bifunctionality to ScGal1p.

Author summaryGalactokinase of S.cerevisiae(ScGal1p), the first enzyme of Leloir pathway of galactose metabolism, phosphorylates galactose using ATP as the phosphate donor. ScGal1p also functions as a signal transducer of GAL regulon wherein galactose and ATP allosterically activate galactokinase. The active form of galactokinase, then sequesters the repressor ScGal80p, to activate the GAL switch. ScGal1p has a single site each for binding to galactose and ATP. How ScGal1p, a monomeric protein, performs the above two mutually exclusive activities using the same set of substrates/ligands, with the same site acting as the active site for enzymatic activity as well as allosteric site for signal transduction activity is unclear. Our findings are that this protein has a distinct conformational state for functioning as a signal transducer and a distinct conformational state for functioning as a kinase. A highly conserved lysine residue (K266) present only in fungal galactokinases, triggers the interconversion between catalysis and signal transduction states. This interconversion is subdued by H bond between Y441 and Y63. These studies suggest that the two activities of ScGal1p are fine tuned by evolution to regulate metabolism through transcriptional control.

sentences: Plant pathogenic bacterium can rapidly evolve tolerance to an antimicrobial plant allelochemical Crop losses to plant pathogens are a growing threat to global food security and more effective control strategies are urgently required. Biofumigation, an agricultural technique where Brassica plant tissues are mulched into soils to release antimicrobial plant allelochemicals called isothiocyanates (ITCs), has been proposed as an environmentally friendly alternative to agrochemicals. While biofumigation has been shown to suppress a range of plant pathogens, its effects on plant pathogenic bacteria remain largely unexplored. Here we used a laboratory model system to compare the efficacy of different types of ITCs against Ralstonia solanacearum plant bacterial pathogen. Additionally, we evaluated the potential for ITC-tolerance evolution under high, intermediate and low transfer frequency ITC exposure treatments. We found that allyl-ITC was the most efficient compound at suppressing R. solanacearum growth, and its efficacy was not improved when combined with other types of ITCs. Despite consistent pathogen growth suppression, ITC tolerance evolution was observed in the low transfer frequency exposure treatment, leading to cross-tolerance to ampicillin beta-lactam antibiotic. Mechanistically, tolerance was linked to insertion sequence movement at four positions in genes that were potentially associated with stress responses (H-NS histone like protein), cell growth and competitiveness (acyltransferase), iron storage ((2-Fe-2S)-binding protein) and calcium ion sequestration (calcium-binding protein). Interestingly, pathogen adaptation to the growth media also indirectly selected for increased ITC tolerance through potential adaptations linked with metabolism and antibiotic resistance (dehydrogenase-like protein) and transmembrane protein movement (Tat pathway signal protein). Together, our results suggest that R. solanacearum can rapidly evolve tolerance to allyl-ITC plant allelochemical which could constrain the long-term efficiency of biofumigation biocontrol and potentially shape pathogen evolution with plants.

sentences: Laboratory evolution of anticipatory gene regulation in Escherichia coli. Environmental cues in an ecological niche are often temporal in nature. For instance, in temperate climates, temperature is higher in daytime compared to during night. In response to these temporal cues, bacteria have been known to exhibit anticipatory regulation, whereby they trigger response to a yet to appear cue, anticipating its actual arrival in the near future. Such an anticipatory response in known to enhance Darwinian fitness, and hence, is likely an important feature of regulatory networks in microorganisms. However, the conditions under which an anticipatory response evolves as an adaptive response are not known. In this work, we develop a quantitative model to study response of a population to two temporal environmental cues, and predict variables which are likely important for evolution of anticipatory regulatory response. We follow this with experimental evolution of E. coli in alternating environments of a pentose sugar, rhamnose, and an oxidative stress molecule, paraquat for more than 800 generations. We demonstrate that growth in this cyclical environment leads to evolution of anticipatory regulation, whereby, exposure to rhamnose leads to partial induction of the oxidative stress response regulon. As a result, pre-exposure to rhamnose leads to a greater fitness in paraquat environment. Overall, we show that in niches where environmental stimuli have a cyclical nature, anticipatory regulation can evolve as an adaptive strategy, in a time course of a few hundred generations. This contributes to our understanding of how environment shapes the topology of regulatory networks in an organism.

sentences: The Gossypium stocksii genome as a novel resource for cotton improvement Cotton is an important textile crop whose gains in production over the last century have been challenged by various diseases. Because many modern cultivars are susceptible to several pests and pathogens, breeding efforts have included attempts to introgress wild, naturally resistant germplasm into elite lines. Gossypium stocksii is a wild cotton species native to Africa, which is part of a clade of vastly understudied species. Most of what is known about this species comes from pest resistance surveys and/or breeding efforts, which suggests that G. stocksii could be a valuable reservoir of natural pest resistance. Here we present a high-quality de novo genome sequence for G. stocksii. We compare the G. stocksii genome with resequencing data from a closely related, understudied species (G. somalense) to generate insight into the relatedness of these cotton species. Finally, we discuss the utility of the G. stocksii genome for understanding pest resistance in cotton, particularly resistance to cotton leaf curl virus.

sentences: Gene Ontology Meta Annotator for Plants (GOMAP) Annotating gene structures and functions to genome assemblies is necessary to make assembly resources useful for biological inference. Gene Ontology (GO) term assignment is the most used functional annotation system, and new methods for GO assignment have improved the quality of GO-based function predictions. The Gene Ontology Meta Annotator for Plants (GOMAP) is an optimized, high-throughput, and reproducible pipeline for genome-scale GO annotation of plants. We containerized GOMAP to increase portability and reproducibility and also optimized its performance for HPC environments. Here we report on the pipelines availability and performance for annotating large, repetitive plant genomes and describe how GOMAP was used to annotate multiple maize genomes as a test case. Assessment shows that GOMAP expands and improves the number of genes annotated and annotations assigned per gene as well as the quality (based on Fmax) of GO assignments in maize. GOMAP has been deployed to annotate other species including wheat, rice, barley, cotton, and soy. Instructions and access to the GOMAP Singularity container are freely available online at https://bioinformapping.com/gomap/. A list of annotated genomes and links to data is maintained at https://dill-picl.org/projects/gomap/.

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Single Cell Atlas of Human Putamen Reveals Disease Specific Changes in Synucleinopathies: Parkinson's Disease and Multiple System Atrophy Understanding disease biology at a cellular level from disease specific tissues is imperative for effective drug development for complex neurodegenerative diseases. We profiled 87,086 nuclei from putamen tissue of healthy controls, Parkinsons Disease (PD), and Multiple System Atrophy (MSA) subjects to construct a comprehensive single cell atlas. Although both PD and MSA are manifestations of -synuclein protein aggregation, we observed that both the diseases have distinct cell-type specific changes. We see a possible expansion and activation of microglia and astrocytes in PD compared to MSA and controls. Contrary to PD microglia, we found absence of upregulated unfolded protein response in MSA microglia compared to controls. Differentially expressed genes in major cell types are enriched for genes associated with PD-GWAS loci. We found altered expression of major neurodegeneration associated genes -- SNCA, MAPT, LRRK2, and APP -- at cell-type resolution. We also identified disease associated gene modules using a network biology approach. Overall, this study creates an interactive atlas from synucleinopathies and provides major cell-type specific disease insights.

Link to interactive atlas will be made available at the time of publication.

sentences: Exocyst stimulates each step of exocytic SNARE complex assembly and vesicle fusion The exocyst is a large multisubunit tethering complex essential for targeting and fusion of secretory vesicles in eukaryotic cells. Although the assembled exocyst complex is proposed to tether vesicles to the plasma membrane and activate the SNARE proteins for membrane fusion, only little is known about the key biochemical steps that exocyst stimulates in the course of SNARE complex assembly, a critical question defining the essential molecular role of the exocyst complex. Here, we use a combination of single molecule and bulk fluorescence assays with purified octameric yeast exocyst complexes to examine the role of exocyst in a reconstituted SNARE assembly and fusion system. We show that the exocyst complex simulates multiple steps spanning from SNARE protein activation to ternary complex assembly, rather than affecting only a specific subset of steps. We also observed that the exocyst has important downstream roles in driving membrane fusion, up to full content mixing of vesicle lumens. Our results suggest that the exocyst complex provides extensive chaperoning functions for the entire process of SNARE complex assembly, presumably using its multi-faceted structure provided by the eight subunits.

sentences: Recirculation of H2, CO2, and ethylene improves carbon fixation and carboxylate yields in anaerobic fermentation Anaerobic fermentation with mixed cultures has gained momentum as a bioprocess for its promise to produce platform carboxylates from low-value biomass feedstocks. Anaerobic fermenters are net carbon emitters and their carboxylate yields are limited by electron donor availability. In a new approach to tackle these two disadvantages, we operated two bioreactors fed with acetate and lactate as a model feedstock while recirculating H2/CO2 to stimulate concomitant autotrophic activity. After 42 days of operation, hydrogenotrophic methanogenesis was predominant and ethylene ([≥]1.3 kPa) was added to one of the reactors, inhibiting methanogenesis completely and recovering net carbon fixation (0.20 g CO2 L-1 d-1). When methanogenesis was inhibited, exogenous H2 accounted for 17% of the consumed electron donors. Lactate-to-butyrate selectivity was 101% (88% in the control without ethylene) and lactate-to-caproate selectivity was 17% (2.3% in the control). Community analysis revealed that ethylene caused Methanobacterium to be washed out, giving room to acetogenic bacteria. In contrast to 2-bromoethanosulfonate, ethylene is a scalable methanogenesis inhibition strategy that did not collaterally block i-butyrate formation. By favoring the bacterial share of the community to become mixotrophic, the concept offers a way to simultaneously increase selectivity to medium-chain carboxylates and to develop a carbon-fixing chain elongation process.

sentences: The endoplasmic reticulum in perisynaptic astrocytic processes: shape, distribution and effect on calcium activity Astrocytes recently emerged as key regulators of information processing in the brain. Ca2+ signals in perisynaptic astrocytic processes (PAPs) notably allow astrocytes to fine-tune neurotransmission at so-called tripartite synapses. As most PAPs are below the diffraction limit, their content in Ca2+ stores and the contribution of the latter to astrocytic Ca2+ activity is unclear. Here, we reconstruct tripartite synapses in 3D from electron microscopy and find that 75% of PAPs contain some endoplasmic reticulum (ER), a major astrocytic Ca2+ store, displaying strikingly diverse geometrical properties. To investigate the role of such spatial properties, we implemented an algorithm that creates 3D PAP meshes of various ER distributions and constant shape. Reaction-diffusion simulations in those meshes reveal that astrocyte activity is shaped by a complex interplay between the location of Ca2+ channels, Ca2+ buffering, ER shape and distribution. Overall, this study sheds new light into mechanisms regulating signal transmission in the brain.

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Semantic representations during language comprehension are affected by context The meaning of words in natural language depends crucially on context. However, most neuroimaging studies of word meaning use isolated words and isolated sentences with little context. Because the brain may process natural language differently from how it processes simplified stimuli, there is a pressing need to determine whether prior results on word meaning generalize to natural language. We investigated this issue by directly comparing the brain representation of semantic information across four conditions that vary in context. fMRI was used to record human brain activity while four subjects (two female) read words presented in four different conditions: narratives (Narratives), isolated sentences (Sentences), blocks of semantically similar words (Semantic Blocks), and isolated words (Single Words). Using a voxelwise encoding model approach, we find two clear and consistent effects of increasing context. First, stimuli with more context (Narratives, Sentences) evoke brain responses with substantially higher SNR across bilateral visual, temporal, parietal, and prefrontal cortices compared to stimuli with little context (Semantic Blocks, Single Words). Second, increasing context increases the representation of semantic information across bilateral temporal, parietal, and prefrontal cortices at the group level. However, in individual subjects, only natural language stimuli (Narratives) consistently evoke widespread representation of semantic information across the cortical surface. These results show that context has large effects on both the quality of neuroimaging data and on the representation of meaning in the brain, and they imply that the results of neuroimaging studies that use stimuli with little context may not generalize well to the natural regime.

Significance StatementContext is an important part of understanding the meaning of natural language, but most neuroimaging studies of meaning use isolated words and isolated sentences with little context. Here we examined whether the results of neuroimaging studies that use out-of-context stimuli generalize to natural language. We find that increasing context improves the quality of neuroimaging data and changes where semantic information is represented in the brain. These results suggest that findings from studies using out-of-context stimuli may not generalize to natural language used in daily life.

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How big should this object be? Perceptual influences on viewing-size preferences When viewing objects depicted in a frame, observers prefer to view large objects like cars in larger sizes and smaller objects like cups in smaller sizes. That is, the visual size of an object that "looks best" is linked to its typical physical size in the world. Why is this the case? One intuitive possibility is that these preferences are driven by semantic knowledge: For example, when we recognize a sofa, we access our knowledge about its real-world size, and this influences what size we prefer to view the sofa within a frame. However, might visual processing play a role in this phenomenon--that is, do visual features that are related to big and small objects look better at big and small visual sizes, respectively, even when observers do not have explicit access to semantic knowledge about the objects? To test this possibility, we used "texform" images, which are synthesized versions of recognizable objects, which critically retain local perceptual texture and coarse contour information, but are no longer explicitly recognizable. To test for visual size preferences, we first used a size adjustment task, and the results were equivocal. However, clear results were obtained using a two-interval forced choice task, in which each texform was presented at the preferred visual size of its corresponding original image, and a visual size slightly bigger or smaller. Observers consistently selected the texform presented at the canonical visual size as the more aesthetically pleasing one. An additional control experiment ruled out alternative explanations related to size priming effects. These results suggest that the preferred visual size of an object depends not only on explicit knowledge of its real-world size, but also can be evoked by mid-level visual features that systematically covary with an objects real-world size.

HighlightsO_LIWe prefer to view large objects like cars large, and small objects like cups small
C_LIO_LIIntuitively, such preferences may be driven by our knowledge of object sizes
C_LIO_LIWe used unrecognizable texforms of objects that preserved mid-level visual features
C_LIO_LISimilar viewing size preferences can be revealed with these texforms
C_LIO_LISuch preferences thus arise not only from knowledge but also from visual processing
C_LI

sentences: Effects of the collagen-glycosaminoglycan mesh on gene expression as determined by using principal component analysis-based unsupervised feature extraction Development of the medical applications for substances or materials that contact the cells is important. Hence, it is necessary to elucidate how substance that surround cells affect the gene expression during incubation. Here, we compared the gene expression profiles of cell lines that were in contact with the collagen-glycosaminoglycan mesh and control cells. Principal component analysis-based unsupervised feature extraction was applied to identify genes with altered expression during incubation in the treated cell lines but not in the controls. The identified genes were enriched in various biological terms. Our method also outperformed a conventional methodology, namely, gene selection based on linear regression with time course.

sentences: Reverse engineering of a mechanistic model of gene expression using metastability and temporal dynamics Differentiation can be modeled at the single cell level as a stochastic process resulting from the dynamical functioning of an underlying Gene Regulatory Network (GRN), driving stem or progenitor cells to one or many differentiated cell types. Metastability seems inherent to differentiation process as a consequence of the limited number of cell types. Moreover, mRNA is known to be generally produced by bursts, which can give rise to highly variable non-Gaussian behavior, making the estimation of a GRN from transcriptional profiles challenging. In this article, we present CARDAMOM (Cell type Analysis from scRna-seq Data achieved from a Mixture MOdel), a new algorithm for inferring a GRN from timestamped scRNA-seq data, which crucially exploits these notions of metastability and transcriptional bursting. We show that such inference can be seen as the successive resolution of as many regression problem as timepoints, after a preliminary clustering of the whole set of cells with regards to their associated bursts frequency. We demonstrate the ability of CARDAMOM to infer a reliable GRN from in silico expression datasets, with good computational speed. To the best of our knowledge, this is the first description of a method which uses the concept of metastability for performing GRN inference.

sentences: Novel genome assemblies and Evolutionary Dynamics of North American Anopheles mosquitoes Anopheles mosquitoes are the principal vectors for malaria and lymphatic filariasis, and evidence for arboviral transmission under laboratory and natural contexts has been demonstrated. Vector management approaches require an understanding of the ecological, epidemiological, and biological context of the species in question, and increased interest in gene drive systems for vector control applications has resulted in an increased need for genome assemblies from understudied mosquito species. While the genomes for many Anopheles species have been sequenced, North American Anopheles taxa have been neglected. In this study we present novel genome assemblies for the North American species An. crucians, An. freeborni, An. albimanus, and An. quadrimaculatus, and examine the evolutionary relationship between these species. We identified 790 shared single copy orthologs between the newly sequenced genomes and created a phylogeny using 673 of the orthologs, identifying 525 orthologs with evidence for positive selection on at least one branch of the phylogeny. Gene ontology terms such as calcium ion signaling, histone binding, and protein acetylation were identified as being biased in the set of selected genes. These novel genome sequences will be useful in developing our understanding of the diverse biological traits that drive vectorial capacity in anophelines.

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Long-term cross-scale comparison of grazing and mowing on plant diversity and community composition in a salt-marsh system O_LILand abandonment is increasing in recent decades in Europe, usually accompanied by a decline in biodiversity. Whether livestock grazing and mowing can safeguard biodiversity across spatial scales in the long term is unclear.
C_LIO_LIUsing a 48-year experiment in a salt marsh, we compared land abandonment (without grazing and mowing) and seven management regimes including cattle grazing, early season mowing, late season mowing, both early and late season mowing, and grazing plus each of the mowing regimes on plant diversity at the local (i.e. plot) and landscape scales (i.e. across plots). Also, we compared their effects on community composition (both in identities and abundances) in time and space.
C_LIO_LIUnder land abandonment, plant diversity declined in the local communities and this decline became more apparent at the landscape scale, particularly for graminoids and halophytes. All management regimes, except the late season mowing, maintained plant diversity at these scales.
C_LIO_LILocal plant communities under all treatments underwent different successional trajectories, in the end, diverged from their initial state except for that under grazing (a cyclic succession). Interannual composition change remained stable over time under land abandonment and grazing plus early season mowing. It increased over time under grazing and late season mowing, it increased in the second half of the experiment under other treatments. Vegetation homogenized in the landscape over time under land abandonment while vegetation was heterogeneous under all management regimes.
C_LIO_LISynthesis. Our experiment suggests that late season mowing may not be sustainable to conserve plant diversity in salt marshes. Other management regimes can maintain plant diversity across scales and vegetation heterogeneity in the landscape in the long term, but local community composition may change over time.
C_LI

sentences: The role of behavioural flexibility in primate diversification Identifying the factors that influence species diversification is fundamental to our understanding of the evolutionary processes underlying extant biodiversity. Behavioural innovation, coupled with the social transmission of new behaviours, has been proposed to increase rates of evolutionary diversification, as novel behaviours expose populations to new selective regimes. Thus, it is believed that behavioural flexibility may play an important role in driving evolutionary diversification across animals. We test this hypothesis within the primates, a taxonomic group with considerable among-lineage variation in both species diversity and behavioural flexibility. We employ a time cut-off in our phylogeny to help account for biases associated with recent taxonomic reclassifications and compare three alternative measures of diversification rate that consider different phylogenetic depths. We find that the presence of behavioural innovation and social learning are positively correlated with diversification rates among primate genera, but not at shallower phylogenetic depths. Given that we find stronger associations when examining older rather than more recent diversification events, we suggest that extinction resistance, as opposed to speciation, may be an important mechanism linking behavioural flexibility and primate diversification. Our results contrast with work linking behavioural flexibility with diversification of birds at various phylogenetic depths. We offer a possible dispersal-mediated explanation for these conflicting patterns, such that the influence behavioural flexibility plays in dictating evolutionary trajectories differs across clades. Our results suggest that behavioural flexibility may act through several different pathways to shape the evolutionary trajectories of lineages.

sentences: Chlorophyll fluorescence-based estimates of photosynthetic electron transport in Arctic phytoplankton assemblages We employed Fast Repetition Rate fluorometry for high-resolution mapping of marine phytoplankton photophysiology and primary productivity in the Lancaster Sound and Barrow Strait regions of the Canadian Arctic Archipelago in the summer of 2019. Continuous ship-board analysis of chlorophyll a variable fluorescence demonstrated relatively low photochemical efficiency over most of the cruise-track, with the exception of localized regions within Barrow Strait where there was increased vertical mixing and proximity to land-based nutrient sources. Along the full transect, we observed strong non-photochemical quenching of chlorophyll fluorescence, with relaxation times longer than the 5-minute period used for dark acclimation. Such long-term quenching effects complicate continuous underway acquisition of fluorescence amplitude-based estimates of photosynthetic electron transport rates, which rely on dark acclimation of samples. As an alternative, we employed a new algorithm to derive electron transport rates based on analysis of fluorescence relaxation kinetics, which does not require dark acclimation. Direct comparison of kinetics- and amplitude-based electron transport rate measurements demonstrated kinetic-based estimates were, on average, 2-fold higher than amplitude-based values. The magnitude of decoupling between the two electron transport rate estimates increased in association with photophysiological diagnostics of nutrient stress. Discrepancies between electron transport rate estimates likely resulted from the use of different photophysiological parameters to derive the kinetics- and amplitude-based algorithms, and choice of numerical model used to fit variable fluorescence curves and analyze fluorescence kinetics under actinic light. Our results highlight environmental and methodological influences on fluorescence-based productivity estimates, and prompt discussion of best-practices for future underway fluorescence-based efforts to monitor phytoplankton photosynthesis.

sentences: DYNAMITE: a phylogenetic tool for identification of dynamic transmission epicenters Molecular data analysis is invaluable in understanding the overall behavior of a rapidly spreading virus population when epidemiological surveillance is problematic. It is also particularly beneficial in describing subgroups within the population, often identified as clades within a phylogenetic tree, that represent individuals connected via direct transmission or transmission via differing risk factors in viral spread. However, transmission patterns or viral dynamics within these smaller groups should not be expected to exhibit homogeneous behavior over time. As such, standard phylogenetic approaches that identify clusters based on summary statistics (e.g., median genetic distance over the clade) would not be expected to capture dynamic clusters of transmission. For this purpose, we have developed DYNAMITE (DYNAMic Identification of Transmission Epicenters), a cluster identification algorithm based on a branch-wise (rather than traditional clade-wise) search for cluster criteria, allowing partial clades to be recognized as clusters. Using simulated viral outbreaks with varying cluster types and dynamics, we show that DYNAMITE is consistently more sensitive than existing tools in detecting both static and dynamic transmission clusters. DYNAMITE has been implemented in R and released as open source at: github.com/ProsperiLab/DYNAMITE.

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Distinct roles for two Caenorhabditis elegans acid-sensing ion channels in an ultradian clock Biological clocks are fundamental to an organisms health, controlling periodicity of behavior and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signaling, the generation of an ultradian oscillator, and its metabolic consequences.

One-Sentence SummaryTwo acid-sensing DEG/ENaC channels play distinct roles in controlling different aspects of rhythmic proton and Ca2+ oscillations in the C. elegans intestine.

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Clonal hematopoiesis is associated with increased toxicity in large B-cell lymphoma patients treated with chimeric antigen receptor T cell therapy To explore the role of clonal hematopoiesis (CH) on chimeric antigen receptor (CAR) T-cell therapy outcomes, we performed targeted deep-sequencing on 114 large B-cell lymphoma patients treated with anti-CD19 CAR T-cells. We detected CH in 42 (36.8%) pre-treatment patient samples, most frequently in PPM1D (19/114) and TP53 (13/114) genes. The incidence of grade [≥]3 immune-effector cell-associated neurotoxicity syndrome (ICANS) was higher in CH-positive patients compared to CH-negative patients (45.2% vs. 25.0%, p=0.038). Higher toxicities with CH were primarily driven by three CH genes, DNMT3A, TET2 and ASXL1 (DTA mutations). The incidence of grade [≥]3 ICANS [58.9% vs. 25%, p=0.02] and grade [≥]3 cytokine release syndrome [17.7% vs. 4.2%, p=0.08] were higher in patients with DTA mutations than those without CH. The estimated 24-month cumulative incidence of therapy-related myeloid neoplasms after CAR-T therapy was higher in patients with CH than those without CH (19% [95%CI: 5.5-38.7] vs. 4.2% [95%CI: 0.3-18.4], p=0.028).

Statement of SignificanceOur study reveals that clonal hematopoiesis mutations, especially those associated with inflammation (DNMT3A, TET2, ASXL1), are associated with severe grade toxicities in lymphoma patients receiving anti-CD19 chimeric antigen receptor therapy. Further studies to investigate the mechanisms and interventions to improve toxicities in the context of CH are warranted.

sentences: A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results suggest that in these cells the variant of concern B.1.1.7, (aka Alpha variant), became the predominant circulating variant in several countries by a clear transmission advantage. In fact, in these cells B.1.1.7 outcompetes its ancestor B.1.177 in terms of a much faster kinetics of entry. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the main role of clathrin as mediator of late-entry endocytosis, reconciling it with the membrane localization of the ACE2 receptor previously attributed to caveolin-enriched rafts. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.

sentences: Virome composition in marine fish revealed by meta-transcriptomics Revealing the determinants of virome composition is central to placing disease emergence in a broader evolutionary context. Fish are the most species-rich group of vertebrates and so provide an ideal model system to study the factors that shape virome compositions and their evolution. We characterised the viromes of 19 wild-caught species of marine fish using total RNA sequencing (meta-transcriptomics) combined with analyses of sequence and protein structural homology to identify divergent viruses that often evade characterisation. From this, we identified 25 new vertebrate-associated viruses and a further 22 viruses likely associated with fish diet or their microbiomes. The vertebrate-associated viruses identified here included the first fish virus in the Matonaviridae (single-strand, negative-sense RNA virus). Other viruses fell within the Astroviridae, Picornaviridae, Arenaviridae, Reoviridae, Hepadnaviridae, Paramyxoviridae, Rhabdoviridae, Hantaviridae, Filoviridae and Flaviviridae and were sometimes phylogenetically distinct from known fish viruses. We also show how key metrics of virome composition - viral richness, abundance and diversity - can be analysed along with host ecological and biological factors as a means to understand virus ecology. Accordingly, these data suggest that that the vertebrate-associated viromes of the fish sampled here are predominantly shaped by the phylogenetic history (i.e. taxonomic order) of their hosts, along with several biological factors including water temperature, habitat depth, community diversity and swimming behaviour. No such correlations were found for viruses associated with porifera, molluscs, arthropods, fungi and algae, that are unlikely to replicate in fish hosts. Overall, these data indicate that fish harbour particularly large and complex viromes and the vast majority of fish viromes are undescribed.

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Idiosyncratic epistasis leads to global fitness-correlated trends Epistasis can dramatically affect evolutionary trajectories. In recent decades, protein-level fitness landscapes have revealed extensive idiosyncratic epistasis among specific mutations. In contrast, other work has found ubiquitous and apparently non-specific patterns of global diminishing-returns and increasing-costs epistasis among mutations across the genome. Here, we use a hierarchical CRISPR gene drive system to construct all combinations of 10 missense mutations from across the genome in budding yeast, and measure their fitness in six environments. We show that the resulting fitness landscapes exhibit global fitness-correlated trends, but that these trends emerge from specific idiosyncratic interactions. This provides the first experimental validation of recent theoretical work that has argued that fitness-correlated trends can emerge as the generic consequence of idiosyncratic epistasis.

One-Sentence SummaryA genome-spanning fitness landscape reveals how idiosyncratic genetic interactions lead to global epistatic patterns.

sentences: Dual brain cortical calcium imaging reveals social interaction-specific wide-spread correlated cortical activity in mice. We employ cortical mesoscale calcium-imaging to observe brain activity in two head-fixed mice in a staged social touch-like interaction. Using a rail system, mice are brought together to a distance where macrovibrissae of each mouse make contact. Cortical signals were recorded from both mice simultaneously before, during, and after the social contact period. When the mice were together, we observed bouts of mutual whisking and cross-mouse correlated cortical activity in the vibrissae cortex. This correlated activity was specific to individual interactions as the correlations fell in trial-shuffled mouse pairs. Whisk-related global GCAMP6s signals were greater in cagemate pairs during the together period. The effects of social interaction extend outside of regions associated with mutual touch and had global synchronizing effects on cortical activity. We present an open-source platform to investigate the neurobiology of social interaction by including mechanical drawings, protocols, and software necessary for others to extend this work.

sentences: Multisite Test-Retest Reliability and Compatibility of Brain Metrics derived from FreeSurfer Versions 7.1, 6.0, and 5.3 Automatic neuroimaging segmentation and parcellation tools provide convenient and systematic methods for extracting numerous features from brain MRI scans, and are becoming standard practice for large-scale coordinated studies. One such tool, FreeSurfer, provides an easy-to-use pipeline to extract metrics describing cortical and subcortical morphometry. Over the past two decades, there have been over 25 stable releases of FreeSurfer, and different versions are used across published works. Despite this, the reliability and compatibility of metrics derived from the most recent major version releases have yet to be assessed empirically. Here, we use test-retest data from three public brain MRI datasets to assess within-version reliability and between-version compatibility across 42 regional outputs from three versions of FreeSurfer: the latest, v7.1, and two previous stable releases - v5.3, and v6.0. We find v7.1 was less compatible with older versions for measuring cortical thickness. In particular, the thickness of the cingulate gyrus had low compatibility (intraclass correlation coefficient (ICC) between 0.37 and 0.61) between versions. Temporal and frontal poles, and the medial orbitofrontal surface area metrics, also showed low to moderate compatibility with v7.1. While our work compares all three versions, our sub-comparisons between the older versions (v5.3 and v6.0) replicates earlier findings of low compatibility of pallidum and putamen volumes. Low between-version compatibility was not always indicative of low within-version reliability - all versions showed good to excellent reliability across most regional measures (ICC>0.8). Age associations, quality control metrics, and Dice coefficients in an independent sample of 106 individual scans, processed with all three versions of FreeSurfer, revealed differences in results of downstream statistical analysis. As neuroimaging studies adopt more recently released software, we provide researchers with a reference to highlight the regions and metrics that may yield findings inconsistent with published works using older FreeSurfer software. An interactive viewer for the results is provided at http://data.brainescience.org/Freesurfer_Reliability/

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Ipsilateral stimulus encoding in primary and secondary somatosensory cortex of awake mice Lateralization is a hallmark of somatosensory processing in the mammalian brain. However, in addition to their contralateral representation, unilateral tactile stimuli also modulate neuronal activity in somatosensory cortices of the ipsilateral hemisphere. The cellular organization and functional role of these ipsilateral stimulus responses in awake somatosensory cortices, especially regarding stimulus coding, are unknown. Here, we targeted silicon probe recordings to the vibrissa region of primary (S1) and secondary (S2) somatosensory cortex of awake head-fixed mice of either sex while delivering ipsilateral and contralateral whisker stimuli. Ipsilateral stimuli drove larger and more reliable responses in S2 than in S1, and activated a larger fraction of stimulus-responsive neurons. Ipsilateral stimulus-responsive neurons were rare in layer 4 of S1, but were located in equal proportion across all layers in S2. Linear classifier analyses further revealed that decoding of the ipsilateral stimulus was more accurate in S2 than S1, while S1 decoded contralateral stimuli most accurately. These results reveal substantial encoding of ipsilateral stimuli in S1 and especially S2, consistent with the hypothesis that higher cortical areas may integrate tactile inputs across larger portions of space, spanning both sides of the body.

S1ignificance StatementTactile information obtained by one side of the body is represented in the activity of neurons of the opposite brain hemisphere. However unilateral tactile stimulation also modulates neuronal activity in the other, or ipsilateral, brain hemisphere. This ipsilateral activity may play an important role in the representation and processing of tactile information, in particular when the sense of touch involves both sides of the body. Our work in the whisker system of awake mice reveals that neocortical ipsilateral activity, in particular that of deep layer excitatory neurons of secondary somatosensory cortex (S2), contains information about the presence and the velocity of unilateral tactile stimuli, which supports a key role for S2 in integrating tactile information across both body sides.

sentences: SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection. Moreover, Nsp14 expression altered the splicing of more than 1,000 genes and resulted in a dramatic increase in the number of circRNAs, which are linked to innate immunity. These effects were independent of the Nsp14 exonuclease activity and required the N7-guanine-methyltransferase domain of the protein. Activation of the NFkB pathway and increased expression of CXCL8 occurred early upon Nsp14 expression. We identified IMPDH2, which catalyzes the rate-limiting step of guanine nucleotides biosynthesis, as a key mediator of these effects. Nsp14 expression caused an increase in GTP cellular levels, and the effect of Nsp14 was strongly decreased in presence of IMPDH2 inhibitors. Together, our data demonstrate an unknown role for Nsp14 with implications for therapy.

sentences: Inter-animal variability in activity phase is constrained by synaptic dynamics in an oscillatory network The levels of voltage-gated and synaptic currents in the same neuron type can vary substantially across individuals. Yet, the phase relationships between neurons in oscillatory circuits are often maintained, even in the face of varying oscillation frequencies. We examined whether synaptic and intrinsic currents are matched to maintain constant activity phases across preparations, using the lateral pyloric (LP) neuron of the stomatogastric ganglion of the crab, Cancer borealis. LP produces stable oscillatory bursts upon release from inhibition, with an onset phase that is independent of oscillation frequency. We quantified the parameters that define the shape of the synaptic current inputs across preparations and found no linear correlations with voltage-gated currents. However, several synaptic parameters were correlated with oscillation period and burst onset phase, suggesting they may play a role in phase maintenance. We used the dynamic clamp to apply artificial synaptic inputs and found that those synaptic parameters correlated with phase and period were ineffective in influencing burst onset. Instead, parameters that showed the least variability across preparations had the greatest influence. Thus, parameters that influence circuit phasing are constrained across individuals, while those that have little effect simply co-vary with phase and frequency.

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Polygenic adaptation, clonal interference, and the evolution of mutators in experimental Pseudomonas aeruginosa populations In bacterial populations, switches in lifestyle from motile, planktonic growth to surface-grown biofilm is associated with persistence in both infections and non-clinical biofilms. Studies have identified the first steps of adaptation to biofilm growth but have yet to replicate the extensive genetic diversity observed in chronic infections or in the natural environment. We conducted a 90-day long evolution experiment with Pseudomonas aeruginosa PA14 in growth media that promotes biofilm formation in either planktonic culture or in a biofilm bead model. Surprisingly, all populations evolved extensive genetic diversity with hundreds of mutations maintained at intermediate frequencies, while fixation events were rare. Instead of the expected few beneficial mutations rising in frequency through successive sweeps, we observe a remarkable 40 genes with parallel mutations spanning both environments and often on coexisting genotypes within a population. Additionally, the evolution of mutator genotypes (mutS or mutL mutator alleles) that rise to high frequencies in as little as 25 days contribute to the extensive genetic variation and strong clonal interference. Parallelism in several transporters (including pitA, pntB, nosD, and pchF) indicate probable adaptation to the arginine media that becomes highly alkaline during growth. Further, genes involved in signal transduction (including gacS, aer2, bdlA, and PA14_71750) reflect likely adaptations to biofilm-inducing conditions. This experiment shows how extensive genetic and phenotypic diversity can arise and persist in microbial populations despite strong selection that would normally purge diversity.

ImportanceHow biodiversity arises and is maintained in clonally reproducing organisms like microbes remains unclear. Many models presume that beneficial genotypes will outgrow others and purge variation via selective sweeps. Environmental structure like biofilms may oppose this process and preserve variation. We tested this hypothesis by evolving P. aeruginosa populations in biofilm-promoting media for three months and found both adaptation and diversification that were mostly uninterrupted by fixation events that eliminate diversity. Genetic variation tended to be greater in lines grown using a bead model of biofilm growth but many lineages also persisted in planktonic lines. Convergent evolution affecting dozens of genes indicates that selection acted on a wide variety of traits to improve fitness, causing many adapting lineages to co-occur and persist. This result demonstrates that some environments may expose a large fraction of the genome to selection and select for many adaptations at once, causing enduring diversity.

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CrowdGO: machine learning and semantic similarity guided consensus Gene Ontology annotation BackgroundCharacterising gene function for the ever-increasing number and diversity of species with annotated genomes relies almost entirely on computational prediction methods. These software are also numerous and diverse, each with different strengths and weaknesses as revealed through community benchmarking efforts. Meta-predictors that assess consensus and conflict from individual algorithms should deliver enhanced functional annotations.

ResultsTo exploit the benefits of meta-approaches, we developed CrowdGO, an open-source consensus-based Gene Ontology (GO) term meta-predictor that employs machine learning models with GO term semantic similarities and information contents. By re-evaluating each gene-term annotation, a consensus dataset is produced with high-scoring confident annotations and low-scoring rejected annotations. Applying CrowdGO to results from a deep learning-based, a sequence similarity-based, and two protein domain-based methods, delivers consensus annotations with improved precision and recall. Furthermore, using standard evaluation measures CrowdGO performance matches that of the communitys best performing individual methods.

ConclusionCrowdGO offers a model-informed approach to leverage strengths of individual predictors and produce comprehensive and accurate gene functional annotations.

Availability and ImplementationCrowdGO is implemented in Python3, and is freely available from https://gitlab.com/mreijnders/CrowdGO, with a Snakemake workflow and pre-trained models.

sentences: Uncovering the ligandome of low-density lipoprotein receptor-related protein 1 in cartilage: a top-down approach to identify therapeutic targets The low-density lipoprotein receptor-related protein 1 (LRP1) is a cell-surface receptor ubiquitously expressed in adult tissues. It plays tissue-specific physiological roles by mediating endocytosis of a diverse range of extracellular molecules. Dysregulation of LRP1 is involved in multiple conditions including Alzheimers disease, atherosclerosis and osteoarthritis (OA). However, little information is available about the specific ligand profile (ligandome) for each tissue, which would lead to better understanding of its role in disease states. Here, we investigated adult articular cartilage where impaired LRP1-mediated endocytosis leads to tissue destruction. We used a top-down approach involving analysis of human chondrocyte secretome, direct binding assays and validation in LRP1-deficient fibroblasts, as well as a novel Lrp1 conditional knockout (KO) mouse model. We found that inhibition of LRP1-mediated endocytosis results in cell death, alteration of the entire secretome and transcriptional modulations in human chondrocytes. We have identified more than 50 novel ligand candidates and confirmed direct LRP1 binding of HGFAC, HMGB1, HMGB2, CEMIP, SLIT2, ADAMTS1, IGFBP7, SPARC and LIF. Our in vitro endocytosis assay revealed the correlation of their affinity for LRP1 and the rate of endocytosis. Moreover, a conditional LRP1 KO mouse model demonstrated a critical role of LRP1 in regulating the high-affinity ligands in cartilage in vivo. This systematic approach revealed the extent of the chondrocyte LRP1 ligandome and identified potential novel therapeutic targets for OA.

sentences: LPS-TLR4 pathway stimulating in acute-on-chronic liver failure by promote NETs formation Backgrounds: Intrahepatic infiltration of neutrophils is a character of alcoholic acute-on-chronic liver failure (AACLF) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. Intestinal bacteria and the gut-liver axis have been thought to play a key role in many liver diseases also including AACLF. However, whether NETs appear in AACLF and play a role in AACLF is still unsure. Methods: WT, NE KO, and TLR4 KO mice were used to build the AACLF model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AACLF related markers were detected. Results: The serum MPO-DNA and LPS concentration was increased in AACLF patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AACLF mice by testing MPO-DNA, Cit H3, and NE. These markers decreased with gut detergent and restored markers with gut detergent plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AACLF related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with combined treatment with antibiotics plus LPS. But the liver injury, intrahepatic fat, fibro deposition, and liver inflammation-related markers did show a significant difference in TLR4 KO mice when they received the same treatment. Conclusion: Intestinal-derived LPS promotes NETs formation in AACLF through the TLR4 pathway and further accelerates the AACLF process by NETs.

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Binocular Mirror-Symmetric Microsaccadic Sampling of Hyperacute 3D-Vision Neural mechanisms behind stereopsis, which requires simultaneous disparity inputs from two eyes, have remained mysterious. Here we show how ultrafast mirror-symmetric photomechanical contractions in the frontal forward-facing left and right eye photoreceptors give Drosophila super-resolution 3D-vision. By interlinking multiscale in vivo assays with multiscale simulations, we reveal how these photoreceptor microsaccades - by verging, diverging and narrowing the eyes overlapping receptive fields - channel depth information, as phasic binocular image motion disparity signals in time. We further show how peripherally, outside stereopsis, microsaccadic sampling tracks a flying flys optic flow field to better resolve the world in motion. These results change our understanding of how insect compound eyes work and suggest a general dynamic stereo-information sampling strategy for animals, robots and sensors.

Significance statementTo move efficiently, animals must continuously work out their x,y,z-positions in respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a new morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila super-resolution 3D-vision and proposes neural computations for accurately predicting these flies depth-perception dynamics, limits, and visual behaviors.

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A reference induced pluripotent stem cell line for large-scale collaborative studies Human induced pluripotent stem cells (iPSCs) are a powerful tool for studying development and disease. However, different iPSC lines show considerable phenotypic variation. The lack of common well-characterised cell lines that are used widely frustrates efforts to integrate data across research groups or replicate key findings. Inspired by model organism communities who addressed this issue by establishing a limited number of widely accepted strains, we characterised candidate iPSC lines in unprecedented detail to select a well-performing line to underpin collaborative studies. Specifically, we characterised the morphology, growth rates, and single-cell transcriptomes of iPSC lines in the pluripotent state and assessed their genomic integrity using karyotyping, DNA microarrays, whole genome sequencing, and functional assays for p53 activity. We further tested their ability to be edited by CRISPR/Cas9 and used single-cell RNA sequencing to compare the efficiency with which they could be differentiated into multiple lineages. We found that there was significant variability in the performance of lines across the tested assays that enabled the rational selection of a lead line, KOLF2.1J, which is a gene-corrected derivative of a publicly available line from the Human Induced Pluripotent Stem Cells Initiative (HipSci) resource. We are now using this line in an initiative from the NIH Center for Alzheimers and Related Dementias to derive hundreds of gene-edited and functionalized sub-clones to be distributed widely throughout the research community along with associated datasets, with the aim of promoting the standardisation required for large-scale collaborative science in the stem cell field.

SummaryThe authors of this collaborative science study describe a deep characterisation of widely available human induced pluripotent stem cell (hiPSC) lines to rationally select a line that performs well in multiple experimental approaches. Analysis of transcriptional patterns in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and differentiation efficiency towards multiple lineages identified KOLF2.1J as an all-around well-performing cell line. The widespread distribution and use of this line makes it an attractive cell line for comparative and collaborative efforts in the stem cell field.

HighlightsO_LIDeep genotyping and phenotyping reveal KOLF2.1J as an all-around well-performing cell line that is readily distributed and could serve as common reference line
C_LIO_LIDespite rare copy-neutral loss of heterozygosity (CN-LOH) events, iPSC lines retain genomic fidelity after CRISPR/Cas9-based gene editing
C_LIO_LIOur multifactorial pipeline serves as a blueprint for future efforts to identify other lead iPSC lines
C_LI

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sentences: MIRA: Joint regulatory modeling of multimodal expression and chromatin accessibility in single cells Rigorously comparing gene expression and chromatin accessibility in the same single cells could illuminate the logic of how coupling or decoupling of these mechanisms regulates fate commitment. Here, we present MIRA: Probabilistic Multimodal Models for Integrated Regulatory Analysis, a comprehensive methodology that systematically contrasts transcription and accessibility to infer the regulatory circuitry driving cells along developmental trajectories. MIRA leverages topic modeling of cell states and regulatory potential modeling of individual gene loci. MIRA thereby represents cell states in an efficient and interpretable latent space, infers high fidelity lineage trees, determines key regulators of fate decisions at branch points, and exposes the variable influence of local accessibility on transcription at distinct loci. Applied to epidermal maintenance differentiation and embryonic brain development from two different multimodal platforms, MIRA revealed that early developmental genes were tightly regulated by local chromatin landscape whereas terminal fate genes were titrated without requiring extensive chromatin remodeling.

sentences: Deep deconvolution of the hematopoietic stem cell regulatory microenvironment reveals a high degree of specialization and conservation between mouse and human. Understanding the regulation of normal and malignant human hematopoiesis requires comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Here, we develop a tailored bioinformatic pipeline to integrate public and proprietary single-cell RNA sequencing (scRNA-seq) datasets. As a result, we robustly identify for the first time 14 intermediate cell states and 11 stages of differentiation in the endothelial and mesenchymal BM compartments, respectively. Our data provide the most comprehensive description to date of the murine HSC-regulatory microenvironment and suggests a higher level of specialization of the cellular circuits than previously anticipated. Furthermore, this deep characterization allows to infer conserved features in human, suggesting that the layers of microenvironmental regulation of hematopoiesis may also be shared between species. Our resource and methodology are a steppingstone towards a comprehensive cell atlas of the BM microenvironment.

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Measuring biodiversity from DNA in the air Impacts of the biodiversity crisis far exceed our ability to monitor changes in terrestrial ecosystems. Environmental DNA has revolutionized aquatic biomonitoring, permitting remote population and diversity assessments. Here we demonstrate that DNA from terrestrial animals can now be collected from the air under natural conditions, a ground-breaking advance for terrestrial biomonitoring. Using air samples from a zoological park, where species are spatially confined and unique compared to native fauna, we show that DNA in air can be used to identify the captive species and their potential interactions with local taxa. Air samples contained DNA from 25 species of mammal and bird including 17 known (and distinct) terrestrial zoo species. We also identified food items from air sampled in enclosures and detected four taxa native to the local area, including the Eurasian hedgehog, endangered in the UK, and the muntjac deer, a locally established invasive species. Our data provide evidence that airDNA is concentrated around recently inhabited areas (e.g., indoor enclosures) but that there is dispersal away from the source suggesting an ecology to airDNA movement which highlights the potential for airDNA sampling at distance. Our data clearly demonstrate the profound potential of air as a source of DNA for global terrestrial biomonitoring and ecological analysis.

Significance StatementThe global decline in biodiversity requires rapid non-invasive biomonitoring tools applicable at a global scale. In this study we collect environmental DNA from mammals and birds from air samples collected in a natural setting. Using only air, we identified 25 species of mammal and bird known to be in the area. Our dataset detected species at risk of local extinction and several confirmed predator-prey interactions. This approach will revolutionize terrestrial biodiversity surveys.

sentences: Stabilising microbial communities by looped mass transfer Creating structurally and functionally stable microbiomes would be greatly beneficial to biotechnology and human health but so far has proven challenging. Here, we propose a looped mass transfer design that keeps microbiomes constant over long periods of time. The effluent of five parallel reactors that began with the same inoculum, was mixed in a reactor that represented a regional pool. Part of this pool was transferred back to the five reactors. Community dynamics were monitored and visualized by quantitative microbial flow cytometry and selected taxonomic sequencing of whole communities and sorted subcommunities. The rescue effect, known from metacommunity theory, was the main stabilizing mechanism that led to the survival of subcommunities with zero netgrowth, especially at high mass transfer rates. The looped mass transfer approach promises to overcome notorious stochastic structural fluctuations in bioreactors and has the potential to design and stabilize communities that can perform desired functions.

sentences: Population modelling insights of extinct environments: the case of the Kem Kem palaeocommunity The Kem Kem beds are well-known palaeontological deposits. Among the species that lived there, there are some large theropods, such as Deltadromeus agilis, Carcharodontosaurus saharicus, and Spinosaurus aegyptiacus. It is possible that these large predators were facultative scavengers, and they could compete for carrion. In the present paper, I simulate a small community module of this environment, consisting of Carrion, Fishes, Spinosaurus, and a functional group composed of large terrestrial Theropods. I assume that these top predators feed on carrion, but they also have exclusive food sources. I show that these exclusive food sources could have assured the possibility of coexistence, and in their absence, one top predator could be locally extinct.

sentences: LiquidCNA: tracking subclonal evolution from longitudinal liquid biopsies using somatic copy number alterations Cell-free DNA (cfDNA) measured via liquid biopsies provides a way for minimally-invasive monitoring of tumour evolutionary dynamics during therapy. Here we present liquidCNA, a method to track subclonal evolution from longitudinally collected cfDNA samples based on somatic copy number alterations (SCNAs). LiquidCNA utilises SCNA profiles derived through cost-effective low-pass whole genome sequencing to automatically and simultaneously genotype and quantify the size of the dominant subclone without requiring prior knowledge of the genetic identity of the emerging clone. We demonstrate the accuracy of liquidCNA in synthetically generated sample sets and in vitro and in silico mixtures of cancer cell lines. Application in vivo in patients with metastatic lung cancer reveals the progressive emergence of a novel tumour sub-population. LiquidCNA is straightforward to use, computationally inexpensive and enables continuous monitoring of subclonal evolution to understand and control therapy-induced resistance.

sentences: Scalable Clustering with Supervised Linkage Methods Data clustering plays a significant role in biomedical sciences, particularly in single-cell data analysis. Researchers use clustering algorithms to group individual cells into populations that can be evaluated across different levels of disease progression, drug response, and other clinical statuses. In many cases, multiple sets of clusters must be generated to assess varying levels of cluster specificity. For example, there are many subtypes of leukocytes (e.g. T cells), whose individual preponderance and phenotype must be assessed for statistical/functional significance. In this report, we introduce a novel hierarchical density clustering algorithm (HAL-x) that uses supervised linkage methods to build a cluster hierarchy on raw single-cell data. With this new approach, HAL-x can quickly predict multiple sets of labels for immense datasets, achieving a considerable improvement in computational efficiency on large datasets compared to existing methods. We also show that cell clusters generated by HAL-x yield near-perfect F1-scores when classifying different clinical statuses based on single-cell profiles. Our hierarchical density clustering algorithm achieves high accuracy in single cell classification in a scalable, tunable and rapid manner. We make HAL-x publicly available at: https://pypi.org/project/hal-x/

sentences: Conditional Freezing, Flight and Darting? Fear conditioning is one of the most frequently used laboratory procedures for modeling learning and memory generally, and anxiety disorders in particular. The conditional response (CR) used in the majority of fear conditioning studies in rodents is freezing. Recently, it has been reported that under certain conditions, running, jumping or darting replaces freezing as the dominant CR. These findings raise both a critical methodological problem and an important theoretical issue. If only freezing is measured but rodents express their learning with a different response, then significant instances of learning, memory, or fear may be missed. In terms of theory, whatever conditions lead to these different behaviors may be a key to how animals transition between different defensive responses and different emotional states. We replicated these past results but along with several novel control conditions. Contrary to the prior conclusions, running and darting were entirely a result of nonassociative processes and were actually suppressed by associative learning. Darting and flight were taken to be analogous to nonassociative startle or alpha responses that are potentiated by fear. On the other hand, freezing was the purest reflection of associative learning. We also uncovered a rule that describes when these movements replace freezing: When afraid, freeze until there is a sudden novel change in stimulation, then burst into vigorous flight attempts. This rule may also govern the change from fear to panic.

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24-Nor-Ursodeoxycholic acid reshapes immunometabolism in CD8+ T cells and alleviates hepatic inflammation Background & Aims24-NorUrsodeoxycholic acid (NorUDCA) is novel therapy for immune-mediated liver diseases such as primary sclerosing cholangitis (PSC) where dysregulated T cells including CD8+ T cells cause liver immunopathology. We hypothesized that NorUDCA may directly modulate CD8+ T cell effector function thus contributing to its therapeutic efficacy independent of anti-cholestatic effects.

MethodsNorUDCA effects on CD8+ T cell function in vivo were investigated in a hepatic injury model system induced by excessive CD8+ T cell immune response upon non-cytolytic lymphocytic choriomeningitis virus (LCMV) infection. Mechanistic studies included molecular and biochemical approaches, flow cytometry and metabolic assays in mouse CD8+ T cells in vitro. Mass spectrometry (MS) was used to identify potential targets modulated by NorUDCA in CD8+ T cells. NorUDCA signaling effects observed in murine systems were validated in peripheral T cells from healthy volunteers and PSC patients.

ResultsIn vivo NorUDCA ameliorated hepatic injury and systemic inflammation upon LCMV infection. Mechanistically, NorUDCA demonstrated a strong immunomodulatory efficacy in CD8+ T cells affecting lymphoblastogenesis, mTORC1 signaling and glycolysis of CD8+ T cells. With MS, we identified that NorUDCA regulates CD8+ T cells via targeting mTORC1. NorUDCAs impact on mTORC1 signaling was further confirmed in circulating human CD8+ T cells.

ConclusionsNorUDCA possesses a yet-unrecognized direct modulatory potency on CD8+ T cells and attenuates excessive CD8+ T cell hepatic immunopathology. These findings may be relevant for treatment of immune-mediated liver diseases such as PSC and beyond.

sentences: Spatiotemporal analysis of glioma heterogeneity reveals Col1A1 as an actionable 1 target to disrupt tumor mesenchymal differentiation, invasion and malignancy Intra-tumoral heterogeneity and diffuse infiltration are hallmarks of glioblastoma that challenge treatment efficacy. However, the mechanisms that set up both tumor heterogeneity and invasion remain poorly understood. Herein, we present a comprehensive spatiotemporal study that aligns distinctive intra-tumoral histopathological structures, oncostreams, with dynamic properties and a unique, actionable, spatial transcriptomic signature. Oncostreams are dynamic multicellular fascicles of spindle-like and aligned cells with mesenchymal properties, detected using ex vivo explants and in vivo intravital imaging. Their density correlates with tumor aggressiveness in genetically engineered mouse glioma models, and high-grade human gliomas. Oncostreams facilitate the intra-tumoral distribution of tumoral and non-tumoral cells, and the invasion of the normal brain. These fascicles are defined by a specific molecular signature that regulates their organization and function. Oncostreams structure and function depend on overexpression of COL1A1. COL1A1 is a central gene in the dynamic organization of glioma mesenchymal transformation, and a powerful regulator of glioma malignant behavior. Inhibition of COL1A1 eliminated oncostreams, reprogramed the malignant histopathological phenotype, reduced expression of the mesenchymal associated genes, induced changes in the tumor microenvironment and prolonged animal survival. Oncostreams represent a novel pathological marker of potential value for diagnosis, prognosis, and treatment.

sentences: Hearing restoration by a low-weight power-efficient multichannel optogenetic cochlear implant system In case of deafness, electrical cochlear implants (eCIs) bypass dysfunctional or lost hair cells by direct stimulation of the auditory nerve. However, spectral selectivity of eCI sound coding is low as the wide current spread from each electrode activates large sets of neurons along the tonotopic axis. As light can be better confined in space, optical cochlear implants (oCIs) promise to overcome this shortcoming of eCIs. This requires appropriate sound processing and control of multiple microscale emitters. Here, we describe the development, characterisation, and application of a preclinical low-weight and wireless LED-based multichannel oCI system for hearing restoration and its companion to its sister eCI system. The head-worn oCI system enabled deafened rats to perform a locomotion task in response to acoustic stimulation proving the concept of multichannel optogenetic hearing restoration in rodents.

sentences: Mechanical bistability of the mesoderm epithelium facilitates mesoderm invagination during Drosophila gastrulation Apical constriction driven by non-muscle myosin II ("myosin") provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of myosin, we find that during Drosophila mesoderm invagination, myosin contractility is critical to prevent tissue relaxation during the early, "priming" stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. The binary response suggests that the mesoderm is mechanically bistable during gastrulation. Combined modeling analysis and experimental measurements suggest that the observed mechanical bistability may arise from apicobasal shrinkage of the surrounding ectoderm, which promotes mesoderm invagination by facilitating a buckling transition. Our results suggest that Drosophila mesoderm invagination requires a joint action of local myosin contractility and mechanical bistability of the epithelium to trigger epithelial buckling.

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Relationship between COVID-19 infection and neurodegeneration: Computational insight into interactions between the SARS-CoV-2 spike protein and the monoamine oxidase enzymes Although COVID-19 has been primarily associated with pneumonia, recent data show that its causative agent, the SARS-CoV-2 coronavirus, can infect many vital organs beyond the lungs, including the heart, kidneys and the brain. The literature agrees that COVID-19 is likely to have long-term mental health effects on infected individuals, which signifies a need to understand the role of the virus in the pathophysiology of brain disorders that is currently unknown and widely debated. Our docking and molecular dynamic simulations show that the affinity of the spike protein from the wild type (WT) and the South African B.1.351 (SA) variant towards the MAO enzymes is comparable to that for its ACE2 receptor. This allows for the WT/SA...MAO complex formation, which changes MAO affinities for their neurotransmitter substrates, thus consequently impacting the rates of their metabolic conversion and misbalancing their levels. Knowing that this fine regulation is strongly linked with the etiology of various brain pathologies, these results are the first to highlight the possibility that the interference with the brain MAO catalytic activity is responsible for the increased neurodegenerative illnesses following a COVID-19 infection, thus placing a neurobiological link between these two conditions in the spotlight. Since the obtained insight suggests that a more contagious SA variant causes even larger disturbances, and with new and more problematic strains likely emerging in the near future, we firmly advise that the presented prospect of the SARS-CoV-2 induced neurological complications should not be ignored, but rather requires further clinical investigations to achieve an early diagnosis and timely therapeutic interventions.

TABLE OF CONTENTS ENTRY

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sentences: Whole-brain in vivo base editing reverses autistic-like behaviors in mice Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder associated with deficits in social communication and stereotypical behaviors. Numerous ASD-related genetic mutations have been identified and genome editing methods have been developed but successful genome editing in the whole-brain scale to alleviate autistic-like behaviors in animal models has not been achieved. Here we report the development of a new CRISPR-mediated cytidine base editor (CBE) system, which converts C{middle dot}G base pairs to T{middle dot}A. We demonstrate the effectiveness of this system by targeting an ASD-associated de novo mutation in the MEF2C gene (c.104T>C, p.L35P). We constructed a Mef2c L35P knock-in mouse and observed that Mef2c L35P heterozygous mice displayed autistic-like behaviors, including deficits in social behaviors and repetitive behaviors. We programmed the CBE to edit the C{middle dot}G base pairs of the mutated Mef2c gene (c.104T>C, p.L35P) to T{middle dot}A base pairs and delivered it via a single dose intravenous injection of blood brain barrier (BBB)-crossing AAV-PHP.eB vector into the mouse brain. This treatment restored MEF2C protein levels and reversed impairments in social interactions and repetitive behaviors in Mef2c L35P heterozygous mice. Together, this work presents an in vivo gene editing strategy in which correcting a single nucleotide mutation in the whole-brain scale could be successfully achieved, further providing a new therapeutic framework for neurodevelopmental disorders.

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recolorize: improved color segmentation of digital images (for people with other things to do) O_LIColor patterns are easy to observe, but difficult to quantify. A necessary first step for most digital color pattern analysis methods is generating color maps, where every pixel in an image is assigned to one of a set of discrete color pattern elements (color segmentation).
C_LIO_LIMost automatic methods for color segmentation rely on color value thresholds or k-means clustering. While these work in specialized cases, these methods often fail due to biological variation in pattern distribution and intensity, technical variation in images, and poor scalability for batch clustering. As a result, color segmentation is the bottleneck step in a wide variety of analyses.
C_LIO_LIHere we present recolorize, an R package for color segmentation that succeeds in many cases where currently used methods fail. Recolorize has three major components: (1) an effective two-part algorithm for color clustering where color distributions are binned and combined according to perceived similarity; (2) a toolkit for making minor manual adjustments to automatic output where needed; and (3) easy export options to a variety of methods that require accurate color maps.
C_LIO_LIThis paper illustrates how to use recolorize, includes examples where we effectively address many of the problems that fail using k-means clustering, and combines the package with two of the most popular R packages for quantifying color pattern variation (patternize and pavo).
C_LI

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Age-Volume Associations in Cerebellar Regions by Sex and Reproductive Stage IntroductionThe cerebellum has established associations with motor function and a well- recognized role in cognition. In advanced age, cognitive and motor impairments contribute to reduced quality of life and are more common. Regional cerebellar volume is associated with performance across these domains and sex hormones may influence this volume. Examining sex differences in regional cerebellar volume in conjunction with age, and in the context of reproductive stage stands to improve our understanding of cerebellar aging and pathology.

MethodsData from 530 healthy adults (ages 18-88; 49% female) from the Cambridge Centre for Ageing and Neuroscience database were used here. CERES was utilized to assess lobular volume in T1-weighted images. We examined sex differences in adjusted regional cerebellar volume while controlling for age. A subgroup (n = 354, 50% female) was used to assess group differences in female reproductive stages as compared to age-matched males.

ResultsSex differences in adjusted volume were seen across most anterior and posterior cerebellar lobules. The majority of cerebellar regions had significant linear relationships with age in males and females. However, there were no interactions between sex and reproductive stage groups (i.e., female reproductive stage did not display a relationship with regional cerebellar volume).

DiscussionWe found sex differences in volume across much of the cerebellum, linear associations with age, and did not find an effect of female reproductive stage on regional cerebellar volume. Longitudinal investigation into hormonal influences on cerebellar structure and function is warranted as hormonal changes with menopause may impact structure over time.

sentences: NMR spectroscopy of a single mammalian embryo The resolving power, chemical sensitivity and non-invasive nature of NMR has made it an established technique for in vivo studies of large organisms both for research and clinical applications. These features would clearly be beneficial at the nanoliter scale (nL), typical of early development of mammalian embryos, microtissues and organoids, the scale where the building blocks of complex organisms could be observed. However, the handling of such small samples (about 100 micrometers) and sensitivity issues have prevented the widespread adoption of NMR. Recently we have shown how these limitations can be overcome with ultra-compact single-chip probes. In this article we show that such probes have sufficient sensitivity to detect and resolve NMR signals from individual bovine pre-implantation embryos. In less than 1 hour these spherical samples of just 130-190 micrometers produce distinct spectral peaks, largely originating from lipids contained inside them. We further observe how the spectral features, namely the peak intensities, vary from one sample to another despite their optical and morphological similarities.

sentences: Activation of the CaMKII-Sarm1-ASK1 MAP kinase pathway protects against axon degeneration caused by loss of mitochondria Mitochondrial defects are tightly linked to axon degeneration, yet the underlying cellular mechanisms remain poorly understood. In C. elegans, PVQ axons that lack mitochondria degenerate spontaneously with age. Using an unbiased genetic screen, we found that cell-specific activation of CaMKII/UNC-43 suppresses axon degeneration due to loss of mitochondria. Unexpectedly, CaMKII/UNC-43 protects against degeneration through the conserved Sarm1/TIR-1-ASK1/NSY-1 MAPK pathway. In addition, we show that disrupting a trafficking complex composed of calsyntenin/CASY-1, Mint/LIN-10, and kinesin suppresses axon degeneration. Further analysis indicates that disruption of this trafficking complex activates the CaMKII-Sarm1-MAPK pathway through L-type voltage-gated calcium channels. Our findings identify CaMKII as a pivot point between mitochondrial defects and axon degeneration, describe how it is regulated in this context, and uncover a surprising neuroprotective role for the Sarm1-ASK1 pathway.

sentences: Sex-biased response to and brain cell infection by SARS-CoV-2 in a highly susceptible human ACE2 transgenic model The COVID-19 pandemic is caused by SARS-CoV-2 infection. Human angiotensin-converting enzyme II (hACE2) has been identified as the receptor enabling SARS-CoV-2 host entry. To establish a mouse model for COVID-19, we generated transgenic mouse lines using the (HS4)2-pCAG-hACE2-HA-(HS4)2 transgene cassette, which expresses HA-tagged hACE2 under control of the CAG promoter and is flanked by HS4 insulators. Expression levels of the hACE2 transgene are respectively higher in lung, brain and kidney of our CAG-hACE2 transgenic mice and relatively lower in duodenum, heart and liver. The CAG-hACE2 mice are highly susceptibility to SARS-CoV-2 infection, with 100 PFU of SARS-CoV-2 being sufficient to induce 87.5% mortality at 9 days post-infection and resulting in a sole (female) survivor. Mortality was 100% at the higher titer of 1000 PFU. At lower viral titers, we also found that female mice exposed to SARS-CoV-2 infection suffered much less weight loss than male mice, implying sex-biased responses to SARS-CoV-2 infection. We subjected neuronal cultures to SARS-CoV-2 pseudovirus infection to ascertain the susceptibilities of neurons and astrocytes. Moreover, we observed that expression of SARS-CoV-2 Spike protein alters the synaptic responses of cultured neurons. Our transgenic mice may serve as a model for severe COVID-19 and sex-biased responses to SARS-CoV-2 infection, aiding in the development of vaccines and therapeutic treatments for this disease.

sentences: Sparse balance: excitatory-inhibitory networks with small bias currents and broadly distributed synaptic weights Cortical circuits generate excitatory currents that must be cancelled by strong inhibition to assure stability. The resulting excitatory-inhibitory (E-I) balance can generate spontaneous irregular activity but, in standard balanced E-I models, this requires that an extremely strong feedforward bias current be included along with the recurrent excitation and inhibition. The absence of experimental evidence for such large bias currents inspired us to examine an alternative regime that exhibits asynchronous activity without requiring unrealistically large feedforward input. In these networks, irregular spontaneous activity is supported by a continually changing sparse set of neurons. To support this activity, synaptic strengths must be drawn from high-variance distributions. Unlike standard balanced networks, these sparse balance networks exhibit robust nonlinear responses to uniform inputs and non-Gaussian statistics. In addition to simulations, we present a mean-field analysis to illustrate the properties of these networks.

sentences: Computational Design and Analysis of Modular Cells for Large Libraries of Exchangeable Product Synthesis Modules Microbial metabolism can be harnessed to produce a large library of useful chemicals from renewable resources such as plant biomass. However, it is laborious and expensive to create microbial biocatalysts to produce each new product. To tackle this challenge, we have recently developed modular cell (ModCell) design principles that enable rapid generation of production strains by assembling a modular (chassis) cell with exchangeable production modules to achieve overproduction of target molecules. Previous computational ModCell design methods are limited to analyze small libraries of around 20 products. In this study, we developed a new computational method, named ModCell-HPC, capable of designing modular cells for large libraries with hundredths of products with a highly-parallel and multi-objective evolutionary algorithm. We demonstrated ModCell-HPC to design Escherichia coli modular cells towards a library of 161 endogenous production modules. From these simulations, we identified E. coli modular cells with few genetic manipulations that can produce dozens of molecules in a growth-coupled manner under different carbons sources. These designs revealed key genetic manipulations at the chassis and module levels to accomplish versatile modular cells. Furthermore, we used ModCell-HPC to identify design features that allow an existing modular cell to be re-purposed towards production of new molecules. Overall, ModCell-HPC is a useful tool towards more efficient and generalizable design of modular cells to help reduce research and development cost in biocatalysis.

sentences: RNA X-ray footprinting reveals the consequences of an in vivo acquired determinant of viral infectivity The secondary structures of the bacteriophage MS2 ssRNA genome, frozen in defined states, were determined with minimal perturbation using constraints from X-ray synchrotron footprinting (XRF). The footprints of the gRNA in the virion and as transcript are consistent with single, dominant but distinct conformations, and reveal the presence of multiple Packaging Signals potentially involved in assembly regulation that have not been detected by other techniques. XRF also reveals the dramatic effect of the unique Maturation Protein (MP) on both the capsid lattice, and the gRNA conformation inside the phage compared with a virus-like-particle composed only of coat protein subunits. Aspects of genome organisation in the phage, their impacts on the capsid shell, and the distortion of lattice geometry by MP, are hallmarks of molecular frustration. Phage assembly therefore appears to prepare the particle for the next step of the infectious cycle.

sentences: Improving GWAS discovery and genomic prediction accuracy in Biobank data Genetically informed and deep-phenotyped biobanks are an important research resource. The cost of phenotyping far outstrips that of genotyping, and therefore it is imperative that the most powerful, versatile and efficient analysis approaches are used. Here, we apply our recently developed Bayesian grouped mixture of regressions model (GMRM) in the UK and Estonian Biobanks and obtain the highest genomic prediction accuracy reported to date across 21 heritable traits. On average, GMRM accuracies were 15% (SE 7%) greater than prediction models run in the LDAK software with SNP annotation marker groups, 18% (SE 3%) greater than a baseline BayesR model without SNP markers grouped into MAF-LD-annotation categories, and 106% (SE 9%) greater than polygenic risk scores calculated from mixed-linear model association (MLMA) estimates. For height, the prediction accuracy R2 was 47% in a UK Biobank hold-out sample, which was 76% of the estimated [Formula]. We then extend our GMRM prediction model to provide MLMA SNP marker estimates for GWAS discovery, which increased the independent loci detected to 7,910 in unrelated UK Biobank individuals, as compared to 5,521 from BoltLMM and 5,727 from Regenie, a 43% and 38% increase respectively. The average{chi} 2 value of the leading markers was 34% (SE 5.11) higher for GMRM as compared to Regenie, and increased by 17% for every 1% increase in prediction accuracy gained over a baseline BayesR model across the traits. Thus, we show that modelling genetic associations accounting for MAF and LD differences among SNP markers, and incorporating prior knowledge of genomic function, is important for both genomic prediction and for discovery in large-scale individual-level biobank-scale studies.

sentences: Reproducible acquisition, management, and meta-analysis of nucleotide sequence (meta)data using q2-fondue The volume of public nucleotide sequence data has blossomed over the past two decades, enabling novel discoveries via re-analysis, meta-analyses, and comparative studies for uncovering general biological trends. However, reproducible re-use and management of sequence datasets remains a challenge. We created the software plugin q2-fondue to enable user-friendly acquisition, re-use, and management of public nucleotide sequence (meta)data while adhering to open data principles. The software allows fully provenance-tracked programmatic access to and management of data from the Sequence Read Archive (SRA). Sequence data and accompanying metadata retrieved with q2-fondue follow a validated format, which is interoperable with the QIIME 2 ecosystem and its multiple user interfaces. To highlight the manifold capabilities of q2-fondue, we present several demonstration analyses using amplicon, whole genome, and shotgun metagenome datasets. These use cases demonstrate how q2-fondue increases analysis reproducibility and transparency from data download to final visualizations by including source details in the integrated provenance graph. We believe q2-fondue will lower existing barriers to comparative analyses of nucleotide sequence data, enabling more transparent, open, and reproducible conduct of meta-analyses. q2-fondue is a Python 3 package released under the BSD 3-clause license at https://github.com/bokulich-lab/q2-fondue.

sentences: M2e-specific monoclonal antibody cocktails against influenza A virus are superior to individual monoclonal antibody treatments, universally effective, and viral escape mutant resistant Influenza virus has pandemic potential, seasonal epidemics burden the human population, and viral resistance has developed to all available treatment options. A universally effective, escape mutant resistant therapeutic agent is desperately needed. We previously described extensive analyses of seven Matrix Protein 2 ectodomain-specific monoclonal antibodies (M2e-mAbs) as the basis for an effective and universal treatment for influenza A virus (IAV) infection. On the strength of these findings, we developed an effective prophylactic cocktail agent using three M2e-mAbs distinct in their M2e epitopes. This cocktail protected mice challenged with laboratory and pandemic IAV strains at a low dose significantly better than single M2e-mAb treatments. Notably, no viral escape mutants developed in immunocompetent and immunodeficient mice after viral passaging in the presence of single, cocktail, or alternating M2e-mAb treatments. Our study reveals the superiority of M2e-mAb cocktails, their universal effectiveness, and resistance to viral escape. It will critically shape future influenza-therapeutic development.

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Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host-factors having implication in the disease pathogenesis and severity Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology that resembles human COVID-19 patients. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples shows a similar type of pathophysiological manifestation of SARS-CoV-2 infection as reported earlier in COVID-19 patients and hamsters infected with other isolates. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14dpi. Here, we carried out quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters at day 4 and day 14 post infection. This resulted in the identification of 1,585 differentially expressed proteins of which 68 proteins were significantly altered among both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in the identification of candidate biomarkers and understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis.

Graphical abstract

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Did Caenorhabditis nematodes recycle transposons to fight pathogens? Animal genomes consist largely of sequences derived from transposons which were previously considered as junk DNA and active transposons can even be deleterious for organismal fitness. Nevertheless, the huge pool of transposon-derived sequences also forms the raw material for molecular innovations. Here, we follow up on the incidental finding of a transposon-derived DNA binding domain in a subset of F-box genes in Caenorhabditis elegans. Based on phylogenetic analysis, we show that a single gene fusion followed by individual losses explains most members of this novel gene family. Phylogenomic data of available Caenorhabditis genomes allowed us to trace this fusion event to the ancestor of the Elegans group. Additional homology searches suggest endogenous Mariner transposons as the likely source of the coopted sequence. Further bioinformatic characterization of different F-box families by Gene Ontology analysis, gene module comparisons, and literature research identified first evidence that some F-box genes might be involved in innate immunity, as it had been proposed previously based on adaptive signatures of molecular evolution. Specifically, the F-box gene fbxa-158 contains one of the transposon-derived domains and was shown to interact with the components of the intracellular pathogen response machinery targeting Microsporidia and viruses. Thus, cooption of transposon-derived sequences likely contributed to the adaptive evolution of the F-box superfamily in Caenorhabditis nematodes.

Significance statementWhen considering transposons as genomic junk or selfish elements that are detrimental for organismal fitness, we often neglect the potential of transposon-derived sequences as a source of molecular innovation. Here, we characterize a case where a transposon-derived sequence has been coopted by one of the largest and fastest evolving gene superfamilies in Caenorhabditis nematodes which include the model organism C. elegans. The resulting chimeric gene family has been stably maintained for about 20 million years and bioinformatic analysis reveals first evidence for potential functions in innate immunity. Thus, strong evolutionary pressure might have forced Caenorhabditis nematodes to recycle transposons in order to fight pathogens.

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Effectiveness of Joint Species Distribution Models in the Presence of Imperfect Detection O_LIJoint species distribution models (JSDMs) are a recent development in biogeography and enable the spatial modelling of multiple species and their interactions and dependencies. However, most models do not consider imperfect detection, which can significantly bias estimates. This is one of the first papers to account for imperfect detection when fitting data with JSDMs and to explore the complications that may arise.
C_LIO_LIA multivariate probit JSDM that explicitly accounts for imperfect detection is proposed, and implemented using a Bayesian hierarchical approach. We investigate the performance of the JSDM in the presence of imperfect detection for a range of factors, including varied levels of detection and species occupancy, and varied numbers of survey sites and replications. To understand how effective this JSDM is in practice, we also compare results to those from a JSDM that does not explicitly model detection but instead makes use of "collapsed data". A case study of owls and gliders in Victoria Australia is also illustrated.
C_LIO_LIUsing simulations, we found that the JSDMs explicitly accounting for detection can accurately estimate intrinsic correlation between species with enough survey sites and replications. Reducing the number of survey sites decreases the precision of estimates, while reducing the number of survey replications can lead to biased estimates. For low probabilities of detection, the model may require a large number of survey replications to remove bias from estimates. However, JSDMs not explicitly accounting for detection may have a limited ability to dis-entangle detection from occupancy, which substantially reduces their ability to accurately infer the species distribution spatially. Our case study showed positive correlation between Sooty Owls and Greater Gliders, despite a low number of survey replications.
C_LIO_LITo avoid biased estimates of inter-species correlations and species distributions, imperfect detection needs to be considered. However, for low probability of detection, the JSDMs explicitly accounting for detection is data hungry. Estimates from such models may still be subject to bias. To overcome the bias, researchers need to carefully design surveys and choose appropriate modelling approaches. The survey design should ensure sufficient survey replications for unbiased inferences on species inter-dependencies and occupancy.
C_LI

sentences: Blood-brain barrier permeability to enrofloxacin in Pengze crucian carp (Carassius auratus var. Pengze) To explore the permeability of blood-brain barrier (BBB) to enrofloxacin (ENR) and the role of brain injury markers in the evaluation of brain damage, crucian carp (Carassius auratus var. Pengze) was employed in this work as the research object treated by a single oral administration of the antibiotic. Results showed that ENR residues could be detected in brain of crucian carp exposed to both half lethal dose (LD50) and safe dose (SD) of ENR throughout the trial period, indicating that ENR had an ability of permeating the BBB of crucian carp. Evans blue (EB) dispersively distributed in brain tissue of fish exposed to SD ENR, but not in the vehicle-treated fish brain, which suggested structural failure of BBB by ENR. Expression levels of BBB-related molecules, occludin and P-glycoprotein (P-gp), were significantly down-regulated by ENR. Brain injury markers, NSE, S100B and GFAP were improved that were demonstrated by elevated contents of proteins in brain and serum. According to the data of correlation analysis, it was observed that ENR residue was closely related to S100B and GFAP, indicating that S100B and GFAP could be used as evaluation indicators for brain injury of crucian carp. All findings mentioned above suggest that ENR may cause structural change of BBB, resulting in biological brain damage in crucian carp.

sentences: Pleiotropic Variability Score: A Genome Interpretation Metric to Quantify Phenomic Associations of Genomic Variants A more complete understanding of phenomic space is critical for elucidating genome-phenome relationships and for assessing disease risk from genome sequencing. To incorporate knowledge of how related a variants associations are, we developed a new genome interpretation metric called Pleiotropic Variability Score (PVS). PVS uses semantic reasoning to score the relatedness of a genetic variants associated phenotypes based on those phenotypes relationships in the human phenotype ontology (HPO) and disease ontology (DO). We tested 78 unique semantic similarity methods and integrated six robust metrics to define the pleiotropy score of SNPs. We computed PVS for 12,541 SNPs which were mapped to 382 HPO and 317 DO unique phenotype terms in a genotype-phenotype catalog (10,021 SNPs mapped to DO phenotypes and 8,569 SNPs mapped to HPO phenotypes). We validated the utility of PVS by computing pleiotropy using an electronic health record linked genomic database (BioME, n=11,210). Further we demonstrate the application of PVS in personalized medicine using "personalized pleiotropy score" reports for individuals with genomic data that could potentially aid in variant interpretation. We further developed a software framework to incorporate PVS into VCF files and to consolidate pleiotropy assessment as part of genome interpretation pipelines. As the genome-phenome catalogs are growing, PVS will be a useful metric to assess genetic variation to find SNPs with highly pleiotropic effects. Additionally, variants with varying degree of pleiotropy can be prioritized for explorative studies to understand specific roles of SNPs and pleiotropic hubs in mediating novel phenotypes and drug development.

sentences: Effects of fine-scale population structure on the distribution of heterozygosity in a long-term study of (Antirrhinum majus) Many studies have quantified the distribution of heterozygosity and relatedness in natural populations, but surprisingly few have examined the demographic processes driving these patterns. In this study we take a novel approach by studying how population structure affects both pairwise identity and the distribution of heterozygosity in a natural population of a self-incompatible plant Antirrhinum majus. We look at a measure of the variance in heterozygosity within a population, identity disequilibrium (g2), together with FST using a panel of 91 SNPs in 22,353 individuals collected over 11 years. We find that pairwise relatedness (FST) declines rapidly over short spatial scales, and the excess variance in heterozygosity between individuals (g2) reflects significant variation in inbreeding. Additionally, we detect an excess of individuals with around half the average heterozygosity, indicating that some are due to selfing or matings between close relatives. We use two types of simulation to ask whether variation in heterozygosity is consistent with fine-scale spatial population structure. First, by simulating offspring using parents drawn from a range of spatial scales, we show that the known pollen dispersal kernel explains g2. Second, we simulate a 1000-generation pedigree using the known dispersal and spatial distribution and find that the resulting g2 is consistent with that observed from the field data. In contrast, a simulated population with uniform density underestimates g2, indicating that heterogeneous density promotes identity disequilibrium. Our study shows that heterogeneous density and leptokurtic dispersal can together explain the distribution of heterozygosity. Furthermore, our study highlights the limitations of making theoretical predictions from simulations that only assume simple density and dispersal distributions.

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Species-specific chromatin landscape determines how transposable elements shape genome evolution Transposable elements (TEs) are selfish genomic parasites that increase their copy number at the expense of host fitness. The "success," or genome-wide abundance, of TEs differs widely between species. Deciphering the causes for this large variety in TE abundance has remained a central question in evolutionary genomics. We previously proposed that species-specific TE abundance could be driven by the inadvertent consequences of host-direct epigenetic silencing of TEs--the spreading of repressive epigenetic marks from silenced TEs into adjacent sequences. Here, we compared this TE-mediated "epigenetic effect" in six species in the Drosophila melanogaster subgroup to dissect step-by-step the role of such effect in determining genomic TE abundance.

We found that TE-mediated spreading of repressive marks is prevalent and substantially varies across and even within species. While this TE-mediated effect alters the epigenetic states of adjacent genes, we surprisingly discovered that the transcription of neighboring genes could reciprocally impact this spreading. Importantly, our multi- species analysis provides the power and appropriate phylogenetic resolution to connect species-specific host chromatin regulation, TE-mediated epigenetic effects, the strength of natural selection against TEs, and genomic TE abundance unique to individual species. Our findings point towards the importance of host chromatin landscapes in shaping genome evolution through the epigenetic effects of a selfish genetic parasite.

sentences: Whole genome analysis sheds light on the genetic origin of Huns, Avars and conquering Hungarians Huns, Avars and conquering Hungarians were Migration Period nomadic groups which arrived in three successive waves in the Carpathian Basin between the 5th and 9th centuries. Based on historical data each of these groups are thought to have arrived from Asia, although their exact origin and relation to other ancient and modern populations has been debated. In this study we have sequenced 9 Hun, 143 Avar and 113 Hungarian conquest period samples, and identified three core populations, representing immigrants from each period, with no recent European ancestry. Our results suggest that this "immigrant core" of both Huns and Avars originated in present day Mongolia, and their origin can be traced back to Xiongnus. On the other hand, the "immigrant core" of the conquering Hungarians derived from an earlier admixture of Mansis, early Sarmatians and descendants of late Xiongnus. In addition, we detected shared Hun-related ancestry in numerous Avar and Hungarian conquest period genetic outliers indicating a genetic link between these successive nomadic groups. Aside from the immigrant core groups we identified that the majority of the individuals from each period were local residents, harboring "native European" ancestry.

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A novel approach to develop wheat chromosome-specific KASP markers for detecting Amblyopyrum muticum segments in doubled haploid introgression lines Many wild relative species are being used in pre-breeding programmes to increase the genetic diversity of wheat. Genotyping tools such as single nucleotide polymorphism (SNP)-based arrays and molecular markers have been widely used to characterise wheat-wild relative introgression lines. However, due to the polyploid nature of the recipient wheat genome, it is difficult to develop SNP-based KASP markers that are codominant to track the introgressions from the wild species. Previous attempts to develop KASP markers have involved both exome- and PCR-amplicon-based sequencing of the wild species. But chromosome-specific KASPs assays have been hindered by homoeologous SNPs within the wheat genome. This study involved whole genome sequencing of the diploid wheat wild relative Amblyopyrum muticum and development of a SNP discovery pipeline that generated [~]38,000 SNPs in single-copy wheat genome sequences. New assays were designed to increase the density of Am. muticum polymorphic KASP markers. With a goal of one marker per 60 Mbp, 335 new KASP assays were validated as functional. Together with assays validated in previous studies, 498 well distributed chromosome-specific markers were used to recharacterize previously genotyped wheat-Am. muticum doubled haploid (DH) introgression lines. The chromosome specific nature of the KASP markers allowed clarification of which wheat chromosomes were involved with recombination events or substituted with Am. muticum chromosomes and the higher density of markers allowed detection of new small introgressions in these DH lines.

Key MessageA novel methodology to generate chromosome-specific SNPs between wheat and its wild relative Amblyopyrum muticum and their use in the development of KASP markers to genotype wheat-Am. muticum introgression lines.

sentences: Alpha and beta rhythms differentially support the effect of symbols on visual object recognition Hearing spoken words can enhance visual object recognition, detection and discrimination. Yet, the mechanisms that underpin this facilitation are incompletely understood. On one account, words do not bias early visual processing, but rather affect later semantic or decision-making stages. However, recent proposals suggest that words can alter early visual processes by activating category-specific priors in sensory regions. A prediction of this account is that top-down priors evoke changes in occipital areas in anticipation of visual stimuli. Here, we tested the hypothesis that neural oscillations serve as a mechanism to activate language-generated visual priors. Participants performed a cue-picture matching task where cues were either spoken words, in their native or second language, or natural sounds, while their EEG and reaction times were recorded. Behaviorally, we replicated the previously reported label-advantage effect, with images cued by words being recognized faster than those cued by natural sounds. A time-frequency analysis of cue-target intervals revealed that this behavioral label-advantage was associated with enhanced power in posterior alpha (9-11 Hz) and beta oscillations (17-19 Hz), both of which were larger when the image was preceded by a word compared to a natural sound. Importantly, object recognition performance improved with high alpha power but slowed down with enhancement of beta synchronization. These results suggest that alpha and beta rhythms play distinct functional roles to support language-mediated visual object recognition: alpha might function to amplify sensory priors in posterior regions, while beta may (re)activate the network states elicited by the auditory cue.

sentences: Genetic Structure and Diversity of Banana Bunchy Top Virus (BBTV) in the Philippines Banana bunchy top virus (BBTV) is an important disease of banana in the Philippines and in other banana-producing countries. This study was conducted to investigate the genetic structure and diversity of Philippine BBTV isolates which remain unexplored in the country. BBTV-infected plant tissues were sampled from banana-growing provinces (i.e., Cagayan, Isabela, Quirino, Batangas, Laguna, Rizal, Quezon, Palawan, Cebu, Leyte, and Davao del Sur) and the partial DNA-R gene of BBTV was sequenced. Analysis of all local BBTV isolates showed a nucleotide diversity ({pi}) of 0.00721, average number of nucleotide differences (k) of 5.51984, and haplotype diversity (hd) of 0.971. Neutrality tests using Fus Fs and Tajimas D showed significant and highly negative values which suggest an excess number of rare alleles due to recent population expansion or from genetic hitchhiking. Haplotype network and phylogenetic analyses revealed that the local BBTV isolates were closely related to Southeast Asian (SEA) group and exhibited a monophyletic clade with distinct haplotype grouping from other SEA sequences. However, some Indonesian and Indian reference sequences were also clustered within the Philippine BBTV group suggesting sequence homology. Results also showed that the local BBTV isolates may be categorized into three major haplotype groups (HA, HB, and HC) but only the HC group remained distinct upon comparison with other Philippine and SEA reference sequences. BBTV isolates from Quezon were the most diverse while isolates from Palawan displayed low genetic diversity indices and belonged only in the HC group. The assessment of the degree of variability among Philippine BBTV isolates will provide a reference towards the development of high-throughput BBTV detection systems as well as enable to devise plant breeding strategies to manage the current BBTV spread and variations.

sentences: Modulating the activities of chloroplasts and mitochondria promotes ATP production and plant growth Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow. A higher rate of ATP and NADPH production from the linear electron flow, a higher capacity of carbon fixation by the Calvin-Benson-Bassham cycle and a greater consumption of NADH in mitochondria, enhance photosynthesis in the chloroplasts, ATP production in the mitochondria, sucrose synthesis in the cytosol, and eventually boosting plant growth and seed yields in the overexpression lines.

sentences: Protein yield is tunable by synonymous codon changes of translation initiation sites Recombinant protein production is a key process in generating proteins of interest in the pharmaceutical industry and biomedical research. However, about 50% of recombinant proteins fail to be expressed in a variety of host cells. To address this problem, we modified up to the first nine codons of messenger RNAs with synonymous substitutions and showed that protein levels can be tuned. These modifications alter the  accessibility of translation initiation sites. We also reveal the dynamics between accessibility, gene expression, and turnovers using a coarse-grained simulation.

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T cells modulate the microglial response to brain ischemia Neuroinflammation after stroke is characterized by the activation of resident microglia and the invasion of circulating leukocytes into the brain. Although lymphocytes infiltrate the brain in small number, they have been consistently demonstrated to be the most potent leukocyte subpopulation contributing to secondary inflammatory brain injury. However, the exact mechanism how this minimal number of lymphocytes can profoundly affect stroke outcome is still largely elusive. Here, using a mouse model for ischemic stroke, we demonstrated that early activation of microglia in response to stroke is differentially regulated by distinct T cell subpopulations. Acute treatment with engineered T cells overexpressing IL-10 administered into the cisterna magna after stroke induces a switch of microglial gene expression to a profile associated with pro-regenerative functions. These findings substantiate the role of T cells in stroke with large impact on the cerebral inflammatory milieu by polarizing the microglial phenotype. Targeting T cell-microglia interactions can have direct translational relevance for further development of immune-targeted therapies for stroke and other neuroinflammatory conditions.

SummaryThe crosstalk between brain infiltrating T cells and microglia in response to stroke remains elusive. Benakis et al. report that transcriptional signature of the stroke-associated microglia is reprogrammed by distinct T cell subpopulations. Engineered T cells overexpressing IL-10 administered four hours after stroke reinitiate microglial function inducing a pro-regenerative environment.

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Aggregation controlled by condensate rheology Condensates in living cells can exhibit a complex rheology including viscoelastic and glassy behaviour. This rheological behavior of condensates was suggested to regulate polymerisation of cytoskeletal filaments and aggregation of amyloid fibrils. Here, we theoretically investigate how the rheological properties of condensates can control the formation of linear aggregates. To this end, we propose a kinetic theory for the aggregate size distribution and the exchange of aggregates between inside and outside of condensates. The rheology of condensates is accounted for by aggregate mobilities that depend on aggregate size. We show that condensate rheology can control whether either aggregates of all sizes or dominantly small aggregates are exchanged between condensate inside and outside on the time-scale of aggregation. As a result, the ratio of aggregate numbers inside to outside of condensates differs significantly. Strikingly, we also find that weak variations in the rheological properties of condensates can lead to a switch-like change of the number of aggregates. These results suggest a possible physical mechanism for how living cells could control linear aggregation in a switch-like fashion through variations in condensate rheology.

SIGNIFICANCEThe intracellular space can be organized through phase-separated condensates that often exhibit rheological properties reminiscent of complex fluids. These condensates can affect biochemical processes such as the formation of linear aggregates, in particular biofilaments or amyloids. Here, we propose a theoretical model for how condensate rheology can control the irreversible formation of linear aggregates. A key finding is that size and number of aggregates change in a switch-like fashion upon weak changes in condensate rheology. Our model paves the way to unravel the physiochemical mechanisms of how the rheology of condensates can control aberrant protein aggregation. Such mechanisms may explain how rheological changes, such as aging or the transition to dormancy, give rise to diseases related to protein aggregation.

sentences: Evolution and genetic basis of the plant-penetrating ovipositor, a key adaptation in the transition to herbivory within the Drosophilidae Herbivorous insects are extraordinarily diverse, yet are found in only one-third of insect orders. This skew may result from barriers to plant colonization, coupled with phylogenetic constraint on plant-colonizing adaptations. Physical barriers have been surmounted through the evolution of key morphological innovations, such as the plant-penetrating ovipositor. Despite their significance, the evolution and genetic basis of such innovations have not been well studied. Ovipositors densely lined with hard bristles have evolved repeatedly in herbivorous lineages within the Drosophilidae. Here, we focus on the evolution of this trait in Scaptomyza, an herbivorous radiation nested in a microbe-feeding clade, sister to Hawaiian Drosophila. Our phylogenetic approach revealed that ovipositor bristle number increased as herbivory evolved. We then dissected the genomic architecture of variation in ovipositor bristle number within S. flava through a genome wide association study. Top associated variants were enriched for transcriptional repressors, and the strongest associations included genes contributing to peripheral nervous system development. Genotyping individual flies replicated the association at a variant upstream of Gi, a neural development gene, contributing to a gain of 0.58 bristles/major allele. These results suggest that regulatory variation involving conserved developmental genes contributes to a key morphological adaptation required for plant colonization.

sentences: Metabolic and transcriptomic profiles of glioblastoma invasion revealed by comparisons between patients and corresponding orthotopic xenografts in mice The invasive behavior of glioblastoma, the most aggressive primary brain tumor, is considered highly relevant for tumor recurrence. However, the invasion zone is difficult to visualize by Magnetic Resonance Imaging (MRI) and is protected by the blood brain barrier, posing a particular challenge for treatment. We report biological features of invasive growth accompanying tumor progression and invasion based on associated metabolic and transcriptomic changes observed in patient derived orthotopic xenografts (PDOX) in the mouse and the corresponding patients tumors. The evolution of metabolic changes, followed in vivo longitudinally by 1H-Magnetic Resonance Spectroscopy (MRS) at ultra-high field, reflected growth and the invasive properties of the human glioblastoma transplanted into the brains of mice (PDOX). Comparison of MRS derived metabolite signatures, reflecting temporal changes of tumor development and invasion in PDOX, revealed high similarity to spatial metabolite signatures of combined multi-voxel MRS analyses sampled across different areas of the patients tumors. Pathway analyses of the transcriptome associated with the metabolite profiles of the PDOX, identified molecular signatures of invasion, comprising extracellular matrix degradation and reorganization, growth factor binding, and vascular remodeling. Specific analysis of expression signatures from the invaded mouse brain, revealed extent of invasion dependent induction of immune response, recapitulating respective signatures observed in glioblastoma. Integrating metabolic profiles and gene expression of highly invasive PDOX provided insights into progression and invasion associated mechanisms of extracellular matrix remodeling that is essential for cell-cell communication and regulation of cellular processes. Structural changes and biochemical properties of the extracellular matrix are of importance for the biological behavior of tumors and may be druggable. Ultra-high field MRS reveals to be suitable for in vivo monitoring of progression in the non-enhancing infiltration zone of glioblastoma.

sentences: Transcription elongation through lac repressor-mediated DNA loops Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible. In support of this, RNA polymerase transcribed a looped segment more slowly than an unlooped segment and paused more briefly at LacI obstacles on positively supercoiled templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase appears to facilitate elongation along topologically-complex, protein-coated templates.

sentences: Repulsive Sema3E-Plexin-D1 signaling coordinates both axonal extension and steering via activating an autoregulatory factor, Mtss1 In the developing nervous system, the axons of newly generated neurons extend toward destination targets following an exquisitely designed program. Axon guidance molecules are critical for neuronal pathfinding because they regulate both directionality and growth pace. However, little is known about the molecular mechanism that coordinates proper axonal extension and turning. Here, we show that Metastasis Suppressor 1 (Mtss1), a membrane protrusion protein, was a molecular facilitator ensuring axonal extension while sensitizing axons to Semaphorin 3E (Sema3E)-Plexin-D1 repulsive guidance cue. We demonstrate that Sema3E-Plexin-D1 signaling regulated Mtss1 expression in projecting striatonigral neurons. Mtss1 in turn induced Plexin-D1 localization to the growth cone where it signaled a repulsive cue to Sema3E. Moreover, Mtss1 was important for neurite extension independent of Sema3E. Ablation of Mtss1 expression reduced growth cone collapse and neurite extension in cultured neurons. Mtss1-knockout mice exhibited fewer striatonigral projections and irregular axonal routes, and these defects were recapitulated in Plxnd1-knockout mice. These findings demonstrate that repulsive axon guidance signaling activates an autoregulatory program to coordinate both axonal extension and steering during neuronal pathfinding.

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Nrg1/ErbB Signaling-Mediated Regulation of Fibrosis After Myocardial Infarction RATIONALEAppropriate fibrotic tissue formation after myocardial infarction (MI) is crucial to maintenance of the hearts structure. Reparative or M2-like macrophages play a vital role in post-MI fibrosis by activating cardiac fibroblasts. However, the mechanism by which post-MI cardiac fibrosis is regulated is not fully understood.

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

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

CONCLUSIONSM2-like macrophage-mediated regulation of Nrg1/ErbB signaling, have a substantial effect on fibrotic tissue formation in the infarcted adult mouse heart, is critical for suppressing the progression of senescence and apoptosis of cardiac fibroblasts.

sentences: Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerases (PARPs), blocks in vitro replication of SARS-CoV-2 variants We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wt (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 M, 8.5 M, 24.1 M, 8.2 M and 13.6 M, respectively. A separate experiment focusing on a combination of 10 M stenoparib and 0.5 M remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha (B.1.1.7) variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir.

sentences: Immune escape of SARS-CoV-2 Omicron variant from mRNA vaccination-elicited RBD-specific memory B cells. Memory B cells (MBCs) represent a second layer of immune protection against SARS-CoV-2. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant is of major concern. We used bio-layer interferometry to assess the affinity against the receptor-binding-domain (RBD) of Omicron spike of 313 naturally expressed monoclonal IgG that were previously tested for affinity and neutralization against VOC prior to Omicron. We report here that Omicron evades recognition from a larger fraction of these antibodies than any of the previous VOCs. Additionally, whereas 30% of these antibodies retained high affinity against Omicron-RBD, our analysis suggest that Omicron specifically evades antibodies displaying potent neutralizing activity against the D614G and Beta variant viruses. Further studies are warranted to understand the consequences of a lower memory B cell potency on the overall protection associated with current vaccines.

sentences: Prenatal Alcohol and THC E-Cigarette Exposure Effects on Motor Development It has been well established that prenatal alcohol exposure can lead to a wide range of neurological and behavioral deficits, including alterations in motor domains. However, much less is known about the effects of prenatal cannabis exposure on motor development, despite the fact that cannabis is the most commonly consumed illicit drug among women. Cannabis use among pregnant women has become increasingly popular given the wide-spread perception that consumption is safe during pregnancy. Moreover, alcohol and cannabis are commonly used together, even among pregnant women. Yet, few studies have explored the potential consequences of combined prenatal exposure on behavioral domains. Using our previously established model, pregnant Sprague-Dawley rats were exposed to vaporized alcohol, THC via e-cigarettes, the combination, or a vehicle from gestational days 5-20. Following birth, offspring were tested on early sensorimotor development, adolescent motor coordination, and adolescent activity levels. Prenatal e-cigarette THC exposure delayed sensorimotor development early in life and impaired motor coordination later in adolescence. However, combined prenatal alcohol and THC exposure produced hyperactivity among male offspring. These data suggest that prenatal cannabis exposure may lead to impaired motor skills throughout early development, and that combined exposure with alcohol during gestation may also lead to hyperactivity in adolescence. These findings have important implications for pregnant women and public policy.

sentences: Experimental sound exposure modifies swimming activities and increases food handling error in zebrafish (Danio rerio) Anthropogenic sound is globally increasing and is recognized as a source of environmental pollution in terrestrial and aquatic habitats. Sound is an important sensory stimulus for aquatic organisms and it may cause fluctuations in stress-related physiological indices and in a broader extent induce behavioural effects in a range of marine and freshwater fishes. However sound exposure may also induce changes in swimming activities, feed efficiency and productivity of available food sources in fish. Here, we experimentally tested sound effects on swimming activities and foraging performance in thirty adult Zebrafish (Danio rerio) individually in captivity. We used adult zebrafish and water flea (Daphnia magna) as model predator and prey, respectively. In terms of acoustic stimuli, we used four sound treatments with different temporal patterns (all in the same frequency range and moderate exposure level). Our results constitute strong evidence for sound-related effects on zebrafish behaviour. All sound treatments led to a significant increase in the number of startle response, brief and prolonged swimming speed for zebrafish (P<0.05). We found partially brief and prolonged sound effects on the spatial distribution of zebrafish; Although there were not any significant sound-related changes for horizontal spatial displacement in all treatments (P>0.05), zebrafish swam significantly more in the lower layer of the tank except for irregular intermittent 1:1-7 in brief sound exposure (P<0.05). The results of foraging performance showed that food discrimination error unaffected by sound treatments and was low for the zebrafish (P>0.05). However, food handling error was affected by sound treatments; all treatments induced a significant rise in handling error (P<0.001). This study highlights the impact of sound on zebrafish swimming activities, and that more feeding bouts are needed to consume the same number of food items increasing energy demand under noisy conditions.

sentences: Functional roles of multiple Ton complex genes in a Sphingobium degrader of lignin-derived aromatic compounds TonB-dependent transporters (TBDTs) mediate outer membrane transport of nutrients using the energy derived from proton motive force transmitted from the TonB-ExbB-ExbD complex localized in the inner membrane. Recently, we discovered ddvT encoding a TBDT responsible for the uptake of a 5,5-type lignin-derived dimer in Sphingobium sp. strain SYK-6. Furthermore, overexpression of ddvT in an SYK-6-derivative strain enhanced its uptake capacity, improving the rate of platform chemical production. Thus, understanding the uptake system of lignin-derived aromatics is fundamental for microbial conversion-based lignin valorization. Here we examined whether multiple tonB-, exbB-, and exbD-like genes in SYK-6 contribute to the outer membrane transport of lignin-derived aromatics. The disruption of tonB2-6 and exbB3 did not reduce the capacity of SYK-6 to convert or grow on lignin-derived aromatics. In contrast, the introduction of the tonB1-exbB1-exbD1-exbD2 operon genes into SYK-6, which could not be disrupted, promoted the conversion of {beta}-O-4-, {beta}-5-, {beta}-1-, {beta}-{beta}-, and 5,5-type dimers and monomers, such as ferulate, vanillate, syringate, and protocatechuate. These results suggest that TonB-dependent uptake involving the tonB1 operon genes is responsible for the outer membrane transport of the above aromatics. Additionally, exbB2/tolQ and exbD3/tolR were suggested to constitute the Tol-Pal system that maintains the outer membrane integrity.

sentences: Quantifying the Tissue-Specific Regulatory Information within Enhancer DNA Sequences Recent efforts to measure epigenetic marks across a wide variety of different cell types and tissues provide insights into the cell type-specific regulatory landscape. We use this data to study if there exists a correlate of epigenetic signals in the DNA sequence of enhancers and explore with computational methods to what degree such sequence patterns can be used to predict cell type-specific regulatory activity. By constructing classifiers that predict in which tissues enhancers are active, we are able to identify sequence features that might be recognized by the cell in order to regulate gene expression. While classification performances vary greatly between tissues, we show examples where our classifiers correctly predict tissue specific regulation from sequence alone. We also show that many of the informative patterns indeed harbor transcription factor footprints.

sentences: Effects of dopamine D2 and opioid receptor antagonism on the trade-off between model-based and model-free behavior in healthy volunteers Our daily behaviour requires a flexible arbitration between actions we do out of habit and actions that are directed towards a specific goal. Drugs that target opioid and dopamine receptors are notorious for inducing maladaptive habitual drug consumption, yet how the opioidergic and dopaminergic neurotransmitter systems contribute to the arbitration between habitual and goal-directed behaviour is poorly understood. By combining pharmacological challenges with a well-established decision-making task and a novel computational model, we show that the administration of the D2 dopamine receptor antagonist amisulpride led to an increase in goal-directed or  model-based relative to habitual or  model-free behaviour, whereas the non-selective opioid receptor antagonist naltrexone had no appreciable effect. These findings highlight the distinct functional contributions of dopamine and opioid receptors to goal-directed and habitual behaviour and support the notion that D2 receptor antagonists promote stabilisation of goal-relevant information.

sentences: All-optical electrophysiology with improved genetically encoded voltage indicators reveals interneuron network dynamics in vivo All-optical electrophysiology can be a powerful tool for studying neural dynamics in vivo, as it offers the ability to image and perturb membrane voltage in multiple cells simultaneously. The "Optopatch" constructs combine a red-shifted archaerhodopsin (Arch)-derived genetically encoded voltage indicator (GEVI) with a blue-shifted channelrhodopsin actuator (ChR). We used a video-based pooled screen to evolve Arch-derived GEVIs with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). By combining optogenetic stimulation of individual cells with high-precision voltage imaging in neighboring cells, we mapped inhibitory and gap junction-mediated connections, in vivo. Optogenetic activation of a single NDNF-expressing neuron in visual cortex Layer 1 significantly suppressed the spike rate in some neighboring NDNF interneurons. Hippocampal PV cells showed near-synchronous spikes across multiple cells at a frequency significantly above what one would expect from independent spiking, suggesting that collective inhibitory spikes may play an important signaling role in vivo. By stimulating individual cells and recording from neighbors, we quantified gap junction coupling strengths. Together, these results demonstrate powerful new tools for all-optical microcircuit dissection in live mice.

sentences: Spatially regulated protease activity in lymph nodes renders B cell follicles a sanctuary for retention of intact antigens The structural integrity of vaccine antigens is critical, as antigen degradation in vivo could eliminate neutralizing epitopes and create competing B cell responses against irrelevant breakdown products. Using FRET imaging and imaging zymography, we found that protease activity and antigen breakdown are spatially heterogeneous in lymph nodes. Following protein immunization, antigens are rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions of the tissue. By contrast, the follicles and follicular dendritic cell (FDC) networks exhibit low protease activity and antigen degradation rates. Immunization regimens targeting antigen rapidly to FDCs led to germinal centers (GCs) where responses to intact antigen were highly dominant, while traditional bolus immunizations led to weaker GC responses where more GC B cells bound to breakdown products than intact antigen. Thus, spatially-compartmentalized antigen proteolysis impacts humoral immunity and can be exploited to enhance vaccine-induced production of antibody responses against key pathogen structural epitopes.

sentences: SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein Histidines Inteins are auto-processing domains that implement a multi-step biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis. Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wild-type precursor protein. Structural analysis and molecular dynamics simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.

sentences: Fear memory-associated synaptic and mitochondrial changes revealed by deep learning-based processing of electron microscopy data Serial section electron microscopy (ssEM) can provide comprehensive 3D ultrastructural information of the brain with exceptional computational cost. Targeted reconstruction of subcellular structures from ssEM datasets is less computationally demanding but still highly informative. We thus developed a Region-CNN-based deep learning method to identify, segment, and reconstruct synapses and mitochondria to explore the structural plasticity of synapses and mitochondria in the auditory cortex of mice subjected to fear conditioning. Upon reconstructing over 135,000 mitochondria and 160,000 synapses, we found that fear conditioning significantly increases the number of mitochondria but decreases their size, and promotes the formation of multi-contact synapses comprising a single axonal bouton and multiple postsynaptic sites from different dendrites. Modeling indicates that such multi-contact configuration increases the information storage capacity of new synapses by over 50%. With high accuracy and speed in reconstruction, our method yields structural and functional insight into cellular plasticity associated with fear learning.

sentences: Somatic mutation rates scale with lifespan across mammals The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans. Comparative analyses can shed light on the diversity of mutagenesis across species and on long-standing hypotheses regarding the evolution of somatic mutation rates and their role in cancer and ageing. Here, we used whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found somatic mutagenesis to be dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans, although the relative contribution of each signature varied across species. Remarkably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied displaying a comparable association. Despite widely different life histories among the species surveyed, including [~]30-fold variation in lifespan and [~]40,000-fold variation in body mass, the somatic mutation burden at the end of lifespan varied only by a factor of [~]3. These data unveil common mutational processes across mammals and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.

sentences: Explosive diversification following continental colonizations by canids Colonization of a new environment may trigger an explosive radiation process, defined as an accelerated accumulation of species in a short period of time. However, how often colonization events trigger explosive radiations is still an open question. We studied the worldwide dispersal of the subfamily Caninae, to investigate whether the invasion of new continents resulted in explosive radiations. We used a combination of phylogenetic analyses and ancestral area reconstructions to estimate ancestral ranges of 56 extant and extinct species of Caninae, as well as variation in speciation and extinction rates through time and across clades. Our findings indicate that canids experienced an explosive radiation event when lineages were able to cross the Bering Strait and the Isthmus of Panama to reach Eurasia and South America, respectively, around 11 million years ago. This large number of species arising in a short period of time suggests that canids experienced ecological opportunity events within the new areas, implying that the differences in the ecological settings between continents may be responsible for the variation in clade dynamics. We argue that interaction with other carnivores probably also affected the diversification dynamics of canids.

sentences: Cis-clustering of cadherin-23 controls the kinetics of cell-cell adhesion Cis and trans-interactions in cadherins are the foundations of multicellularity. While the trans-interaction mediate cell-cell adhesion, the cis-interaction is postulated as strengthening to trans by clustering. The well-accepted model in cadherin-adhesion is that the  trans precedes cis via a diffusion-trap kinetic model. Here we report that cadherin-23, a non-classical cadherin with an extended extracellular region, undergoes clustering in solution via lateral interactions independent of trans and phase separate as liquid droplets. In cellulo using fluorescence-recovery after the photobleaching, we noticed a significantly slow-diffusion of cadherin-23 at the intercellular junctions, indicating the diffusion of a cluster. The cis-clustering accelerates the cell-cell adhesion and, thus, kinetically controls cell-adhesion via  cis precedes trans model. Though the connection of cis-clustering with the rapid adhesion is yet to explore, M2-macrophages that predominantly express cadherin-23 undergo fast attachments to circulatory tumor cells during metastasis.

sentences: Asgard ESCRT-III and VPS4 reveal evolutionary conserved chromatin binding properties of the ESCRT machinery The ESCRT machinery drive membrane remodeling in numerous processes in eukaryotes. Genes encoding for ESCRT proteins have been identified in Asgard archaea, a newly discovered superphylum, currently recognized as the ancestor of all eukaryotes. This begs the question of the functional evolutionary origin of this machinery and its conservation across lineages. Here, we find that Asgard-ESCRTs exhibit conserved DNA-binding properties, which is derived from recruitment of specific members. We show that Asgard-ESCRT-III/VPS4 homologs interact with one another inside mammalian cells, associate with chromatin, and recruit their counterparts to organize in discrete foci in the mammalian nucleus. This is congruent with human-ESCRT-III homologs. We find that human- and Asgard-ESCRT-IIIs associate with chromatin via the same N terminal domain, and that human-ESCRT-III can recruit Asgard-VPS4 to the nucleus to form foci. Therefore, ESCRTs possess chromatin binding properties that were preserved through the billion years of evolution that separate Asgard and human cells.

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The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis Clathrin-mediated endocytosis in plants is an essential process but the underlying mechanisms are poorly understood, not least because of the extreme intracellular turgor pressure acting against the formation of endocytic vesicles. In contrast to other models, plant endocytosis is independent of actin, indicating a mechanistically distinct solution. Here, by using biochemical and advanced microscopy approaches, we show that the plant-specific TPLATE complex acts outside of endocytic vesicles as a mediator of membrane bending. Cells with disrupted TPLATE fail to generate spherical vesicles, and in vitro biophysical assays identified protein domains with membrane bending capability. These results redefine the role of the TPLATE complex as a key component of the evolutionarily distinct mechanism mediating membrane bending against high turgor pressure to drive endocytosis in plant cells.

One Sentence SummaryWhile plant CME is actin independent, we identify that the evolutionarily ancient octameric TPLATE complex mediates membrane bending against high turgor pressure in plant clathrin-mediated endocytosis.

sentences: Reconstructing unobserved cellular states from paired single-cell lineage tracing and transcriptomics data AO_SCPLOWBSTRACTC_SCPLOWNovel experimental assays now simultaneously measure lineage relationships and transcriptomic states from single cells, thanks to CRISPR/Cas9-based genome engineering. These multimodal measurements allow researchers not only to build comprehensive phylogenetic models relating all cells but also infer transcriptomic determinants of consequential subclonal behavior. The gene expression data, however, is limited to cells that are currently present ("leaves" of the phylogeny). As a consequence, researchers cannot form hypotheses about unobserved, or "ancestral", states that gave rise to the observed population. To address this, we introduce TreeVAE: a probabilistic framework for estimating ancestral transcriptional states. TreeVAE uses a variational autoencoder (VAE) to model the observed transcriptomic data while accounting for the phylogenetic relationships between cells. Using simulations, we demonstrate that TreeVAE outperforms benchmarks in reconstructing ancestral states on several metrics. TreeVAE also provides a measure of uncertainty, which we demonstrate to correlate well with its prediction accuracy. This estimate therefore potentially provides a data-driven way to estimate how far back in the ancestor chain predictions could be made. Finally, using real data from lung cancer metastasis, we show that accounting for phylogenetic relationship between cells improves goodness of fit. Together, TreeVAE provides a principled framework for reconstructing unobserved cellular states from single cell lineage tracing data.

sentences: Existence of log-phase Escherichia coli persisters and lasting memory of a starvation pulse The vast majority of a bacterial population is quickly killed when treated with a lethal concentration of antibiotics. The time scale of this killing is often comparable with the bacterial generation time before addition of antibiotics. Yet, a small subpopulation typically survives for an extended period. However, the long-term killing dynamics of bacterial cells has not been fully quantified even in well-controlled laboratory conditions. We constructed a week-long killing assay and followed the survival fraction of Escherichia coli K12 exposed to a high concentration of ciprofloxacin. We found that long-term survivors were formed during exponential growth, with some cells surviving at least 7 days. The long-term dynamics contained at least three timescales, which greatly enhances predictions of the population survival time compared to the biphasic extrapolation from the short term behavior. Furthermore, we observed a surprisingly long memory effect of a brief carbon starvation pulse, which was dependent on the (p)ppGpp synthase relA. Specifically, one hour of carbon starvation prior to antibiotics exposure increased the surviving fraction by nearly 100-fold even after 4 days of ciprofloxacin treatment.

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Identification of PAX6 and NFAT4 as the transcriptional regulators of lncRNA Mrhl in neuronal progenitors LncRNA Mrhl has been shown to be involved in regulating meiotic commitment of mouse spermatogonial progenitors and coordinating differentiation events in mouse embryonic stem cells. Here we have characterized the interplay of Mrhl with lineage-specific transcription factors during mouse neuronal lineage development. Our results demonstrate that Mrhl is predominantly expressed in the neuronal progenitor populations in mouse embryonic brains and in retinoic acid derived radial-glia like neuronal progenitor cells. Mrhl levels are significantly down regulated in postnatal brains and in maturing neurons. In neuronal progenitors, a master transcription factor, PAX6, acts to regulate the expression of Mrhl through direct physical binding at a major site in the distal promoter, located at 2.9kb usptream of the TSS of Mrhl. Furthermore, NFAT4 occupies the Mrhl proximal promoter at two sites, at 437bp and 143bp upstream of the TSS. ChIP studies reveal that PAX6 and NFAT4 interact with each other, suggesting co-regulation of lncRNA Mrhl expression in neuronal progenitors. Our studies herewith are crucial towards understanding how lncRNAs are regulated by major lineage-specific TFs towards defining specific development and differentiation events.

Summary statementTranscriptional regulation of lncRNA Mrhl by multiple lineage-specific transcription factors in neuronal progenitors highlights context-dependent regulation important for lineage specification.

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Longitudinal Auditory Pathophysiology Following Mild Blast Induced Trauma Blast-induced hearing difficulties affect thousands of veterans and civilians. The long-term impact of even a mild blast exposure on the central auditory system is hypothesized to contribute to lasting behavioral complaints associated with mild blast traumatic brain injury (bTBI). Although recovery from mild blast has been studied separately over brief or long time windows, few, if any, studies have investigated recovery longitudinally over short-term and longer-term (months) time windows. Specifically, many peripheral measures of auditory function either recover or exhibit subclinical deficits, masking deficits in processing complex, real-world stimuli that may recover differently. Thus, examining the acute time course and pattern of neurophysiological impairment using appropriate stimuli is critical to better understanding and intervention of bTBI-induced auditory system impairments. Here, we compared auditory brainstem response, middle-latency auditory evoked potentials, and envelope following responses. Stimuli were clicks, tone pips, amplitude modulated tones in quiet and in noise, and speech-like stimuli (iterated rippled noise pitch contours) in adult male rats subjected to mild blast and sham exposure over the course of two months. We found that blast animals demonstrated drastic threshold increases and auditory transmission deficits immediately after blast exposure, followed by substantial recovery during the window of 7-14 days post-blast, though with some deficits remaining even after two months. Challenging conditions and speech-like stimuli can better elucidate mild bTBI-induced auditory deficit during this period. Our results suggest multiphasic recovery and therefore potentially different time windows for treatment, and deficits can be best observed using a small battery of sound stimuli.

New and NoteworthyFew studies on blast-induced hearing deficits go beyond simple sounds and sparsely track post-exposure. Therefore, the recovery arc for potential therapies and real-world listening is poorly understood. Evidence suggested multiple recovery phases over 2 months post-exposure. Hearing thresholds largely recovered within 14 days and partially explained recovery. However, mid-latency responses, responses to AM in noise, and speech-like pitch sweeps exhibited extended changes, implying persistent central auditory deficits and the importance of subclinical threshold shifts.

sentences: Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an enzyme that mediates N-terminal modification of several subtrates, including the monocyte-chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody blockade of the receptor CSF1R, which more broadly eliminates tissue macrophages, reversed tumor growth inhibition in Qpctl-/- mice, and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

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Metabolic-imaging of human glioblastoma explants: a new precision-medicine model to predict tumor treatment response early BackgroundGlioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) and radiotherapy (RT) remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical models that do not reflect the complexity of GB cell biology and fail to test anti-cancer treatments valuably. Better preclinical models for compound screening are therefore needed.

MethodsIn-vitro 3D glioblastoma live explants (GB-EXPs) were derived from patients resected tumors. We then applied metabolic imaging by fluorescence lifetime imaging microscopy (FLIM) on to GB-EXPs to assess drug response, using TMZ as our benchmark drug. Whole-transcriptome and whole-exome analyses were performed.

ResultsUsing FLIM we were able to stratify samples in Responder and Non-Responder tumors. Our functional precision medicine approach was successfully completed within 1 week of surgery. FLIM-based tumor samples stratification was well reflected at the molecular level, highlighting new targets associated with TMZ treatment and identifying a molecular signature associated with survival.

ConclusionsTo the best of our knowledge, this is the first time that FLIM-based metabolic imaging is used on live glioblastoma 3D explants to test anti-neoplastic drugs rapidly. FLIM-based readouts of drug response in GB explants could improve precision treatment of patients with GB and the identification of new anti-GB drugs.

O_LIPatients tumors can be tested for several individual anti-cancer treatments early after surgery
C_LIO_LIFLIM metabolic imaging is an accurate and rapid in vitro tumor treatment response assessment tool
C_LIO_LIOur model can be exploited to identify new drug molecular targets and applied to other cancers
C_LI

Importance of the studyIn this article, we propose an accurate glioblastoma anti-cancer treatment response 3D-approach consisting of FLIM-based metabolic imaging applied directly to in-vitro vital patient-derived glioblastoma explants. Unlike other models, drug response of live explants is assessed within 1 week from patients surgery, therefore with minimal tampering of natural tumor cyto-architecture, preventing the conventional structural and molecular divergence that the tumor undergoes after long time of in vitro culturing and with no animal involvement. This novel in vitro approach for glioblastoma treatment assessment could be exploited as a tool, in a range of clinical and laboratory settings to improve patients clinical management. Selection of the optimal drug regimen for glioblastoma patients remains a clinical challenge and a method to assess the tumor specific response to several therapies at an early stage could be transformative for glioblastoma patients management. Furthermore, this model can be easily used for large-scale screening of new pharmaceutical compounds.

sentences: Population dynamics of multi-host communities attacked by a common parasitoid We model population dynamics of two host species attacked by a common parasitoid using a discrete-time formalism that captures their population densities from year to year. It is well known starting from the seminal work of Nicholson and Bailey that a constant parasitoid attack rate leads to an unstable host-parasitoid interaction. However, a Type III functional response, where the parasitoid attack rate accelerates with increasing host density stabilizes the population dynamics. We first consider a scenario where both host species are attacked by a parasitoid with the same Type III functional response. Our results show that sufficient fast acceleration of the parasitoid attack rate stabilizes the population dynamics of all three species. For two symmetric host species, the extent of acceleration needed to stabilize the three-species equilibrium is exactly the same as that needed for a single host-parasitoid interaction. However, asymmetry can lead to scenarios where the removal of a host species from a stable interaction destabilizes the interaction between the remaining host species and the parasitoid. Next, we consider a situation where one of the host species is attacked at a constant rate (i.e., Type I functional response), and the other species is attacked via a Type III functional response. We identify parameter regimes where a Type III functional response to just one of the host species stabilizes the three species interaction. In summary, our results show that a generalist parasitoid with a Type III functional response to one or many host species can play a key role in stabilizing population dynamics of host-parasitoid communities in apparent competition.

sentences: ACE2 binding is an ancestral and evolvable trait of sarbecoviruses Two different sarbecoviruses have caused major human outbreaks in the last two decades1,2. Both these sarbecoviruses, SARS-CoV-1 and SARS-CoV-2, engage ACE2 via the spike receptor-binding domain (RBD)2-6. However, binding to ACE2 orthologs from humans, bats, and other species has been observed only sporadically among the broader diversity of bat sarbecoviruses7-11. Here, we use high-throughput assays12 to trace the evolutionary history of ACE2 binding across a diverse range of sarbecoviruses and ACE2 orthologs. We find that ACE2 binding is an ancestral trait of sarbecovirus RBDs that has subsequently been lost in some clades. Furthermore, we demonstrate for the first time that bat sarbecoviruses from outside Asia can bind ACE2. In addition, ACE2 binding is highly evolvable: for many sarbecovirus RBDs there are single amino-acid mutations that enable binding to new ACE2 orthologs. However, the effects of individual mutations can differ markedly between viruses, as illustrated by the N501Y mutation which enhances human ACE2 binding affinity within several SARS-CoV-2 variants of concern12 but severely dampens it for SARS-CoV-1. Our results point to the deep ancestral origin and evolutionary plasticity of ACE2 binding, broadening consideration of the range of sarbecoviruses with spillover potential.

sentences: Mutational effects of ubiquitously present gamma radiation on Arabidopsis thaliana: insight into radiosensitivity in the reproductive stage Earth has always been exposed to ionizing radiation from natural sources, and man-made sources have added to this radiation. In order to assess mutational effects of ubiquitously present radiation on plants, we performed a whole-genome resequencing analysis of mutations induced by chronic irradiation throughout the life-cycle of Arabidopsis thaliana under controlled conditions. Resequencing data from 12 M1 lines and 36 M2 progeny derived under gamma-irradiation conditions ranging from 0.0 to 2.0 Gy/d were obtained to identify de novo mutations, including single base substitutions (SBSs) and small insertions/deletions (INDELs). The relationship between de novo mutation frequency and a low-to-middling dose of radiation was assessed by statistical modeling. The increasing of de novo mutations in response to doses of irradiation fit the negative binomial model, accounting for the high variability of mutation frequency observed. Among the different types of mutations, SBSs were more prevalent than INDELs, with deletions being more frequent than insertions. Furthermore, we observed that the mutational effects of chronic radiation are more intensive during the reproductive stage. These outcomes could provide valuable insights into practical strategies for environmental radioprotection of plants on Earth and in space.

sentences: Multivalent Interactions between Molecular Components Involved in Clathrin Independent Endocytosis Drive Protein Phase Separation Endocytosis of transmembrane receptors initiates via molecular interactions between the activated receptor and the endocytic machinery. A specific group of receptors, including the {beta}1-adrenergic receptor ({beta}1-AR), is internalized through a non-clathrin pathway known as Fast Endophilin Mediated Endocytosis (FEME). A key question is: how does the endocytic machinery assemble and how is it modulated by activated receptors during FEME. Here we show that endophilin, a major regulator of FEME, undergoes a phase transition into liquid-like condensates, which facilitates the formation of multi-protein assemblies by enabling the phase partitioning of endophilin binding proteins. The phase transition can be triggered by specific multivalent binding partners of endophilin in the FEME pathway such as the third intracellular loop (TIL) of the {beta}1-AR, and the proline-rich-motifs of lamellipodin (LPD-PRMs). Other endocytic accessory proteins can either partition into, or target interfacial regions of, these condensate droplets. On the membrane, TIL promotes protein clustering in the presence of endophilin and LPD-PRMs. Our results demonstrate how the multivalent interactions between endophilin, LPD-PRMs and TIL regulate protein assembly formation on the membrane, providing mechanistic insights into the priming and initiation steps of FEME.

sentences: On the origin and evolution of Drosophila new genes during spermatogenesis Origin of functional new genes is a basic biological process that has significant contribution to organismal diversity. Previous studies in both Drosophila and mammals showed that new genes tend to be expressed in testis, and avoid the X chromosome presumably because meiotic sex chromosome inactivation (MSCI). Here we analyze the published single-cell transcriptome data of Drosophila adult testis and find an enrichment of male germline mitotic genes, but an underrepresentation of meiotic genes on the X chromosome. This can be attributed to an excess of autosomal meiotic genes that were derived from their X-linked mitotic progenitors, which provides direct cell-level evidence for MSCI in Drosophila. We reveal that new genes, particularly those produced by retrotransposition, tend to exhibit an expression shift toward late spermatogenesis compared to their parental copies, probably due to the more intensive sperm competition or sexual conflict. Our results dissect the complex factors including the age, the origination mechanisms and the chromosomal locations that influence the new gene origination and evolution in testis, and identify new gene cases that show divergent cell-level expression pattern from their progenitors for future functional studies.

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The cold-drought tolerance trade-off in temperate woody plants constrains range size, but not range filling Interspecific differences in plant species ranges are shaped by complex mechanistic interactions, which have so far remained largely beyond the reach of comprehensive models and explanations. Previous attempts to find underlying mechanisms by examining physiological tolerances to cold and heat separately have yielded contradictory results. Here we test the hypothesis that, instead of examining single stressors, abiotic stress tolerance syndromes that involve trade-offs between multiple abiotic stressors (namely drought, cold, waterlogging and shade), will provide reliable explanations.

We compiled a dataset of actual range size and range filling (the ratio between actual and potential species range) as range metrics for 331 temperate woody plants species from Europe and North America. Tolerance syndromes were expressed as two PCA axes. One axis reflects a drought-cold/waterlogging tolerance trade-off (cold/wet-drought trade-off), the second axis represents a shade tolerance spectrum. Phylogenetic generalized linear mixed models were used to model the range metric-tolerance axes relationships using latitude as an additional main effect, and phylogeny and plant functional type as random effects.

Actual range scaled negatively with the cold/wet-drought tolerance trade-off axis, mostly independently of latitude and continent. Thus, cold/wet-tolerant species had the largest ranges and drought tolerant species the smallest. The sign (-) of the relationship was independent of phylogeny and plant functional type. In contrast, range filling depended on latitude. However, deciduous and evergreen species displayed different distributions of range metrics and tolerance syndromes. No significant relationships with the shade tolerance spectrum were found.

Our findings demonstrate that the cold/wet-drought trade-off partly explains interspecific range size differences. However, this trade-off did not explain range filling. We also showed that fundamental adaptations of species also significantly influence range sizes - stress avoidance through the deciduous habit also explained interspecific differences in range size.

sentences: Genome-wide analysis of DNA methylation reveals selection signatures of the grass carp during domestication With the rapid development of aquaculture, more and more fish species from wild environments are artificially domesticated and cultured. In the process of domestication, the fish develop some adaptations and phenotypic traits, namely selection signatures. However, it is still unclear about the biological process underlying these selection signatures. Here, we used grass carp (Ctenopharyngodon idellus), an aquaculture fish with the largest production worldwide, to detect its selection signatures and investigate the roles of DNA methylation in the emergence of selection signatures during domestication based on whole-genome bisulfite sequencing technology. Our results showed that domesticated grass carp demonstrated four selection signatures, including growth and metabolism, immunity, foraging and learning behaviors, and 38 candidate genes were associated with these traits. 16 of candidate genes, such as IGF-1, GK, GYS1, etc., were found to play major roles in the growth and metabolism. Immunity signature was related to 11 of candidate genes, including MHCI, MHCII, C1QA, etc. The GRM1, TAS1R1 and TAS1R3 genes were essential for the adaptation of domesticated grass carp to commercial feed in artificial rearing condition. The C-FOS, POMC and CBP genes might be responsible for the acquisition of novel feeding habits and contribute to faster growth indirectly by enhancing food intake. These findings would provide new insights to expand our understanding on the role of DNA methylation in shaping physiological phenotypes in fish, and also contribute to efficient breeding of aquaculture stocks and restocking programs.

sentences: Genetic basis of a spontaneous mutation's expressivity Genetic background often influences the phenotypic consequences of mutations, resulting in variable expressivity. How standing genetic variants collectively cause this phenomenon is not fully understood. Here, we comprehensively identify loci in a budding yeast cross that impact the growth of individuals carrying a spontaneous missense mutation in the nuclear-encoded mitochondrial ribosomal gene MRP20. Initial results suggested that a single large effect locus influences the mutations expressivity, with one allele causing inviability in mutants. However, further experiments revealed this simplicity was an illusion. In fact, many additional loci shape the mutations expressivity, collectively leading to a wide spectrum of mutational responses. These results exemplify how complex combinations of alleles can produce a diversity of qualitative and quantitative responses to the same mutation.

sentences: Genetic diversity loss in the Anthropocene More species than ever before are at risk of extinction due to anthropogenic habitat loss and climate change. But even species that are not threatened have seen reductions in their populations and geographic ranges, likely impacting their genetic diversity. Although preserving genetic diversity is key to maintaining adaptability of species, we lack predictive tools and global estimates of genetic diversity loss across ecosystems. By bridging theories of biodiversity and population genetics, we introduce a mathematical framework to understand the loss of naturally occurring DNA mutations within decreasing habitat within a species. Analysing genome-wide variation data of 10,095 geo-referenced individuals from 20 plant and animal species, we show that genome-wide diversity follows a power law with geographic area (the mutations-area relationship), which can predict genetic diversity loss in spatial computer simulations of local population extinctions. Given pre-21st century values of ecosystem transformations, we estimate that over 10% of genetic diversity may already be lost, surpassing the United Nations targets for genetic preservation. These estimated losses could rapidly accelerate with advancing climate change and habitat destruction, highlighting the need for forecasting tools that facilitate implementation of policies to protect genetic resources globally.

sentences: Vectofusin-1 based T-cell transduction approach for developing murine CAR-T cells for cancer. Gene transfer into human and murine T-cells using viral-based approaches has several promising therapeutic applications including the production of chimeric antigen receptor T-cell (CAR-T) therapy. The generation of murine CAR-T is paramount to test and validate immunocompetent mouse models for CAR-T therapy. Several viral transduction enhancers already exist for gene therapy with few limitations. In this study, we tested vectofusin-1, a short cationic peptide, as a soluble transduction enhancer for gammaretroviral transduction for the generation of anti-CD19 murine CAR-T. We found that in comparison to Retronectin, Vectofusin-1 is an equally optimal transduction enhancer for the generation of murine CAR-T cells.

sentences: SOX4 facilitates PGR protein stability and FOXO1 expression conducive for human endometrial decidualization The establishment of receptive endometrium in human necessitates appropriate decidualization of stromal cells, which involves steroids regulated periodic transformation of endometrial stromal cells during menstrual cycle. Insufficient decidualization of endometrium contributes to not only the failure of embryo implantation and unexplained infertility, but also the occurrence of recurrent spontaneous abortion, intrauterine growth retardation, preeclampsia, and other clinical gynecological diseases. However, the potential molecular regulatory mechanism underlying the initiation and maintenance of decidualization in humans is yet to be fully elucidated. In this investigation, we document that SOX4 is a key regulator of human endometrial stromal cells (hESCs) decidualization by directly regulating PRL and FOXO1 expression as revealed by whole genomic binding of SOX4 assay and RNA-Seq. Besides, our immunoprecipitation and mass spectrometry results unravel that SOX4 modulates progesterone receptor (PGR) stability through repressing E3 ubiquitin ligase HERC4 mediated degradation. More importantly, we provide evidence that dysregulated SOX4-HERC4-PGR axis is a potential cause of defective decidualization and recurrent implantation failure (RIF) in IVF patients. In summary, this study evidences that SOX4 is a new and critical regulator for human endometrial decidualization, and provides insightful information for the pathology of decidualization-related infertility and will pave the way for pregnancy improvement.

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Plant detection and counting from high-resolution RGB images acquired from UAVs: comparison between deep-learning and handcrafted methods with application to maize, sugar beet, and sunflower crops Progresses in agronomy rely on accurate measurement of the experimentations conducted to improve the yield component. Measurement of the plant density is required for a number of applications since it drives part of the crop fate. The standard manual measurements in the field could be efficiently replaced by high-throughput techniques based on high-spatial resolution images taken from UAVs. This study compares several automated detection of individual plants in the images from which the plant density can be estimated. It is based on a large dataset of high resolution Red/Green/Blue (RGB) images acquired from Unmanned Aerial Vehicules (UAVs) during several years and experiments over maize, sugar beet and sunflower crops at early stages. A total of 16247 plants have been labelled interactively on the images. Performances of handcrafted method (HC) were compared to those of deep learning (DL). The HC method consists in segmenting the image into green and background pixels, identifying rows, then objects corresponding to plants thanks to knowledge of the sowing pattern as prior information. The DL method is based on the Faster Region with Convolutional Neural Network (Faster RCNN) model trained over 2/3 of the images selected to represent a good balance between plant development stage and sessions. One model is trained for each crop.

Results show that simple DL methods generally outperforms simple HC, particularly for maize and sunflower crops. A significant level of variability of plant detection performances is observed between the several experiments. This was explained by the variability of image acquisition conditions including illumination, plant development stage, background complexity and weed infestation. The image quality determines part of the performances for HC methods which makes the segmentation step more difficult. Performances of DL methods are limited mainly by the presence of weeds. A hybrid method (HY) was proposed to eliminate weeds between the rows using the rules developed for the HC method. HY improves slightly DL performances in the case of high weed infestation. When few images corresponding to the conditions of the testing dataset were complementing the training dataset for DL, a drastic increase of performances for all the crops is observed, with relative RMSE below 5% for the estimation of the plant density.

sentences: Natural variation in fertility is correlated with species-wide levels of divergence in Caenorhabditis elegans Life history traits underlie the fitness of organisms and are under strong natural selection in the face of environmental challenges. A new mutation that positively impacts a life history trait will likely increase in frequency and become fixed in a population (e.g. selective sweep). The identification of the beneficial alleles that underlie selective sweeps provides insights into the mechanisms that occurred during the evolution of species. In the global population of Caenorhabditis elegans, we previously identified selective sweeps that have drastically reduced chromosomal-scale genetic diversity in the species. Here, we measured the fertility (viable offspring) of a collection of wild C. elegans strains, including many recently isolated divergent strains from the Hawaiian islands and found that strains with larger swept genomic regions on multiple chromosomes have significantly higher fertility than strains that do not have evidence of the recent selective sweeps. We used genome-wide association (GWA) mapping to identify three quantitative trait loci (QTL) underlying the fertility variation. Additionally, we mapped previous fertility data of wild C. elegans strains and C. elegans recombinant inbred advanced intercross lines (RIAILs) that were grown in various conditions and detected eight QTL across the genome using GWA and linkage mappings. These QTL show the genetic complexity of life history traits such as fertility across this species. Moreover, the haplotype structure in each GWA QTL region revealed correlations with recent selective sweeps in the C. elegans population. North American and European strains had significantly higher fertility than most strains from Hawaii, a hypothesized origin of the C. elegans species, suggesting that beneficial alleles that cause increased fertility could underlie the selective sweeps during the worldwide expansion of C. elegans.

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ACES: Analysis of Conservation with Expansive Species MotivationAn abundance of new reference genomes are becoming available through large-scale sequencing efforts. While the reference FASTA for each genome is available, there is currently no automated mechanism to query a specific sequence across all new reference genomes.

ResultsWe developed ACES (Analysis of Conservation with Expansive Species) as a computational workflow to query specific sequences of interest (e.g., enhancers, promoters, exons) against reference genomes with an available reference FASTA. This automated workflow generates BLAST hits against each of the reference genomes, a multiple sequence alignment file, a graphical fragment assembly file, and a phylogenetic tree file. These data files can then be used by the researcher in several ways to provide key insights into conservation of the query sequence.

AvailabilityACES is available at https://github.com/TNTurnerLab/ACES

Contacttychele@wustl.edu

Supplementary informationSupplementary Figure 1 is available on bioRxiv.

sentences: Global change differentially modulates coral physiology and suggests future shifts in Caribbean reef assemblages Global change driven by anthropogenic carbon emissions is altering ecosystems at unprecedented rates, especially coral reefs, whose symbiosis with algal endosymbionts ise particularly vulnerable to increasing ocean temperatures and altered carbonate chemistry. Here, we assess the physiological responses of the coral holobiont (animal host + algal symbiont) of three Caribbean coral species from two reef environments after exposure to simulated ocean warming (28, 31 {degrees}C), acidification (300 - 3290 atm), and the combination of stressors for 93 days. We used multidimensional analyses to assess how multiple coral holobiont physiological parameters respond to ocean acidification and warming. Our results demonstrate significantly diminishing holobiont physiology in S. siderea and P. astreoides in response to projected ocean acidification, while future warming elicited severe declines in P. strigosa. Offshore S. siderea fragments exhibited higher physiological plasticity than inshore counterparts, suggesting that this offshore population has the capacity to modulate their physiology in response to changing conditions, but at a cost to the holobiont. Plasticity of P. strigosa and P. astreoides was not clearly different between natal reef environments, however, temperature evoked a greater plastic response in both species. Interestingly, while these species exhibit unique physiological responses to ocean acidification and warming, when data from all three species are modeled together, convergent stress responses to these conditions are observed, highlighting the overall sensitivities of tropical corals to these stressors. Our results demonstrate that while ocean warming is a severe acute stressor that will have dire consequences for coral reefs globally, chronic exposure to acidification may also impact coral physiology to a greater extent than previously assumed. The variety of responses to global change we observe across species will likely manifest in altered Caribbean reef assemblages in the future.

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Atypical genomic cortical patterning in autism with poor early language outcome Cortical regional identities develop through anterior-posterior (A-P) and dorsal-ventral (D-V) prenatal genomic patterning gradients. Here we find that A-P and D-V genomic patterning of cortical surface area (SA) and thickness (CT) is intact in typically developing and autistic toddlers with good language outcome, but is absent in autistic toddlers with poor early language outcome. Genes driving this effect are prominent in midgestational A-P and D-V gene expression gradients and prenatal cell types driving SA and CT variation (e.g., progenitor cells versus excitatory neurons). These genes are also important for vocal learning, human-specific evolution, and prenatal co-expression networks enriched for high-penetrance autism risk genes. Autism with poor early language outcome may be linked to atypical genomic cortical patterning starting in prenatal periods and which impacts later development of regional functional specialization and circuit formation.

One Sentence SummaryGenomic patterning of the cortex is atypical in autistic toddlers with poor early language outcome.

sentences: Integrating melt-electrowriting and inkjet bioprinting for engineering structurally organized articular cartilage Successful cartilage engineering requires the generation of biological grafts mimicking the structure, composition and mechanical behaviour of the native tissue. Here melt-electrowriting (MEW) was used to produce arrays of polymeric structures whose function was to orient the growth of cellular aggregates spontaneously generated within these structures, and to provide tensile reinforcement to the resulting tissues. Inkjeting was used to deposit defined numbers of cells into MEW structures, which self-assembled into an organized array of spheroids within hours, ultimately generating a hybrid tissue that was hyaline-like in composition. Structurally, the engineered cartilage mimicked the histotypical organization observed in skeletally immature synovial joints. This biofabrication framework was then used to generate scaled-up (50mm x 50mm) cartilage implants containing over 3,500 cellular aggregates in under 15 minutes. After 8 weeks in culture, a 50-fold increase in the compressive properties of these MEW reinforced tissues were observed, while the tensile properties were still dominated by the polymer network, resulting in a composite construct demonstrating tension-compression nonlinearity mimetic of the native tissue. Helium ion microscopy further demonstrated the development of an arcading collagen network within the engineered tissue. This hybrid bioprinting strategy provides a versatile and scalable approach to engineer cartilage biomimetic grafts for biological joint resurfacing.

sentences: Transition to invasive breast cancer is associated with progressive changes in the structure and composition of tumor stroma Ductal carcinoma in situ (DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand how the tumor microenvironment (TME) changes with transition to IBC, we used Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to analyze 140 clinically annotated surgical resections covering the full spectrum of breast cancer progression. We compared normal, DCIS, and IBC tissues using machine learning tools for multiplexed cell segmentation, pixel-based clustering, and object morphometrics. Transition from DCIS to IBC was found to occur along a trajectory marked by coordinated shifts in location and function of myoepithelium, fibroblasts, and infiltrating immune cells in the surrounding stroma. Taken together, this comprehensive study within the HTAN Breast PreCancer Atlas offers insight into the etiologies of DCIS, its transition to IBC, and emphasizes the importance of the TME stroma in promoting these processes.

sentences: Brain transcriptome analysis reveals gene expression differences associated with dispersal behaviour between range-front and range-core populations of invasive cane toads in Australia Understanding the mechanisms underlying rapid adaptation of invasive species in novel environments is key to improving our ability to manage these species. Many invaders demonstrate rapid evolution of behavioural traits involved in range expansion such as locomotor activity, exploration and risk-taking. However, the molecular mechanisms that underpin these changes are poorly understood. In 86 years, invasive cane toads (Rhinella marina) in Australia have drastically expanded their geographic range westward from coastal Queensland to Western Australia. During their range expansion, toads have undergone extensive phenotypic changes, particularly in behaviours that enhance the toads dispersal ability. Common-garden experiments have shown that some changes in behavioural traits related to dispersal are heritable. However, genetic diversity is greatly reduced across the invasive range due to a strong founder effect, and the genetic basis underlying dispersal-related behavioural changes remains unknown. Here we used RNA-seq to compare the brain transcriptomes of toads from the Hawaiian source population, as well as three distinct populations from across the Australian invasive range. We found markedly different gene expression profiles between the source population and Australian toads. By contrast, cane toads from across the Australian invasive range had very similar transcriptomic profiles. Yet, key genes with functions putatively related to dispersal behaviour showed differential expression between range-core and range-front populations. These genes could play an important role in the behavioural changes characteristic of range expansion in Australian cane toads.

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De novo brain-computer interfacing deforms manifold of populational neural activity patterns in human cerebral cortex Human brains are capable of modulating innate activities to adapt to novel environmental stimuli; for sensorimotor cortices (SM1) this means acquisition of a rich repertoire of motor behaviors. We investigated the adaptability of human SM1 motor control by analyzing net neural population activity during the learning of brain-computer interface (BCI) operations. We found systematic interactions between the neural manifold of cortical population activities and BCI classifiers; the neural manifold was stretched by rescaling motor-related features of electroencephalograms with model-based fixed classifiers, but not with adaptive classifiers that were constantly recalibrated to user activity. Moreover, operation of a BCI based on a de novo classifier with a fixed decision boundary based on biologically unnatural features, deformed the neural manifold to be orthogonal to the boundary. These principles of neural adaptation at a macroscopic level may underlie the ability of humans to learn wide-ranging behavioral repertoires and adapt to novel environments.

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Hinokitiol alters Gene Expression in Aspergillus fumigatus, protects against fungal keratitis by Reducing Fungal Load, LOX-1, Proinflammatory cytokines and Neutrophil Infiltration Hinokitiol (HK), a tropolone-related compound found in cupressaceous plants, has antifungal and anti-inflammatory properties. But its roles in Aspergillus fumigatus ( A.F. ) keratitis remains unknown. This study examined the antifungal activity of HK against A.F. and investigated its possible mechanisms at the ultrastructural and transcriptional levels. We tested the minimum inhibitory concentration (MIC) of HK against A.F.. Our data showed that the MIC50 was 2 g/ml and MIC90 was 8 g/ml, and HK could significantly inhibit the spore germination and mycelial growth of A.F.. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations showed that HK induced significant changes in hyphal morphology and microstructure, including cell membrane rupture and intracellular structural disorder. Adhesion assay and biofilm assay experiments showed that HK reduced the adhesion of spores to human corneal epithelial cells (HCECs) and inhibited the formation of biofilms. The genetic changes of A.F. after HK treatment were analyzed by RNA sequencing, and a total of 2487 differentially expressed genes (DEGs) were identified, including the down-regulated DEGs related to carbohydrate and various amino acid metabolism, ribosome biogenesis, asexual reproduction, and up-regulated DEGs related to steroid biosynthesis through GO and KEGG enrichment analysis. RNA-seq results suggested that HK may play an antifungal effect by destroying the structural integrity of its wall and membrane, inhibiting protein biosynthesis and the growth and reproduction of A.F..10 g/ml HK, which had no effect on proliferation and migration of HCECs, was used in the treatment of murine fungal keratitis (FK). We found that HK eyedrop treatment could reduce the severity of FK by reducing corneal fungal burden, neutrophil infiltration, and the expression of LOX-1 and pro-inflammatory factors. HK is expected to be a safe and effective new drug for FK treatment.

Author summaryFK is a serious common blind-causing eye disease. The current clinical antifungal drugs have the disadvantages of low bioavailability and high corneal toxicity, which greatly limit the clinical effectiveness. Therefore, there is an urgent need for a new, safe and effective treatment for FK. HK had shown great antifungal capacity against A.F., amazingly well anti-inflammatory effect and real improvement in murine FK in our research. These findings provide a theoretical basis for the application of HK as an antifungal drug and reveal the potential of HK to be applied in the clinical treatment of FK.

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Spatial differences in stoichiometry of EGR phosphatase and Microtubule-Associated Stress Protein 1 control root meristem activity during drought stress. During moderate severity drought and low water potential ({psi}w) stress, poorly understood signaling mechanisms restrict both meristem cell division and subsequent cell expansion. We found that the Clade E Growth-Regulating 2 (EGR2) protein phosphatase and Microtubule Associated Stress Protein 1 (MASP1) differed in their stoichiometry of expression across the root meristem and had opposing effects on root meristem activity at low {psi}w. Ectopic MASP1 or EGR expression increased or decreased, respectively, root meristem size and root elongation during low {psi}w stress. This, along with the ability of phosphomimic MASP1 to overcome EGR suppression of root meristem size and observation that ectopic EGR expression had no effect on unstressed plants, indicated that during low {psi}w EGR activation and attenuation of MASP1 phosphorylation in their overlapping zone of expression determines root meristem size and activity. Ectopic EGR expression also decreased root cell size at low {psi}w. Conversely, both the egr1-1egr2-1 and egr1-1egr2-1masp1-1 mutants had similarly increased root cell size; but, only egr1-1egr2-1 had increased cell division. These observations demonstrated that EGRs affect meristem activity via MASP1 but affect cell expansion via other mechanisms. Interestingly, EGR2 was highly expressed in the root cortex, a cell type important for growth regulation and environmental response.

One Sentence SummarySpatial differences in EGR-MASP1 expression and control of MASP1 phosphorylation adjust root meristem activity to regulate growth during drought stress.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Paul E. Verslues (paulv@gate.sinica.edu.tw).

sentences: Physicochemical Insights into Microscopic Events Driven by GTP Hydrolysis Reaction in Ras-GAP system ATPase and GTPase have been widely found as chemical energy-mechanical work transducers, whereas the physicochemical mechanisms are not satisfactorily understood. We addressed the problem by examining John Ross conjecture that repulsive Coulomb interaction between ADP/GDP and inorganic phosphate (Pi) does the mechanical work upon the system. We effectively simulated the consequence of GTP hydrolysis reaction in a complex system of Rat sarcoma (Ras) and GTPase activation protein (GAP) in the framework of classical molecular dynamics by switching force field parameters between the reactant and product systems. We then observed ca. 5 kcal/mol raise of potential energy about the phosphate-binding loop (P-loop) in Ras protein, indicating that the mechanical work generated via the GTP hydrolysis is converted into the local interaction energy and stored in the P-loop. Interestingly, this local energy storage in the P-loop depends on neither impulsive nor consecutive collisions of GDP and Pi with P-loop. Instead, GTP-GDP conversion itself does work on the Ras system, elevating the potential energy. These observations encourage us to challenge a conjecture previously given by Ross. We assert that triphosphate nucleotide hydrolyses do mechanical work by producing emergent steric interaction accompanied with relaxation, namely, a shift of biomolecular system to non-equilibrium state on the reshaped potential energy landscape. Recalling the universality of the P-loop motif among GTPases and ATPases, the observations that we obtained through this study would progress physicochemical understanding for the operating principles of GTP/ATP hydrolysis-driven biological nano-machines.

sentences: The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to {beta}-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated that this could occur either through multiple D-loop formation from one donor DNA molecule, or by the integration of multiple DNA fragments. We also show that the final minimum inhibitory concentration (MIC) differs depending on recipient genome, explained by differences in the extent of recombination at key loci. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we confirm that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake is important for successful resistance evolution. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates.

sentences: Genomic patterns of malignant peripheral nerve sheath tumour (MPNST) evolution correlate with clinical outcome and are detectable in cell-free DNA Malignant peripheral nerve sheath tumour (MPNST) is an aggressive soft-tissue sarcoma that arises in peripheral nerves. MPNST occurs either sporadically or in people with neurofibromatosis type 1 (NF1), a common cancer predisposition syndrome caused by germline pathogenic variants in NF1. Although MPNST is the most common cause of death and morbidity for individuals with NF1, the molecular underpinnings of MPNST pathogenesis remain unclear. Here, we report the analysis of whole-genome sequencing, multi-regional exome sequencing, transcriptomic and methylation profiling data for 95 MPNSTs and precursor lesions (64 NF1-related; 31 sporadic) from 77 individuals. Early events in tumour evolution include biallelic inactivation of NF1 followed by inactivation of CDKN2A and in some cases also TP53 and polycomb repressive complex 2 (PRC2) genes. Subsequently, both sporadic and NF1-related MPNSTs acquire a high burden of somatic copy number alterations (SCNAs). Our analysis revealed distinct pathways of tumour evolution and immune infiltration associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumours with loss of H3K27me3 evolve through extensive chromosomal losses with retention of chromosome 8 heterozygosity followed by whole genome doubling and chromosome 8 amplification. These tumours show lower levels of immune cell infiltration with low cytotoxic activity and low expression of immune checkpoints. In contrast, tumours with retention of H3K27me3 evolve through extensive genomic instability in the absence of recurrent alterations and exhibit an immune cell-rich phenotype. Specific SCNAs detected in both tumour samples and cell-free DNA (cfDNA) act as a surrogate for loss of H3K27me3 and immune infiltration, and predict prognosis. Our results suggest that SCNA profiling of tumour or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups.

sentences: Cryo-EM structure of the Smc5/6 holo-complex The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral integration. Here, we report the cryo-EM structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing a full overview of the complex in its apo / non-liganded form, and revealing how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the  W-loop of Smc4 or  F-loop of Smc1, mediates an essential interaction with Nse1. Taken together, these data confirm a degree of functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.

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PD1+ T-cells correlate with Nerve Fiber Density as a prognostic biomarker in patients with resected perihilar cholangiocarcinoma. Background & AimsPerihilar cholangiocarcinoma (pCCA) is a rare hepatobiliary malignancy. Nerve fiber invasion (NFI) shows cancer invading the nerve and is considered an aggressive feature. Nerve fiber density (NFD) consists of small nerve fibers without cancer invasion and is divided into high NFD (high numbers of small nerve fibers) or low NFD (low numbers of small nerve fibers). We aim to explore differences in immune cell populations and survival.

MethodsWe applied multiplex immunofluorescence (mIF) on 47 pCCA surgically resected patients and investigated the immune cell composition in the tumor microenvironment (TME) of different nerve fiber phenotypes (NFI, high and low NFD). Extensive group comparison was carried out and the association with overall survival (OS) was assessed.

ResultsThe NFI ROI was measured with highest CD68+ macrophage levels among 3 ROIs (NFI compared to tumor free p= 0.016 and to tumor p=0.034). Further, for NFI patients the density of co-inhibitory markers CD8+PD1+ and CD68+PD1+ were more abundant in the tumor rather than NFI ROI (p= 0.004 and p= 0.0029 respectively). Comparison between patients with NFD and NFI groups, the signals of co-expression of CD8+PD1+ as well as CD68+PD1+ were significantly higher in the high NFD group (p= 0.027 and p= 0.044, respectively). The OS for high NFD patients was 92 months median OS (95% CI:41-142), for low NFD patients 20 months ((95% CI: 4-36) and for NFI group of patients 19 months (95% CI 7-33). The OS for high NFD patients was significantly better compared to low NFD (p= 0.046) and NFI (p= 0.032).

ConclusionsPD1+ T-cells correlate with high NFD as a prognostic biomarker, the biological pathway behind this needs to be investigated.

Lay summaryNerve fibers play a dual role in the tumor microenvironment in pCCA. In our previous study, we showed that the presence of high numbers of small nerve fibers is associated with a better overall survival. In addition, we found that in high NFD patients PD1+ T-cells are significantly overexpressed. Therefore, we present high NFD as a promising prognostic biomarker.

Graphical abstract

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sentences: Quantification and image-derived phenotyping of retinal ganglion cell nuclei in the nee mouse model of congenital glaucoma The nee mouse model exhibits characteristic features of congenital glaucoma, a common cause of childhood blindness. The current study of nee mice had two components. First, the time course of neurodegeneration in nee retinal flat-mounts was studied over time using a retinal ganglion cell (RGC)-marker, BRN3A; a pan-nuclear marker, TO-PRO-3; and H&E staining. Based on segmentation of nuclei using ImageJ and RetFM-J, this analysis identified a rapid loss of BRN3A+ nuclei from 4-15 weeks of age, with the first statistically significant difference in average density compared to age-matched controls detected in 8-week-old cohorts (49% reduction in nee). Consistent with a model of glaucoma, no reductions in BRN3A- nuclei were detected, but the combined analysis indicated that some RGCs lost BRN3A marker expression prior to actual cell loss. These results have a practical application in the design of experiments using nee mice to study mechanisms or potential therapies for congenital glaucoma. The second component of the study pertains to a discovery-based analysis of the large amount of image data with 748,782 segmented retinal nuclei. Using the automatedly collected region of interest feature data captured by ImageJ, we tested whether RGC density of glaucomatous mice was significantly correlated to average nuclear area, perimeter, Feret diameter, or MinFeret diameter. These results pointed to two events influencing nuclear size. For variations in RGC density above approximately 3,000 nuclei/mm2 apparent spreading was observed, in which BRN3A- nuclei--regardless of genotype--became slightly larger as RGC density decreased. This same spreading occurred in BRN3A+ nuclei of wild-type mice. For variation in RGC density below 3,000 nuclei/mm2, which only occurred in glaucomatous nee mutants, BRN3A+ nuclei became smaller as disease was progressively severe. These observations have relevance to defining RGCs of relatively higher sensitivity to glaucomatous cell death and the nuclear dynamics occurring during their demise.

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Heritable functional architecture in human visual cortex How much of the functional organization of our visual system is inherited? Here we tested the heritability of retinotopic maps in human visual cortex using functional magnetic resonance imaging. We demonstrate that retinotopic organization shows a closer correspondence in monozygotic (MZ) compared to dizygotic (DZ) twin pairs, suggesting a partial genetic determination. Using population receptive field (pRF) analysis to examine the preferred spatial location and selectivity of these neuronal populations, we estimate a heritability around 30% for polar angle preferences and spatial selectivity, as quantified by pRF size, in extrastriate areas V2 and V3. Our findings are consistent with heritability in both the macroscopic arrangement of visual regions and stimulus tuning properties of visual cortex. This could constitute a neural substrate for variations in a range of perceptual effects, which themselves have been found to be at least partially genetically determined. These findings also add convergent evidence for the hypothesis that functional map topology is linked with cortical morphology.

HighlightsO_LIWe analyzed retinotopic maps from monozygotic and dizygotic twin pairs
C_LIO_LIVisual field maps in V1-V3 are more similar in monozygotic twins
C_LIO_LIHeritability is greater in V1 and V3 for polar angle and population receptive field sizes
C_LIO_LIEccentricity maps show lesser degree of heritability
C_LIO_LIFurther evidence for link between cortical morphology and topology of retinotopic maps
C_LI

Graphical Abstract

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HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism and immunity. The class IB PI3K, PI3K{gamma}, is a heterodimeric complex composed of a catalytic p110{gamma} subunit bound to a p101 or p84 regulatory subunit. PI3K{gamma} is a critical component in multiple immune signaling processes and is dependent on activation by Ras and GPCRs to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3K{gamma} single chain camelid nanobodies using hydrogen deuterium exchange mass spectrometry (HDX-MS) for structural and biochemical studies. This allowed us to identify nanobodies that stimulated lipid kinase activity, blocked Ras activation and specifically inhibited p101-mediated GPCR activation. Overall, this reveals novel insight into PI3K{gamma} regulation and identifies sites that may be exploited for therapeutic development.

Highlights- HDX-MS rapidly identifies epitopes of camelid single-chain nanobodies raised against Class IB PI3K complexes, p110{gamma}/p101 and p110{gamma}/p84
- A nanobody targeting p101 improves local resolution in EM studies with p110{gamma}/p101 facilitating structural characterization of the complex
- Nanobodies that bind at the interfaces with the lipidated activators Ras and G{beta}{gamma} can prevent activation of p110{gamma}/p101 and p110{gamma}/p84

sentences: CMTR1 is recruited to transcription start sites and promotes ribosomal protein and histone gene expression in embryonic stem cells CMTR1 (cap methyltransferase 1) catalyses methylation of the first transcribed nucleotide of RNAPII transcripts (N1 2-O-Me), creating part of the mammalian RNA cap structure. In addition to marking RNA as self, N1 2-O-Me has ill-defined roles in RNA expression and translation. Here we investigated the gene specificity of CMTR1 and its impact on RNA expression in embryonic stem cells. Using chromatin immunoprecipitation, CMTR1 was found to bind to transcription start sites (TSS) correlating with RNAPII levels, predominantly binding at histone genes and ribosomal protein (RP) genes. Repression of CMTR1 expression resulted in repression of RNAPII binding at the TSS and repression of RNA expression, particularly of histone and RP genes. In correlation with regulation of histones and RP genes, CMTR1 repression resulted in repression of translation and induction of DNA replication stress and damage. Indicating a direct role for CMTR1 in transcription, addition of recombinant CMTR1 to purified nuclei increased transcription of the histone and RP genes. CMTR1 was found to be upregulated during neural differentiation and there was an enhanced requirement for CMTR1 for gene expression and proliferation during this process. We highlight the distinct roles of the cap methyltransferases RNMT and CMTR1 in target gene expression and differentiation.

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Complete sequence of a 641-kb insertion of mitochondrial DNA in the Arabidopsis thaliana nuclear genome Intracellular transfers of mitochondrial DNA continue to shape nuclear genomes. Chromosome 2 of the model plant Arabidopsis thaliana contains one of the largest known nuclear insertions of mitochondrial DNA (numts). Estimated at over 600 kb in size, this numt is larger than the entire Arabidopsis mitochondrial genome. The primary Arabidopsis nuclear reference genome contains less than half of the numt because of its structural complexity and repetitiveness. Recent datasets generated with improved long-read sequencing technologies (PacBio HiFi) provide an opportunity to finally determine the accurate sequence and structure of this numt. We performed a de novo assembly using sequencing data from recent initiatives to span the Arabidopsis centromeres, producing a gap-free sequence of the Chromosome 2 numt, which is 641-kb in length and has 99.933% nucleotide sequence identity with the actual mitochondrial genome. The numt assembly is consistent with the repetitive structure previously predicted from fiber-based fluorescent in situ hybridization. Nanopore sequencing data indicate that the numt has high levels of cytosine methylation, helping to explain its biased spectrum of nucleotide sequence divergence and supporting previous inferences that it is transcriptionally inactive. The original numt insertion appears to have involved multiple mitochondrial DNA copies with alternative structures that subsequently underwent an additional duplication event within the nuclear genome. This work provides insights into numt evolution, addresses one of the last unresolved regions of the Arabidopsis reference genome, and represents a resource for distinguishing between highly similar numt and mitochondrial sequences in studies of transcription, epigenetic modifications, and de novo mutations.

Significance statementNuclear genomes are riddled with insertions of mitochondrial DNA. The model plant Arabidopsis has one of largest of these insertions ever identified, which at over 600-kb in size represents one of the last unresolved regions in the Arabidopsis genome more than 20 years after the insertion was first identified. This study reports the complete sequence of this region, providing insights into the origins and subsequent evolution of the mitochondrial DNA insertion and a resource for distinguishing between the actual mitochondrial genome and this nuclear copy in functional studies.

sentences: CGRP-receptor family reveals endogenous GPCR agonist bias and its significance in primary human cardiovascular cells Agonist bias at G protein-coupled receptors has attracted considerable interest, although its relevance for physiologically-produced agonists is not always clear. Here, using primary human cells and gene editing techniques, we demonstrate for the first time, endogenous agonist bias with physiological consequences for the calcitonin-like receptor (CLR). We reveal that by switching the accessory protein: receptor activity-modifying protein (RAMP) associated with CLR we can re-route the physiological pathways activated by the stimulating peptide agonists. These results have revealed a unique role in calcium-mediated nitric oxide signalling for the little-understood peptide adrenomedullin 2 and distinct pro-proliferative effects of calcitonin-gene related peptide (CGRP) and adrenomedullin in cardiovascular cells. This work reveals that CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and its importance in endogenous GPCR signalling.

sentences: Bioinformatics analysis of SARS-CoV-2 RBD mutant variants and insights into antibody and ACE2 receptor binding Prevailing COVID-19 vaccines are based on the spike protein of earlier SARS-CoV-2 strain that emerged in Wuhan, China. The continuously evolving nature of SARS-CoV-2 resulting emergence of new variants raises the risk of immune absconds. During the last few months, several RBD (receptor-binding domain) variants have been reported to affect the vaccine efficacy considerably. Soon after reporting of a new double mutant variant (L452R & E484Q) in India, the country facing a deadlier second wave of infections which prompts researchers to suspects this variant to be accountable. To address the relevant concerns about this new variant affecting vaccine efficacy, we performed molecular simulation dynamics based structural analysis of spike protein of double mutant (L452R & E484Q) along with K417G variants and earlier reported RBD variants and found structural changes in RBD region after comparing with the wild type. Comparison of the binding affinity of the double mutant and earlier reported RBD variant for ACE2 (angiotensin 2 altered enzymes) receptor and CR3022 antibody with the wild-type strain revealed the lowest binding affinity of the double mutant for CR3022 among all other variants. These findings suggest that the newly emerged double mutant could significantly reduce the impact of the current vaccine which threatens the protective efficacy of current vaccine therapy.

sentences: White matter connections of high-level visual areas predict cytoarchitecture better than category-selectivity in childhood, but not adulthood Ventral temporal cortex (VTC) consists of high-level visual regions that are arranged in consistent anatomical locations across individuals. This consistency has led to several hypotheses about the factors that constrain the functional organization of VTC. A prevailing theory is that white matter connections influence the organization of VTC, however, the nature of this constraint is unclear. Here, we test two hypotheses: (1) white matter tracts are specific for each category or (2) white matter tracts are specific to cytoarchitectonic areas of VTC. To test these hypotheses, we used diffusion magnetic resonance imaging (dMRI) to identify white matter tracts and functional magnetic resonance imaging (fMRI) to identify category-selective regions in VTC in children and adults. We find that in childhood, white matter connections are linked to cytoarchitecture rather than category-selectivity. In adulthood, however, white matter connections are linked to both cytoarchitecture and category-selectivity. These results suggest a rethinking of the view that category-selective regions in VTC have category-specific white matter connections early in development. Instead, these findings suggest that the neural hardware underlying the processing of categorical stimuli may be more domain-general than previously thought, particularly in childhood.

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Discovery of UPSTREAM OF FLOWERING LOCUS C (UFC) and FLOWERING LOCUS C EXPRESSOR (FLX) in Gladiolus xhybridus, G. dalenii Gladiolus is a geophytic floricultural crop, cultivated for cut flower and garden ornamental uses. Ornamental geophytes such as gladiolus, lily, tulip and daffodil are examples of floral crops that are currently being investigated to understand the flowering pathway. While the environmental and hormonal factors leading to flowering are established in Arabidopsis. However, the lack of genetic regulation is poorly understood. Thus, the importance of such an ornamental crop that relies on flowers (flowering) for economic purposes encourages researchers to discover the flowering genes to breed vigorous flowering cultivars. The understanding of the flowering mechanisms in the flowering pathway is also paramount. Herein we show the discovery of UPSTREAM OF FLOWERING LOCUS C (UFC) and FLOWERING LOCUS C EXPRESSOR (FLX) genes in Gladiolus xhybridus and G. dalenii. The UFC gene is adjacent to FLOWERING LOCUS C (FLC) which is a floral repressor in many temperate species. FLX gene upregulates FRIGIDA (FRI) which upregulates FLC expression. The discovery of both genes is a step forward in finding the FLC gene in gladiolus, provided they are linked. Seventeen gladiolus genotypes, consisting of early flowering and commercial cultivars, have the UFC gene, consisting of four exons in two allelic forms. The UFC gene sequenced when translated into amino acid sequence and set in pair-alignment to other species, has up to 57% in amino acid identity to Musa acuminata. The UFC protein ranges in identity with pair-alignment to other species, reaching up to 57% in amino acid identity to Musa acuminata. The FLX gene in gladiolus has 3/5 (60%) exons in relative to Ananas comosus, i.e. lacking 2 exons and a partially complete gene sequence; the pair-alignment of the three exons shows up over all ~65% identity of FLX to Ananas comosus. The UFC protein consists of a conserved domain, DUF966, which is higher in identity and pair-alignment, with up to 86% identity in Elaeis guineensis. The discovered FLX gene in gladiolus has 3/5 (60%) exons, i.e. lacking 2 exons and a partially complete gene sequence; the pair-alignment of the 3 exons shows up to ~65% of identity of FLX to Ananas comosus. These discovered two genes in gladiolus provide insight to further our understanding of the flowering and vernalization response in ornamental geophytes.

Summary StatementTwo gladiolus flowering genes (UFC; FLX) were discovered which will aid research in understanding flowering and vernalization in geophytes

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Inexpensive and easy method for 6 fragment Golden Gate Assembly of a modular S/MARs mammalian expression vector and its variants BackgroundA basic requirement for synthetic biology is the availability of efficient DNA assembly methods. Numerous methods have been previously reported to accomplish this task. One such method has been reported, which allows parallel assembly of multiple DNA fragments in a one-tube reaction, called Golden Gate Assembly. This study aims to further simplify that method and make it more suitable for small labs and students.

MethodsPrior to amplification of the parental plasmids used in building the modules were domesticated using a variation of SDM (Site Directed Mutagenesis) called SPRIP. After careful design and amplification of the desired modules, using a high-fidelity polymerase, amplified PCR fragments that enter the one-step-one-pot reaction were stored in Zymo DNA/RNA Shield at -20 degrees C and thawed whenever needed to be used as fragments or modules in the assembly. The fragments were designed to posses unique overhangs using NEB Golden Gate assembly tool and Snapgene, amplification of modules was performed using a Q5 high fidelity polymerase from preexisting plasmids or gene fragments, clean-up of the PCR products (fragments) was performed in one tube per assembly using Zymo DNA Clean and Concentrator-5, assembled using BsaI and T4 ligase, DpnI digestion performed for eliminating the background plasmids that remain after the PCR reaction and the resulting assembled product was transformed into competent E.coli cells. Transformants were screened using diagnostic digest, transfected into HEK293T cells and the fluorescence was evaluated using fluorescent microscopy and flow cytometry.

ResultsHerein presented is a simple and inexpensive alternate protocol to build modular plasmids using the Golden Gate Assembly method. A total of p37 S/MARs mammalian expression vectors were designed and constructed using 6 modules previously amplified by PCR and stored in the appropriate buffer to eliminate exo- and endonuclease activity and to protect the DNA from freeze thaw cycles. The existing modules were interchangeable and new modules were easily amplified and stored for use when needed. The mammalian expression vectors constructed showed the desired restriction pattern and GFP expression in bacteria and in mammalian cells. A comparison of 7 pNoname variants was conducted using flow cytometry. Interestingly, no pNoname variant harbouring the SV40 promoter showed expression in tested HEK293T cells. It appears that using the Ef1a promoter in combination with the BGH polyA signal provides the best expression in S/MARS vectors harboring the DTS40 region, as measured by flow cytometry.

ConclusionsProvided the design steps are respected and the fragments are stored and labeled appropriately, multiple plasmid variants and combinations of the pre-designed modules can be assembled in one day, easier and using less resources than the established protocols, with good efficiency. The simplicity of the design and the affordability of the method could make modular cloning of plasmid constructs more accessible to small labs and students.

sentences: Multi-omics integration and regulatory inference for unpaired single-cell data with a graph-linked unified embedding framework With the ever-increasing amount of single-cell multi-omics data accumulated during the past years, effective and efficient computational integration is becoming a serious challenge. One major obstacle of unpaired multi-omics integration is the feature discrepancies among omics layers. Here, we propose a computational framework called GLUE (graph-linked unified embedding), which utilizes accessible prior knowledge about regulatory interactions to bridge the gaps between feature spaces. Systematic benchmarks demonstrated that GLUE is accurate, robust and scalable. We further employed GLUE for various challenging tasks, including triple-omics integration, model-based regulatory inference and multi-omics human cell atlas construction (over millions of cells) and found that GLUE achieved superior performance for each task. As a generalizable framework, GLUE features a modular design that can be flexibly extended and enhanced for new analysis tasks. The full package is available online at https://github.com/gao-lab/GLUE for the community.

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Development of a novel connectivity-based electric-field modelling approach for individualized targeting of transcranial magnetic stimulation treatment. BackgroundResting state functional connectivity (rsFC) offers promise for individualizing stimulation targets for transcranial magnetic stimulation (TMS) treatments. However current targeting approaches do not account for non-focal TMS effects or large-scale connectivity patterns. To overcome these limitations, we propose a novel targeting optimization approach that combines whole-brain rsFC and electric-field (e-field) modelling to identify single-subject, symptom-specific TMS targets.

MethodsIn this proof of concept study, we recruited 91 anxious misery (AM) patients and 25 controls. We measured depression symptoms (MADRS/HAMD) and recorded rsFC. We used a PCA regression to predict symptoms from rsFC and estimate the parameter vector, for input into our e-field augmented model. We modeled 17 left dlPFC and 7 M1 sites using 24 equally spaced coil orientations. We computed single-subject predicted {Delta}MADRS/HAMD scores for each site/orientation using the e-field augmented model, which comprises a linear combination of the following elementwise products 1) the estimated connectivity/symptom coefficients, 2) a vectorized e-field model for site/orientation, 3) the pre-treatment rsFC matrix, scaled by a proportionality constant.

ResultsIn AM patients, our pre-stimulation connectivity-based model predicted a significant decrease depression for sites near BA46, but not M1 for coil orientations perpendicular to the cortical gyrus. In control subjects, no site/orientation combination showed a significant predicted change.

DiscussionThese results corroborate previous work suggesting the efficacy of left dlPFC stimulation for depression treatment, and predict better outcomes with individualized targeting. They also suggest that our novel connectivity-based e-field modelling approach may effectively identify potential TMS treatment responders and individualize TMS targeting to maximize the therapeutic impact.

sentences: An approach to gene-based testing accounting for dependence of tests among nearby genes In genome-wide association studies (GWAS), it has become commonplace to test millions of SNPs for phenotypic association. Gene-based testing can improve power to detect weak signal by reducing multiple testing and pooling signal strength. While such tests account for linkage disequilibrium (LD) structure of SNP alleles within each gene, current approaches do not capture LD of SNPs falling in different nearby genes, which can induce correlation of gene-based test statistics. We introduce an algorithm to account for this correlation. When a genes test statistic is independent of others, it is assessed separately; when test statistics for nearby genes are strongly correlated, their SNPs are agglomerated and tested as a locus. To provide insight into SNPs and genes driving association within loci, we develop an interactive visualization tool to explore localized signal. We demonstrate our approach in the context of weakly powered GWAS for autism spectrum disorder, which is contrasted to more highly powered GWAS for schizophrenia and educational attainment. To increase power for these analyses, especially those for autism, we use adaptive p-value thresholding (AdaPT), guided by high-dimensional metadata modeled with gradient boosted trees, highlighting when and how it can be most useful. Notably our workflow is based on summary statistics.

sentences: Cell Rearrangement Generates Pattern Emergence as a Function of Temporal Morphogen Exposure. As tissues elongate, cell rearrangement alters positional information in manner that must be accounted for to generate gene expression pattern. How this is achieved during paraxial mesoderm elongation is unknown. By reverse-engineering gene regulatory networks that predict single cell expression trajectories across the tissue, we find a network capable of recapitulating the full range of dynamic differentiation profiles observed both in vivo and in vitro. Simulating gene expression profiles on in toto cell tracking data sets reveal that temporal exposure to Wnt and FGF is generated by cell movement. The absence of reversal in gene expression towards a more premature gene expression state predicts the generation of aberrant tbx6 expression in the posterior progenitor zone that we then confirm by quantitative single cell imaging. Taken together, these results demonstrate cell rearrangement tunes the dynamics of mesoderm progenitor differentiation to generate pattern emergence as a function of temporal Wnt and FGF exposure.

sentences: BinChecker: a new algorithm for quality assessment of microbial draft genomes In the reconstruction of microbial genomes from metagenomic sequence data, the estimation of the final completeness and possible contamination is crucial for quality control. In metagenomics candidate genomes are usually obtained from a metagenome assembly and a subsequent binning of the assembled contigs. BinChecker provides a novel approach to quality assessment that is based on a fast protein domain search and a clustering approach for identification of marker domain ("feature") sets. The feature sets that are used for estimation are not pre-computed for a given database of reference genomes, but are individually found for each bin by adaptive clustering and feature selection. In particular, the adaptivity facilitates the creation and extension of the underlying database, which just requires to add protein feature profiles of reference genomes. Tests with simulated bins indicate that the prediction accuracy of BinChecker meets the current state of the art while providing significant advantages in terms of speed and flexibility.

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Mild and severe SARS-CoV-2 infection induces respiratory and intestinal microbiome changes in the K18-hACE2 transgenic mouse model Transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and declining economies around the world. K18-hACE2 mice develop disease resembling severe SARS-CoV-2 infection in a virus dose-dependent manner. The relationship between SARS-CoV-2 and the intestinal or respiratory microbiome is not fully understood. In this context, we characterized the cecal and lung microbiome of SARS-CoV-2 challenged K18-hACE2 transgenic mice in the presence or absence of treatment with the Mpro inhibitor GC376. Cecum microbiome showed decreased Shannon and Inv Simpson diversity index correlating with SARS-CoV-2 infection dosage and a difference of Bray-Curtis dissimilarity distances among control and infected mice. Bacterial phyla such as Firmicutes, particularly Lachnospiraceae and Oscillospiraceae, were significantly less abundant while Verrucomicrobiota, particularly the family Akkermansiaceae, were increasingly more prevalent during peak infection in mice challenged with a high virus dose. In contrast to the cecal microbiome, the lung microbiome showed similar microbial diversity among the control, low and high challenge virus groups, independent of antiviral treatment. Bacterial phyla in the lungs such as Bacteroidota decreased while Firmicutes and Proteobacteria were significantly enriched in mice challenged with a high dose of SARS-CoV-2. In summary, we identified changes in the cecal and lung microbiome of K18-hACE2 mice with severe clinical signs of SARS-CoV-2 infection.

IMPORTANCEThe COVID-19 pandemic has resulted in millions of deaths. The hosts respiratory and intestinal microbiome can affect directly or indirectly the immune system during viral infections. We characterized the cecal and lung microbiome in a relevant mouse model challenged with a low and high dose of SARS-CoV-2 in the presence or absence of an antiviral Mpro inhibitor, GC376. Decreased microbial diversity and taxonomic abundances of the phyla Firmicutes, particularly Lachnospiraceae, correlating with infection dosage was observed in the cecum. In addition, microbes within the family Akkermansiaceae were increasingly more prevalent during peak infection, which is observed in other viral infections. The lung microbiome showed similar microbial diversity to the control, independent of antiviral treatment. Decreased Bacteroidota and increased Firmicutes and Proteobacteria were observed in the lungs in a virus dose-dependent manner. These studies add to a better understanding of the complexities associated with the intestinal microbiome during respiratory infections.

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Harvesting electrical current from intact plant leaves Efforts to replace fossil fuels with renewable energy technologies, especially solar energy conversion, continue to improve the potential to produce useful amounts of energy without significant pollution. Utilization of photosynthetic organisms in bio-photo electrochemical cells (BPECs) are a potentially important source of clean energy. Here, we show that it is possible to harvest photocurrent directly from unprocessed plant tissues in specialized BPECs. The source of electrons are shown to originate from the Photosystem II water-oxidation reaction that results in oxygen evolution. In addition to terrestrial and crop plants, we further demonstrate the ability of the desert plant Corpuscularia lehmannii to produce bias-free photocurrent without the addition of an external electrolyte. Finally, we show the use of pond-grown water lilies to generate photocurrent. Different leaves produce photocurrent densities in the range of [~] 1 - 10 mA / cm2 which is significantly higher than microorganism-based BPECs. The relatively high photocurrent and the simplicity of the plants BPEC may pave the way toward the establishment of first applicative photosynthetic based energy technologies.

Broader ContextIt is no secret human society is experiencing an energy and environmental crisis due to our reliance on fossil fuels. In order to promote alternative, cleaner, and more sustainable approaches to energy production, we wish to explore the possibility of using natures method of solar energy conversion in the simplest, least polluting, most sustainable fashion possible. Photosynthesis provides a remarkable example of molecular system for solar energy conversion to storable fuels. Many studies have strived to merge natural photosynthesis (as isolated complexes, isolated membranes, or intact microorganisms) with a variety of electrochemical harvesting technologies. In this paper we show that we can directly couple the power of water oxidation by Photosystem II in intact plants to bio-electrochemical cells without the need to perform expensive, complicated, and polluting isolation. We show that current harvesting (up to current densities of 10 mA / cm2) can be performed using plants of different types: plants of agricultural importance, succulents with internal water-based reservoirs and aquatic plants, used in situ in their growth ponds. We also show that with minimal external bias, hydrogen can be obtained, to be used as a clean fuel. We believe that these results can lead to the development of localized clean energy technologies, where the benefits of plant growth for any purpose can be enhanced by obtaining significant amounts of clean energy.

sentences: Molecular basis of ocean acidification sensitivity and adaptation in Mytilus galloprovincialis One challenge in global change biology is to identify the mechanisms underpinning physiological sensitivities to environmental change and to predict their potential to adapt to future conditions. Using ocean acidification as the representative stressor, molecular pathways associated with abnormal larval development of a globally distributed marine mussel are identified. The targeted developmental stage was the trochophore stage, which is, for a few hours, pH sensitive and is the main driver of developmental success. RNA sequencing and in situ RNA hybridization were used to identify processes associated with abnormal development, and DNA sequencing was used to identify which processes evolve when larvae are exposed to low pH for the full duration of their larval stage. Trochophores exposed to low pH exhibited 43 differentially expressed genes. Thirteen genes, none of which have previously been identified in mussel trochophores, including three unknown genes, were expressed in the shell field. Gene annotation and in situ hybridization point to two core processes associated with the response to low pH: development of the trochophore shell field and the cellular stress response. Encompassing both of these processes, five genes demonstrated changes in allele frequency that are indicative of rapid adaptation. Thus, genes underpinning the most pH-sensitive developmental processes also exhibit scope to adapt via genetic variation currently maintained in the mussel population. These results provide evidence that protecting species existing genetic diversity is a critical management action to maximize the potential for rapid adaptation under a changing environment.

sentences: SARS-CoV-2 variants show a gradual declining pathogenicity and pro-inflammatory cytokine stimulation and an increasing antigenic and anti-inflammatory cytokine induction Hyper-transmissibility with decreased disease severity are typical characteristics of Omicron variant. To understand this phenomenon, we used various bioinformatics approaches to analyze randomly selected genome sequences (one each) of the Gamma, Delta, and Omicron variants submitted to NCBI from 15 to 31 December 2021. We show that: (i) Pathogenicity of SARS-CoV-2 variants decreases in the order: Wuhan > Gamma > Delta > Omicron; however, the antigenic property follows the order: Omicron > Gamma > Wuhan > Delta. (ii) Omicron Spike RBD has lower pathogenicity but higher antigenicity than other variants. (iii) Decreased disease severity by Omicron variant may be due to its decreased pro-inflammatory and IL-6 stimulation and increased IFN-{gamma} and IL-4 induction efficacy. (iv) Mutations in N protein are associated with decreased IL-6 induction and human DDX21-mediated increased IL-4 production in Omicron. (v) Due to mutations, the stability of S, M, N, and E proteins decreases in the order: Omicron > Gamma > Delta > Wuhan. (vi) Stronger Spike RBD-hACE2 binding in Omicron is associated with increased transmissibility. However, the lowest stability of the Omicron Spike protein makes Spike RBD-hACE2 interaction weak for systemic infection and for causing severe disease. Finally (vii), the highest instability of Omicron E protein may also be associated with decreased viral maturation and low viral load leading to less severe disease and faster recovery. Our method may be used for other similar viruses, and these findings will contribute to the understanding of the dynamics of SARS-CoV-2 variants and the management of emerging variants.

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Comparative genomics of Chinese and international isolates of Escherichia albertii: population structure and evolution of virulence and antimicrobial resistance Escherichia albertii is a newly recognized species in the genus Escherichia that causes diarrhea. The population structure, genetic diversity and genomic features has not been fully examined. Here, 169 E. albertii isolates from different sources and regions in China were sequenced and combined with 312 publicly available genomes for phylogenetic and genomic analyses. The E. albertii population was divided into 2 clades and 8 lineages, with lineage 3 (L3), L5 and L8 more common in China. Clinical isolates were observed in all clades/lineages. Virulence genes were found to be distributed differently among lineages: subtypes of the intimin encoding gene eae and the cytolethal distending toxin (Cdt) gene cdtB were lineage associated, the second type three secretion system (ETT2) island was truncated in L3 and L6. Seven new eae subtypes and 1 new cdtB subtype (cdtB-VI) were found. Alarmingly, 85.9% of the Chinese E. albertii isolates were predicted to be multidrug resistant (MDR) with 35.9% harboured genes capable of conferring resistance to 10 to 14 different drug classes. By in silico multi-locus sequence typing, majority of the MDR isolates belonged to 4 STs (ST4638, ST4479, ST4633 and ST4488). Thirty-four intact plasmids carrying MDR and virulence genes, and 130 intact prophages were identified from 17 complete E. albertii genomes. Ten plasmid replicon types were found to be significantly associated with MDR. The 130 intact prophages were clustered into 5 groups, with group 5 prophages harbouring more virulence genes. Our findings provided fundamental insights into the population structure, virulence variation and MDR of E. albertii.

Impact statementE. albertii is newly recognized foodborne pathogen causing diarrhea. Elucidation of its genomic features is important for surveillance and control of E. albertii infections. In this work, 169 E. albertii genomes from difference sources and regions in China were collected and sequenced, which contributed to the currently limited genomic data pool of E. albertii. In combination with 312 publicly available genomes from 14 additional countries, the population structure of E. albertii was defined. The presence and subtypes of virulence genes in different lineages were significantly different, indicating potential pathogenicity variation. Additionally, the presence of multidrug resistance (MDR) genes was alarmingly high in the Chinese dominated lineages. MDR associated STs and plasmid subtypes were identified, which could be used as sentinels for MDR surveillance. Moreover, the subtypes of plasmids and prophages were distributed differently across lineages, and were found to contribute to the acquisition of virulence and MDR genes of E. albertii. Altogether, this work reveals the diversity of E. albertii and characterized its genomic features in unprecedented detail.

Data SummaryAll newly sequenced data in this work were deposited in National Center for Biotechnology Information (NCBI) under the BioProject of PRJNA693666, including 6 complete genomes and raw reads of 164 E. albertii isolates.

sentences: Unification of Sparse Bayesian Learning Algorithms for Electromagnetic Brain Imaging with the Majorization Minimization Framework Methods for electro- or magnetoencephalography (EEG/MEG) based brain source imaging (BSI) using sparse Bayesian learning (SBL) have been demonstrated to achieve excellent performance in situations with low numbers of distinct active sources, such as event-related designs. This paper extends the theory and practice of SBL in three important ways. First, we reformulate three existing SBL algorithms under the majorization-minimization (MM) framework. This unification perspective not only provides a useful theoretical framework for comparing different algorithms in terms of their convergence behavior, but also provides a principled recipe for constructing novel algorithms with specific properties by designing appropriate bounds of the Bayesian marginal likelihood function. Second, building on the MM principle, we propose a novel method called LowSNR-BSI that achieves favorable source reconstruction performance in low signal-to-noise-ratio (SNR) settings. Third, precise knowledge of the noise level is a crucial requirement for accurate source reconstruction. Here we present a novel principled technique to accurately learn the noise variance from the data either jointly within the source reconstruction procedure or using one of two proposed cross-validation strategies. Empirically, we could show that the monotonous convergence behavior predicted from MM theory is confirmed in numerical experiments. Using simulations, we further demonstrate the advantage of LowSNR-BSI over conventional SBL in low-SNR regimes, and the advantage of learned noise levels over estimates derived from baseline data. To demonstrate the usefulness of our novel approach, we show neurophysiologically plausible source reconstructions on averaged auditory evoked potential data.

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The Codon Statistics Database: a Database of Codon Usage Bias MotivationMost amino acids can be encoded by a set of synonymous codons. Often, for any given amino acid, certain codons are significantly more used than others, a phenomenon known as codon usage bias. The genomes of different species differ in the frequencies at which they use each codon (e.g., a codon that is highly used in one species may be lowly used in another species). In addition, within any given genome, genes differ in their degree of codon bias, with highly expressed genes being more likely to use preferred codons. Knowing the codons that are preferred by a certain genome, and the amount of codon bias exhibited by each gene, has multiple applications (e.g., in heterologous expression, gene prediction, or phylogenetic inference).

ResultsWe have developed the Codon Statistics Database, an online database that contains codon usage statistics for all the species with reference or representative genomes in RefSeq. The user can search for any species and access two sets of tables. One set lists, for each codon, the frequency, the Relative Synonymous Codon Usage (RSCU), and whether the codon is preferred. Another set of tables lists, for each gene, its GC content, Effective Number of Codons (ENC), Codon Adaptation Index (CAI), and frequency of optimal codons (Fop). Equivalent tables can be accessed for 1) all nuclear genes, 2) nuclear genes encoding ribosomal proteins, 3) mitochondrial genes and 4) chloroplastic genes (if available in the relevant assembly). The user can also search for any taxonomic group (e.g., "primates") and obtain a table comparing all the species in the group.

AvailabilityThe database is free to access without registration at http://codonstatsdb.unr.edu.

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Three orphan histidine kinases inhibit Clostridioides difficile sporulation The ability of the anaerobic gastrointestinal pathogen, Clostridioides difficile, to survive outside the host relies on the formation of dormant endospores. Spore formation is contingent on the activation of a conserved transcription factor, Spo0A, by phosphorylation. Multiple kinases and phosphatases regulate Spo0A activity in other spore-forming organisms; however, these factors are not well conserved in C. difficile. Previously, we discovered that deletion of a conserved phosphotransfer protein, CD1492, increases sporulation, indicating that CD1492 inhibits C. difficile spore formation. In this study, we investigate the functions of additional conserved orphan phosphotransfer proteins, CD2492, CD1579, and CD1949 which are hypothesized to regulate Spo0A phosphorylation. Disruption of the conserved phosphotransfer protein, CD2492, also increased sporulation frequency, similarly to the CD1492 mutant, and in contrast to a previous study. A CD1492 CD2492 mutant phenocopied the sporulation and gene expression patterns of the single mutants, suggesting that these proteins function in the same genetic pathway to repress sporulation. Deletion of the conserved CD1579 phosphotransfer protein also variably increased sporulation frequency; however, knockdown of CD1949 expression did not influence sporulation. We provide evidence that CD1492, CD2492 and CD1579 function as phosphatases, as mutation of the conserved histidine residue for phosphate transfer abolished CD2492 function, and expression of the CD1492 or CD2492 histidine site-directed mutants or the wild-type CD1579 allele in a parent strain resulted in a dominant negative hypersporulation phenotype. Altogether, at least three phosphotransfer proteins, CD1492, CD2492 and CD1579 (herein, PtpA, PtpB and PtpC) repress C. difficile sporulation initiation by regulating activity of Spo0A.

IMPORTANCEThe formation of inactive spores is critical for the long-term survival of the gastrointestinal pathogen Clostridioides difficile. The onset of sporulation is controlled by the master regulator of sporulation, Spo0A, which is activated by phosphorylation. Multiple kinases and phosphatases control Spo0A phosphorylation; however, this regulatory pathway is not defined in C. difficile. We show that two conserved phosphotransfer proteins, CD1492 (PtpA) and CD2492 (PtpB), function in the same regulatory pathway to repress sporulation by preventing Spo0A phosphorylation. We show that another conserved phosphotransfer protein, CD1579 (PtpC), also represses sporulation, and we eliminate the possibility that a fourth orphan histidine kinase protein, CD1949, impacts C. difficile sporulation. These results support the idea that C. difficile inhibits sporulation initiation through multiple phosphatases.

sentences: Methylothon: a versatile course-based high school research experience in microbiology and bioinformatics-- with pink bacteria Methylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here we present an overview of the modules learning goals, describe course resources (available for public use on http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from Spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees, and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

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Oncogenic ZMYND11-MBTD1 fusion protein anchors the NuA4/TIP60 histone acetyltransferase complex to the coding region of active genes A chromosomal translocation found in cannibalistic acute myeloid leukemia (AML) leads to an in-frame fusion of the transcription elongation repressor ZMYND11 to MBTD1, a subunit of the NuA4/TIP60 histone acetyltransferase (HAT) complex. In contrast to the NuA4/TIP60 complex, ZMYND11 is linked to repression of actively transcribed genes through recognition of H3.3K36me3. To understand the abnormal molecular events that expression of this ZMYND11-MBTD1 fusion protein can create, we performed its biochemical and functional characterization in comparison to each individual fusion partner. ZMYND11-MBTD1 is stably incorporated into the endogenous NuA4/TIP60 complex but does not bring any additional interactors as the fusion lacks the MYND domain of ZMYND11. Nevertheless, this truncated ZMYND11 moiety in the fusion leads to mislocalization of the NuA4/TIP60 complex on the body of genes normally bound by ZMYND11 in the genome, in a PWWP-H3.3K36me3 interaction-dependent manner. This can be correlated to increased chromatin acetylation and altered gene transcription, most notably on the MYC oncogene, and alternative splicing. Importantly, expression of ZMYND11-MBTD1, but not the individual fusion partners, during embryonic stem cell differentiation, leads to decreased expression of specific differentiation markers, while favoring Myc-driven pluripotency. It also favors self-renewal of hematopoietic stem/progenitor cells. Altogether, these results indicate that the ZMYND11-MBTD1 fusion protein functions primarily by mistargeting the NuA4/TIP60 complex to the body of genes, altering normal transcription of specific genes, likely driving oncogenesis in part through the Myc regulatory network.

Highlights-A recurrent chromosomal translocation detected in cannibalistic acute myeloid leukemia leads to the production of a ZMYND11-MBTD1 fusion protein.
-The ZMYND11-MBTD1 fusion protein is stably incorporated into the endogenous NuA4/TIP60 complex.
-ZMYND11-MBTD1 leads to mistargeting of NuA4/TIP60 activity to the coding region of ZMYND11-target genes, altering gene expression and splicing.
-ZMYND11-MBTD1 binds the MYC gene leading to its upregulation, favoring growth and pluripotency while inhibiting differentiation markers.

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Fish diversity drives regional productivity but not stability in southeastern United States coastal marine fishes AimEcosystem-based management requires accurate predictions on how biotic and environmental factors interact to deliver ecosystem services. Biodiversity-ecosystem function (BEF) theory predicts that as diversity increases, the ecosystem will become more productive (positive diversity-productivity relationship - DPR) and more stable (positive diversity-stability relationship - DSR). Support for BEF has been primarily derived from fine-grained, non-harvested systems. The purpose of this study is to examine the robustness of BEF predictions for the DPR and DSR by examining how well fish diversity predicts productivity and stability of fish, shrimp, and flounder at a regional scale.

LocationSoutheast coast of United States.

Time Period1989 - 2015

Major Taxa StudiedMarine Fishes

MethodsWe used 27 years of the SEAMAP-SA Coastal Trawl Survey database to derive estimates of fish, shrimp, and flounder biomass (i.e., productivity), temporal stability of biomass (i.e., invariability of productivity), and fish community species richness. We pooled trawls into 22 km x 22 km raster cells and 3-year time bins. We controlled for variation in sampling effort using sample-based rarefaction. We compared the ability of fish species richness, water salinity, and water temperature to predict biomass and stability of all fish, shrimp, and flounder using multiple linear regression.

ResultsBoth the DPR and DSR exhibited positive log-log linear trends as expected, but the DPR had a much stronger signal. Species richness outperformed the environmental covariates in both the fish and shrimp DPR models. Surface temperature was the most important variable in both flounder models. Overall, our models better explained productivity than stability.

Main ConclusionsThe DPR and DSR are relevant at regional scales in a commercially important fishery although support for the DSR is less justified than DPR. Further investigation into the underlying mechanisms driving the DPR and DSR are necessary to design management around BEF theory.

sentences: Single-cell RNA-seq analysis reveals lung epithelial cell-specific contributions of Tet1 to allergic inflammation Tet1 protects against house dust mite (HDM)-induced lung inflammation in mice and alters the lung methylome and transcriptome. In order to explore the role of Tet1 in individual lung epithelial cell types in HDM-induced inflammation, we established a model of HDM-induced lung inflammation in Tet1 knockout and littermate wildtype mice and studied EpCAM+ lung epithelial cells using single-cell RNA-seq analysis. We identified eight EpCAM+ lung epithelial cell types, among which AT2 cells were the most abundant. HDM challenge increased the percentage of alveolar progenitor cells (AP), broncho alveolar stem cells (BAS), and goblet cells, and decreased the percentage of AT2 and ciliated cells. Bulk and cell-type-specific analysis identified genes subject to Tet1 regulation and linked to augmented lung inflammation, including alarms, detoxification enzymes, oxidative stress response genes, and genes in tissue repair. The transcriptomic regulation was accompanied by alterations in TF activities. Trajectory analysis supports that HDM may enhance the differentiation of AP and BAS cells into AT2 cells, independent of Tet1. Collectively, our data showed that lung epithelial cells had common and unique transcriptomic signatures of allergic lung inflammation. Tet1 deletion altered transcriptomic networks in various lung epithelial cells, with an overall effect of promoting allergen-induced lung inflammation.

sentences: Phylogenomic assessment of the role of hybridization and introgression in trait evolution Trait evolution among a set of species--a central theme in evolutionary biology--has long been understood and analyzed with respect to a species tree. However, the field of phylogenomics, which has been propelled by advances in sequencing technologies, has ushered in the era of species/gene tree incongruence and, consequently, a more nuanced understanding of trait evolution. For a trait whose states are incongruent with the branching patterns in the species tree, the same state could have arisen independently in different species (homoplasy) or followed the branching patterns of gene trees, incongruent with the species tree (hemiplasy). Another evolutionary process whose extent and significance are better revealed by phylogenomic studies is gene flow between different species. In this work, we present a phylogenomic method for assessing the role of hybridization and introgression in the evolution of polymorphic or monomorphic binary traits. We apply the method to simulated evolutionary scenarios to demonstrate the interplay between the parameters of the evolutionary history and the role of introgression in a binary traits evolution (which we call xenoplasy). Very importantly, we demonstrate, including on a biological data set, that inferring a species tree and using it for trait evolution analysis in the presence of gene flow could lead to misleading hypotheses about trait evolution.

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Sensory and cognitive factors affecting multi-digit touch: a perceptual and modeling study Whilst everyday interactions with objects often involve multiple tactile contacts, integration of tactile signals remains poorly understood. Here we characterise the integration process of tactile motion on multiple fingerpads. Across four experiments, participants averaged the direction of two simultaneous tactile motion trajectories delivered to different fingerpads. Averaging performance differed between within- and between-hands in terms of sensitivity and precision but was unaffected by somatotopic proximity between stimulated fingers. The sensitivity to the average direction was influenced by the discrepancy between individual motion signals, but only for within-hand conditions. This was explained by a model, in which the  virtually leading finger received a higher perceptual weighting. Precision was greater in between-hand compared to within-hand conditions. While biased weighting accounted for differences in sensitivity, it was not sufficient to explain the difference in precision, implying additional sensory limitations during within-hand integration. We suggest that unimanual integration is limited and thus exploits a  natural cognitive prior involving a single object moving relative to the hand to maximise information gain.

Author summaryTactile stimulation is always on. Yet little is known about how the brain combines widespread tactile inputs for perception. Most tactile studies emphasize a single point of tactile stimulation (e.g., location or intensity of a static stimulus) and minimal units of tactile perception (e.g., acuity or selectivity). However, our daily interactions with the world involve encoding spatially and temporally extended tactile signals. Perceiving tactile objects and events as coherent entities requires the somatosensory system to aggregate tactile afferent signals across separate skin regions (i.e., separate digits). Across four experiments we asked participants to average direction of two tactile motion trajectories delivered simultaneously to two different fingerpads, either on the same, or on different hands. Our results show strong integration between multiple tactile inputs, but subject to limitations for inputs delivered within a hand. Our model suggests that tactile inputs are weighted according to an integrative model of hand-object interaction that operates within-hands on purely geometric information to prioritise  novel information from a  virtually leading finger (VLF).

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Differential richness inference for 16S rRNA marker gene surveys Individual and environmental health outcomes are frequently linked to changes in the diversity of associated microbial communities. This makes deriving health indicators based on microbiome diversity measures essential.

While microbiome data generated using high throughput 16S rRNA marker gene surveys are appealing for this purpose, 16S surveys also generate a plethora of spurious microbial taxa. When this artificial inflation in the observed number of taxa (i.e., richness, a diversity measure) is ignored, we find that changes in the abundance of detected taxa confound current methods for inferring differences in richness.

Here we argue that the evidence of our own experiments, theory guided exploratory data analyses and existing literature, support the conclusion that most sub-genus discoveries are spurious artifacts of clustering 16S sequencing reads. We proceed based on this finding to model a 16S surveys systematic patterns of sub-genus taxa generation as a function of genus abundance to derive a robust control for false taxa accumulation.

Such controls unlock classical regression approaches for highly flexible differential richness inference at various levels of the surveyed microbial assemblage: from sample groups to specific taxa collections. The proposed methodology for differential richness inference is available through an R package, Prokounter.

Package availability: https://github.com/mskb01/prokounter

sentences: An Hourglass Circuit Motif Transforms a Motor Program via Subcellularly Localized Muscle Calcium Signaling and Contraction Neural control of muscle function is fundamental to animal behavior. Many muscles can generate multiple distinct behaviors. Nonetheless, individual muscle cells are generally regarded as the smallest units of motor control. We report that muscle cells can alter behavior by contracting subcellularly. We previously discovered that noxious tastes reverse the net flow of particles through the C. elegans pharynx, a neuromuscular pump, resulting in spitting. We now show that spitting results from the subcellular contraction of the anterior region of the pm3 muscle cell. Subcellularly localized calcium increases accompany this contraction. Spitting is controlled by an "hourglass" circuit motif: parallel neural pathways converge onto a single motor neuron that differentially controls multiple muscles and the critical subcellular muscle compartment. We conclude that subcellular muscle units enable modulatory motor control and propose that subcellular muscle contraction is a fundamental mechanism by which neurons can reshape behavior.

sentences: Biases in demographic modelling affect our understanding of recent divergence Testing among competing demographic models of divergence has become an important component of evolutionary research in model and non-model organisms. However, the effect of unaccounted demographic events on model choice and parameter estimation remains largely unexplored. Using extensive simulations, we demonstrate that under realistic divergence scenarios, failure to account for population size (Ne) changes in daughter and ancestral populations leads to strong biases in divergence time estimates as well as model choice. We illustrate these issues reconstructing the recent demographic history of North Sea and Baltic Sea turbots (Schopthalmus maximus) by testing 16 Isolation with Migration (IM) and 16 Secondary Contact (SC) scenarios, modelling changes in Ne as well as the effects of linked selection and barrier loci. Failure to account for changes in Ne resulted in selecting SC models with long periods of isolation and divergence times preceding the formation of the Baltic Sea. In contrast, models accounting for Ne changes suggest recent (<6 kya) divergence with constant gene flow. We further show how interpreting genomic landscapes of differentiation can help discerning among competing models. For example, in the turbots data islands of differentiation show signatures of recent selective sweeps, rather than old divergence resisting secondary introgression. The results have broad implications for the study of population divergence by high-lighting the potential effects of unmodeleld changes in Ne on demographic inference. Tested models should aim at representing realistic divergence scenarios for the target taxa, and extreme caution should always be exercised when interpreting results of demographic modelling.

sentences: BORCS6 is involved in the enlargement of lung lamellar bodies in Lrrk2 knockout mice Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson disease. It has been shown that Lrrk2 knockout (KO) rodents have enlarged lamellar bodies (LBs) in their alveolar epithelial type II cells, although the underlying mechanisms remain unclear. Here we performed proteomic analyses on LBs isolated from Lrrk2 KO mice and found that the LB proteome is substantially different in Lrrk2 KO mice compared with wild-type mice. In Lrrk2 KO LBs, several Rab proteins were increased, and subunit proteins of BLOC-1-related complex (BORC) were decreased. The amount of surfactant protein C was significantly decreased in the bronchoalveolar lavage fluid obtained from Lrrk2 KO mice, suggesting that LB exocytosis is impaired in Lrrk2 KO mice. We also found that the enlargement of LBs is recapitulated in A549 cells upon KO of LRRK2 or by treating cells with LRRK2 inhibitors. Using this model, we show that KO of BORCS6, a BORC subunit gene, but not other BORC genes, causes LB enlargement. Our findings implicate the LRRK2-BORCS6 pathway in the maintenance of LB morphology.

sentences: Cold shock induces a terminal investment reproductive response in C. elegans Challenges from environmental stressors have a profound impact on many life-history traits of an organism, including reproductive strategy. Examples across multiple taxa have demonstrated that maternal reproductive investment resulting from stress can improve offspring survival; a form of matricidal provisioning when death appears imminent is known as terminal investment. Here we report a reproductive response in the nematode Caenorhabditis elegans upon exposure to acute cold shock at 2{degrees}C, whereby vitellogenic lipid movement from the soma to the germline appears to be massively upregulated at the expense of parental survival. This response is dependent on functional TAX-2;TAX-4 cGMP-gated channels that are part of canonical thermosensory mechanisms in worms and can be prevented in the presence of activated SKN-1/Nrf2, the master stress regulator. Increased maternal provisioning promotes improved embryonic cold shock survival, which is notably suppressed in animals with impaired vitellogenesis. These findings suggest that cold shock in C. elegans triggers terminal investment to promote progeny fitness at the expense of parental survival and may serve as a tractable model for future studies of stress-induced progeny plasticity.

sentences: mRNA-1273 and Ad26.COV2.S vaccines protect against the B.1.621 variant of SARS-CoV-2 Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, viral variants with greater transmissibility or immune evasion properties have arisen, which could jeopardize recently deployed vaccine and antibody-based countermeasures. Here, we evaluated in mice and hamsters the efficacy of preclinical non-GMP Moderna mRNA vaccine (mRNA-1273) and the Johnson & Johnson recombinant adenoviral-vectored vaccine (Ad26.COV2.S) against the B.1.621 (Mu) South American variant of SARS-CoV-2, which contains spike mutations T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N. Immunization of 129S2 and K18-human ACE2 transgenic mice with mRNA-1273 vaccine protected against weight loss, lung infection, and lung pathology after challenge with B.1.621 or WA1/2020 N501Y/D614G SARS-CoV-2 strain. Similarly, immunization of 129S2 mice and Syrian hamsters with a high dose of Ad26.COV2.S reduced lung infection after B.1.621 virus challenge. Thus, immunity induced by mRNA-1273 or Ad26.COV2.S vaccines can protect against the B.1.621 variant of SARS-CoV-2 in multiple animal models.

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A new sugar for an old phage: A c-di-GMP dependent polysaccharide pathway sensitizes E. coli for bacteriophage infection Bacteriophages are ubiquitous parasites of bacteria and major drivers of bacterial ecology and evolution. Despite an ever-growing interest in their biotechnological and therapeutic applications, detailed knowledge of the molecular mechanisms underlying phage-host interactions remains scarce. Here, we show that bacteriophage N4 exploits a novel surface glycan, NGR, as a receptor to infect its host Escherichia coli. We demonstrate that this process is regulated by the second messenger c-di-GMP and that N4 infection is specifically stimulated by the diguanylate cyclase DgcJ while the phosphodiesterase PdeL effectively protects E. coli from N4-mediated killing. PdeL-mediated protection requires its catalytic activity to reduce c-di-GMP and includes a secondary role as a transcriptional repressor. We demonstrate that PdeL binds to and represses the promoter of the wec operon, which encodes components of the ECA exopolysaccharide pathway. However, only the acetylglucosamine epimerase WecB but none of the other ECA components is required for N4 infection. Based on this, we postulate that NGR is an N-acetylmannosamine-based carbohydrate polymer that is produced and exported to the cell surface of E. coli in a c-di-GMP dependent manner where it serves as a receptor for N4. This novel carbohydrate pathway is conserved in E. coli and other bacterial pathogens, serves as the primary receptor for a range of N4-like bacteriophages, and is induced at elevated temperature and by specific amino acid-based nutrients. These studies provide an entry point into understanding how bacteria use specific regulatory mechanisms to balance costs and benefits of highly conserved surface structures.

ImportanceBecause bacterial surface glycans are in direct contact with the environment they can provide essential protective functions during infections or against competing bacteria. But such structures are also  Achilles heels as they can serve as primary receptors for bacteriophages. Bacteria thus need to carefully control the exposure of conserved surface glycans to balance costs and benefits. Here, we identify a novel exopolysaccharide that is widely conserved in E. coli and is used by N4 and related bacteriophages as primary receptor. We demonstrate that the synthesis of NGR (N4 glycan receptor) is tightly controlled by the second messenger c-di-GMP in a highly specific manner and by a single diguanylate cyclase. These studies provide an example of how bacteria can alleviate the strong selective pressure imposed on them by bacteriophages entering through conserved surface structures by carefully regulating their synthesis and secretion.

sentences: Prediction of Inefficient BCI Users based on Cognitive Skills and Personality Traits BCI inefficiency is one of the major challenges of motor imagery brain-computer interfaces (MI-BCI). Past research suggests that certain cognitive skills and personality traits correlate with MI-BCI real-time performance. Other studies have examined sensorimotor rhythm changes (also known as  suppression) as a valuable indicator of successful execution of the MI task. This research aims to combine these insights by investigating whether cognitive factors and personality traits can make predictions of a users ability to modulate  rhythms during a MI-BCI task. Data containing 55 subjects who completed a MI task was employed, and a stepwise linear regression model was implemented to select the most relevant features for  suppression prediction. The most accurate model was based on these factors: Spatial Ability, Visuospatial Memory, Autonomy, and Vividness of Visual Imagery. Further correlation analyses showed that a novice users  suppression during a MI-BCI task can be predicted based on their visuospatial memory ability, as measured by the Design Organization Test (DOT).

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A Co-abundant Gene Analysis for Identification of Gut Microbial Strains Associated with Immune Responses to Oral Cholera Vaccine BackgroundOral cholera vaccines (OCVs) are an important tool for reduction of the worldwide cholera burden, but some individuals who receive an OCV do not develop protective immune responses. The gut microbiota is a potential explanation for these differences. Components of the gut microbiota associated with differences in OCV response have not been identified.

ResultsWe used metagenomic sequencing to identify predicted protein-coding genes in the gut microbiota at the time of OCV administration, and then measured immune responses to vaccination. Vaccine recipients were classified as OCV "responders" if they developed a post{-}vaccination increase in memory B cell populations that produce IgA or IgG specific for cholera toxin and the V. cholerae O-specific polysaccharide. We next analyzed microbial genes seen at similar abundances across individual samples and classified these into co-abundant gene groupings (CAGs), and correlated CAGs with OCV responses. Next, to identify specific bacterial strains associated with OCV responses, we mapped CAGs to bacterial genomes and generated a "priority score" for each strain detected in the study population. This score reflects both the number of CAGs aligning to a specific bacterial genome and the strength of the association between the CAGs and the vaccine response. This strain-level analysis revealed relationships between the gut microbiota and immune response to OCV that were not detected at the genus or species level. Bacterial strains which produce short-chain fatty acids and those with sphingolipid-containing cell membranes were correlated with more robust immune responses to vaccination.

ConclusionOur study demonstrates a method for translating metagenomic sequencing data into strain-specific results associated with a biological outcome. Using this approach, we identified strains for the study of bacterial-derived molecules or metabolites associated with immune responses; such agents might have potential utility as vaccine adjuvants.

sentences: Rv3737 is required for Mycobacterium tuberculosis growth in vitro and in vivo and correlates with bacterial load and disease severity in human tuberculosis Rv3737 is the sole homologue of multifunctional transporter ThrE in Mycobacterium tuberculosis (Mtb). In this study, we aimed to investigate whether this transporter participates in vitro and in vivo survival of Mtb. To characterize the role of Rv3737, we constructed and characterized an Mtb H37Rv{Delta}Rv3737. This strain was evaluated for altered growth rate and macrophage survival using cell model of infection. In addition, the comparative analysis was conducted to determine the association between Rv3737 mRNA expression and disease severity in active pulmonary TB patients. The H37Rv{Delta}Rv3737 strain exhibited significant slow growth rate compared to H37Rv-WT strain in standard culture medium. Additionally, the survival rate of H37Rv-WT strain in macrophages was 2 folds higher than that of H37Rv{Delta}Rv3737 at 72 h. A significant higher level of TNF- and IL-6 mRNA expression was observed in macrophages infected with H37Rv{Delta}Rv3737 as compared to H37Rv-WT. Of note, Rv3737 expression was significantly increased in clinical Mtb isolates than H37Rv-WT. The relative expression level of Rv3737 was positively correlated with lung cavity number in TB patients. Similarly, the higher Rv3737 mRNA level resulted in lower C(t) value by Xpert MTB/RIF assay, demonstrating that a positive correlation between Rv3737 expression and bacterial load in TB patients. In conclusion, our data is the first to demonstrate that the transporter Rv3737 is required for in vitro growth and survival of bacteria inside macrophages. In addition, the expression level of Rv3737 is associated with bacterial load and disease severity in pulmonary tuberculosis patients.

sentences: Photoswitchable epothilone-based microtubule stabilisers allow GFP imaging-compatible, optical control over the microtubule cytoskeleton Optical methods to modulate microtubule stability and dynamics are promising approaches to reach the micron- and millisecond-scale resolution needed to decrypt the diverse roles of the microtubule cytoskeleton in biology. However, such optical methods have until now focussed nearly exclusively on microtubule destabilisation. Here, we introduce "STEpos" as light-responsive epothilone reagents, designed to photoswitchably bind to tubulin and stabilise lateral contacts in the microtubule lattice. Using a novel styrylthiazole photoswitch, designed to allow the hydrogen-bonding that is key to epothilone potency, we have created the first set of GFP-orthogonal photoswitchable microtubule stabilisers. The STEpos can photocontrol microtubule polymerisation, cell division, and cellular microtubule dynamics with micron- and second-scale spatiotemporal precision. STEpos offer substantial improvements of potency, solubility, and ease-of-use compared to the only previous photopharmaceuticals for microtubule stabilisation. The intriguing structure-photoswitching-activity relationship insights from this work will also assist future developments of improved STEpo reagents, and we anticipate that these will contribute greatly to high-precision cytoskeleton research across the fields of biophysics, cargo transport, cell motility, cell division, development, and neuroscience.

sentences: Super-human cell death detection with biomarker-optimized neural networks Cell death is an essential process in biology that must be accounted for in live microscopy experiments. Nevertheless, cell death is difficult to detect without perturbing experiments with stains, dyes or biosensors that can bias experimental outcomes, lead to inconsistent results, and reduce the number of processes that can be simultaneously labelled. These additional steps also make live microscopy difficult to scale for high-throughput screening because of the cost, labor, and analysis they entail. We address this fundamental limitation of live microscopy with biomarker-optimized convolutional neural networks (BO-CNN): computer vision models trained with a ground truth biosensor that detect live cells with superhuman, 96% accuracy more than 100 times faster than previous methods. Our models learn to identify important morphological characteristics associated with cell vitality without human input or additional perturbations, and to generalize to other imaging modalities and cell types for which they have no specialized training. We demonstrate that we can interpret decisions from BO-CNN models to gain biological insight into the patterns they use to achieve superhuman accuracy. The BO-CNN approach is broadly useful for live microscopy, and affords a powerful new paradigm for advancing the state of high-throughput imaging in a variety of contexts.

sentences: Cross-dataset reproducibility of human retinotopic maps Population receptive field (pRF) models fit to fMRI data are used to non-invasively measure retinotopic maps in human visual cortex, and these maps are a fundamental component of visual neuroscience experiments. Here, we examined the reproducibility of retinotopic maps across two datasets: a newly acquired retinotopy dataset from New York University (NYU) (n=44) and a public dataset from the Human Connectome Project (HCP) (n=181). Our goal was to assess the degree to which pRF properties are similar across datasets, despite substantial differences in their experimental protocols. The two datasets simultaneously differ in their stimulus apertures, participant pool, fMRI protocol, MRI field strength, and preprocessing pipeline. We assessed the cross-dataset reproducibility of the two datasets in terms of the similarity of vertex-wise pRF estimates and in terms of large-scale polar angle asymmetries in cortical magnification. Within V1, V2, V3, and hV4, the group-median NYU and HCP vertex-wise polar angle estimates were nearly identical. Both eccentricity and pRF size estimates were also strongly correlated between the two datasets, but with a slope different from 1; the eccentricity and pRF size estimates were systematically greater in the NYU data. Next, to compare large-scale map properties, we quantified two polar angle asymmetries in V1 cortical magnification previously identified in the HCP data. The NYU dataset confirms earlier reports that more cortical surface area represents horizontal than vertical visual field meridian, and lower than upper vertical visual field meridian. Together, our findings show that the retinotopic properties of V1, V2, V3, and hV4 can be reliably measured across two datasets, despite numerous differences in their experimental design. fMRI-derived retinotopic maps are reproducible because they rely on an explicit computational model of the fMRI response. In the case of pRF mapping, the model is grounded in physiological evidence of how visual receptive fields are organized, allowing one to quantitatively characterize the BOLD signal in terms of stimulus properties (i.e., location and size). The new NYU Retinotopy Dataset will serve as a useful benchmark for testing hypotheses about the organization of visual areas and for comparison to the HCP 7T Retinotopy Dataset.

sentences: Population-scale long-read sequencing uncovers transposable elements contributing to gene expression variation and associated with adaptive signatures in Drosophila melanogaster High quality reference genomes are crucial to understanding genome function, structure and evolution. The availability of reference genomes has allowed us to start inferring the role of genetic variation in biology, disease, and biodiversity conservation. However, analyses across organisms demonstrate that a single reference genome is not enough to capture the global genetic diversity present in populations. In this work, we generated 32 high-quality reference genomes for the well-known model species D. melanogaster and focused on the identification and analysis of transposable element variation as they are the most common type of structural variant. We showed that integrating the genetic variation across natural populations from five climatic regions increases the number of detected insertions by 58%. Moreover, 26% to 57% of the insertions identified using long-reads were missed by short-reads methods. We also identified hundreds of transposable elements associated with gene expression variation and new TE variants likely to contribute to adaptive evolution in this species. Our results highlight the importance of incorporating the genetic variation present in natural populations to genomic studies, which is essential if we are to understand how genomes function and evolve.

sentences: Pangenome evolution in environmentally transmitted symbionts of deep-sea mussels is governed by vertical inheritance Microbial pangenomes vary across species; their size and structure are determined by genetic diversity within the population and by gene loss and horizontal gene transfer (HGT). Many bacteria are associated with eukaryotic hosts where the host colonization dynamics may impact bacterial genome evolution. Host-associated lifestyle has been recognized as a barrier to HGT in parentally transmitted bacteria. However, pangenome evolution of environmentally acquired symbionts remains understudied, often due to limitations in symbiont cultivation. Using high-resolution metagenomics, here we study pangenome evolution of two co-occurring endosymbionts inhabiting Bathymodiolus brooksi mussels from a single cold seep. The symbionts, sulfur-oxidizing (SOX) and methane-oxidizing (MOX) gamma-proteobacteria, are environmentally acquired at an early developmental stage and individual mussels may harbor multiple strains of each species. We found differences in the accessory gene content of both symbionts across individual mussels, which are reflected by differences in symbiont strain composition. Compared to core genes, accessory genes are enriched in functions involved in genome integrity. We found no evidence for recent horizontal gene transfer between both symbionts. A comparison between the symbiont pangenomes revealed that the MOX population is less diverged and contains fewer accessory genes, supporting that the MOX association with B. brooksi is more recent than that of SOX. Our results show that the pangenomes of both symbionts evolved mainly by vertical inheritance. We conclude that environmentally transmitted symbionts that associate with individual hosts over their lifetime show features of narrow symbioses, where the frequency of HGT is constrained.

sentences: GhostiPy: an efficient signal processing and spectral analysis toolbox for large data Recent technological advances have enabled neural recordings consisting of hundreds to thousands of channels. As the pace of these developments continues to grow rapidly, it is imperative to have fast, flexible tools supporting the analysis of neural data gathered by such large scale modalities. Here we introduce ghostipy (general hub of spectral techniques in Python), a Python open source software toolbox implementing various signal processing and spectral analyses including optimal digital filters and time-frequency transforms. ghostipy prioritizes performance and efficiency by using parallelized, blocked algorithms. As a result, it is able to outperform commercial software in both time and space complexity for high channel count data and can handle out-of-core computation in a user-friendly manner. Overall, our software suite reduces frequently encountered bottlenecks in the experimental pipeline, and we believe this toolset will enhance both the portability and scalability of neural data analysis.

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From adverse to beneficial - contrasting dietary effects of freshwater mixotrophs on zooplankton O_LIThe importance of mixotrophic algae as key bacterivores in microbial food webs is increasingly acknowledged, but their effects on the next trophic level remain poorly understood. Their high stoichiometric food quality is contrasted by anti-grazing strategies.
C_LIO_LIWe tested the quality of freshwater mixotrophs as prey for zooplankton, using four non-colonial chrysophyte species and a cryptophyte as a high quality reference food. We (1) analyzed the stoichiometric and biochemical (fatty acid) composition of the mixotrophs, and (2) quantified their dietary effects on Daphnia longispina survival.
C_LIO_LISurvival of D. longispina significantly depended on the identity of species provided as food, ranging from higher to lower as compared to starvation. This was not reflected in differences in cellular stoichiometry or fatty acid profiles of the mixotrophs. We suggest that toxicity may be the driver for the observed differences.
C_LIO_LIGeneralization of the dietary effects of mixotrophic chrysophytes does not appear straightforward. Besides fundamental species-specific differences, potential toxic effects may vary depending on environmental cues or physiological strategies. Notably in our study, Ochromonas tuberculata, a species previously reported to be deleterious turned out to be a beneficial food source, in terms of enabling high survival of D. longispina.
C_LIO_LIWe challenge the generality of the assumption that chrysophytes are of low value as food for zooplankton. We recommend that future studies test how environmental conditions and physiological strategies shape the quality of mixotrophs as food for consumers at higher trophic levels, specifically focusing on effects of dietary toxicity.
C_LI

sentences: Genetic immune escape landscape in primary and metastatic cancer Immune surveillance escape is a hallmark of tumorigenesis1. Multiple studies have characterized the immune escape landscape across several untreated early-stage primary cancer types2-4. However, whether late-stage treated metastatic tumors present differences in genetic immune escape (GIE) prevalence and dynamics remains unclear. Here, we performed a pan-cancer characterization of GIE prevalence across six immune escape pathways in 6,457 uniformly processed Whole Genome Sequencing (WGS) tumor samples including 58 cancer types from 1,943 primary untreated patients and 4,514 metastatic patients. To effectively address the complexity of the Human Leukocyte Antigen (HLA-I) locus and to characterize its tumor status, we developed LILAC, an open-source integrative framework. We demonstrate that one in four tumors harbor GIE alterations, with high mechanistic and frequency variability across cancer types. GIE prevalence is highly consistent between primary and metastatic tumors for most cancer types with few exceptions such as prostate and thyroid carcinomas that have increased immune evasion frequencies in metastatic tumors. Positive selection analysis revealed that GIE alterations are frequently selected for in tumor evolution and that focal LOH of HLA-I, unlike non-focal LOH of HLA-I, tends to lose the HLA allele that presents the largest neoepitope repertoire. We also unraveled tumor genomic features contributing to immune escape incidence, including DNA repair deficiency, APOBEC activity, tobacco associated mutation load and viral DNA integration. Finally, there is a strong tendency for mid and high tumor mutation burden (TMB) tumors to preferentially select LOH of HLA-I for GIE whereas hypermutated samples favor global immune evasion strategies. Our results indicate that genetic immune escape is generally a pre-metastatic event during tumor evolution and that tumors adapt different strategies depending on their neoepitope burden.

sentences: Microbiome divergence of marine gastropod species separated by the Isthmus of Panama The rise of the Isthmus of Panama ~3.5 mya separated populations of many marine organisms, which then diverged into new geminate sister species currently living in the Eastern Pacific Ocean and Caribbean Sea. However, we know very little about how such evolutionary divergences of host species have shaped their microbiomes. Here, we compared the microbiomes of whole-body and shell-surface samples of geminate species of marine gastropods in the genera Cerithium and Cerithideopsis to those of congeneric outgroups. Our results show that the effects of the Isthmus on microbiome composition varied among host genera and between sample types within the same hosts. In the whole-body samples, microbiome compositions of geminate species pairs in the focal genera tended to be similar, likely due to host filtering, although the strength of this relationship varied among the two groups and across similarity metrics. Shell-surface communities showed contrasting patterns, with co-divergence between the host taxa and a small number of microbial clades evident in Cerithideopsis, but not Cerithium. These results suggest that (i) the rise of the Isthmus of Panama affected microbiomes of geminate hosts in a complex and clade-specific manner and (ii) host-associated microbial taxa respond differently to vicariance events than the hosts themselves.

sentences: Different neural codes serve long and short-term memory functions in primate Hippocampus and Lateral Prefrontal Cortex during virtual navigation The hippocampus (HPC) and the lateral prefrontal cortex (LPFC) are two cortical areas of the primate brain deemed essential to cognition. Here we hypothesize that the codes mediating neuronal communication in HPC and LPFC microcircuits have distinctively evolved to serve plasticity and memory function at different spatiotemporal scales. We used a virtual reality task in which animals navigated through a maze using a joystick and selected one of two targets in the arms of the maze according to a learned context-color rule. We found that neurons and neuronal populations in both regions encode similar information about the task. Moreover, we demonstrate that many HPC neurons concentrate spikes into bursts, whereas most layer II/III LPFC neurons sparsely distribute spikes over time. As the animals learned the task HPC neurons, but not LPFC neurons, increased their burst rate as a function of performance. When integrating spike rates over short intervals, HPC neuronal ensembles reached maximum decoded information with fewer neurons than LPFC ensembles. Our results show that during associative learning HPC principal cells concentrate spikes in bursts enabling temporal summation and fast synaptic plasticity in small populations of neurons and ultimately facilitating rapid encoding of associative memories. On the other hand, layers II/III LPFC pyramidal cells fire spikes more sparsely distributed in time and over a larger number of neurons. The latter would facilitate broadcasting of signals loaded in short term memory across neuronal populations without necessarily triggering fast synaptic plasticity.

sentences: FGF/ERK autocrine signaling is enhanced by NANOG in a subpopulation of pluripotent stem cells to execute autoregulation and induce heterogeneity The self-renewal and differentiation potential of Embryonic stem cells (ESCs) is maintained by the regulated expression of core pluripotency factors. The expression level of core pluripotency factor Nanog is tightly regulated by a negative feedback autorepression loop. However, it remains unclear how the ESCs perceive the NANOG levels and execute autorepression. Here, we show that a dose-dependent induction of Fgfbp1 and Fgfr2 by NANOG activates an autocrine mediated ERK signaling in high-Nanog cells to trigger autorepression. pERK recruits NONO to Nanog locus to repress transcription by preventing POL2 loading. The Nanog autorepression process establishes a self-perpetuating NANOG-pERK reciprocal regulatory circuit. We further demonstrate that the reciprocal regulatory circuit induces the pERK heterogeneity and ERK signaling dynamics in pluripotent stem cells.

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Two Types of Cinnamoyl-CoA Reductase Function Divergently in Tissue Lignification, Phenylpropanoids Flux Control, and Inter-pathway Cross-talk with Glucosinolates as Revealed in Brassica napus Cinnamoyl-CoA reductase (CCR) is the entry point of lignin pathway and a crucial locus in dissection and manipulation of associated traits, but its functional dissection in Brassicaceae plants is largely lagged behind though Arabidopsis thaliana CCR1 has been characterized to certain extent. Here, 16 CCR genes are identified from Brassica napus and its parental species B. rapa and B. oleracea. Brassicaceae CCR genes are divided into CCR1 subfamily and CCR2 subfamily with divergent organ-specificity, yellow-seed trait participation and stresses responsiveness. CCR1 is preferential in G- and H-lignins biosynthesis and vascular development, while CCR2 has a deviation to S-lignin biosynthesis and interfascicular fiber development. CCR1 has stronger effects on lignification-related development, lodging resistance, phenylpropanoid flux control and seed coat pigmentation, whereas CCR2 controls sinapates levels. CCR1 upregulation could delay bolting and flowering time, while CCR2 upregulation weakens vascular system in leaf due to suppressed G lignin accumulation. Besides, CCR1 and CCR2 are deeply but almost oppositely linked with glucosinolates metabolism through inter-pathway crosstalk. Strangely, upregulation of both CCR1 and CCR2 could not enhance resistance to UV-B and S. sclerotiorum though CCR2 is sharply induced by them. These results provide systemic dissection on Brassica CCRs and CCR1-CCR2 divergence in Brassicaceae.

HighlightBrassicaceae contains two types of Cinnamoyl-CoA reductase. As revealed in Brassica napus, they are divergently involved in lignin monomer biosynthesis, tissue lignification, phenylpropanoid flux control, and inter-pathway crosstalk with glucosinolates.

sentences: Acidovorax pan-genome reveals specific functional traits for plant beneficial and pathogenic plant-associations Beta-proteobacteria belonging to the genus Acidovorax have been described from various environments. Many strains can interact with a range of hosts, including humans and plants, forming neutral, beneficial or detrimental associations. In the frame of this study we investigated genomic properties of 52 bacterial strains of the genus Acidovorax, isolated from healthy roots of Lotus japonicus, with the intent of identifying traits important for effective plant-growth promotion. Based on single-strain inoculation bioassays with L. japonicus, performed in a gnotobiotic system, we could distinguish 7 robust plant-growth promoting strains from strains with no significant effects on plant-growth. We could show that the genomes of the two groups differed prominently in protein families linked to sensing and transport of organic acids, production of phytohormones, as well as resistance and production of compounds with antimicrobial properties. In a second step, we compared the genomes of the tested isolates with those of plant pathogens and free-living strains of the genus Acidovorax sourced from public repositories. Our pan-genomics comparison revealed features correlated with commensal and pathogenic lifestyle. We could show that commensals and pathogens differ mostly in their ability to use plant-derived lipids and in the type of secretion-systems being present. Most free-living Acidovorax strains did not harbour any secretion-systems. Overall, our data indicated that Acidovorax strains undergo extensive adaptations to their particular lifestyle by horizontal uptake of novel genetic information and loss of unnecessary genes.

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Rhythmic neuronal activities of the rat nucleus of the solitary tract are impaired by short-term high fat diet - implications for daily control of satiety by the brainstem Temporal partitioning of daily food intake is crucial for survival and involves the integration of internal circadian states and external influences such as the light-dark cycle and the composition of diet. These intrinsic and extrinsic factors are interdependent with misalignment of circadian rhythms promoting body weight gain, while consumption of a calorie dense diet elevates the risk of obesity and blunts circadian rhythms. Since cardiovascular disease, metabolic disorders, and cancer are comorbid with obesity, understanding the relationships between brain activity and diet is of pivotal importance. Recently, we defined for the first time the circadian properties of the dorsal vagal complex of the brainstem, a structure implicated in the control of food intake and autonomic tone, but if and how 24 h rhythms in this area are influenced by diet remains unresolved. Here we focused on a key structure of this complex, the nucleus of the solitary tract, and using a range of approaches, we interrogated how its neuronal and cellular rhythms are affected by high-fat diet. We report that short term consumption of this diet increases food intake during the day and blunts daily rhythms in gene expression and neuronal discharge in the nucleus of the solitary tract. These alterations in this structure occur without prominent body weight gain, suggesting that high-fat diet acts initially to reduce activity in the nucleus of the solitary tract, thereby disinhibiting mechanisms that suppress daytime feeding.

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Functional and ultrastructural analysis of reafferent mechanosensation in larval zebrafish All animals need to differentiate between exafferent stimuli, which are caused by the environment, and reafferent stimuli, which are caused by their own movement. In the case of mechanosensation in aquatic animals, the exafferent inputs are water vibrations in the animals proximity, which need to be distinguished from the reafferent inputs arising from fluid drag due to locomotion. Both of these inputs are detected by the lateral line, a collection of mechanosensory organs distributed along the surface of the body.

In this study, we characterize in detail how the hair cells, which are the receptor cells of the lateral line, discriminate between such reafferent and exafferent signals in zebrafish larvae. Using dye labeling of the lateral line nerve, we visualize two parallel descending inputs that can influence lateral line sensitivity. We combine functional imaging with ultra-structural EM circuit reconstruction to show that cholinergic signals originating from the hindbrain transmit efference copies that cancel out self-generated reafferent stimulation during locomotion, and that dopaminergic signals from the hypothalamus may have a role in threshold modulation both in response to locomotion and salient stimuli. We further gain direct mechanistic insight into the core components of this circuit by loss-of-function perturbations using targeted ablations and gene knockouts.

We propose that this simple circuit is the core implementation of mechanosensory reafferent suppression in these young animals and that it might form the first instantiation of state-dependent modulation found at later stages in development.

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A calibrated cell-based functional assay to aide classification of MLH1 DNA mismatch repair gene variants PURPOSEFunctional assays provide important evidence for classifying the disease significance of germline variants in the DNA mismatch repair genes. We sought to develop a cell-based approach for testing the function of variants of uncertain significance (VUS) in the MLH1 gene.

METHODSUsing CRISPR gene editing, we knocked-in MLH1 VUS into the endogenous MLH1 loci in human embryonic stem cells. We examined their impact at the RNA and protein level, including their ability to maintain stability of microsatellite sequences and instigate a DNA damage response. We calibrated these assays by testing well-established pathogenic and benign control variants.

RESULTSFive VUS resulted in functionally abnormal protein, 15 VUS resulted in functionally normal protein, and one VUS showed mixed results. Furthermore, we converted the functional outputs into a single odds in favor of pathogenicity score for each VUS.

CONCLUSIONOur CRISPR-based functional assay successfully models phenotypes observed in patients in a cellular context. Using this approach, we generated evidence for or against pathogenicity for utilization by variant classification expert panels. Ultimately, this information will assist in proper diagnosis and disease management for suspected Lynch syndrome patients.

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Host EPAC1 modulates rickettsial adhesion to vascular endothelial cells via regulation of ANXA2 Y23 phosphorylation Recently we have identified that endothelial surface annexin A2 (ANAX2) functions as a receptor for spotted fever group rickettsial adhesin outer membrane protein B (OmpB), which binds to the endothelial cell (EC) surface. Moreover, we reported that intracellular cAMP receptor EPAC1 modulates ANXA2 tyrosine (Y) 23 phosphorylation, and inactivation of EPAC1 suppresses ANXA2 expression on the EC luminal surface by downregulating Y23 phosphorylation. Since we reported that EPAC1 plays a critical role in the initial step to successfully establish rickettsial infection of ECs, this work aims to answer the following: (a) What is the mechanism underlying the regulatory role of EPAC1 in ECs during the initial step of bacterial infection? (b) Is the EPAC1-ANXA2 signaling pathway involved in the regulation of rickettsial adhesion to ECs?

In the present study, an established system that is anatomically-based and quantifies bacterial adhesion to ECs in vivo was combined with novel fluidic force microscopy (FluidFM) to dissect the functional role of the EPAC1-ANXA2 signaling pathway in rickettsiae-EC adhesion. We reveal that the deletion of the EPAC1 gene impedes rickettsial binding to endothelium in vivo. In addition, single living brain microvascular EC study that employs FluidFM and site-directed mutagenesis provides evidence that supports our finding that EPAC1 governs rickettsial adhesion to EC surfaces via regulation of ANXA2 Y23 phosphorylation.

sentences: Do Motoneurons Slow With Aging? A Systematic Review And Meta-Analysis With Meta-Regression Nervous system maladaptation is linked to the loss of maximal strength production and motor control with aging. Motor unit discharge rates are a critical determinant of force production; thus, lower discharge rates could be a mechanism underpinning maximal strength and motor control losses during aging. This meta-analysis summarized the findings of studies comparing motor unit discharge rates between young and older adults, and examined the effect of distinct muscles and contraction intensities on the magnitude of discharge rates difference between these two groups. Eligible studies were combined in a meta-analysis, including tested contraction intensities and muscles in different levels, to investigate whether there were differences in discharge rates between younger and older adults. Motor unit discharge rates were higher in younger adults compared to older adults, with a pooled standardized mean difference (SMD) for all studies of 0.63 (95%CI= 0.27 to 0.99). Contraction intensity had a significant effect on the pooled SMD, with a 1% increase in intensity associated with a 0.009 (95%CI= 0.003 to 0.015) change in the pooled SMD. These findings suggest that the reductions in motor unit discharge rates, especially at higher contraction intensities, may be an important mechanism underpinning age-related losses in maximal strength production.

sentences: Beyond Pairwise Interactions: Higher-Order Dynamics in Protein Interaction Networks Protein interactions form a complex dynamic system that shapes cell phenotype and function; in this regard, network analysis is a powerful tool for studying the dynamics of cellular processes. Graph-based models are limited, however, in that these models consider only pairwise relationships. Higher-order interactions are well-characterized in biology, including protein complex formation and feedback or feedforward loops. These higher-order relationships are better represented by a hypergraph as a generalized network model. Here, we present an approach to analyzing dynamic gene expression data using a hypergraph model and quantify network heterogeneity via Forman-Ricci curvature. We observe, on a global level, increased network curvature in pluripotent stem cells and cancer cells. Further, we use local curvature to conduct pathway analysis in a melanoma dataset, finding increased curvature in several oncogenic pathways and decreased curvature in tumor suppressor pathways. We compare this approach to a graph-based model and a differential gene expression approach.

sentences: SPDEF promotes the classical subtype of pancreatic ductal adenocarcinoma. Pancreatic ductal adenocarcinoma (PDA) samples reveal extensive cellular heterogeneity. Using single-cell RNA sequencing, we uncover multiple tumor cell populations distinguished by their differentiation state and associated with different stages of tumor progression in a mouse model of PDA. We identify Spdef as a factor required for tumorigenesis in pancreatic cancer cells of epithelial and mucinous nature. By comparative analysis of cell differentiation states in mice and humans, we find that the Spdef program is highly expressed by human PDAs of the classical subtype. Mouse and human PDA cells expressing elevated levels of Spdef are dependent upon this transcription factor for tumor progression in vivo. The tumor-promoting function of Spdef is recapitulated by two Spdef target genes that regulate protein folding and endoplasmic reticulum activity, Agr2 and Ern2/Ire1{beta}. These findings offer insights into the factors controlling differentiation states in PDA and identify new vulnerabilities in the most common subtype of pancreatic cancer.

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Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug-drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilize conditional Mtb knockdown mutants of essential genes as an experimentally-tractable surrogate for drug treatment, and probe the relationship between Mtb carbon metabolism and chemical-genetic interactions (CGI). We examined the anti-tubercular drugs isoniazid, rifampicin and moxifloxacin, and found that CGI are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo-relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGI could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of novel chemical-genetic-environmental interactions that can be used to optimize drug-drug interactions as well as provide a framework for understanding in vitro correlates of in vivo efficacy.

SignificanceEfforts to improve tuberculosis therapy include optimizing multi-drug regimens to take advantage of drug-drug synergies. However, the complex host environment has a profound effect on bacterial metabolic state and drug activity, making predictions of optimal drug combinations difficult. In this study, we leverage a newly developed library of conditional knockdown Mycobacterium tuberculosis mutants in which genetic depletion of essential genes mimics the effect of drug therapy. This tractable system allowed us to assess the effect of growth condition on predicted drug-drug interactions. We found that these interactions can be differentially sensitive to the metabolic state and select in vitro-defined interactions can be leveraged to accelerate bacterial killing during infection. These findings suggest new strategies for optimizing tuberculosis therapy.

sentences: A phenotypic screen of Marfan syndrome iPSC-derived vascular smooth muscle cells uncovers GSK3β as a new target Marfan syndrome (MFS) is a rare connective tissue disorder caused by mutations in FBN1. Patients with MFS notably suffer from aortic aneurysm and dissection. Despite considerable effort, animal models have proven to be poorly predictive for therapeutic intervention in human aortic disease. Using a "humanised" model system may be more appropriate in identifying new therapeutic targets. Patient-derived induced pluripotent stem cells can be differentiated into vascular smooth muscle cells (VSMCs) and recapitulate major features of MFS. We have screened 1,022 small molecules in our in vitro model, exploiting the highly-proteolytic nature of MFS-VSMCs, and identified 36 effective compounds. Further analysis identified GSK3{beta} as a recurring target in the compound screen. GSK3{beta} inhibition/knockdown did not ameliorate the proliferation defect in MFS-VSMCs but improved MFS-VSMC apoptosis and proteolysis. To conclude, we have identified GSK3{beta} as a novel target for MFS, forming the foundation for future work in MFS and other aortic diseases.

sentences: Improved prediction of behavioral and neural similarity spaces using pruned DNNs Deep Neural Networks (DNNs) have become an important tool for modeling brain and behaviour. One key area of interest has been to apply these networks to model human similarity judgements. Several previous works have used the embeddings from the penultimate layer of vision DNNs and showed that a reweighting of these features improves the fit between human similarity judgments and DNNs. These studies underline the idea that these embeddings form a good basis set but lack the correct level of salience. Here we re-examined the grounds for this idea and on the contrary, we hypothesized that these embeddings, beyond forming a good basis set, also have the correct level of salience to account for similarity judgments. It is just that the huge dimensional embedding needs to be pruned to select those features relevant for the considered domain for which a similarity space is modeled. In Study 1 we supervised DNN pruning based on a subset of human similarity judgments. We found that pruning: i) improved out-of-sample prediction of human similarity judgments from DNN embeddings, ii) produced better alignment with WordNet hierarchy, and iii) retained much higher classification accuracy than reweighting. Study 2 showed that pruning by neurobiological data is highly effective in improving out-of-sample prediction of brain-derived representational dissimilarity matrices from DNN embeddings, at times fleshing out isomorphisms not otherwise observable. Pruning supervised by human brain/behavior therefore effectively identifies alignable dimensions of semantic knowledge between DNNs and humans and constitutes an effective method for understanding the organization of knowledge in neural networks.

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Rapid phenotypic differentiation and local adaptation in Japanese knotweed s.l. (Reynoutria japonica and R. x bohemica, Polygonaceae) invading novel habitats PREMISEMany plant invaders like the Japanese knotweeds are thought to colonize new habitats with low genetic diversity. Such species provide an opportunity to study rapid adaptation to complex environmental conditions.

METHODSUsing replicate reciprocal transplants of clones across three habitats, we described patterns of phenotypic response and assessed degree of local adaptation.

KEY RESULTSWe found plants from beach habitats had decreased height, number of leaves, leaf area, and biomass allocation to roots and shoots compared to plants from marsh and roadside habitats when grown in their home habitat. In the marsh habitat, marsh plants were generally larger than beach plants, but not different from roadside plants. There were no differences among plants from different habitats grown in the roadside habitat. Despite this evidence of differentiation in beach and marsh habitats, we found mixed evidence for local adaptation. In their "home site" plants from the marsh habitat had greater biomass than plants from the beaches but not compared to plants from roadsides. Biomass comparisons in other habitats were either maladaptive or not significant. However, plants from the roadside had greater survival in their "home site" compared to foreign plants. There were no differences in survival in the other habitats.

CONCLUSIONSWe found phenotypic differentiation associated with habitats despite the low reported genetic diversity for these populations. Our results partially support the hypothesis of local adaptation in marsh and roadside habitats. Identifying whether these patterns of differentiation result from genetic or heritable non-genetic mechanisms will require further work.

sentences: The Innate Immune Response to Invasive Pulmonary Aspergillosis: A Systems Modeling Approach Aspergillus species are ubiquitous environmental molds, the spores of which are inhaled daily by most humans. Immunocompromised hosts can develop an invasive infection resulting in high mortality. There is, therefore, a pressing need for host-centric therapeutics for this infection. To address this need, we created a multi-scale computational model of the infection, focused on its interaction with the innate immune system and iron, a critical nutrient for the pathogen. The model, parameterized using published data, was found to recapitulate a wide range of biological features and was experimentally validated in vivo. Conidial swelling was identified as critical in fungal strains with high growth, whereas the siderophore secretion rate is an essential prerequisite for establishment of the infection in low-growth strains. In immunocompetent hosts, high growth, high swelling probability, and impaired leukocyte activation lead to a high conidial germination rate. Similarly, in neutropenic mice, high fungal growth was achieved through synergy between high growth rate, high swelling probability, slow leukocyte activation, and high siderophore secretion. In summary, the model reveals a small set of parameters related to fungal growth, iron acquisition, and leukocyte activation as key determinants of the fate of the infection.

sentences: Computer-Assisted Mitotic Count Using a Deep Learning-based Algorithm Improves Inter-Observer Reproducibility and Accuracy in canine cutaneous mast cell tumors The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intra-observer discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying/classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, the computer-assisted review by pathologists may ensure reliability. In the present study we have compared partial (MC-ROI preselection) and full (additional visualization of MF candidate proposal and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MF and improving classification against imposters. The inter-observer consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study prove that computer assistance may lead to a more reproducible and accurate MCs in ccMCTs.

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Adipogenic activity of chemicals used in plastic consumer products Bisphenols and phthalates, chemicals frequently used in plastic products, promote obesity in cell and animal models. However, these well-known metabolism disrupting chemicals (MDCs) represent only a minute fraction of all compounds found in plastics. To gain a comprehensive understanding of plastics as a source of exposure to MDCs, we characterized all chemicals present in 34 everyday products using nontarget high-resolution mass spectrometry and analyzed their joint adipogenic activities by high-content imaging. We detected 55,300 chemical features and tentatively identified 629 unique compounds, including 11 known MDCs. Importantly, chemicals that induced proliferation, growth, and triglyceride accumulation in 3T3-L1 adipocytes were found in one third of the products. Since the majority did not target peroxisome proliferator-activated receptor {gamma}, the effects are likely to be caused by unknown MDCs. Our study demonstrates that daily-use plastics contain potent mixtures of MDCs and can, therefore, be a relevant yet underestimated environmental factor contributing to obesity.

TeaserPlastics contain a potent mixture of chemicals promoting adipogenesis, a key process in developing obesity.

sentences: Infant brain regional cerebral blood flow dynamics supporting emergence of vital functional networks Human infancy is characterized by most rapid cerebral blood flow (rCBF) increases across lifespan and emergence of a fundamental brain system default-mode network (DMN). However, how infant rCBF change spatiotemporally across the brain and how the rCBF dynamics support emergence of vital functional networks such as DMN remains unknown. Here, by acquiring cutting-edge multi-modal MRI including pseudo-continuous arterial-spin-labeled perfusion MRI and resting-state functional MRI of infants aged 0 to 24 months, we elucidated unprecedented 4D spatiotemporal infant rCBF framework and region-specific physiology-function coupling across infancy. We found faster rCBF increases in the DMN than other regions. We also found strongly coupled increases of rCBF and network strength specifically in the DMN, suggesting faster local blood flow increase to meet extra neuronal metabolic demands in the DMN maturation. These results offer insights into physiological mechanism of brain functional network emergence and have important implications in altered network maturation in brain disorders.

sentences: Humans optimally anticipate and compensate for an uneven step during walking The simple task of walking up a sidewalk curb is actually a dynamic prediction task. The curb is a disturbance that causes a loss of momentum, to be anticipated and compensated for. For example, the compensation might regain momentum and ensure undisturbed time of arrival. But without a selection criterion, there are infinite possible strategies. Here we show that humans compensate with an anticipatory pattern of forward speed adjustments, with a criterion of minimizing mechanical energy input. This is predicted by optimal control for a simple model of walking dynamics, with each legs push-off work as input. Optimization predicts a tri-phasic trajectory of speed (and thus momentum) adjustments, including an anticipatory, feedforward phase. In experiment, human subjects successfully regain time relative to undisturbed walking, with the predicted tri-phasic trajectory. They also scale the pattern with up- or down-steps, and inversely with average speed, as also predicted by model. Humans can reason about the dynamics of walking to plan anticipatory and economical control, even with a sidewalk curb in the way.

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Metal transporter SLC39A14/ZIP14 modulates regulation between the gut microbiome and host metabolism Zinc (Zn) plays a critical role in maintaining intestinal homeostasis by regulating intestinal epithelial cells, host immune cells, and gut microbiome community composition. Deletion of metal transporter Slc39a14/Zip14 causes spontaneous intestinal permeability with low-grade chronic inflammation, mild hyperinsulinemia, and greater body fat with insulin resistance in adipose, suggesting a role for ZIP14-mediated intestinal metal transport in regulating both intestinal homeostasis and systemic metabolism. Here, we showed the function of ZIP14-mediated Zn transport in the gut microbiome composition and how ZIP14-linked changes to gut microbiome community composition are correlated with changes in host metabolism. Deletion of Zip14 generated Zn-deficient epithelial cells and luminal content in the entire intestinal tract; reduced bacterial diversity and Saccharomyces cerevisiae (S. cerevisiae) overgrowth; altered host metabolome; and shifted host energy metabolism toward glucose utilization. This work provides evidence for the regulation of gut microbiome composition, host metabolome, and energy metabolism by metal transporter ZIP14.

SignificanceIntestinal permeability, gut dysbiosis, and Zn dyshomeostasis are emerging signatures of inflammatory bowel diseases and metabolic disorders such as type-2-diabetes and obesity. Zn deficiency is a common clinical finding among these diseases. Zn is essential for the regulation of the intestinal epithelial cells, host immune cells, and the gut microbiome. Transporter-mediated mobilization of Zn plays a critical role in maintaining intestinal homeostasis by facilitating the targeted tissue/cell-specific function. However, studies are lacking in linking transporter-mediated Zn mobilization, gut microbiome, hosts intestinal health, and metabolism. Using the systems-level approach, this study revealed novel findings that deletion of Slc39a14/Zip14 resulted in altered intestinal Zn homeostasis, gut microbiome composition, host metabolome and energy metabolism.

sentences: The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: the case of the spike A222V mutation The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has now reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.

sentences: Probabilistic Brain Extraction in MR Images via Conditional Generative Adversarial Networks Brain extraction, which refers to the task of segmenting brain tissue in an MR image of a subject, forms an essential first step for many quantitative neuroimaging applications. These include quantifying grey and white matter volumes, monitoring neurological diseases like multiple sclerosis (MS) and Alzheimers disease, and estimating brain atrophy. Over the years several algorithms that automate brain extraction have been proposed. More recently, novel image-to-image deep learning methods have been implemented for this task, and have demonstrated significant gains in accuracy and robustness. However, to our knowledge, none of these algorithms account for the uncertainty that is inherent in brain extraction. Motivated by this, we propose a novel, probabilistic deep learning algorithm for brain extraction that recasts this task as a Bayesian inference problem, and then utilizes a conditional generative adversarial network (cGAN) to solve it. The input to the generator network is an MR image of the head and the output is a collection of images of the brain that are drawn from a probability density conditioned on the input image. These images are used to generate a pixel-wise mean image, which serves as the best guess for an image of the brain, and a pixel-wise standard deviation image, which quantifies the uncertainty in the prediction. We test this algorithm on head MR images of fifty subjects and demonstrate that it is more accurate than a commonly used brain extraction tool, and that its performance compares well with the current state of the art in deep learning algorithms. We also demonstrate the utility of the estimates of uncertainty generated by the algorithm.

sentences: Region-specific alterations of perineuronal net expression in postmortem autism brain tissue Genetic variance in ASD is often associated with mechanisms that broadly fall into the category of neuroplasticity. Parvalbumin positive neurons and their surrounding perineuronal nets (PNNs) are important factors in critical period plasticity and have both been implicated in ASD. PNNs are found in high density within output structures of the cerebellum and basal ganglia, two regions that are densely connected to many other brain areas and have the potential to participate in the diverse array of symptoms present in an ASD diagnosis. The dentate nucleus and globus pallidus were therefore assessed for differences in PNN expression in human postmortem ASD brain tissue. While Purkinje cell loss is a consistent neuropathological finding in ASD, in this cohort, the Purkinje cell targets within the dentate nucleus did not show differences in number of cells with or without a PNN. However, the density of parvalbumin positive neurons with a PNN were significantly reduced in the globus pallidus internus and externus of ASD cases, which was not dependent on seizure status. It is unclear whether these alterations manifest during development or are a consequence of activity-dependent mechanisms that lead to altered network dynamics later in life.

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Application of a novel haplotype-based scan for local adaptation to study high-altitude adaptation in rhesus macaques When natural populations split and migrate to different environments, they may experience different selection pressures that can lead to local adaptation. To capture the genomic patterns of a local selective sweep, we develop XP-nSL, a genomic scan for local adaptation that compares haplotype patterns between two populations. We show that XP-nSL has power to detect ongoing and recently completed hard and soft sweeps, and we then apply this statistic to search for evidence of adaptation to high altitude in rhesus macaques. We analyze the whole genomes of 23 wild rhesus macaques captured at high altitude (mean altitude > 4000m above sea level) to 22 wild rhesus macaques captured at low altitude (mean altitude < 500m above sea level) and find evidence of local adaptation in the high-altitude population at or near 303 known genes and several unannotated regions. We find the strongest signal for adaptation at EGLN1, a classic target for convergent evolution in several species living in low oxygen environments. Furthermore, many of the 303 genes are involved in processes related to hypoxia, regulation of ROS, DNA damage repair, synaptic signaling, and metabolism. These results suggest that, beyond adapting via a beneficial mutation in one single gene, adaptation to high altitude in rhesus macaques is polygenic and spread across numerous important biological systems.

Impact SummaryWhen positive selection is ongoing or a beneficial mutation has recently fixed in a population, genetic diversity is reduced in the vicinity of the adaptive allele, and we expect to observe long homozygous haplotypes at high frequency. Here we develop a statistic that summarizes these expected patterns and compares between two populations in order to search for evidence of adaptation that may have occurred in one but not the other. We implement this statistic in a popular and easy-to-use software package, and then apply it to study adaptation to high altitude in rhesus macaques.

Extreme environments pose a challenge to life on multiple fronts. Very high-altitude environments are one such example, with low atmospheric oxygen, increased ultraviolet light exposure, harsh temperatures, and reduced nutrition availability. In spite of these challenges, many plants and animals, including humans, have genetically adapted to cope with these hardships. Here we study two populations of rhesus macaques, one living at high altitude and one living close to sea level. We apply our novel statistic to compare their haplotype patterns between them to search for evidence of genetic changes that are indicative of adaptation to their environment.

We find evidence for adaptation at a critical gene that helps control physiological response to low-oxygen, one that has been the target of repeated convergent evolution across many species. We also find evidence for positive selection across a range of traits, including metabolic and neurological. This work helps to explain the evolutionary history of the rhesus macaque and furthers our understanding about the ways organisms genetically adapt to high-altitude environments.

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Recombinant binder of sperm protein1 (rec-BSP1) as a potent fertility factor mediates its effect on embryo production ObjectiveTo understand the effect of recombinant BSP1 (rec-BSP1) on in vitro capacitation of sperm and fertilization study

Method(s)Articles were screened for reports including rec-BSP1, Capacitation, in vitro fertilization

InterventionNone

Main Outcome Measure(s)Reproductive outcomes, effect on gametes and embryos

Result(s)Here we report an optimization of condition for rec-BSP1 production which was used for in vitro capacitation and enhancement of buffalo embryo production. The sequence of the protein was used for multiple sequence alignment which has 99% similarity with PDC 109 protein. The expression of rec-BSP1 was carried out successfully with 1 mM IPTG at 160 C for 22 hrs and purified it in soluble form. The structure of rec-BSP1 was generated using 3D modelling and analysed its mode of binding with heparin and PC by molecular docking and the structural stability of rec-BSP1-PC and rec-BSP1-heparin complexes by using molecular dynamic (MD) simulation. The effect of rec-BSP1 was observed on in vitro capacitation of spermatozoa and buffalo blastocyst production. It was found that the rec-BSP1 enhanced the sperm motility at a concentration of 50 g/ml for 1 h of incubation without having any detrimental effect on the sperm morphology and a significant increase in blastocyst production at concentration of 50 g/ml rec-BSP1. Hence this finding represents a new insight and advance the prospective approach to develop a potential fertility factor in reproduction.

Conclusion(s)The purified rec-BSP1 may enhance on male fertility and mediated its effect on in vitro blastocyst production in buffalo.

sentences: Discovering Rare Cell Types through Information-based Dimensionality Reduction Dimensionality reduction is crucial to summarizing the complex transcriptomic landscape of single cell datasets for downstream analyses. However, current dimensionality reduction approaches favor large cellular populations defined by many genes, at the expense of smaller and more subtly-defined populations. Here, we present surprisal component analysis (SCA), a technique that leverages the information-theoretic notion of surprisal for dimensionality reduction, and demonstrate its ability to improve the representation of clinically important populations that are indistinguishable using existing pipelines. For example, in cytotoxic T-cell data, SCA cleanly separates the gamma-delta and MAIT cell subpopulations, which are not detectable via PCA, ICA, scVI, or a wide array of specialized rare cell recovery tools. We also show that, when used instead of PCA, SCA improves downstream imputation to more accurately restore mRNA dropouts and recover important gene-gene relationships. SCAs information-theoretic paradigm opens the door to more meaningful signal extraction, with broad applications to the study of complex biological tissues in health and disease.

sentences: Linking individual differences in human primary visual cortex to contrast sensitivity around the visual field A central question in neuroscience is how the organization of cortical maps relates to perception, for which human primary visual cortex (V1) is an ideal model system. V1 nonuniformly samples the retinal image, with greater cortical magnification (surface area per degree of visual field) at the fovea than periphery and at the horizontal than vertical meridian. Moreover, the size and cortical magnification of V1 varies greatly across individuals. Here, we used fMRI and psychophysics in the same observers to quantify individual differences in V1 cortical magnification and contrast sensitivity at the four polar angle meridians. Across observers, the overall size of V1 and localized cortical magnification positively correlated with contrast sensitivity. Moreover, greater cortical magnification and higher contrast sensitivity at the horizontal than the vertical meridian were strongly correlated. These data reveal a link between cortical anatomy and visual perception at the level of individual observer and stimulus location.

sentences: A novel class of long small RNAs associates with Argonaute1 and is up-regulated by nutrient deprivation in the alga Chlamydomonas Small RNAs (sRNAs) associate with Argonaute (AGO) proteins forming effector complexes with key roles in gene regulation and defense responses against molecular parasites. In multicellular eukaryotes, extensive duplication and diversification of RNA interference (RNAi) components have resulted in intricate pathways for epigenetic control of gene expression. The unicellular alga Chlamydomonas reinhardtii also has a complex RNAi machinery, including three AGOs and three Dicer-like (DCL) proteins. However, little is known about the biogenesis and function of most endogenous sRNAs. We demonstrate here that Chlamydomonas contains uncommonly long sRNAs (>26 nt), which associate preferentially with AGO1. Somewhat reminiscent of animal PIWI-interacting RNAs, these long sRNAs are derived from moderately repetitive genomic clusters and their biogenesis appears to be Dicer-independent. Interestingly, long sRNA encoding sequences have been conserved and amplified in phylogenetically related Chlamydomonas species. Additionally, expression of several long sRNAs increases substantially under nutrient deprivation, correlating with the downregulation of predicted target transcripts. We hypothesize that the transposon-like sequences encoding long sRNAs might have been ancestrally targeted for silencing by the RNAi machinery but, during evolution, some long sRNAs might have fortuitously acquired endogenous target genes and become integrated into gene regulatory networks.

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Neither wood ash application nor phosphorus-fertilisation mitigated the negative effects of whole-tree harvesting on a temperate oligotrophic forest. Background and AimsConcerns about climate change and carbon economy have prompted the promotion of alternative energy sources, including forest-based bioenergy. An evaluation of the environmental consequences of intensive harvests (stumps and roots, and also branches and foliage) for energy wood supply, and use of wood-ash recycling as a compensatory practice, helps in the evaluation of the use of forest biomass for energy production.

MethodsWe made use of records from a split-plot experimental site crossing four different intensities of biomass harvesting (Stem-Only Harvest [SOH], Aboveground Additional Harvest [AAH], Belowground Additional Harvest [BAH], and Whole-Tree Harvest [WTH]) and three compensation methods (control [C], wood ash application [A] and phosphorus fertilisation [P]) to evaluate, in the 11-years-old stand (maritime pine: Pinus pinaster) that followed the biomass exports of the former stand, their effects on nutrient budgets, tree growth, soil fertility, chemical properties and soil carbon. This site is located in a forest on a poor soil, under a warm temperate climate (SW France).

Key resultsDespite their low additional biomass exports (+10% for AAH to +34% for WTH), the non-conventional harvest practices exported much higher quantities of nutrients than the conventional SOH technique (for example +145% for N and K in WTH). Consequently, these treatments had negative effects on the soil nutritive status. Additional biomass harvests impacted the soil organic matter content, with negative effects on P-organic, soil cation exchange capacity, exchangeable Ca, and most extractible nutrients. However, data suggested that tree growth and foliage nutrient content had not yet been significantly impacted by harvest treatments, whereas tree nutritional status was improved by P-fertiliser or wood ash. As expected, we observed a positive effect of wood ash application on soil pH and nutrient content but, like additional harvests, wood ash application decreased the pool of soil organic carbon (~10% of the initial stock with ~7% of N-total losses).

ConclusionsOverall, this factorial experiment showed that exporting more forest biomass due to the additional harvesting of tree canopies, stumps and roots had negative consequences on the ecosystem biogeochemistry. Additional harvests have aggravated the poverty of the already oligotrophic soil, and decreased the soil organic carbon content. Importantly, applying nutrients as fertiliser or wood ash did not compensate for all the negative impacts of biomass exports and the method of wood ash recycling in forests could even decrease the soil organic carbon.

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Mitochondrial One-Carbon Flux has a Growth-Independent Role in Promoting Breast Cancer Metastasis Progression of primary cancer to metastatic disease is the most common cause of death in cancer patients with minimal treatment options available. Canonical drugs target mainly the proliferative capacity of cancer cells, which often leaves slow-proliferating, persistent cancer cells unaffected. Metabolic determinants that contribute to growth-independent functions supporting resistance and metastatic dissemination are still poorly understood.

In the present study, we revealed that antifolate treatment results in an uncoupled and autarkic mitochondrial one-carbon (1C) metabolism allowing sustained serine catabolism and formate overflow when cytosolic 1C metabolism is impaired. Interestingly, antifolate dependent growth-arrest did not correlate with decreased migration capacity. Therefore, using the antifolate Methotrexate as a tool compound allowed us to disentangle proliferation and migration to profile the metabolic phenotype of migrating (growth-arrested) cells. Supported by an increased NAD/NADH ratio, we observed increased serine de novo synthesis and increased serine catabolism to formate.

Consequently, inhibition of serine de novo synthesis using the competitive PHGDH-inhibitor BI-4916 or direct inhibition of mitochondrial 1C metabolism reduced cancer cell migration. Using an orthotopic breast cancer model, we show that sole inhibition of mitochondrial serine catabolism does not affect primary tumor growth but strongly inhibits pulmonary metastasis.

We conclude that mitochondrial 1C metabolism, despite being dispensable for proliferative capacities, confers an advantage to cancer cells by supporting their motility potential.

Our results improve our understanding of 1C metabolism and of metabolic determinants that support the process of cancer cell migration and metastasis.

sentences: The Geometry of Concept Learning Understanding the neural basis of the remarkable human cognitive capacity to learn novel concepts from just one or a few sensory experiences constitutes a fundamental problem. We propose a simple, biologically plausible, mathematically tractable, and computationally powerful neural mechanism for few-shot learning of naturalistic concepts. We posit that the concepts that can be learnt from few examples are defined by tightly circumscribed manifolds in the neural firing rate space of higher order sensory areas. We further posit that a single plastic downstream readout neuron learns to discriminate new concepts based on few examples using a simple plasticity rule. We demonstrate the computational power of our proposal by showing it can achieve high few-shot learning accuracy on natural visual concepts using both macaque inferotemporal cortex representations and deep neural network models of these representations, and can even learn novel visual concepts specified only through linguistic descriptors. Moreover, we develop a mathematical theory of few-shot learning that links neurophysiology to behavior by delineating several fundamental and measurable geometric properties of high-dimensional neural representations that can accurately predict the few-shot learning performance of naturalistic concepts across all our numerical simulations. We discuss testable predictions of our theory for psychophysics and neurophysiological experiments.

sentences: Parallel mechanisms of visual memory formation across distinct regions of the honey bee brain Visual learning is vital to the behavioral ecology of the Western honey bee (Apis mellifera). Honey bee workers forage for floral resources, a behavior that requires the learning and long-term memory of visual landmarks, but how these memories are mapped to the brain remains poorly understood. To address this gap in our understanding, we collected bees that successfully learned visual associations in a conditioned aversion paradigm and compared gene expression correlates of memory formation in the mushroom bodies, a higher-order sensory integration center classically thought to contribute to learning, as well as the optic lobes, the primary visual neuropil responsible for sensory transduction of visual information. We quantitated expression of CREB and CaMKii, two classical genetic markers of learning and fen-1, a gene specifically associated with punishment learning in vertebrates. As expected, we report substantial involvement of the mushroom bodies for all three markers but additionally demonstrate an unexpected involvement of the optic lobes across a similar time course. Our findings imply the molecular involvement of a sensory neuropil during visual associative learning parallel to a higher-order brain region, furthering our understanding of how a tiny brain processes environmental signals.

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Early Dynamics of Chronic Myeloid Leukemia on Nilotinib Predicts Deep Molecular Response Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by the BCR-ABL1 tyrosine kinase1,2. ABL1-selective tyrosine kinase inhibitors (TKIs) including nilotinib have dramatically improved the prognosis of patients with CML3-7. The ultimate goal of CML treatment is likely to be TKI-free maintenance of deep molecular response (DMR), which is defined by quantitative measurement of BCR-ABL1 transcripts on the international scale (IS)8, and durable DMR is a prerequisite to reach this goal9. Thus, an algorithm to enable the early prediction of DMR achievement on TKI therapy is quite valuable. Here, we show that our mathematical framework based on a clinical trial dataset10 can accurately predict the response to frontline nilotinib. We found that our simple dynamical model can predict nilotinib response by using two common laboratory findings (observation values): IS11,12 and white blood cell (WBC) count. Furthermore, our proposed method identified patients who failed to achieve DMR with high fidelity according to the data collected only at three initial time points during nilotinib therapy. Since our model relies on the general properties of TKI response, our framework would be applicable to CML patients who receive frontline nilotinib or other TKIs in clinical practice.

Significance StatementChronic myeloid leukemia (CML) is a myeloproliferative disorder caused by the BCR-ABL1 tyrosine kinase. The goal of this treatment is the sequential achievement of deep molecular response (DMR). Tyrosine kinase inhibitors (TKIs) are effective in the reduction because they inhibit CML cell proliferation. However, because of individual differences in the TKI efficacy, some patients are unable to achieve DMR, so that early prediction of DMR reachability is necessary for personalized medicine. By combining time series analysis and mathematical modeling, we developed a mathematical method that accurately predicts patients who do not achieve DMR in the early stages of TKI administration. Our prediction method gives a basis of effective personalized treatments for CML patients.

sentences: Dynamically Linking Influenza Virus Infection Kinetics, Lung Injury, Inflammation, and Disease Severity Influenza viruses cause a significant amount of morbidity and mortality. Understanding host immune control efficacy and how different factors influence lung injury and disease severity are critical. Here, we established dynamical connections between viral loads, infected cells, CD8+ T cell-mediated clearance, lung injury, inflammation, and disease severity using an integrative model-experiment exchange. The model was validated through CD8 depletion and whole lung histomorphometry, which showed that the infected area matched the model-predicted infected cell dynamics and that the resolved area paralleled the relative CD8 dynamics. Inflammation could further be predicted by the infected cell dynamics, and additional analyses revealed nonlinear relations between lung injury, inflammation, and disease severity. These links between important pathogen kinetics and host pathology enhance our ability to forecast disease progression, potential complications, and therapeutic efficacy.

sentences: DLG2 knockout reveals neurogenic transcriptional programs underlying neuropsychiatric disorders and cognition Brain development requires a complex choreography of cell proliferation, specialisation, migration and network formation, guided by the activation and repression of gene expression programs. It remains unclear how this process is disrupted in neuropsychiatric disorders. Here we integrate human genetics with transcriptomic data from the differentiation of human embryonic stem cells into cortical excitatory neurons. This reveals a cascade of transcriptional programs, activated during early corticoneurogenesis in vitro and in vivo, in which genetic variation is robustly associated with neuropsychiatric disorders and cognitive function. Within these early neurogenic programs, genetic risk is concentrated in loss-of-function intolerant (LoFi) genes, capturing virtually all LoFi disease association. Down-regulation of these programs in DLG2 knockout lines delays expression of cell-type identity alongside marked deficits in neuronal migration, morphology and action potential generation, validating computational predictions. These data implicate specific cellular pathways and neurodevelopmental processes in the aetiology of multiple neuropsychiatric disorders and cognition.

sentences: Versatile genetic toolbox for Prevotella copri enables studying polysaccharide utilization systems Prevotella copri is a prevalent inhabitant of the human gut and has been associated with plant-rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable to P. copri strains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs), and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate that P. copri has evolved two distinct types of arabinan-processing PULs (PULAra) and that the type-II PULAra is significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies for P. copri in the future.

sentences: Demographic feedbacks can hamper the spatial spread of a gene drive AO_SCPLOWBSTRACTC_SCPLOWThis paper is concerned with a reaction-diffusion system modeling the fixation and the invasion in a population of a gene drive (an allele biasing inheritance, increasing its own transmission to offspring). In our model, the gene drive has a negative effect on the fitness of individuals carrying it, and is therefore susceptible of decreasing the total carrying capacity of the population locally in space. This tends to generate an opposing demographic advection that the gene drive has to overcome in order to invade. While previous reaction-diffusion models neglected this aspect, here we focus on it and try to predict the sign of the traveling wave speed. It turns out to be an analytical challenge, only partial results being within reach, and we complete our theoretical analysis by numerical simulations. Our results indicate that taking into account the interplay between population dynamics and population genetics might actually be crucial, as it can effectively reverse the direction of the invasion and lead to failure. Our findings can be extended to other bistable systems, such as the spread of cytoplasmic incompatibilities caused by Wolbachia.

sentences: SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma SARS-CoV-2 501Y.V2 (B.1.351), a novel lineage of coronavirus causing COVID-19, contains substitutions in two immunodominant domains of the spike protein. Here, we show that pseudovirus expressing 501Y.V2 spike protein completely escapes three classes of therapeutically relevant antibodies. This pseudovirus also exhibits substantial to complete escape from neutralization, but not binding, by convalescent plasma. These data highlight the prospect of reinfection with antigenically distinct variants and foreshadows reduced efficacy of spike-based vaccines.

sentences: A Lattice Model on the Rate of DNA Hybridization We develop a lattice model on the rate of hybridization of the complementary single-stranded DNAs (c-ssDNAs). Upon translational diffusion mediated collisions, c-ssDNAs interpenetrate each other to form correct (cc), incorrect (icc) and trap-correct contacts (tcc) inside the reaction volume. Correct contacts are those with exact registry matches which leads to nucleation and zipping. Incorrect contacts are the mismatch contacts which are less stable compared to tcc which can occur in the repetitive c-ssDNAs. Although tcc possess registry match within the repeating sequences, they are incorrect contacts in the view of the whole c-ssDNAs. The nucleation rate (kN) is directly proportional to the collision rate and the average number of correct-contacts (<ncc>) formed when both the c-ssDNAs interpenetrate each other. Detailed lattice model simulations suggest that [<]ncc[>] {propto} L/V where L is the length of c-ssDNAs and V is the reaction volume. Further numerical analysis revealed the scaling for the average radius of gyration of c-ssDNAs (Rg) with their length as [Formula]. Since the reaction space will be approximately a sphere with radius equals to 2Rg and V {propto} L3/2, one obtains [Formula]. When c-ssDNAs are nonrepetitive, then the overall renaturation rate becomes as kR {propto} kNL and one finally obtains [Formula] in line with the experimental observations. When c-ssDNAs are repetitive with a complexity of c, then earlier models suggested the scaling [Formula][Formula] which breaks down at c = L. This clearly suggested the existence of at least two different pathways of renaturation in case of repetitive c-ssDNAs viz. via incorrect contacts and trap correct contacts. The trap correct contacts can lead to the formation of partial duplexes which can keep the complementary strands in the close vicinity for a prolonged timescale. This is essential for the extended 1D slithering, inchworm movements and internal displacement mechanisms which can accelerate the searching for the correct contacts. Clearly, the extent of slithering dynamics will be inversely proportional to the complexity. When the complexity is close to the length of c-ssDNAs, then the pathway via incorrect contacts will dominate. When the complexity is much lesser than the length of c-ssDNA, then pathway via trap correct contacts would be the dominating one.

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Turning movements in real life capture subtle longitudinal and preataxic changes in cerebellar ataxia OBJECTIVESClinical and regulatory acceptance of upcoming molecular treatments in degenerative ataxias might greatly benefit from ecologically valid endpoints which capture change in ataxia severity in patients real life. This longitudinal study aimed to unravel quantitative motor biomarkers in degenerative ataxias in real life turning movements which are sensitive for changes both longitudinally and at the preataxic stage.

METHODSCombined cross-sectional (n=30) and longitudinal (n=14, 1-year interval) observational study in degenerative cerebellar disease (including 8 pre-ataxic mutation carriers) compared to 23 healthy controls. Turning movements were assessed by three body-worn inertial sensors in three conditions: (1) instructed laboratory assessment, (2) supervised free walking, and (3) unsupervised real-life movements.

RESULTSMeasures which quantified dynamic balance during turning - lateral velocity change (LVC) and outward acceleration -, but not general turning measures such as speed, allowed differentiating ataxic against healthy subjects in real life with high effect size ({delta}=0.68), with LVC also differentiating preataxic against healthy subjects ({delta}=0.53). LVC was highly correlated with clinical ataxia severity (SARA score, effect size {rho}=0.79) and subjective balance confidence (ABC score, {rho}=0.66). Moreover, LVC in real life - but not general turning measures, gait measures, or the SARA score - allowed detecting significant longitudinal change in one-year follow-up with high effect size (rprb=0.66).

CONCLUSIONSMeasures of turning allow to capture specific changes of dynamic balance in degenerative ataxia in real life, with high sensitivity to longitudinal differences in ataxia severity and to the preataxic stage. They thus present promising ecologically valid motor biomarkers for capturing change in real life, even in the highly treatment-relevant early stages of degenerative cerebellar disease.

sentences: Predicting the retinotopic organization of human visual cortex from anatomy using geometric deep learning Whether it be in a single neuron or a more complex biological system like the human brain, form and function are often directly related. The functional organization of human visual cortex, for instance, is tightly coupled with the underlying anatomy with cortical shape having been shown to be a useful predictor of the retinotopic organization in early visual cortex. Although the current state-of-the-art in predicting retinotopic maps is able to account for gross individual differences, such models are unable to account for any idiosyncratic differences in the structure-function relationship from anatomical information alone due to their initial assumption of a template. Here we developed a geometric deep learning model capable of exploiting the actual structure of the cortex to learn the complex relationship between brain function and anatomy in human visual cortex such that more realistic and idiosyncratic maps could be predicted. We show that our neural network was not only able to predict the functional organization throughout the visual cortical hierarchy, but that it was also able to predict nuanced variations across individuals. Although we demonstrate its utility for modeling the relationship between structure and function in human visual cortex, our approach is flexible and well-suited for a range of other applications involving data structured in non-Euclidean spaces.

sentences: Coordinated changes in glycosylation regulate the germinal centre through CD22 Germinal centres (GC) are sites of B-cell expansion and selection, which are essential for antibody affinity maturation. Compared to naive follicular B-cells, GC B-cells have several notable changes in their cell surface glycans. While these changes are routinely used to identify the GC, functional roles for these changes have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a reduction in the glycan ligands for CD22, which is an inhibitory co-receptor of the B-cell receptor (BCR). To test a functional role for downregulated CD22 ligands in the GC, we generated a mouse model that maintains CD22 ligands on GC B-cells. With this model, we demonstrate that glycan remodeling is crucial for proper GC B-cell response, including plasma cell output and affinity maturation of antibodies. The defect we observe in this model is dependent on CD22, highlighting that coordinated downregulation of CD22 ligands on B cells plays a critical function in the GC. Collectively, our study uncovers a crucial role for glycan remodeling and CD22 in B-cell fitness in the GC.

sentences: CD8+ T Cell Metabolic Rewiring Defined by Single-Cell RNA-Sequencing Identifies a Critical Role of ASNS Expression Dynamics in T Cell Differentiation Cytotoxic T cells dynamically rewire their metabolism during the course of an immune response. While T-cell metabolism has been extensively studied at phenotypic endpoints of activation and differentiation, the underlying dynamics remain largely elusive. Here, we leverage on single-cell RNA-sequencing (scRNA-seq) measurements of in vitro activated and differentiated CD8+ T cells cultured in physiological media to resolve these metabolic dynamics. We find that our scRNA-seq analysis identifies most metabolic changes previously defined in in vivo experiments, such as a rewiring from an oxidative to an anabolism-promoting metabolic program during activation to an effector state, which is later reverted upon memory polarization. Importantly, our scRNA-seq data further provide a dynamic description of these changes. In this sense, our data predict a differential time-dependent reliance of CD8+ T cells on the synthesis versus uptake of various non-essential amino acids during T-cell activation, which we corroborate with additional functional in vitro experiments. We further exploit our scRNA-seq data to identify metabolic genes that could potentially dictate the outcome of T-cell differentiation, by ranking them based on their expression dynamics. Among the highest-ranked hits, we find asparagine synthetase (Asns), whose expression sharply peaks for effector CD8+ T cells and further decays towards memory polarization. We then confirm that these in vitro Asns expression dynamics are representative of an in vivo situation in a mouse model of viral infection. Moreover, we find that disrupting these expression dynamics in vitro, by depleting asparagine from the culture media, delays central-memory polarization. Accordingly, we find that preventing the decay of ASNS by stable overexpression at the protein level in vivo leads to a significant increase in effector CD8+ T-cell expansion, and a concomitant decrease in central-memory formation, in a mouse model of viral infection. This shows that ASNS expression dynamics dictate the fate of CD8+ T-cell differentiation. In conclusion, we provide a resource of dynamic expression changes during CD8+ T-cell activation and differentiation that is expected to increase our understanding of the dynamic metabolic requirements of T cells progressing along the immune response cascade.

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MetaFun: Unveiling sex differences in multiple omics studies through comprehensive functional meta-analysis SummarySex and gender differences in different health scenarios has been thoroughly acknowledged in the literature, and yet, very scarcely analyzed. To fill the gap, here we present MetaFun, which allows to meta-analyze multiple omics datasets with a sex-based perspective, and to combine different datasets to gain major statistical power and to assist the researcher in understanding these sex differences in the diseases under study. Metafun is freely available at bioinfo.cipf.es/metafun Availability and implementation: MetaFun is available under http://bioinfo.cipf.es/metafun. The backend has been implemented in R and Java and the frontend has been developed using Angular.

Supplementary informationR code available at https://gitlab.com/ubb-cipf/metafunr

sentences: Evaluation of an optimized protocol and Illumina ARTIC V4 primer pool for sequencing of SARS-CoV-2 using COVIDSeq™ and DRAGEN™ COVID Lineage App workflow Next-Generation Sequencing based genomic surveillance has been widely implemented for identification and tracking of emerging SARS-CoV-2 variants to guide the Public Health response to the COVID-19 pandemic. Amplicon-based assays, such as the Illumina(R) COVIDSeq Test (RUO) and COVIDSeq Assay (RUO), enable scalable sequencing of SARS-CoV-2, leveraging V3 and V4 primer designs from the ARTIC community and DRAGEN COVID Lineage App analysis available on Illumina BaseSpace. We report here a comparison of COVIDSeq performance for SARS-CoV-2 genome reporting using the ARTIC V3 based primer pool (including primers for human control genes) that is provided with the COVIDSeq kit versus the ARTIC V4 based Illumina COVIDSeq V4 primer pool, using an optimized protocol and DRAGEN COVID Lineage App analysis. The data indicates that both primer pools enable robust reporting of SARS-CoV-2 variants. The Illumina COVIDSeq V4 primer pool has superior performance for SARS-CoV-2 genome reporting, particularly in samples with low virus load, and is therefore the recommended primer pool for genomic surveillance of SARS-CoV-2 for research use using COVIDSeq.

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Metastatic Single Tumor Cells Evade NK Cell-mediated Killing by Thrombin-mediated Loss of the Activating Ligand CD155/PVR/Necl-5 Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is not known. Here we have combined ultra-sensitive bioluminescence whole body imaging with intravital two-photon microscopy involving genetically-encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hrs of arrival, but not thereafter. Intravital dynamic imaging revealed that a disseminated tumor cell in a pulmonary capillary interacts with an NK cell every 2 hrs on average. In the first 4 hrs after tumor cell arrival, 50% of such encounters lead to tumor cell death but after 24 hrs of arrival, nearly 100% of the interactions result in the survival of the tumor cell. This evasion of NK cell surveillance is mediated by thrombin-dependent loss of cell surface CD155/PVR/Necl-5, a ligand for the NK cell activating receptor DNAM-1. This loss prevents the NK cell signaling needed for effective killing of tumor targets. By quantitatively visualizing the evasion of NK cell surveillance, we have uncovered a molecular mechanism for cancer evasion and provided an explanation for the anti-metastatic effect of anticoagulants.

SUMMARYIntravital functional two-photon microscopy reveals that metastatic tumor cells lodged in pulmonary capillaries acquire resistance to patrolling NK cells. Protease-mediated loss of the activating ligand CD155/PVR/Necl-5 on tumor cells prevents NK cells from ERK activation and tumor cell killing.

sentences: A genome in flux: homoeologous exchanges, subgenome dominance, and gene dosage balance constraints in resynthesized allopolyploid Brassica napus Allopolyploidy involves the hybridization of two evolutionary diverged species and the doubling of genomic material. Frequently, allopolyploids exhibit genomic rearrangements that recombine, duplicate, or delete homoeologous regions of the newly formed genome. While decades of investigation have focused on how genome duplication leads to systematic differences in the retention and expression of duplicate genes, the impact of genomic rearrangements on genome evolution has received less attention. We used genomic and transcriptomic data for six independently resynthesized, isogenic Brassica napus lines in the first, fifth, and tenth generation to identify genomic rearrangements and assess their impact on gene expression dynamics related to subgenome dominance and gene dosage constraint. We find that dosage constraints on the gene expression response to polyploidy begin to loosen within the first ten generations of evolution and systematically differ between dominant and non-dominant subgenomes. We also show that genomic rearrangements can bias estimation of homoeolog expression bias, but fail to fully obscure which subgenome is dominantly expressed. Finally, we demonstrate that dosage-sensitive genes exhibit the same kind of coordinated response to homoeologous exchange as they do for genome duplication, suggesting constraint on dosage balance also acts on these changes to gene dosage.

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Changes in Iris Stiffness and Permeability in Primary Angle Closure Glaucoma PurposeTo evaluate the biomechanical properties of the iris by evaluating iris movement during pupil constriction and to compare such properties between healthy and primary angle-closure glaucoma (PACG) subjects.

MethodsA total of 140 subjects were recruited for this study. In a dark room, the anterior segments of one eye per subject were scanned using anterior segment optical coherence tomography (AS-OCT, SS-1000 CASIA, Tomey Corporation, Nagoya, Japan) imaging during induced pupil constriction with an external white light source of 1700 lux. Using a custom segmentation code, we automatically isolated the iris segments from the AS-OCT images, which were then discretized and transformed into a three-dimensional point cloud. For each iris, a finite element (FE) mesh was constructed from the point cloud, and an inverse FE simulation was performed to match the clinically observed iris constriction in the AS-OCT images. Through this optimization process, we were able to identify the elastic modulus and permeability of each iris.

ResultsFor all 140 subjects (95 healthy and 45 PACG of Indian/Chinese ethnicity, Age: 60.2{+/-}8.7 for PACG subjects and 57.7{+/-}10.1 for healthy subjects), the simulated deformation pattern of the iris during pupil constriction matched well with OCT images. We found that the iris stiffness was higher in PACG than in healthy controls (24.5{+/-}8.4 kPa vs 17.1{+/-}6.6 kPa with 40 kPa of active stress specified in the sphincter region; p < 0.001), whereas iris permeability was lower (0.41{+/-}0.2 mm2/kPa.s vs 0.55{+/-}0.2 mm2/kPa.s; p = 0.142).

ConclusionThis study suggests that the biomechanical properties of the iris in PACG are different from those in healthy controls. An improved understanding of the biomechanical behavior of the iris may have implications for the understanding and management of angle-closure glaucoma.

sentences: Scarf: A toolkit for memory efficient analysis of large-scale single-cell genomics data The increasing capacity to perform large-scale single-cell genomic experiments continues to outpace the computational requirements to efficiently handle growing datasets. Herein we present Scarf, a modularly designed Python package that seamlessly interoperates with other single-cell toolkits and allows for memory-efficient single-cell analysis of millions of cells on a laptop or low-cost devices like single board computers. We demonstrate Scarfs memory and compute-time efficiency by applying it to the largest existing single-cell RNA-Seq and ATAC-Seq datasets. Scarf wraps memory-efficient implementations of a graph-based t-stochastic neighbour embedding and hierarchical clustering algorithm. Moreover, Scarf performs accurate reference-anchored mapping of datasets while maintaining memory efficiency. By implementing a novel data downsampling algorithm, Scarf additionally can generate representative sampling of cells from a given dataset wherein rare cell populations and lineage differentiation trajectories are conserved. Together, Scarf provides a framework wherein any researcher can perform advanced processing, downsampling, reanalysis, and integration of atlas-scale datasets on standard laptop computers.

sentences: Synthetic glabridin derivatives mitigate steatohepatitis in a diet-induced biopsy-confirmed non-alcoholic steatosis hepatitis mouse model through paraoxonase-2 Limited therapeutic agents have been developed for non-alcoholic steatohepatitis (NASH), a common immunometabolic disease that can progress to hepatic cirrhosis and cancer. Glabridin and its derivatives are potential therapeutics for some metabolic diseases. However, the therapeutic effects of glabridin and its derivatives on NASH and their biological functions are unclear. This study demonstrated the role of synthetic glabridin derivatives (SGDs) in alleviating hepatic steatosis and inflammation in a biopsy-confirmed rodent NASH model. SGDs exerted therapeutic effects by activating autophagy and the antioxidant defense system, which mitigate NASH pathogenesis. The cellular target of HSG4112, an SGD, was paraoxonase 2. These findings will enable the development of novel therapeutics for NASH in the future.

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Inverse design of cholesterol attracting transmembrane helices reveals a paradoxical role of hydrophobic length The occurrence of linear cholesterol-recognition motifs in alpha-helical transmembrane domains has long been debated. Here, we demonstrate the ability of a genetic algorithm guided by coarse-grained molecular dynamics simulations--a method coined evolutionary molecular dynamics (evo-MD)--to directly resolve the sequence which maximally attracts/sorts cholesterol within a single-pass alpha-helical transmembrane domain (TMDs). We illustrate that the evolutionary landscape of cholesterol attraction in membrane proteins is characterized by a sharp, well-defined global optimum. Surprisingly, this optimal solution features an unusual short hydrophobic block, consisting of typically only eight short chain hydrophobic amino acids, surrounded by three successive lysines. Owing to the membrane thickening effect of cholesterol, cholesterol-enriched ordered phases favor TMDs characterized by a long rather than a short hydrophobic length. However, this short hydrophobic pattern evidently offers a pronounced net advantage for the binding of free cholesterol in both coarse-grained and atomistic simulations. Attraction is mediated by the unique ability of cholesterol to snorkel within the hydrophobic core of the membrane and thereby shield deeply located lysines from the unfavorable hydrophobic surrounding. Since this mechanism of attraction is of a thermodynamic nature and is not based on molecular shape specificity, a large diversity of sub-optimal cholesterol attracting sequences can exist. The puzzling sequence variability of proposed linear cholesterol-recognition motifs is thus consistent with sub-optimal, unspecific binding of cholesterol. Importantly, since evo-MD uniquely enables the targeted design of recognition motifs for distinct fluid lipid membranes, we foresee wide applications for evo-MD in the biological and biomedical fields.

Significance StatementOur work demonstrates how a synergy between evolutionary algorithms and high-throughput coarse-grained molecular dynamics can yield fundamentally new insights into the evolutionary fingerprints of protein-mediated lipid sorting. We illustrate that the evolutionary landscape of cholesterol attraction in isolated transmembrane domains is characterized by a sharp, well-defined global optimum. In contrast, sub-optimal attraction of cholesterol is associated with a diverse solution space and features a high sequence variability despite binding the same unique molecule. The contrasting physicochemical nature of the resolved attraction optimum suggests that cholesterol attraction/binding via linear motifs does not pose a dominant pressure/constrain on the evolution of transmembrane proteins.

sentences: Viscoelasticity and Noise Properties Reveal the Formation of Biomemory in Cells Living cells are neither perfectly elastic nor liquid and return a viscoelastic response to external stimuli. Nanoindentation provides force distance curves allowing the investigation of cell mechanical properties, and yet, these curves can differ from point to point on cell surface revealing its inhomogeneous character. In the present work, we propose a mathematical method to estimate both viscoelastic and noise properties of cells, as these are depicted on the values of the scaling exponents of relaxation function and power spectral density respectively. The method uses as input the time derivative of the response force in a nanoindentation experiment. Generalized moments method and/or rescaled range analysis are used to study the resulting time series depending on their non-stationary or stationary nature. We conducted experiments in living Ulocladium Chartarum spores. We found that spores, in the approaching phase present a viscoelastic behavior with the corresponding scaling exponent in the range 0.25-0.52, and in the retracting phase present a liquid-like behavior with exponents in the range 0.67-0.85. This substantial difference of the scaling exponents in the two phases suggests the formation of biomemory as response of the spores to the indenting AFM mechanical stimulus. The retracting phase may be described as a process driven by bluish noises, while the approaching one is driven by persistent noise.

sentences: Microgravity enhances the phenotype of Arabidopsis zigzag-1 and reduces the Wortmannin-induced vacuole fusion in root cells The spaceflight environment of the International Space Station poses a multitude of stresses on plant growth including reduced gravity. Plants exposed to microgravity and other conditions on the ISS display root skewing, changes in gene expression and protein abundance that may result in changes in cell wall composition, antioxidant accumulation and modification of growth anisotropy. Systematic studies that address the effects of microgravity on cellular organelles are lacking but altered numbers and sizes of vacuoles have been detected in previous flights. The prominent size of plant vacuoles makes them ideal models to study organelle dynamics in space. Here, we used Arabidopsis zigzag1 (zig-1) as a sensitized genotype to study the effect of microgravity on plant vacuole fusion. Wortmannin was used to induce vacuole fusion in seedlings and a formaldehyde-based fixation protocol was developed to visualize plant vacuole morphology after sample return, using confocal microscopy. Our results indicate that microgravity enhances the zig-1 phenotype by reducing hypocotyl growth and vacuole fusion in some cells. This study demonstrates the feasibility of chemical inhibitor treatments for plant cell biology experiments in space.

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Damaraland mole-rats are not obligate cooperative breeders Damaraland mole-rats (Fukomys damarensis) are usually viewed as a eusocial or obligate cooperative breeder in which successful reproduction is dependent on help from closely related group members. However, because longitudinal studies of mole-rats in their natural environment are uncommon, the extent to which successful reproduction by breeders relies on assistance from other group members is unclear, and for non-breeders, the immediate and delayed fitness consequences of staying and helping are poorly understood. Using data from a 7-year field study of marked individuals, we investigate whether increasing group size confers reproductive and survival benefits to breeders and non-breeders, and explore the different routes through which individuals acquire breeding positions. We show that solitary natal dispersal was the most common route to breeding for both sexes and that the inheritance of dominant breeding positions was uncommon in both sexes. After dispersing, females typically settled alone in new burrow systems where they enjoyed high survival rates and remained in good body condition - often for several years - before being joined by males. In contrast to most obligately cooperative species, pairs of potential breeders reproduced successfully without helpers and experimentally formed pairs had the same reproductive success as larger established groups. Though larger breeding groups recruited slightly more pups on average, our data suggest that neither survival nor reproduction depend on the presence of non-breeding helpers, indicating that Damaraland mole-rats are not obligate cooperative breeders. We suggest that extended philopatry and group living in Damaraland mole-rats have evolved because of the high costs and constraints of dispersal rather than because of strong indirect benefits accrued through cooperative behaviour and that similarities between their breeding systems and those of obligatorily eusocial insects have been over-emphasized.

Significance StatementThe social mole-rats are often considered eusocial mammals in which successful reproduction depends on assistance from non-breeding helpers. In this study we show that in wild Damaraland mole-rats, the presence of non-breeders is associated with both costs and benefits and that nascent breeding pairs show high reproductive success despite the lack of non-breeding helpers. These findings indicate that Damaraland mole-rats are not obligate cooperative breeders and suggest that similarities between their breeding systems and those of obligatorily eusocial insects have been over-emphasized.

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Microbiota-dependent inflammation promotes metabolic disorder via NF-kB-mediated inhibition of SREBP in Drosophila adipocytes Bacteria that colonize eukaryotic gut have profound influences on the physiology of their host. In Drosophila, many of these effects are mediated by the adipocytes that combine immune and metabolic functions. We show here that gut colonization by specific bacteria species stimulate lipogenesis in surrounding enterocytes but also in remote fat body cells and ovaries. This bacteria-dependent lipid production is mediated by SREBP and requires a functional insulin signaling. However, it is antagonized by microbiota-born peptidoglycan which by activating NF-{kappa}B signaling, cell-autonomously represses SREPP activation in adipocytes but not in enterocytes. We finally show that by reducing microbiota-derived PGN, the gut-produced PGRP-LB amidase balances host immune and metabolic responses of the fat body to gut-associated bacteria. In the absence of such modulation, uncontrolled immune pathway activation prevents lipid production by the fat body resulting in infection-dependent host death.

BulletsO_LIGut microbiota activates lipogenesis locally in enterocytes and remotely in adipocytes
C_LIO_LIBacteria-dependent activation of SREBP in adipocytes is mediated by insulin signaling
C_LIO_LIActivation of lipid synthesis by microbiota is antagonized by NF-{kappa}B signaling triggered by gut-born peptidoglycan
C_LIO_LIBy reducing peptidoglycan circulating levels, PGRP-LB maintains a balance between immune and metabolic response to microbiota
C_LI

sentences: Colour polymorphism associated with a gene duplication in male wood tiger moths Colour is often used as an aposematic warning signal, with predator learning expected to lead to a single colour pattern within a population. However, there are many puzzling cases where aposematic signals are also polymorphic. The wood tiger moth, Arctia plantaginis, uses bright hindwing colours as a signal of unpalatability, and males have discrete colour morphs which vary in frequency geographically. In Finland, both white and yellow morphs can be found, and these colour morphs also differ in behavioural and life-history traits. Complex polymorphisms such as these are often explained by supergenes. Here, we show that male colour is linked to an extra copy of a yellow family gene that is only present in the white morphs. This white-specific duplication, which we name valkea, is highly upregulated during wing development, and could act to reduce recombination, thus potentially representing a supergene. We also characterise the pigments responsible for yellow, white and black colouration, showing that yellow is partly produced by pheomelanins, while black is dopamine-derived eumelanin. The yellow family genes have been linked to melanin synthesis and behavioural traits in other insect species. Our results add to only a few examples of seemingly paradoxical and complex polymorphisms which are associated with single genes.

sentences: Caulobacter requires anionic sphingolipids and deactivation of fur to lose lipid A Lipid A, the membrane-anchored portion of lipopolysaccharide, is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here, we identify regulatory and structural factors that together permit Caulobacter crescentus to eliminate lipid A from its OM. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog which we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to eliminate lipid A. Fitness of lipid A-deficient Caulobacter requires a previously uncharacterized anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

sentences:
Defining the scope of the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet): a bottom-up and One Health approach BackgroundBuilding the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) was proposed to strengthen the European One Health antimicrobial resistance (AMR) surveillance approach.

ObjectivesThe objectives were to (i) define the combinations of animal species, production types, age categories, bacterial species, specimens and antimicrobials to be monitored in EARS-Vet and to (ii) determine antimicrobial test panels able to cover most combinations.

MethodsThe EARS-Vet scope was defined by consensus between 26 European experts. Decisions were guided by a survey of the combinations that are relevant and feasible to monitor in diseased animals in 13 European countries (bottom-up approach). Experts also considered the One Health approach and the need for EARS-Vet to complement existing European AMR monitoring systems coordinated by the European Centre for Disease Prevention and Control (ECDC) and the European Food Safety Authority (EFSA).

ResultsEARS-Vet would monitor AMR in six animal species (cattle, swine, chicken (broiler and laying hen), turkey, cat and dog), for 11 bacterial species (Escherichia coli, Klebsiella pneumoniae, Mannheimia haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus hyicus, Streptococcus uberis, Streptococcus dysgalactiae and Streptococcus suis). Relevant antimicrobials for their treatment were selected (e.g. tetracyclines) and complemented with antimicrobials of more specific public health interest (e.g. carbapenems). Three test panels of antimicrobials were proposed covering most EARS-Vet combinations of relevance for veterinary antimicrobial stewardship.

ConclusionsWith this scope, EARS-Vet would enable to better address animal health in the strategy to mitigate AMR and better understand the multi-sectoral AMR epidemiology in Europe.

sentences: Phylogeny and Metadata Network Database for Epidemiologic Surveillance AO_SCPLOWBSTRACTC_SCPLOWThe ongoing SARS-CoV-2 pandemic has highlighted the difficulty in integrating disparate data sources for epidemiologic surveillance. To address this challenge, we have created a graph database to integrate phylogenetic trees, associated metadata, and community surveillance data for phylodynamic inference. As an example use case, we divided 22,713 SARS-CoV-2 samples into 5 groups, generated maximum likelihood trees, and inferred a potential transmission network from a forest of minimum spanning trees built on patristic distances between samples. We then used Cytoscape to visualize the resultant graphs.

sentences: A Single Cell Atlas of Lung Development Lung organogenesis requires precisely timed shifts in the spatial organization and function of parenchymal cells, especially during the later stages of lung development. To investigate the mechanisms governing lung parenchymal dynamics during development, we performed a single cell RNA sequencing (scRNA-seq) time-series yielding 92,238 epithelial, endothelial, and mesenchymal cells across 8 time points from embryonic day 12 (E12) to postnatal day 14 (P14) in mice. We combined new computational analyses with RNA in situ hybridization to explore transcriptional velocity, fate likelihood prediction, and spatiotemporal localization of cell populations during the transition between the saccular and alveolar stages. We interrogated this atlas to illustrate the complexity of type 1 pneumocyte function during the saccular and alveolar stages, and we demonstrate an integrated view of the cellular dynamics during lung development.

sentences: Bacterial filamentation drives colony chirality Chirality is ubiquitous in nature, with consequences at the cellular and tissue scales. As Escherichia coli colonies expand radially, an orthogonal component of growth creates a pinwheel-like pattern that can be revealed by fluorescent markers. To elucidate the mechanistic basis of this colony chirality, we investigated its link to left-handed, single-cell twisting during E. coli elongation. While chemical and genetic manipulation of cell width altered single-cell twisting handedness, colonies ceased to be chiral rather than switching handedness, and anaerobic growth altered colony chirality without affecting single-cell twisting. Chiral angle increased with increasing temperature even when growth rate decreased. Unifying these findings, we discovered that colony chirality was associated with the propensity for cell filamentation. Inhibition of cell division accentuated chirality under aerobic growth and generated chirality under anaerobic growth. Thus, regulation of cell division is intrinsically coupled to colony chirality, providing a mechanism for tuning macroscale spatial patterning.

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PDZ-containing proteins targeted by the ACE2 receptor Angiotensin converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residues segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein interacting domains called PSD-95/Dlg/ZO-1 (PDZ).

Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants values ranging from 3 to 81 M. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role on ACE2 internalization and recycling that could benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in neurological symptoms of COVID-19.

sentences: Exploring Parallel MPI Fault Tolerance Mechanisms for Phylogenetic Inference with RAxML-NG Phylogenetic trees are now routinely inferred on large scale HPC systems with thousands of cores as the parallel scalability of phylogenetic inference tools has improved over the past years to cope with the molecular data avalanche. Thus, the parallel fault tolerance of phylogenetic inference tools has become a relevant challenge. To this end, we explore parallel fault tolerance mechanisms and algorithms, the software modifications required, and the performance penalties induced via enabling parallel fault tolerance by example of RAxML-NG, the successor of the widely used RAxML tool for maximum likelihood based phylogenetic tree inference. We find that the slowdown induced by the necessary additional recovery mechanisms in RAxML-NG is on average 2%. The overall slowdown by using these recovery mechanisms in conjunction with a fault tolerant MPI implementation amounts to 8% on average for large empirical datasets. Via failure simulations, we show that RAxML-NG can successfully recover from multiple simultaneous failures, subsequent failures, failures during recovery, and failures during checkpointing. Recoveries are automatic and transparent to the user. The modified fault tolerant RAxML-NG code is available under GNU GPL at https://github.com/lukashuebner/ft-raxml-ng Contact: lukas.huebner@{kit.edu,h-its.org};, alexey.kozlov@h-its.org, hespe@kit.edu, sanders@kit.edu, alexandros.stamatakis@hits.org Supplementary information: Supplementary data are available at bioR{chi}iv.

sentences: Host stress hormones affect host, but not vector, infectiousness for West Nile virus Hormones that help hosts cope with stressors also affect how hosts regulate the processes that influence their susceptibility to parasites as well as their propensity to transmit pathogens to other hosts and vectors. In birds, corticosterone (CORT), influences timing of activity, feeding behaviors, and various immune defenses that influence the number and outcomes of host interactions with vectors and parasites. No study to our knowledge, though, has investigated whether CORT in hosts affects the extrinsic incubation period (EIP) of a vector for a virus, one of the strongest drivers of vector-borne disease cycles. Our goal here was to discern whether experimental CORT alterations in zebra finches (Taeniopygia guttata) affected EIP for West Nile virus (WNV) in the mosquito, Culex quinquefasciatus, a common vector of WNV and other infections in the southern US. We experimentally manipulated CORT in birds, infected them with WNV, and then investigated whether EIP differed between vectors fed on CORT-treated or control birds. Although CORT enhanced WNV viremia in hosts, as we have observed previously, we found no effects of CORT on vector EIP or post-feeding mortality rates, another important component of epidemiological models. These results, plus our prior observations that CORT enhances host attractiveness, indicate that some but not all stages of host-vector-virus interactions are sensitive to host stress.

sentences: Two-pore channel 2 is a key regulator of adipocyte differentiation via the cAMP signaling pathway with calpain as downstream effector We investigated whether the endolysosomal two-pore channel TPC2 is a mediator of adipocyte differentiation. We show that Tpcn2 mRNA is expressed transiently during induction of C3H10T1/2 mesenchymal stem cells to differentiate into adipocytes, and that this expression is triggered by cAMP. This is the first demonstration of a cell signaling pathway that can regulate TPC gene expression. We also identified an important functional role for TPC2 in adipocyte differentiation. First, ectopic TPC2 expression in C3H10T1/2 cells partially rescued the block to adipocyte differentiation caused by cAMP absence. Second, inhibition of endogenous TPC2 expression in primary preadipocytes substantially reduced their ability to differentiate into adipocytes. Finally, genetic variation at the Tpcn2 locus is associated with increased upper-body fat distribution in women concomitant with reduced Tpcn2 expression in abdominal adipose tissue. Our findings implicate TPC2 as an important mediator of adipogenesis and may aid identification of new drug targets for treatment of obesity.

sentences: Effectiveness and relationship between biased and unbiased measures of dopamine release and clearance Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine (DA) release and clearance throughout the brain, including the ventral and dorsal striatum. Striatal DA terminals are abundant with signals heavily regulated by release machinery and the dopamine transporter (DAT). Peak height is a common method for measuring release but can be affected by changes in clearance. The Michaelis-Menten model has been a standard in measuring DA clearance, but requires experimenter fitted modeling subject to experimenter bias. The current study presents the use of the first derivative (velocity) of evoked DA signals as an alternative approach for measuring dopamine release and clearance and can be used to distinguish the two measures. Maximal upwards velocity predicts reductions in DA peak height due to D2 and GABAB receptor stimulation and by alterations in calcium concentrations. The Michaelis-Menten maximal velocity (Vmax) measure, an approximation for DAT numbers, predicted maximal downward velocity in slices and in vivo. Dopamine peak height and upward velocity were similar between wildtype C57 (WT) and DAT knock out (DATKO) mice. In contrast, downward velocity was considerably reduced and exponential decay (tau) was increased in DATKO mice, supporting use of both measures for changes in DAT activity. In slices, the competitive DAT inhibitors cocaine, PTT and WF23 increased peak height and upward velocity differentially across increasing concentrations, with PTT and cocaine reducing these measures at high concentrations. Downward velocity and tau values decreased and increased respectively across concentrations, with greater potency and efficacy observed with WF23 and PTT. In vivo recordings demonstrated similar effects of WF23 and PTT on measures of release and clearance. Tau was a more sensitive measure at low concentrations, supporting its use as a surrogate for the Michaelis-Menten measure of apparent affinity (Km). Together, these results inform on the use of these measures for DA release and clearance.

sentences: Decoding silence in free recall In experiments on free recall from lists of items, not all memory retrievals are necessarily reported. Previous studies investigated unreported retrievals by attempting to induce their externalization. We show that, without any intervention, their statistics may be directly estimated through a model-free analysis of inter-response times - the silent intervals between recalls. A delay attributable to unreported recalls emerges in three situations: if the final item was already recalled ("silent recency effect"); if the item that, within the list, follows the latest recalled item was already recalled ("silent contiguity effect"); and in sequential recalls within highly performing trials ("sequential slowdown"). We then turn to reproducing all these effects by a minimal model where the discarding of memories ("bouncing") occurs either if they are repetitious or, in strategically organized trials, if they are not sequential. Based on our findings, we propose various approaches to further probing the submerged dynamics of memory retrieval.

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Seed morphological traits as a tool to quantify variation maintained in ex situ collections: a case study in Pinus torreyana (Parry) BackgroundUnderstanding the within- and among-population distribution of trait variation within seed collections may provide a means to approximate standing genetic variation and inform plant conservation.

AimsThis study aimed to estimate population- and family-level seed trait variability for existing seed collections of Torrey pine (Pinus torreyana), and to use these data to guide sampling of future collections.

MethodsWe quantified variation in 14 seed morphological traits and seedling emergence within and among Torrey pine populations. Using a simulation-based approach, we used estimates of within-population variance to assess the number of maternal families required to capture 95% of trait variation within each existing seed collection.

ResultsSubstantial structure was observed both within and among Torrey pine populations, with island and mainland seeds varying in seed size and seed coat thickness. Despite morphological differences, seedling emergence was similar across populations. Simulations revealed that 83% and 71% of all maternal families within island and mainland seed collections respectively needed to be resampled to capture 95% of seed trait variation within existing collections.

ConclusionsFrom a conservation perspective, our results indicate that to optimize genetic diversity captured in Torrey pine seed collections, maximizing the number of maternal families sampled within each population will be necessary.

sentences: Persistence of Hippocampal and Striatal Multivoxel Patterns during Awake Rest after Motor Sequence Learning Memory consolidation, the process by which newly encoded and fragile memories become more robust, is thought to be supported by the reactivation of brain regions - including the hippocampus - during post-learning rest. While hippocampal reactivations have been demonstrated in humans in the declarative memory domain, it remains unknown whether such a process takes place after motor learning. Using multivariate analyses of task-related and resting state fMRI data, here we show that patterns of brain activity within both the hippocampus and striatum elicited during motor learning persist into post-learning rest, indicative of reactivation of learning-related neural activity patterns. Moreover, results indicate that hippocampal reactivation reflects the spatial representation of the learned motor sequence. These results thus provide insights into the functional significance of neural reactivation after motor sequence learning.

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Akkermansia muciniphila secretome promotes α-synuclein aggregation in enteroendocrine cells. The notion that the gut microbiota play a role in neurodevelopment, behavior and outcome of neurodegenerative disorders is recently taking place. A number of studies have consistently reported a greater abundance of Akkermansia muciniphila in Parkinsons disease (PD) fecal samples. Nevertheless, a functional link between A.muciniphila and sporadic PD remained unexplored. Here, we investigated whether A.muciniphila secretome could initiate the misfolding process of -synuclein (Syn) in enteroendocrine cells (EECs), which are part of the gut epithelium and possess many neuron-like properties. We found that A.muciniphila secretome is directly modulated by mucin, induces intracellular calcium (Ca2+) release, and causes increased mitochondrial Ca2+ uptake in EECs, which in turn leads to production of reactive oxygen species (ROS) and Syn aggregation. However, these events were efficiently inhibited once we buffered mitochondrial Ca2+. Thereby, these molecular insights provided here offer evidence that bacterial secretome is capable of inducing Syn aggregation in enteroendocrine cells.

SYNOPSIS FIGURE DESCRIPTIONThe secretome isolated from the commensal gut bacterium Akkermansia muciniphila triggers intracellular Ca2+ signaling in enteroendocrine cells, leading to increased mitochondrial Ca2+ uptake. Mitochondrial Ca2+ overload leads to ROS generation culminating with Syn phosphorylation and aggregation (left panel). All these events were inhibited once mitochondrial Ca2+ is buffered (right panel).

O_LIGram-negative gut bacterium Akkermansia muciniphila is consistently found more abundant in Parkinsons disease patients.
C_LIO_LIAkkermansia muciniphila protein secretome composition is directly modulated by mucin and induces an IP3-independent endoplasmic reticulum (ER)-calcium release in enteroendocrine cells.
C_LIO_LIThis Ca2+ release is triggered by direct activation of Ryanodine Receptors leading to increased mitochondrial Ca2+ uptake.
C_LIO_LIMitochondrial Ca2+ overload leads to ROS generation culminating with Syn aggregation.
C_LIO_LIBuffering mitochondrial Ca2+ efficiently inhibits A.muciniphila-induced Syn aggregation in enteroendocrine cells.
C_LI

sentences: Genetic and pharmacological manipulations of glyoxalase 1 mediate ethanol withdrawal seizure susceptibility in mice Central nervous system (CNS) hyperexcitability is a clinically significant feature of acute ethanol withdrawal. There is evidence for a genetic contribution to withdrawal severity, but specific genetic risk factors have not be identified. The gene glyoxalase 1 (Glo1) has been previously implicated in ethanol consumption in mice, and GLO1 inhibition can attenuate drinking in mice and rats. Here, we investigated whether genetic and pharmacological manipulations of GLO1 activity can also mediate ethanol withdrawal seizure severity in mice. Mice from two transgenic lines overexpressing Glo1 on different genetic backgrounds (C57BL/6J [B6] and FVB/NJ [FVB]) were tested for handling induced convulsions (HICs) as a measure of acute ethanol withdrawal. Following an injection of 4 g/kg alcohol, both B6 and FVB mice overexpressing Glo1 showed increased HICs compared to wild type littermates, though only the FVB line showed a statistically significant difference. We also administered daily ethanol injections (2 g/kg + 9 mg/kg 4-methylpyrazole) to wild type B6 mice for 10 days and tested them for HICs on the 10th day, following treatment with either vehicle or a GLO1 inhibitor (S-bromobenzylglutathione cyclopentyl diester [pBBG]). Treatment with pBBG reduced HICs, although this effect was only statistically significant following two 10-day cycles of ethanol exposure and withdrawal. These results provide converging genetic and pharmacological evidence that GLO1 can mediate ethanol withdrawal seizure susceptibility. We discuss the possible role of GLO1s substrate, methylglyoxal, acting as an agonist at GABAA receptors.

sentences: Palmerolide PKS-NRPS gene clusters characterized from the microbiome of an Antarctic ascidian Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the [~]75 kb trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicohabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.

sentences: A cattle graph genome incorporating global breed diversity Despite only 8% of cattle being found in Europe, European breeds dominate current genetic resources. This adversely impacts cattle research in other important global cattle breeds. To mitigate this issue, we have generated the first assemblies of African breeds, which have been integrated with genomic data for 294 diverse cattle into the first graph genome that incorporates global cattle diversity. We illustrate how this more representative reference assembly contains an extra 116.1Mb (4.2%) of sequence absent from the current Hereford sequence and consequently inaccessible to current studies. We further demonstrate how using this graph genome increases read mapping rates, reduces allelic biases and improves the agreement of structural variant calling with independent optical mapping data. Consequently, we present an improved, more representative, reference assembly that will improve global cattle research.

sentences: A CRISPRi/a platform in iPSC-derived microglia uncovers regulators of disease states Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human iPSC-derived microglia. We developed an efficient eight-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the "druggable genome". These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by SPP1 expression was selectively depleted by CSF1R inhibition. Thus, our platform can systematically uncover regulators of microglia states, enabling their functional characterization and therapeutic targeting.

sentences: Programmable large DNA deletion, replacement, integration, and inversion with twin prime editing and site-specific recombinases The targeted deletion, replacement, integration, or inversion of DNA sequences at specified locations in the genome could be used to study or treat many human genetic diseases. Here, we describe twin prime editing (twinPE), a method for the programmable replacement or excision of DNA sequence at endogenous human genomic sites without requiring double-strand DNA breaks. TwinPE uses a prime editor (PE) protein and two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences. We show that twinPE in human cells can perform precise deletions of at least 780 bp and precise replacements of genomic DNA sequence with new sequences of at least 108 bp. By combining single or multiplexed twinPE with site-specific serine recombinases either in separate steps or in a single step, we demonstrate targeted integration of gene-sized DNA plasmids (>5,000 bp) into safe-harbor loci including AAVS1, CCR5, and ALB in human cells. To our knowledge, these results represent the first RNA-programmable insertion of gene-sized DNA sequences into targeted genomic sites of unmodified human cells without requiring double-strand breaks or homology-directed repair. Twin PE combined with recombinases also mediated a 40,167-bp inversion at IDS that corrects a common Hunter syndrome allele. TwinPE expands the capabilities of precision gene editing without requiring double-strand DNA breaks and synergizes with other tools to enable the correction or complementation of large or complex pathogenic alleles in human cells.

sentences: Gluconobacter oxydans Knockout Collection Finds Improved Rare Earth Element Extraction Rare earth elements (REE) are critical components of our technological society and essential for renewable energy technologies. Traditional thermochemical processes to extract REE from mineral ores or recycled materials are costly and environmentally harmful1, and thus more sustainable extraction methods require exploration. Bioleaching offers a promising alternative to conventional REE extraction2-4, and is already used to extract 5% of the worlds gold, and {approx} 15% of the worlds copper supply5,6. However, the performance of REE bioleaching lags far behind thermochemical processes2,7-9. Despite this, to the best of our knowledge no genetic engineering strategies have yet been used to enhance REE bioleaching, and little is known of the genetics that confer this capability. Here we build a whole genome knockout collection for Gluconobacter oxydans B58, one of the most promising organisms for REE bioleaching10, and use it to comprehensively characterize the genomics of REE bioleaching. In total, we find 304 genes that notably alter production of G. oxydans acidic biolixiviant, including 165 that hold up under statistical comparison with wild-type. The two most impactful groups of genes involved in REE bioleaching have opposing influences on acid production and REE bioleaching. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase all but eliminates bioleaching. In contrast, disruption of the phosphate-specific transport system accelerates acid production and enhances bioleaching. We identified 6 disruption mutants, that increase bioleaching by at least 11%. Most significantly, disruption of pstC, encoding part of the phosphate-specific transporter, pstSCAB, enhances bioleaching by 18%. Taken together, these results give a comprehensive roadmap for engineering multiple sites in the genome of G. oxydans to further increase its bioleaching efficiency.

sentences: FECAL MICROBIOME OF CHILDREN WITH NOROVIRUS GASTROENTERITIS The human fecal microbiome is composed of endogenous bacteria, eukaryotic viruses, bacteriophages and retroviruses. Several pathological conditions, including gastroenteritis, may be characterized by imbalance of gastrointestinal functions, with alteration in the diversity and composition of the fecal microbiota. Were analyzed twenty-seven fecal microbiome in children hospitalized with gastroenteritis (norovirus positive) from northern region of Brazil. After sequencing, was verified the presence of the domains Bacteria (95%) and Eukaryota (3.1%), the viruses represented 1.9%. Among the pathogenic viruses were found in addition to noroviruses the picornaviruses, enterovirus and parechovirus. The bacteriophages detected were of Caudovirales order, families Siphoviridae, Podoviridae and Myoviridae. In 22.2% (6/27) of the samples was observed co-infection between norovirus, enterovirus B and echovirus. As for the others components of the microbiome, we can highlight the presence of the taxonomic groups: Terrabacteria (50.2%), composed mainly of Actinobacteria and Firmicutes; Proteobacteria (34.5%) represented by the Enterobacteriaceae family; and FCB group (22%) whose most abundant microorganisms were those of the phylum Bacterioidetes. We performed a metagenomic approach to analyze the fecal microbiota of children with viral gastroenteritis, it was observed that the bacterias (Enterobacteriaceae) deserve attention in a possible association with noroviruses, as they were found in large quantities in infections. In addition, other enteric viruses were observed, such as enteroviruses.

sentences: Effortless integration of probabilistic visual input Current theories of perception suggest that the brain represents features of the world as probability distributions, but can such uncertain foundations provide the basis for everyday vision? Perceiving objects and scenes requires knowing not just how features (e.g., colors) are distributed but also where they are and which other features they are combined with. Using a Bayesian computational model, we recover probabilistic representations used by human observers to search for odd stimuli among distractors. Importantly, we found that the brain integrates information between feature dimensions and spatial locations, leading to more precise representations compared to when information integration is not possible. We also uncover representational asymmetries and biases, showing their spatial organization and arguing against simplified "summary statistics" accounts. Our results confirm that probabilistically encoded visual features are bound with other features and to particular locations, proving how probabilistic representations can be a foundation for higher-level vision.

sentences: Reconstruction of hundreds of reference ancestral genomes across the eukaryotic kingdom Ancestral sequence reconstruction is a fundamental aspect of molecular evolution studies, and can trace small-scale sequence modifications through the evolution of genomes and species. In contrast, fine-grained reconstructions of ancestral genome organisations are still in their infancy, limiting our ability to draw comprehensive views of genome and karyotype evolution. Here we reconstruct the detailed gene contents and organisations of 624 ancestral vertebrate, plant, fungi, metazoan and protist genomes, 183 of which are near-complete chromosomal reconstructions. Reconstructed ancestral genomes are similar to their descendants in terms of gene content as expected and agree precisely with reference cytogenetic and in silico reconstructions when available. By comparing successive ancestral genomes along the phylogenetic tree, we estimate the intra- and inter-chromosomal rearrangement history of all major vertebrate clades at high resolution. This freely available resource introduces the possibility to follow evolutionary processes at genomic scales in chronological order, across multiple clades and without relying on a single extant species as reference.

sentences: Detecting cell-of-origin and cancer-specific features of cell-free DNA with Nanopore sequencing The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for clinical sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNA) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell-type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy, especially real-time medical applications.

sentences:
pkgndep: a tool for heaviness analysis on R package dependencies SummaryNumerous R packages have been developed for bioinformatics analysis in the last decade and dependencies among packages have become critical issues to consider. In this work, we proposed a new metric named dependency heaviness that measures the number of unique dependencies that a parent brings to a package, and we proposed possible solutions for reducing the complexity of dependencies by optimizing the use of heavy parents. We implemented the metric in a new R package pkgndep which provides an intuitive way for dependency heaviness analysis. Based on pkgndep, we additionally performed a global analysis of dependency heaviness on CRAN and Bioconductor ecosystems and we revealed top packages that have significant contributions of high dependency heaviness to their child packages.

Availability and implementationThe package pkgndep and documentations are freely available from the Comprehensive R Archive Network (CRAN) https://cran.r-project.org/package=pkgndep. The dependency heaviness analysis for all 21,741 CRAN and Bioconductor packages retrieved on 2021-10-28 are available at https://pkgndep.github.io/.

sentences: Sex differences and risk factors for bleeding in Alagille syndrome Spontaneous lethal bleeds are major cause of death in the pediatric liver disease Alagille syndrome (ALGS), yet risk factors and screening methods have not been established. We performed a systematic review and identified significantly more female than male patients with idiopathic intracranial hemorrhage (10:1). We investigated bleeding and vasculature in patients and a mouse model for ALGS (Jag1Ndr/Ndr mice) and asked whether phenotypes identified in mice could be detected in patients non-invasively. Jag1Ndr/Ndr mice bled spontaneously, exhibiting a thin skull and vascular defects including artery-vein crossings, tortuous vessels, capillary breakdown and CADASIL-like sparse vascular smooth muscle cell coverage which was aggravated by hypertension. Retinographs from patients confirmed tortuous blood vessels and artery-vein crossings in ALGS. In conclusion, Jag1Ndr/Ndr mice could be used to develop interventions for vascular defects in ALGS, and retinography could provide a non-invasive method for vascular analysis in these pediatric patients.

sentences: Knockout of zebrafish desmin genes does not cause skeletal muscle degeneration but alters calcium flux Desmin is a muscle-specific intermediate filament protein that has fundamental role in muscle structure and force transmission. Whereas human desmin protein is encoded by a single gene, two desmin paralogs (desma and desmb) exist in zebrafish. Desma and desmb show differential spatiotemporal expression during zebrafish embryonic and larval development, being similarly expressed in skeletal muscle until hatching, after which expression of desmb shifts to gut smooth muscle. We generated knockout (KO) mutant lines carrying loss-of-function mutations for each gene by using CRISPR/Cas9. Mutants are viable and fertile, and lack obvious skeletal muscle, heart or intestinal defects. In contrast to morphants, knockout of each gene did not cause any overt muscular phenotype, but did alter calcium flux in myofibres. These results point to a possible compensation mechanism in these mutant lines generated by targeting nonsense mutations to the first coding exon.

sentences: Bridging brain and cognition: A multilayer network analysis of brain structural covariance and general intelligence in a developmental sample of struggling learners Network analytic methods that are ubiquitous in other areas, such as systems neuroscience, have recently been used to test network theories in psychology, including intelligence research. The network or mutualism theory of intelligence proposes that the statistical associations among cognitive abilities (e.g., specific abilities such as vocabulary or memory) stem from causal relations among them throughout development. In this study, we used network models (specifically LASSO) of cognitive abilities and brain structural covariance (grey and white matter) to simultaneously model brain-behavior relationships essential for general intelligence in a large (behavioral, N=805; cortical volume, N=246; fractional anisotropy, N=165), developmental (ages 5-18) cohort of struggling learners (CALM). We found that mostly positive, small partial correlations pervade our cognitive, neural, and multilayer networks. Moreover, using community detection (Walktrap algorithm) and calculating node centrality (absolute strength and bridge strength), we found convergent evidence that subsets of both cognitive and neural nodes play an intermediary role  between brain and behavior. We discuss implications and possible avenues for future studies.

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Spatiotemporal cytokinin signaling imaging reveals IPT3 function in nodule development in Medicago truncatula Most legumes can establish a symbiotic association with soil rhizobia that triggers the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharide (LCO) signal in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) acts as an essential positive regulator of nodule organogenesis, and specific CK receptors are required for nodule formation. Temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In the present study, using a fluorescence-based CK sensor (TCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the CK responses sequential activation during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYL TRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::tdTOMATO and the CK sensor showed that IPT3 induction in the root stele at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

One-sentence summaryHigh-resolution spatiotemporal imaging of cytokinin signaling reveals IPT3 function during indeterminate nodule development in Medicago truncatula

sentences: Local translation provides the asymmetric distribution of CaMKII required for associative memory formation How compartment-specific local proteomes are generated and maintained is inadequately understood, particularly in neurons, which display extreme asymmetries. Here we show that local enrichment of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in axons of Drosophila mushroom body neurons is necessary for cellular plasticity and associative memory formation. Enrichment is achieved via enhanced axoplasmic translation of CaMKII mRNA, through a mechanism requiring the RNA-binding protein Mub and a 23-base Mub-recognition element in the CaMKII 3UTR. Perturbation of either dramatically reduces axonal, but not somatic, CaMKII protein without altering the distribution or amount of mRNA in vivo and both are necessary and sufficient to enhance axonal translation of reporter mRNA. Together, these data identify elevated levels of translation of an evenly distributed mRNA as a novel strategy for generating subcellular biochemical asymmetries. They further demonstrate the importance of distributional asymmetry in the computational and biological functions of neurons.

sentences: Lack of evidence for participation of TMEM150c/TENTONIN3 in sensory mechanotransduction. The membrane protein TMEM150c has been proposed to form a mechanosensitive ion channel that is required for normal proprioceptor function. Here we examined whether expression of TMEM150c in neuroblastoma cells lacking Piezo1 is associated with the appearance of mechanosensitive currents. Using three different modes of mechanical stimuli, indentation, membrane stretch and substrate deflection we could not evoke mechanosensitive currents in cells expressing TMEM150c. We next asked if TMEM150c is necessary for the normal mechanosensitivity of cutaneous sensory neurons. We used an available mouse model in which the Tmem150c locus was disrupted through the insertion of a LacZ cassette with a splice acceptor that should lead to transcript truncation. Analysis of these mice indicated that ablation of the Tmem150c gene was not complete in sensory neurons of the dorsal root ganglia (DRG). Using a Crispr/cas9 strategy we made a second mouse model in which a large part of the Tmem150c gene was deleted and established that these Tmem150c-/- mice completely lack TMEM150c protein in the DRGs. We used an ex vivo skin nerve preparation to characterize the mechanosenstivity of mechanoreceptors and nociceptors in the glabrous skin of the Tmem150c-/- mice. We found no quantitative alterations in the physiological properties of any type of cutaneous sensory fiber in Tmem150c-/- mice. Since it has been claimed that TMEM150c is required for normal proprioceptor function we made a quantitative analysis of locomotion in Tmem150c-/- mice. Here again we found no indication that there was altered gait in Tmem150c-/- mice compared to wild type controls. In summary, we conclude that existing mouse models that have been used to investigate TMEM150c function in vivo are problematic. Furthermore, we could find no evidence that TMEM150c forms a mechanosensitive channel or that it is necessary for the normal mechanosensitivity of cutaneous sensory neurons.

sentences: Neutrophils contribute to ER stress in lung epithelial cells in the pristane-induced diffuse alveolar hemorrhage mouse model Diffuse alveolar hemorrhage (DAH), although rare, is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans, although increasingly neutrophils, NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model, we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress, namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model, with accompanying reduction in IFN-driven genes and pathology. Lastly, coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH.

sentences: Probabilistic modeling of ambient noise in single-cell omics data Droplet-based single-cell omics, including single-cell RNA sequencing (scRNAseq), single-cell CRISPR perturbations (e.g., CROP-seq), and single-cell protein and transcriptomic profiling (CITE-seq) hold great promise for comprehensive cell profiling and genetic screening at the single-cell resolution. However, these technologies suffer from substantial noise, among which ambient signals present in the cell suspension may be the predominant source. Current models to address this issue are highly technology-specific and relatively scRNAseq-centric. while a universal model to describe the noise across these technologies may reveal this common source, improving the denoising accuracy. To this end, we explicitly examined these unexpected signals in multiple datasets across droplet-based technologies, summarised a predictable pattern, and developed single-cell Ambient Remover (scAR) - a hypothesis-driven machine learning model to predict and remove ambient signals (including mRNA counts, protein counts, and sgRNA counts) at the molecular level. We benchmarked scAR on three technologies - single-cell CRISPR screens, CITE-seq, and scRNAseq along with the state-of-the-art single-technology-specific approaches. scAR showed high denoising accuracy for each type of dataset.

sentences: Cortex-wide fast activation of VIP-expressing inhibitory neurons by reward and punishment Reward and punishment powerfully inform ongoing behaviors and drive learning throughout the brain, including neocortex. Yet it remains elusive how these global signals are represented and impact local cortical computations. Previously we found that in auditory cortex, VIP-expressing interneurons are recruited by reinforcement feedback. Here, we used 3D random-access two-photon microscopy and fiber photometry to monitor VIP neural activity in dozens of cortical areas while mice learned an auditory decision task. We show that reward and punishment evoke a rapid, cortex-wide activation of most VIP interneurons. This global recruitment mode of VIP interneurons showed variations in temporal dynamics in individual neurons and across areas. Neither their weak sensory tuning in visual cortex, nor their arousal state modulation was predictive of reinforcer responses of VIP interneurons. We suggest that VIP-expressing cortical inhibitory neurons transduce global reinforcement signals to provide disinhibitory control over local circuit computations and their plasticity.

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Selection on early survival does not explain germination rate clines in Mimulus cardinalis (Phrymaceae) PremiseMany traits covary with environmental gradients to form phenotypic clines. While local adaptation to the environment can generate phenotypic clines, other nonadaptive processes may also. If local adaptation causes phenotypic clines, then the direction of genotypic selection on traits should shift from one end of the cline to the other. Traditionally genotypic selection on non-Gaussian traits like germination rate have been hampered because it is challenging to measure their genetic variance.

MethodsHere we used quantitative genetics and reciprocal transplants to test whether a previously discovered cline in germination rate showed additional signatures of adaptation in the scarlet monkeyflower (Mimulus cardinalis). We measured genotypic and population level covariation between germination rate and early survival, a component of fitness. We developed a novel discrete log-normal model to estimate genetic variance in germination rate.

ResultsContrary to our adaptive hypothesis, we found no evidence that genetic variation in germination rate contributed to variation in early survival. Across populations, southern populations in both gardens germinated earlier and survived more.

ConclusionsSouthern populations have higher early survival but this is not caused by faster germination. This pattern is consistent with nonadaptive forces driving the phenotypic cline in germination rate, but future work will need to assess whether there is selection at other life stages. The statistical framework should help expand quantitative genetic analyses for other waiting-time traits.

sentences: β-Catenin and canonical Wnts control two separate pattern formation systems in Hydra: Insights from mathematical modelling Formation of the body axes and the subsequent formation of the apical termini are fundamental steps during animal development. Nuclear {beta}-catenin and canonical Wnt3 have been identified as major players in both these processes in Hydra. Based on molecular knowledge of canonical Wnt signalling directly linking nuclear {beta}-catenin and Wnt3 activity, de novo axis formation and head formation have frequently been assumed to be part of the same pattern formation system. In this study, we investigated this relationship by combining new model simulations with available experimental results. We demonstrated that nuclear {beta}-catenin and Wnt3 likely contribute to two distinct de novo pattern formation systems in Hydra, organizing development and differentiation on two different spatial scales. In particular, the results suggested that nuclear {beta}-catenin acts at the scale of the whole body to control axis formation, whereas canonical Wnt3 signalling is involved in a downstream pathway responsible for small-scale patterning of the head. These results suggest that the underlying mechanisms may also be more complex than previously assumed in other animals where axis formation has been ascribed to canonical Wnt signalling.

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Development of white matter tracts between and within the dorsal and ventral streams The degree of interaction between the ventral and dorsal visual streams has been discussed in multiple scientific domains for decades. Recently, several white matter tracts that directly connect cortical regions associated with the dorsal and ventral streams have become possible to study due to advancements in automated and reproducible methods. The developmental trajectory of this set of tracts, here referred to as the posterior vertical pathway (PVP), has yet to be described. We propose an input-driven model of white matter development and provide evidence for the model by focusing on the development of the PVP. We used reproducible, cloud-computing methods and diffusion imaging from adults and children (ages 5-8 years) to compare PVP development to that of tracts within the ventral and dorsal pathways. PVP microstructure was more adult-like than dorsal stream microstructure, but less adult-like than ventral stream microstructure. Additionally, PVP microstructure was more similar to the microstructure of the ventral than the dorsal stream and was predicted by performance on a perceptual task in children. Overall, results suggest a potential role for the PVP in the development of the dorsal visual stream that may be related to its ability to facilitate interactions between ventral and dorsal streams during learning. Our results are consistent with the proposed model, suggesting that the microstructural development of major white matter pathways is related, at least in part, to the propagation of sensory information within the visual system.

Significance StatementUnderstanding white matter development is important to building predictive models that can inform interventions and targeted educational methods. We propose and provide evidence for an input-driven model of white matter development. We tested an uncharacterized aspect of human brain development. Namely, how the recently described posterior vertical white matter tracts develop. Our results suggest a developmental progression along the known, direct anatomical connections from posterior visual areas to anterior ventral and dorsal areas. Our results suggest fundamental biological mechanisms that clarify the role of white matter in predicting human learning and behavior.

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Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with Pol α - Primase The molecular mechanisms that drive the infection by the SARS-CoV-2 coronavirus - the causative agent of the COVID-19 (Coronavirus disease-2019) pandemic - are under intense current scrutiny, to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated.

Recent cell-based analyses of SARS-CoV-2 protein - protein interactions have mapped the human proteins targeted by the virus. The DNA polymerase  - primase complex or primosome - responsible for initiating DNA synthesis in genomic duplication - was identified as a target of nsp1 (non structural protein 1), a major virulence factor in the SARS-CoV-2 infection.

Here, we report the biochemical characterisation of the interaction between nsp1 and the primosome and the cryoEM structure of the primosome - nsp1 complex. Our data provide a structural basis for the reported interaction between the primosome and nsp1. They suggest that Pol  - primase plays a part in the immune response to the viral infection, and that its targeting by SARS-CoV-2 aims to interfere with such function.

sentences: Global emergence and dissemination of Neisseria gonorrhoeae ST-9363 isolates with reduced susceptibility to azithromycin Neisseria gonorrhoeae multi-locus sequence type (ST) 9363 genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the U.S. Here we analyze a global collection of ST-9363 genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009-2011 and again during the 2012-2013 time, facilitating the clonal expansion of this genogroup with AZMrs in the U.S. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the U.S. and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers.

sentences: Nucleosome sliding by the Chd1 chromatin remodeler relies on theintegrity of the DNA duplex Chromatin remodelers use a helicase-type ATPase motor to shift DNA around the histone core. Although not directly reading out the DNA sequence, some chromatin remodelers are biased by DNA sequences, suggesting that they may be sensitive to properties of the DNA duplex. Here, we present a high-throughput method for determining nucleosome positioning in vitro using site-specific DNA cleavage coupled with next-generation sequencing. This method allowed us to systematically test how the introduction of poly(dA:dT) tracts and other perturbations affected the distribution of nucleosomes remodeled by the Chd1 remodeler. We found that Chd1 is sensitive to poly(dA:dT) tracts as short as 3 bp, and that its nucleosome sliding activity is severely perturbed by DNA mismatches and single-nucleotide insertions. These results suggest that remodelers rely on the integrity of duplex DNA for nucleosome sliding. We also discovered that DNA on the nucleosome can shift in the absence of a remodeler when multiple mismatches are placed at superhelix location 2 (SHL2). This DNA movement in response to a disruption of the double helix may explain why SHL2 is the preferred site of engagement by most chromatin remodelers.

sentences: Towards Novel Herbicide Modes of Action by Inhibiting Lysine Biosynthesis in Plants Weeds are becoming increasingly resistant to our current herbicides, posing a significant threat to agricultural production. Therefore, new herbicides are urgently needed. In this study, we exploited a novel herbicide target, dihydrodipicolinate synthase (DHDPS), which catalyses the first and rate-limiting step in lysine biosynthesis. Using a high throughput chemical screen, we identified the first class of plant DHDPS inhibitors that have micromolar potency against Arabidopsis thaliana DHDPS isoforms. Employing X-ray crystallography, we determined that this class of inhibitors binds to a novel and unexplored pocket within DHDPS, which is highly conserved across plant species. We also demonstrated that the inhibitors attenuated the germination and growth of A. thaliana seedlings and confirmed their pre-emergence herbicidal activity in soil-grown plants. These results provide proof-of-concept that lysine biosynthesis represents a promising target for the development of herbicides with a novel mode of action to tackle the global rise of herbicide resistant weeds.

sentences: A conserved role of Parkinson-associated DJ-1 metabolites in sperm motility, mitosis, and embryonic development Fertility rates in the developing world have dramatically dropped in the last decades. This drop is likely due to a decline in sperm quality and women having children at older ages. Loss of function mutations in DJ-1, a Parkinsons associated gene, are linked to alterations in multiple cellular processes such as mitochondrial activity, ROS production or sperm motility and lead to an early onset of Parkinsons disease and male infertility in humans and other species. Glycolate (GA) and D-lactate (DL), products of DJ-1 glyoxalase activity, sustain mitochondrial function and protect against environmental aggressions. We, therefore, tested whether these substances could also have a rescue effect on these phenotypes. Here, we show that DJ-1 loss of function not only affects sperm motility but also leads to defects in mitosis and an age-dependent increase in the abortion rate. Remarkably, whereas DL was only able to rescue embryonic lethality in C. elegans, GA rescued these phenotypes in all model systems tested and even increased sperm motility in wild-type sperm. These positive effects seem to be mediated through an increase in NAD(P)H production and the regulation of intracellular calcium. These findings not only strongly suggest GA as a new therapeutic candidate to improve male and female fertility but also show its potential to treat diseases associated with a decline in mitochondrial function or to improve mitochondrial function in aging.

sentences: Conformational Change of Syntaxin-3b in Regulating SNARE Complex Assembly in the Ribbon Synapses Neurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigate the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

sentences: Lipid membranes modulate the activity of RNA through sequence-specific interactions RNA is a ubiquitous biomolecule that can serve as both catalyst and information carrier. Understanding how RNA bioactivity is controlled is crucial for elucidating its physiological roles and potential applications in synthetic biology. Here we show that lipid membranes can act as RNA organization platforms, introducing a novel mechanism for ribo-regulation. The activity of R3C ribozyme can be modified by the presence of lipid membranes, with direct RNA-lipid interactions dependent on RNA nucleotide content, base pairing and length. In particular, the presence of guanine in short RNAs is crucial for RNA-lipid interactions, and G-quadruplex formation further promotes lipid binding. Lastly, by artificially modifying the R3C substrate sequence to enhance membrane binding we generated a lipid-sensitive ribozyme reaction with riboswitch-like behavior. These findings introduce RNA-lipid interactions as a tool for developing synthetic riboswitches and novel RNA-based lipid biosensors, and bear significant implications for RNA World scenarios for the origin of life.

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Unique genomic sequences in novel Mycobacterium avium subsp. hominissuis lineage enabled fine scale tracing the transmission route during pig movement Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial (NTM) disease in human and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig and human MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. We conducted a variable number of tandem repeat (VNTR), genome phylogenetic tree analysis, historical recombination analysis and genome content analysis. Consistent with the results of previous studies, VNTR revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human. Recombination analysis indicated that most pig isolates could be classified into SC2/4 and SC3. To our surprise, four isolates belonged to a new lineage in the global MAH population. Members of this group had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. As the members of this new lineage were rare, we were able to trace the putative transmission route via their host pigs. Together, these results reveal the diversity in the pig MAH population and clarify the difference of source of infection and the vector of transmission between humans and pigs.

ImportanceMycobacterium avium subsp. hominissuis (MAH) is a dominant cause of non-tuberculous mycobacterial (NTM) disease in human and animals. In this study, in order to clarify the epidemiological relatedness between MAH isolates from human and pigs, and to determine the MAH transmission route among pigs, we conducted molecular and genomic-based epidemiological research. The pig isolates were genetically distant from those of humans in Japan, however, they were closely related to human isolates from North America, Europe and Russia, suggesting a proportion of pig MAH isolates are imported from abroad. Importantly, we identified a novel lineage of MAH that was distributed in a specific area in Japan. Epidemiological survey and its unique genomic features allowed us to trace putative transmission routes via pig movement. Our study provides critical information for understanding the diversity of MAH and will be useful for countermeasures of pig and human NTM disease.

sentences: Geometric effects of volume to surface mapping of fMRI data In this work, we identify a problem with the process of volume-to-surface mapping of functional Magnetic Resonance Imaging (fMRI) data when interested in investigating local connectivity. We show that neighborhood correlations on the surface of the brain vary spatially with anatomical patterns (gyral structure), even when the underlying volumetric data are uncorrelated noise. This could potentially have impacted studies focusing upon local neighborhood connectivity. We explore the effects of this anomaly across varying data resolutions and surface mesh densities, and propose an approach to mitigate these unwanted effects.

sentences: Cannabinoids accumulate in mouse breast milk and differentially regulate lipid signaling molecules involved in infant development Maternal cannabis use during lactation may expose developing infants to cannabinoids (CBs) such as tetrahydrocannabinol (THC) and cannabidiol (CBD). CBs modulate lipid signaling molecules in the central nervous system in age- and cell-dependent ways, but their influence on the lipid composition of breastmilk has yet to be established. This study investigates the effects of THC, CBD, or their combination on milk lipids by analyzing the stomach contents of CD1 mouse pups that have been nursed by dams injected with CBs on postnatal days (PND) 1-10 collected 2 hours after the last injection on PND10. HPLC/MS/MS was used to identify and quantify over 80 endogenous lipid species and cannabinoids in pup stomach contents. We show that CBs differentially accumulate in milk, lead to widespread decreases in free fatty acids, decreases in N-acyl methionine species, increases N-linoleoyl species, as well as modulate levels of endogenous CBs (eCBs) AEA, 2-AG, and their structural congeners. Our data indicate the passage of CBs to pups through breast milk and that maternal CB exposure alters breast milk lipid compositions.

sentences: Integrated single-cell analysis of enteric glial cells reveals a molecular basis for postnatal neurogenesis and its therapeutic application The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to assess EGCs undergoing spontaneous neurogenesis in culture, as well as freshly isolated small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. Multiome analysis of freshly isolated EGCs reveals transcriptional diversity, with open chromatin at neuron-associated genomic elements. A subset of EGCs, highly enriched within the myenteric ganglia, has a gene expression program and chromatin state consistent with neurogenic potential.

sentences: Systematic analysis of CD39, CD103, CD137 and PD-1 as biomarkers for naturally occurring tumor antigen-specific TILs The detection of tumor-specific T cells in solid tumors is integral to the interrogation of endogenous antitumor responses and to the advancement of downstream therapeutic applications, such as checkpoint immunotherapy and adoptive cell transfer. A number of biomarkers are reported to identify endogenous tumor-specific tumor infiltrating lymphocytes (TILs), namely CD137, PD-1, CD103, and CD39, however a direct comparison of these molecules has yet to be performed. Here, we evaluate these biomarkers in primary human high-grade serous ovarian tumor samples using single-cell mass cytometry to characterize and compare their relative phenotypic profiles, as well as their response to autologous tumor cells ex vivo. CD137+, PD-1+, CD103+, and CD39+ TILs are all detectable in tumor samples with CD137+ TILs being the least abundant. PD-1+, CD103+, and CD39+ TILs all express a subset of CD137+ cells, while CD137+ TILs highly co-express the aforementioned markers. CD137+ TILs exhibit the highest expression of cytotoxic effector molecules, such as IFN{gamma} and Granzyme B, compared to PD-1+, CD103+ or CD39+ TILs. Removal of CD137+ TILs from PD-1+, CD103+, or CD39+ TILs results in lower secretion of IFN{gamma} in response to autologous tumor stimulation, while CD137+ TILs highly secrete IFN{gamma} in an HLA-dependent manner. CD137+ TILs exhibited an exhausted phenotype with CD28 co-expression, suggestive of antigen recognition and receptiveness to reinvigoration via immune checkpoint blockade. Together, our findings demonstrate that the antitumor abilities of PD-1+, CD103+, and CD39+ TILs are mainly derived from a subset of TILs expressing CD137, implicating CD137 is a more selective biomarker for naturally occurring tumor-specific TILs.

sentences: Glycogen Synthase Kinase 3 regulates the genesis of the rare displaced ganglion cell retinal subtype Glycogen Synthase Kinase 3 (GSK) proteins (GSK3 and GSK3{beta}) are key mediators of signaling pathways, with crucial roles in coordinating fundamental biological processes during neural development. Here we show that the complete loss of GSK3 signaling in mouse retinal progenitors leads to microphthalmia with broad morphological defects. Both proliferation of retinal progenitors and neuronal differentiation are impaired and result in enhanced cell death. A single wild-type allele of either Gsk3 or Gsk3{beta} is able to rescue these phenotypes. In this genetic context, all cell types are present with a functional retina. However, we unexpectedly detect a large number of cells in the inner nuclear layer expressing retinal ganglion cell (RGC)-specific markers (called displaced RGCs, dRGCs) when at least one allele of Gsk3 is expressed. Excess dRGCs lead to increased number of axons projecting into the ipsilateral medial terminal nucleus, an area of the brain belonging to the non-image-forming visual circuit and poorly targeted by RGCs in wild-type retina. Transcriptome analysis and optomotor response assay suggest that at least a subset of dRGCs in Gsk3 mutant mice are direction-selective RGCs. Our study thus uncovers a unique role of GSK3 in controlling the genesis of dRGCs, a rare and poorly characterized retinal cell type.

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Sensitive Detection of Site-wise Convergent Evolution in Large Protein Alignments with ConDor Evolutionary convergences are observed at all levels, from phenotype to DNA and protein sequences, and the changes observed at these different levels tend to be strongly correlated. Here we propose a simulation-based method to detect positions under convergent evolution in large protein alignments, without prior knowledge on the phenotype and environmental constraints. A phylogeny is inferred from the data and used in simulations to estimate the expected number of amino-acid changes in stable evolutionary constraints (null model) for each position. Similarly, we count the number of mutations towards the same amino acid in the data and test if they are occurring more often than expected.

We applied our method to two real datasets: HIV reverse transcriptase and fish rhodopsin, and to HIV-like simulated data. On the latter, with known convergent events and substitution model, we detected on average two third of these events, with a low fraction of false positives. With HIV data, one knows that drug resistance mutations (DRMs) are convergent. Even without any knowledge of patient treatment status, we retrieved more than 70% of positions corresponding to known DRMs. On the rhodopsin dataset, four substitutions are supposed to be convergent, as they change the maximum wavelength absorption of the photoreceptor and occurred several times independently during evolution. We detected three of them.

These results demonstrate the potential of the method to target specific mutations to be further studied experimentally or, for example, using a nonsynonymous/synonymous rate ratio approach. Our software named ConDor is available at http://condor.pasteur.cloud.

sentences: Larger but younger fish when growth compensates for higher mortality in heated ecosystem Ectotherms are often predicted to "shrink" with global warming, in line with general growth models and the temperature-size rule (TSR), both predicting smaller adult sizes with warming. However, they also predict faster juvenile growth rates, leading to larger size-at-age. Hence, the result of warming on population size-structure also depends on mortality rates and how much also adult size changes. We used data from an artificially heated (+8C) bay in comparison with an unheated area, to analyze how warming has affected body growth, mortality rates and population size-structure of Eurasian perch (Perca fluviatilis). In the heated bay, body size was larger for all ages and growth faster for all sizes, resulting in larger size-spectrum exponent (greater proportion of large fish), despite higher mortality. Hence, to understand how warming alters species size-structure, and thus ecological interactions and dynamics, it is critical to account for warming-induced changes in both mortality and growth rate.

sentences: A Computational Model of Phosphene Appearance for Epiretinal Prostheses Retinal neuroprostheses are the only FDA-approved treatment option for blinding degenerative diseases. A major outstanding challenge is to develop a computational model that can accurately predict the elicited visual percepts (phosphenes) across a wide range of electrical stimuli. Here we present a phenomenological model that predicts phosphene appearance as a function of stimulus amplitude, frequency, and pulse duration. The model uses a simulated map of nerve fiber bundles in the retina to produce phosphenes with accurate brightness, size, orientation, and elongation. We validate the model on psychophysical data from two independent studies, showing that it generalizes well to new data, even with different stimuli and on different electrodes. Whereas previous models focused on either spatial or temporal aspects of the elicited phosphenes in isolation, we describe a more comprehensive approach that is able to account for many reported visual effects. The model is designed to be flexible and extensible, and can be fit to data from a specific user. Overall this work is an important first step towards predicting visual outcomes in retinal prosthesis users across a wide range of stimuli.

sentences: Structural basis for sterol sensing by Scap and Insig The sterol regulatory element-binding protein (SREBP) pathway senses the cellular cholesterol level through sterol regulated association between Scap and Insig. Despite the recent structural determination of the transmembrane domains of human Scap and Insig-2 bound to 25-hydroxycholesterol (25HC), the structure and regulatory mechanism of the luminal domains of Scap by cholesterol remains elusive. Here, combining cryo-EM analysis and artificial intelligence-facilitated structural prediction, we report the structure of the human Scap/Insig-2 complex in the presence of digitonin instead of 25HC. Despite the lack of sequence similarity, the structure of the luminal domain Loop 1 and a co-folded segment in Loop 7 of Scap resembles that of the luminal/extracellular domain in NPC1 and related proteins. Comparison of the sterol-loaded structures of these proteins provides clues of the regulation of Loop 1/7 interaction by cholesterol. We also show that the structure of Scap(D428A), which suppresses SREBP activation under sterol depletion, is identical to WT when complexed with Insig-2, although the gain of function may also involve a later step in protein trafficking.

sentences: Ikaros family proteins regulate developmental windows in the mouse retina through convergent and divergent transcriptional programs Temporal identity factors regulate the competence of neural progenitors to generate specific cell types in a time-dependent manner, but how they operate remains poorly defined. In the developing mouse retina, the Ikaros zinc finger transcription factor Ikzf1 regulates the production of early-born cell types, except cone photoreceptors. In this study we show that Ikzf4, another Ikaros family protein, cooperates with Ikzf1 to control cone photoreceptor production during early stages of retinal development, whereas at late stages, when Ikzf1 is no longer expressed in progenitors, Ikzf4 is instead required for Muller glia production. Using CUT&RUN sequencing, we find that both Ikzf1 and Ikzf4 generally bind to the same genes involved in cone development and other early-born fates, but at different cis-regulatory elements. In late-stage progenitors, Ikzf4 re-localizes to bind target genes involved in Muller glia development and regulate their expression. Specifically, we show that Ikzf4 maintains Hes1 expression in differentiating cells using two Ikzf GGAA binding sites at the Hes1 promoter, thereby favouring Muller glia fate commitment. These results uncover a combinatorial role for Ikaros family members in nervous system development and provide mechanistic insights on how they temporally regulate cell fate output.

sentences: Heritable and climatic sources of variation in juvenile tree growth in an Austrian common garden experiment of Central European Norway spruce populations We leveraged publicly available data on juvenile tree height of 299 Central European Norway spruce populations grown in a common garden experiment across 24 diverse trial locations in Austria and weather data from the trial locations and population provenances to parse the heritable and climatic components of juvenile tree height variation. Principal component analysis of geospatial and weather variables demonstrated high interannual variation among trial environments, largely driven by differences in precipitation, and separation of population provenances based on altitude, temperature, and snowfall. Tree height was highly heritable and modeling the covariance between populations and trial environments based on climatic data led to more stable estimation of heritability and population x environment variance. Climatic similarity among population provenances was highly predictive of population x environment estimates for tree height.

sentences: CLCd and CLCf act redundantly at the TGN/EE and prevent acidification of the Golgi stack The trans-Golgi network/early endosome (TGN/EE) serves as the central hub in which exo- and endocytic trafficking pathways converge and specificity of cargo routing needs to be achieved. Acidification is a hallmark of the TGN/EE and is maintained by the vacuolar H+-ATPase (V-ATPase) with support of proton-coupled antiporters. We show here that CLCd and CLCf, two distantly related members of the Arabidopsis chloride channel (CLC)-family that colocalize in the TGN/EE act redundantly and are essential for male gametophyte development. Combining an inducible knock-down approach and in vivo pH-measurements, we show here that reduced CLC-activity does not affect pH in the TGN/EE but causes accumulation of the V-ATPase in trans-Golgi cisternae leading to their hyper-acidification. Taken together, our results show that CLC-mediated anion transport into the TGN/EE is essential and affects spatio-temporal aspects of TGN/EE-maturation as well as its functional separation from the Golgi stack.

sentences: Deficient thalamo-cortical networks dynamics and sleep homeostatic processes in a redox dysregulation model relevant to schizophrenia. A growing body of evidence implicates thalamo-cortical oscillations with the neuropathophysiology of schizophrenia (SZ) in both mice and humans. Yet, the precise mechanisms underlying sleep perturbations in SZ remain unclear. Here, we characterised the dynamics of thalamo-cortical networks across sleep-wake states in a mouse model carrying a mutation in the enzyme glutathione synthetase gene (Gclm-/-) associated with SZ in humans. We hypothesised that deficits in parvalbumin immunoreactive cells in the thalamic reticular nucleus (TRN) and the anterior cingulate cortex (ACC) - caused by oxidative stress - impact thalamocortical dynamics, thus affecting non-rapid eye movement (NREM) sleep and sleep homeostasis. Using polysomnographic recordings in mice, we showed that KO mice exhibited a fragmented sleep architecture, similar to SZ patients and altered sleep homeostasis responses revealed by an increase in NREM latency and slow wave activities during the recovery period (SR). Although NREM sleep spindle rate during spontaneous sleep was similar in Gclm-/- and Gcml +/+, KO mice lacked a proper homeostatic response during SR. Interestingly, using multisite electrophysiological recordings in freely-moving mice, we found that high order thalamic network dynamics showed increased synchronisation, that was exacerbated during the sleep recovery period subsequent to SD, possibly due to lower bursting activity in TRN-antero dorsal thalamus circuit in KO compared to WT littermates. Collectively, these findings provide a mechanism for SZ associated deficits of thalamo-cortical neuron dynamics and perturbations of sleep architecture.

sentences: Home literacy environment mediates the relationship between socioeconomic status and white matter structure in infants The home literacy environment (HLE) in infancy has been associated with subsequent pre-literacy skill development and HLE at pre-school age has been shown to correlate with white matter organization in tracts that subserve prereading and reading skills. Furthermore, childhood socioeconomic status (SES) has been linked with both HLE and white matter organization. It is also important to understand whether the relationships between environmental factors such as HLE and SES and white matter organization can be detected as early as infancy, as this period is characterized by rapid brain development that may make white matter pathways particularly susceptible to these early experiences. Here, we hypothesized (1) an association between HLE and white matter organization in pre-reading and reading-related tracts in infants, and (2) that this association mediates a link between SES and white matter organization. To test these hypotheses, infants (mean age: 9.2 {+/-} 2.5 months, N = 18) underwent diffusion-weighted imaging MRI during natural sleep. Fractional anisotropy (FA) was estimated from the left superior longitudinal fasciculus (SLF) and left arcuate fasciculus using the automated fiber-tract quantification method. HLE was measured with the Reading subscale of the StimQ and SES was measured with years of maternal education. Self-reported maternal reading ability was also quantified and applied to all statistical models to control for confounding genetic effects. The Reading subscale of the StimQ positively related to FA in left SLF and mediated the association between maternal education and FA in the left SLF. Taken together, these findings underscore the importance of considering HLE from the start of life and may inform novel prevention and intervention strategies targeted at low-SES families to support developing infants during a period of heightened brain plasticity.

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Robustness in regulatory networks of Epithelial Mesenchymal Plasticity as a function of positive and negative feedback loops Epithelial-Mesenchymal plasticity (EMP) is a key arm of cancer metastasis and is observed across many contexts. Cells undergoing EMP can reversibly switch between three classes of phenotypes: Epithelial (E), Mesenchymal (M), and Hybrid E/M. While a large number of multistable regulatory networks have been identified to be driving EMP in various contexts, the exact mechanisms and design principles that enable robustness in driving EMP across contexts are not yet fully understood. Here we investigated dynamic and structural robustness in EMP networks with regards to phenotypic distribution and plasticity. We use two different approaches to simulate these networks: a computationally inexpensive, parameter-independent continuous state space boolean model, and an ODE-based parameter-agnostic framework (RACIPE), both of which yield similar phenotypic distributions. Using perturbations to network topology and by varying network parameters, we show that multistable EMP networks are structurally and dynamically more robust as compared to their randomized counterparts, thereby highlighting their topological hallmarks. These features of robustness are governed by a balance of positive and negative feedback loops embedded in these networks. Using a combination of the number of negative and positive feedback loops weighted by their lengths and sign, we identified a metric that can explain the structural and dynamical robustness of these networks. This metric enabled us to compare networks across multiple sizes, and the network principles thus obtained can be used to identify fragilities in large networks without simulating their dynamics. Our analysis highlights a network topology-based approach to quantify robustness in multistable EMP networks.

Significance StatementEpithelial-Mesenchymal plasticity (EMP) is a key arm of cancer metastasis. Despite extensive intra- and inter-tumor heterogeneity, the characteristics of EMP have been observed to be robust across multiple contexts. We hypothesize that topology of EMP regulatory networks contributes towards this robustness. Here, we measure the robustness of EMP in the form of its phenotypic heterogeneity and multistability and show that EMP networks are more robust to dynamical (change in kinetic parameters) and structural (change in network topology) perturbations as compared to their random network counterparts. Furthermore, we propose a network topology-based metric using the nature and length of feedback loops that explains the observed robustness. Our metric hence serves to quantify robustness in multistable EMP networks without simulating their dynamics.

sentences: Planning the potential future during multi-item visual working memory Working memory allows us to retain visual information to guide upcoming future behavior. In line with this future-oriented purpose of working memory, recent studies have shown that action planning occurs during encoding and retention of a single visual item, for which the upcoming action is certain. We asked whether and how this extends to multi-item visual working memory, when visual representations serve the potential future. Human participants performed a visual working memory task with a memory-load manipulation (one/two/four items), and a delayed orientation-reproduction report (of one item). We measured EEG to track 15-25 Hz beta activity in electrodes contralateral to the required response hand - a canonical marker of action planning. We show an attenuation of beta activity, not only in load one (with one certain future action), but also in load two (with two potential future actions), compared to load four (with low prospective-action certainty). Moreover, in load two, potential action planning occurs regardless whether both visual items afford similar or dissimilar manual responses; and it predicts the speed of ensuing memory-guided behavior. This shows that potential action planning occurs during multi-item visual working memory, and brings the perspective that working memory helps us prepare for the potential future.

sentences: CRY-BARs: Versatile light-gated molecular tools for the remodeling of membrane architectures BAR (Bin, Amphiphysin and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR domain containing tools ( CRY-BARs) with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate that the CRY-BAR optogenetic tool reports membrane dynamic changes associated with cellular activity. Moreover, we provide evidence that Ezrin acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.

sentences: Exposure to 1800 MHz LTE electromagnetic fields under proinflammatory conditions decreases the response strength and increases the acoustic threshold of auditory cortical neurons. Increased needs in mobile phone communications have raised successive generations (G) of wireless technologies, which could differentially affect biological systems. We have assessed how a single 2h head-only exposure to a 4G long term evolution (LTE)-1800 MHz electromagnetic field (EMF) impacts on the microglial space coverage and the electrophysiological neuronal activity in the primary auditory cortex (ACx) in rats submitted to an acute neuroinflammation induced by lipopolysaccharide. The mean specific absorption rate in the ACx was 0.5 W/kg. Multiunit recording revealed that LTE-EMF triggered reduction in the response strength to pure tones and to natural vocalizations, together with an increase in acoustic threshold in the low and medium frequencies. Iba1 immunohistochemistry showed no change in the area covered by microglia cell bodies and processes. In healthy rats, the same LTE-exposure induced no change in response strength and acoustic threshold. Our data indicate that an acute neuroinflammation sensitize neuronal responses to LTE-EMF, which leads to an altered processing of acoustic stimuli in the ACx.

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Genomic diversity across Candida auris clinical isolates shapes rapid development of antifungal resistance in vitro and in vivo Antifungal drug resistance and tolerance poses a serious threat to global public health. In the human fungal pathogen, Candida auris, resistance to triazole, polyene, and echinocandin antifungals is rising, resulting in multidrug resistant isolates. Here, we use genome analysis and in vitro evolution of seventeen new clinical isolates of C. auris from clades I and IV to determine how quickly resistance mutations arise, the stability of resistance in the absence of drug, and the impact of genetic background on evolutionary trajectories. We evolved each isolate in the absence of drug as well as in low and high concentrations of fluconazole. In just three passages, we observed genomic and phenotypic changes including karyotype alterations, aneuploidy, acquisition of point mutations, and increases in MIC values within the populations. Fluconazole resistance was stable in the absence of drug, indicating little to no fitness cost associated with resistance. Importantly, two isolates substantially increased fluconazole resistance to [≥]256{micro}g/ml fluconazole. Multiple evolutionary pathways and mechanisms to increase fluconazole resistance occurred simultaneously within the same population. Strikingly, the sub-telomeric regions of C. auris were highly dynamic as deletion of multiple genes near the sub-telomeres occurred during the three passages in several populations. Finally, we discovered a mutator phenotype in a clinical isolate of C. auris. This isolate had elevated mutation rates compared to other isolates and acquired substantial resistance during evolution in vitro and in vivo supporting that the genetic background of clinical isolates can have a significant effect on evolutionary potential.

ImportanceDrug resistant Candida auris infections are recognized by the CDC as an urgent threat. Here, we obtained and characterized a set of clinical isolates of C. auris including multiple isolates from the same patient. To understand how drug resistance arises, we evolved these isolates and found that resistance to fluconazole, the most commonly prescribed antifungal, can occur rapidly and that there are multiple pathways to resistance. During our experiment, resistance was gained, but it was not lost, even in the absence of drug. We also found that some C. auris isolates have higher mutation rates than others and are primed to acquire antifungal resistance mutations. Furthermore, we found that multidrug resistance can evolve within a single patient. Overall, our results highlight the high stability and high rates of acquisition of antifungal resistance of C. auris that allow evolution of pan-resistant, transmissible isolates in the clinic.

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Carbon nanotube biocompatibility in plants is determined by their surface chemistry Agriculture faces significant global challenges including climate change and an increasing food demand due to a growing population. Addressing these challenges will require the adoption of transformative innovations into biotechnology practice, such as nanotechnology. Recently, nanomaterials have emerged as unmatched tools for their use as biosensors, or as biomolecule delivery vehicles. Despite their increasingly prolific use, plant-nanomaterial interactions remain poorly characterized, drawing into question the breadth of their utility and their broader environmental compatibility. Herein, we characterize Arabidopsis thaliana transcriptional response to single walled carbon nanotubes (SWNTs) with two different surface chemistries commonly used for biosensing and nucleic acid delivery: oligonucleotide adsorbed-pristine SWNTs, and polyethyleneimine-SWNTs loaded with plasmid DNA (PEI-SWNTs), both introduced by leaf infiltration. We observed that SWNTs elicit a mild stress response almost undistinguishable from the infiltration process, indicating that these nanomaterials are well-tolerated by the plant. However, PEI-SWNTs induce a much larger transcriptional reprogramming that involves stress, immunity, and senescence responses. PEI-SWNT-induced transcriptional profile is very similar to that of mutant plants displaying a constitutive immune response or treated with stress-priming agrochemicals. We selected molecular markers from our transcriptomic analysis and identified PEI as the main cause of this reaction. We show that PEI-SWNT response is concentration-dependent and, when persistent over time, leads to cell death. We probed a panel of PEI variant-functionalized SWNTs across two plant species and identified biocompatible SWNT surface functionalizations. Our results highlight the importance of nanoparticle surface chemistry on their biocompatibility and will facilitate the use of functionalized nanomaterials for agricultural improvement.

Significance statementNanomaterials can be used in agriculture as biosensors to monitor plant health, as fertilizers or growth regulators, and as delivery vehicles for genome engineering reagents to improve crops. However, the interactions between nanoparticles and plant cells are not well understood. Here, we characterize the plant transcriptomic response to single-walled carbon nanotubes (SWNTs) commonly used for sensing and nucleic acid delivery. While SWNTs themselves are well tolerated by plants, SWNTs surface-functionalized with positively charged polymers become toxic and produce cell death. We identify molecular markers of this toxic response to create biocompatible SWNT formulations. These results highlight the significance of nanoparticle surface chemistry, perhaps more than the nanoparticles themselves, on downstream interactions of nanoparticles with the environment.

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Heritable changes in division speed accompany the diversification of single T cell fate Rapid clonal expansion of antigen specific T cells is a fundamental feature of adaptive immune responses. It enables the outgrowth of an individual T cell into thousands of clonal descendants that diversify into short-lived effectors and long-lived memory cells. Clonal expansion is thought to be programmed upon priming of a single naive T cell and then executed by homogenously fast divisions of all of its descendants. However, the actual speed of cell divisions in such an emerging  T cell family has never been measured with single-cell resolution. Here, we utilize continuous live-cell imaging in vitro to track the division speed and genealogical connections of all descendants derived from a single naive CD8+ T cell throughout up to ten divisions of activation-induced proliferation. This comprehensive mapping of T cell family trees identifies a short burst phase, in which division speed is homogenously fast and maintained independent of external cytokine availability or continued T cell receptor stimulation. Thereafter, however, division speed diversifies and model-based computational analysis using a novel Bayesian inference framework for tree-structured data reveals a segregation into heritably fast and slow dividing branches. This diversification of division speed is preceded already during the burst phase by variable expression of the interleukin-2 receptor alpha chain. Later it is accompanied by selective expression of memory marker CD62L in slower dividing branches. Taken together, these data demonstrate that T cell clonal expansion is structured into subsequent burst and diversification phases the latter of which coincides with specification of memory vs. effector fate.

SignificanceRapid clonal expansion of antigen-specific T cells is a fundamental feature of adaptive immune responses. Here, we utilize continuous live-cell imaging in vitro to track the division speed and genealogical connections of all descendants derived from a single naive CD8+ T cell throughout up to ten divisions of activation-induced proliferation. Bayesian inference of tree-structured data reveals that clonal expansion is divided into a homogenously fast burst phase encompassing two to three divisions and a subsequent diversification phase during which T cells segregate into quickly dividing effector T cells and more slowly cycling memory precursors. Our work highlights cell cycle speed as a major heritable property that is regulated in parallel to key lineage decisions of activated T cells.

sentences: A rapid and reliable multiplexed LC-MS/MS method for simultaneous analysis of six monoamines from brain tissues Monoamines are a class of neuromodulators that are crucial for a variety of brain functions, including control of mood, movement, sleep and cognition. From mammals to insects, the nervous system is enriched in monoamines such as dopamine, serotonin and melatonin, analytes which range from being highly polar to non-polar. Here we developed a method using liquid chromatography coupled with mass spectrometry (LC-MS) to quantify in a single run the amounts of six distinct monoamines in extracts from dissected Drosophila and mouse brain tissues. The measured monoamines were dopamine (DA), serotonin (also known as 5-hydroxytryptamine (5-HT)), octopamine (OA, an insect equivalent of norepinephrine), tyramine (TA), melatonin (MT) and N-acetyl-hydroxy-serotonin (NAS). The analytical range of these monoamines was between 0.25 to 5.0 ng/mL.

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Mycorrhizal symbiosis alleviates plant drought stress within and across plant generations via plasticity O_LIPhenotypic plasticity is essential for organisms to adapt to local ecological conditions. It is expected that mutualistic interactions, such as arbuscular mycorrhizal (AM) symbiosis, mediate plant phenotypic plasticity, although it is not clear to what extent this plasticity may be heritable (i.e., transgenerational plasticity).
C_LIO_LIWe tested for plant plasticity within- and across-generations in response to AM symbiosis and varying water availability in a full factorial experiment over two generations, using a genetically uniform line of a perennial apomictic herb, Taraxacum brevicorniculatum. We examined changes in phenotype, performance, and AM fungal colonization of the offspring throughout plant development.
C_LIO_LIAM symbiosis and water availability triggered phenotypic changes during the life cycle of plants. Additionally, both triggered adaptive transgenerational effects, especially detectable during the juvenile stage of the offspring. Water deficit and absence of AM fungi caused concordant plant phenotypic modifications towards a "stress-coping phenotype", both within- and across-generations. AM fungal colonization of offspring was also affected by the parental environment.
C_LIO_LIAM symbiosis can trigger within-generation and transgenerational plasticity in terms of functional traits related to resource-use acquisition and AM fungal colonization. Thus, transgenerational effects of mycorrhizal symbiosis are not limited to plant fitness, but also improve plants ability to cope with environmental stress.
C_LI

sentences: Aurora B phosphorylates Bub1 to promote spindle assembly checkpoint signaling Accurate chromosome segregation during cell division requires amphitelic chromosome attachment to the spindle apparatus. It is ensured by the combined activity of the Spindle Assembly Checkpoint1 (SAC), a signaling mechanism that delays anaphase onset in response to unattached chromosomes, and an error correction mechanism that eliminates syntelic attachments2. The SAC becomes active when Mps1 kinase sequentially phosphorylates the kinetochore protein Spc105/KNL1 and the signaling proteins that Spc105/KNL1 recruits to facilitate the production of the Mitotic Checkpoint Complex3-8 (MCC). The error correction mechanism is regulated by the Aurora B kinase, but Aurora B also promotes SAC signaling via indirect mechanisms9-12. Here we present evidence that Aurora B kinase activity directly promotes MCC production by working downstream of Mps1 in budding yeast and human cells. Using the ectopic SAC activation (eSAC) system, we find that the conditional dimerization of Aurora B in budding yeast, and an Aurora B recruitment domain in HeLa cells, with either Bub1 or Mad1, but not the phosphodomain of Spc105/KNL1, leads to ectopic MCC production and mitotic arrest13-16. Importantly, Bub1 must recruit both Mad1 and Cdc20 for this ectopic signaling activity. These and other data show that Aurora B cooperates with Bub1 to promote MCC production, but only after Mps1 licenses Bub1 recruitment to the kinetochore. This direct involvement of Aurora B in SAC signaling may maintain SAC signaling even after Mps1 activity in the kinetochore is lowered.

sentences: Does non-invasive vagus nerve stimulation affect heart rate variability? A living and interactive Bayesian meta-analysis Non-invasive brain stimulation techniques, such as transcutaneous auricular vagus nerve stimulation (taVNS), have considerable potential for clinical use. Beneficial effects of taVNS have been demonstrated on symptoms in patients with mental or neurological disorders as well as transdiagnostic dimensions, including mood and motivation. However, since taVNS research is still an emerging field, the underlying neurophysiological processes are not yet fully understood, and the replicability of findings on biomarkers of taVNS effects has been questioned. Here, we perform a living Bayesian random effects meta-analysis to synthesize the current evidence concerning the effects of taVNS on heart rate variability (HRV), a candidate biomarker that has, so far, received most attention in the field. To keep the synthesis of evidence transparent and up to date as new studies are being published, we developed a Shiny web app that regularly incorporates new results and enables users to modify study selection criteria to evaluate the robustness of the inference across potential confounds. Our analysis focuses on 17 single-blind studies comparing taVNS versus sham in healthy participants. These newly synthesized results provide strong evidence for the null hypothesis (g = 0.011, CIshortest = [-0.103, 0.125], BF01 = 25.587), indicating that acute taVNS does not alter HRV compared to sham. To conclude, based on a synthesis of the available evidence to date, there is no support for the hypothesis that HRV is a robust biomarker for acute taVNS. By increasing transparency and timeliness, we believe that the concept of living meta-analyses can lead to transformational benefits in emerging fields such as non-invasive brain stimulation.

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Desiccation-driven senescence and its repression in Xerophyta schlechteri are regulated at extremely low water contents O_LIVegetative desiccation tolerance, the ability to survive loss of over 90% of cellular water, is an extremely rare trait in Angiosperms. Xerophyta schlechteri survives such extreme water deficit by entering prolonged quiescence and suppressing drought-induced senescence in most of the leaf area, except the apical tip. Information on the molecular regulation of senescence in such plants is scarce and this is the first study to investigate such regulation in senescing and non-senescing tissues of the same leaf.
C_LIO_LIGenome-wide RNA sequencing enabled comparison of senescent and non-senescent tissues during desiccation and early rehydration, establishment of the water content range in which senescence is initiated and identification of molecular mechanisms employed to bring about cellular death.
C_LIO_LISenescence-associated genes (XsSAG) specific to this species were identified and two potential regulatory sites were enriched in regions upstream to these XsSAGs, allowing us to create a model of senescence regulation in X. schlechteri based on homology with known Arabidopsis senescence regulators.
C_LIO_LIWe hypothesise that desiccation-driven senescence occurs as a result of a convergence of signals around MAPK6 to trigger WRKY-mediated ethylene synthesis and XsSAG expression, not unlike aging and stress-related senescence in Arabidopsis, but at remarkably lower water contents (<35% RWC).
C_LI

sentences: The anti-angiogenic compound dimethyl fumarate inhibits the serine synthesis pathway and increases glycolysis in endothelial cells A pathological and persistent angiogenesis is observed in several diseases like retinopathies, diabetes, psoriasis and cancer. Dimethyl fumarate, an ester from the Krebs cycle intermediate fumarate, is approved as a drug for the treatment of psoriasis and multiple sclerosis, and its anti-angiogenic activity has been reported in vitro and in vivo. However, it is not known whether dimethyl fumarate is able to modulate endothelial cell metabolism, considered an essential feature for the angiogenic switch. By means of different experimental approximations, including proteomics, isotope tracing and metabolomics experimental approaches, in this work we studied the possible role of dimethyl fumarate in endothelial cell energetic metabolism. We demonstrate for the first time that dimethyl fumarate promotes glycolysis and diminishes cell respiration, which could be a consequence of a down-regulation of serine and glycine synthesis through inhibition of PHGDH activity in endothelial cells. This new target can open a new field of study regarding the mechanism of action of dimethyl fumarate.

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A bacteria-based assay to study SARS-CoV-2 protein-protein interactions Methods for detecting and dissecting the interactions of virally encoded proteins are essential for probing basic viral biology and providing a foundation for therapeutic advances. The dearth of targeted therapeutics for the treatment of COVID-19, an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of SARS-CoV-2-encoded proteins. Here we describe the use of a convenient bacteria-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome. We identify sixteen distinct intraviral protein-protein interactions (PPIs), involving sixteen proteins. We find that many of the identified proteins interact with more than one partner. We further show how our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified B2H system that permits the detection of disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm and we use this system to detect the interaction of the SARS-CoV-2 spike protein receptor-binding domain (RBD) with its cognate cell surface receptor ACE2. We then examine how the RBD-ACE2 interaction is perturbed by several RBD amino acid substitutions found in currently circulating SARS-CoV-2 variants. Our findings illustrate the utility of a genetically tractable bacterial system for probing the interactions of viral proteins and investigating the effects of emerging mutations. In principle, the system could also facilitate the identification of potential therapeutics that disrupt specific interactions of virally encoded proteins. More generally, our findings establish the feasibility of using a B2H system to detect and dissect disulfide bond-dependent interactions of eukaryotic proteins.

ImportanceUnderstanding how virally encoded proteins interact with one another is essential in elucidating basic viral biology, providing a foundation for therapeutic discovery. Here we describe the use of a versatile bacteria-based system to investigate the interactions of the protein set encoded by SARS-CoV-2, the virus responsible for the current pandemic. We identify sixteen distinct intraviral protein-protein interactions, involving sixteen proteins, many of which interact with more than one partner. Our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified version of our bacteria-based system that permits detection of the interaction between the SARS-CoV-2 spike protein (specifically its receptor binding domain) and its cognate human cell surface receptor ACE2 and we investigate the effects of spike mutations found in currently circulating SARS-CoV-2 variants. Our findings illustrate the general utility of our system for probing the interactions of virally encoded proteins.

sentences: Predicting Dog Phenotypes from Genotypes We analyze dog genotypes (i.e., positions of dog DNA sequences that often vary between different dogs) in order to predict the corresponding phenotypes (i.e., unique observed characteristics). More specifically, given chromosome data from a dog, we aim to predict the breed, height, and weight. We explore a variety of linear and non-linear classification and regression techniques to accomplish these three tasks. We also investigate the use of a neural network (both in linear and non-linear modes) for breed classification and compare the performance to traditional statistical methods. We show that linear methods generally outperform or match the performance of non-linear methods for breed classification. However, we show that the reverse is true for height and weight regression. Finally, we evaluate the results of all of these methods based on the number of input features used in the analysis. We conduct experiments using different fractions of the full genomic sequences, resulting in input sequences ranging from 20 SNPs to [~]200k SNPs. In doing so, we explore the impact of using a very limited number of SNPs for prediction. Our experiments demonstrate that these phenotypes in dogs can be predicted with as few as 0.5% of randomly selected SNPs (i.e., 992 SNPs) and that dog breeds can be classified with 50% balanced accuracy with as few as 0.02% SNPs (i.e., 40 SNPs).

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Sialophorin is an essential host element for vaccine immunity against pulmonary fungal infections The global burden of fungal infections is alarming, primarily due to the increasing immune-compromised population. The immuno-preventive/therapeutic measures, including vaccines, are necessary to prevent or control fungal diseases. Identifying a protective host element as a functional phenotypic marker is immensely valuable. We identified a host element, sialophorin, preferentially associated with antifungal memory T cells. We investigated its role in vaccine immunity using a mouse model of pulmonary fungal infection. We found that sialophorin was essential to bolster CD8+ T-cell responses to the vaccine by enhancing their differentiation and expanding cytokine-producing cells required for immunity. Using a gain-of-function approach, activating sialophorin using mAb augmented the CD8+ T cell responses, and sialophorin-sufficient CD8+ T cells were competitively superior in differentiation and expansion to the deficient cells. Sialophorin-mediated vaccine immunity was independent of the T cell trafficking effect. Finally, we show that sialophorin is a potential functional phenotypic marker of fungal vaccine-potency and immunity. Our study revealed that sialophorin is an essential host-target element to bolster vaccine responses and serves as a potential biomarker of fungal immunity.

Author SummaryFungal infections have been rising in recent years due to increased immunocompromised individuals. Vaccination of at-risk individuals helps counter the infections. Thus, suitable vaccine platforms are needed with apt adjuvants, and a phenotypic marker of vaccine immunity will bolster the efforts. We identified a phenotypic marker, sialophorin, associated with T cell vaccine immunity to fungal infection. Our findings show an essential role of sialophorin for fungal immunity, as a target of adjuvanticity, and as a potential biomarker of vaccine immunity against many fungal infections.

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A standardized non-visual behavioral event is broadcasted homogeneously across cortical visual areas without modulating visual responses Multiple recent studies have shown that motor activity greatly impacts the activity of primary sensory areas like V1. Yet, the role of this motor related activity in sensory processing is still unclear.

Here we dissect how these behavior signals are broadcast to different layers and areas of the visual cortex. To do so, we leveraged a standardized and spontaneous behavioral fidget event in passively viewing mice. Importantly, this behavior event had no relevance to any ongoing task allowing us to compare its neuronal correlate with visually relevant behavior like running.

A large two-photon Ca2+ imaging database of neuronal responses uncovered four neural response types during fidgets that were surprisingly consistent in their proportion and response patterns across all visual areas and layers of the visual cortex. Indeed, the layer and area identity could not be decoded above chance level based only on neuronal recordings. In contrast to running behavior, fidget evoked neural responses were independent to visual processing.

The broad availability of visually orthogonal standardized behavior signals could be a key component in how the cortex selects, learns and binds local sensory information with motor outputs. Contrary to relevant motor outputs, irrelevant motor signals would use a separate neural subspaces.

Significance StatementRecent studies have shown contextual and behavioral variables to dominate brain-wide activity, but yet it is unknown how this information is broadcast across cortical layers and areas. Using a large two-photon dataset collected in mice passively viewing a battery of visual stimuli, we characterized the neuronal response of neurons of the visual cortex to a standardized fidget behavior. We found that as much as 47% of excitatory neurons show significant co-activity with fidgets. Throughout all areas and layers we recorded from, those responses were distributed across surprisingly consistent three neural response types. Further analysis showed no interaction between fidget neural events and cells visual stimulus responses, contrasting with feedback neural signals induced by running. These contrasting response distribution patterns suggest that behavioral neuronal correlates are broadly available but will modulate sensory responses depending on their relevance to local sensory inputs.

sentences: Global mammalian zooregions reveal a signal of past human impacts Ecologists have long documented that the worlds biota is spatially organized in regions with boundaries shaped by processes acting on geological and evolutionary timescales. Although growing evidence suggests that human impact has been key in how biodiversity is currently assembled, its role as a driver of the geographical organization of biodiversity remains unclear. Here, we quantify the relative importance of human land use from [~]5000 years ago to predict the current assemblage of terrestrial mammals in biogeographical regions across the Earth. Results show that past anthropogenic land use has left an imprint on the taxonomic differentiation of some of the largest biogeographical realms, whereas land use at present stands out as a driver of the taxonomic differences between medium-sized subregions, i.e., within and among continents. Our findings highlight the far-reaching effect that past anthropogenic actions have had on the organization of biodiversity globally.

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Exploring cell-specific miRNA regulation with single-cell miRNA-mRNA co-sequencing data BackgroundExisting computational methods for studying miRNA regulation are mostly based on bulk miRNA and mRNA expression data. However, bulk data only allows the analysis of miRNA regulation regarding a group of cells, rather than the miRNA regulation unique to individual cells. Recent advance in single-cell miRNA-mRNA co-sequencing technology has opened a way for investigating miRNA regulation at single-cell level. However, as currently single-cell miRNA-mRNA co-sequencing data is just emerging and only available at small-scale, there is a strong need of novel methods to exploit existing single-cell data for the study of cell-specific miRNA regulation.

ResultsIn this work, we propose a new method, CSmiR (Cell-Specific miRNA regulation) to use single-cell miRNA-mRNA co-sequencing data to identify miRNA regulatory networks at the resolution of individual cells. We apply CSmiR to the miRNA-mRNA co-sequencing data in 19 K562 single-cells to identify cell-specific miRNA-mRNA regulatory networks for understanding miRNA regulation in each K562 single-cell. By analyzing the obtained cell-specific miRNA-mRNA regulatory networks, we observe that the miRNA regulation in each K562 single-cell is unique. Moreover, we conduct detailed analysis on the cell-specific miRNA regulation associated with the miR-17/92 family as a case study. Finally, through exploring cell-cell similarity matrix characterized by cell-specific miRNA regulation, CSmiR provides a novel strategy for clustering single-cells to help understand cell-cell crosstalk.

ConclusionsTo the best of our knowledge, CSmiR is the first method to explore miRNA regulation at a single-cell resolution level, and we believe that it can be a useful method to enhance the understanding of cell-specific miRNA regulation.

sentences: Two NLR immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain A subset of plant NLR immune receptors carry unconventional integrated domains in addition to their canonical domain architecture. One example is rice Pik-1 that comprises an integrated heavy metal-associated (HMA) domain. Here, we reconstructed the evolutionary history of Pik-1 and its NLR partner, Pik-2, and tested hypotheses about adaptive evolution of the HMA domain. Phylogenetic analyses revealed that the HMA domain integrated into Pik-1 before Oryzinae speciation over 15 million years ago and has been under diversifying selection. Ancestral sequence reconstruction coupled with functional studies showed that two Pik-1 allelic variants independently evolved from a weakly binding ancestral state to high-affinity binding of the blast fungus effector AVR-PikD. We conclude that for most of its evolutionary history the Pik-1 HMA domain did not sense AVR-PikD, and that different Pik-1 receptors have recently evolved through distinct biochemical paths to produce similar phenotypic outcomes. These findings highlight the dynamic nature of the evolutionary mechanisms underpinning NLR adaptation to plant pathogens.

sentences: Recombination patterns in coronaviruses As shown during the SARS-CoV-2 pandemic, phylogenetic and phylodynamic methods are essential tools to study the spread and evolution of pathogens. One of the central assumptions of these methods is that the shared history of pathogens isolated from different hosts can be described by a branching phylogenetic tree. Recombination breaks this assumption. This makes it problematic to apply phylogenetic methods to study recombining pathogens, including, for example, coronaviruses. Here, we introduce a Markov chain Monte Carlo approach that allows inference of recombination networks from genetic sequence data under a template switching model of recombination. Using this method, we first show that recombination is extremely common in the evolutionary history of SARS-like coronaviruses. We then show how recombination rates across the genome of the human seasonal coronaviruses 229E, OC43 and NL63 vary with rates of adaptation. This suggests that recombination could be beneficial to fitness of human seasonal coronaviruses. Additionally, this work sets the stage for Bayesian phylogenetic tracking of the spread and evolution of SARS-CoV-2 in the future, even as recombinant viruses become prevalent.

sentences: Transcriptomic analysis reveals that severity of infectious bursal disease in White Leghorn inbred chicken lines is associated with greater bursal inflammation in vivo and more rapid induction of pro-inflammatory responses in primary bursal cells stimulated ex vivo. In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p<0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

sentences: Functional differences drive the selection of NRAS mutants in melanoma Distinct NRAS mutants are enriched in various tumor types. Here, we generated a suite of fully congenic, conditional, Nras knock-in mouse models (LSL-Nras Q61R, -K, -L, -H, -P, -Q; G12D and G13D, -R) to test the hypothesis that melanocyte transformation requires functions specific to the NRAS mutants enriched in human melanoma (Q61R and Q61K). Consistent with the rarity of NRAS codon 12 and 13 mutants in human melanoma, spontaneous melanomas were rare or absent in mice expressing NRAS G12D, G13D or G13R. Mice expressing less common codon 61 alleles (Q61H, Q61P) also developed few or no tumors. NRAS Q61R, Q61K, or Q61L expression, by contrast, induced rapid melanoma onset with high penetrance. Cohorts of heterozygous mice containing one LSL-Nras Q61R and one LSL-Nras Q61K, -L, -H, -P, or -Q allele were generated to assess potential interactions between NRAS mutants. The ability of each Nras variant to substitute for an Nras Q61R allele was consistent with its own ability to drive spontaneous melanoma formation. However, LSL-Nras Q61Q/Q61R mice rarely developed tumors. In vitro experiments in mouse embryonic fibroblasts (MEFs) highlighted activation of the MAPK pathway as a defining difference between tumorigenic and non-tumorigenic NRAS mutants. Enhanced MAPK activation was associated with the promotion of BRAF-BRAF and BRAF-CRAF dimers. These results support the development of cancer preventative strategies specific to the properties of the commonly observed RAS mutants in each tumor type.

sentences: Facilitation between ecosystem engineers, salt marsh grass and mussels, produces pattern formation on salt marsh shorelines Interspecific facilitation between ecosystem engineers, such as salt marsh grass and mussel aggregations, is a key process that structures communities and enhances biodiversity. Scale-dependent pattern formation via self-organization is ubiquitous in terrestrial, aquatic and marine ecosystems. Despite their prevalence and ecological importance, these two phenomena have rarely been linked. We provide empirical evidence that the facilitative interaction in salt marshes between smooth cordgrass Spartina alterniflora and the ribbed mussel Geukensia demissa produces distinct spatial patterns along marsh shorelines. These findings advance our understanding of linkages between facilitation and pattern formation in nature, and are particularly relevant to conservation and restoration of salt marshes threatened by climate change and sea-level rise.

sentences: Lpnet: Reconstructing Phylogenetic Networks from Distances Using Integer Linear Programming We present Lpnet, a variant of the widely used Neighbor-net method that approximates pairwise distances between taxa by a circular phylogenetic network. We first apply standard methods to construct a binary phylogenetic tree and then use integer linear programming to compute an optimal circular orderings that agrees with all tree splits. This approach achieves an improved approximation of the input distance for the clear majority of experiments that we have run for simulated and real data. We release an implementation in R that can handle up to 94 taxa and usually needs about one minute on a standard computer for 80 taxa. For larger taxa sets, we include a top-down heuristic which also tends to perform better than Neighbor-net.

sentences: Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it is structurally identical to the protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer construct with the purpose of increasing RBD immunogenicity. We produced multimeric particles by a transpeptidation reaction between the RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric protein of 170 kDa. We vaccinated mice with two doses 30 days apart, and then we measured humoral immune response. When the number of RBD copies coupled to BLS was high (6-7 RBD molecules per BLS decamer, in average), the immune response was significantly better than that elicited by RBD alone or even by RBD-BLS comprising low number of RBD copies (1-2 RBD molecules per BLS decamer). Remarkably, the construct with high number of RBD copies induced high IgG titers with high neutralizing capacity. Furthermore, a superior immune response was observed when Al(OH)3 adjuvant was added to this formulation, exhibiting a higher titer of neutralizing antibodies. Altogether our results suggest that RBD covalent coupled to BLS forming a multimer-particle shows an advantageous architecture to the antigen-presentation to the immune system which enhances immune responses. This new antigen should be considered a potent candidate for a protein-based vaccine.

sentences: Developmental Venous Anomalies are a Genetic Primer for Cerebral Cavernous Malformations Cerebral cavernous malformations (CCM) are a neurovascular anomaly that may occur sporadically in otherwise healthy individuals, or be inherited by autosomal dominant mutations in the genes that encode the proteins of the CCM signaling complex (KRIT1, CCM2, or PDCD10)1-4. CCMs have long been known to follow a genetic two-hit model where lesion formation is initiated by somatic mutations resulting in biallelic loss of a CCM complex gene5-8. Recent studies have shown that somatic mutations in MAP3K3 and PIK3CA also contribute to CCM pathogenesis9-11; however, it remains unclear how these mutations contribute to sporadic versus familial cases. Here we show that somatic mutations in MAP3K3 are mutually exclusive with mutations in CCM complex genes and that mutations in MAP3K3 contribute to sporadic, but not familial CCM. Using single-nucleus DNA sequencing, we show that co-occurring MAP3K3 and PIK3CA mutations are present within the same clonal population of cells. Furthermore, we identify PIK3CA mutations in CCM-associated developmental venous anomalies (DVA). It has long been known that sporadic CCM often develop in the vicinity of a DVA. However, the underlying cause of this association is unknown12-14. In this first report of the molecular pathology of CCM-associated DVA, we find that the identical PIKC3A mutation is found in both the DVA and its associated CCM, but that an activating MAP3K3 mutation appears only in the CCM. These results support a mechanism where DVA develop as the result of a PIK3CA mutation, creating a region of the brain vasculature that functions as a genetic primer for CCM development following acquisition of an additional somatic mutation.

sentences: Novel Generic Models for Differentiating Stem Cells Reveal Oscillatory Mechanisms Understanding cell fate selection remains a central challenge in developmental biology. We present a class of simple yet biologically-motivated mathematical models for cell differentiation that generically generate oscillations and hence suggest alternatives to the standard framework based on Waddingtons epigenetic landscape. The models allow us to suggest two generic dynamical scenarios that describe the differentiation process. In the first scenario gradual variation of a single control parameter is responsible for both entering and exiting the oscillatory regime. In the second scenario two control parameters vary: one responsible for entering, and the other for exiting the oscillatory regime. We analyse the standard repressilator and four variants of it and show the dynamical behaviours associated with each scenario. We present a thorough analysis of the associated bifurcations and argue that gene regulatory networks with these repressilator-like characteristics are promising candidates to describe cell fate selection through an oscillatory process.

sentences: Novel bisubstrate inhibitors for protein N-terminal acetyltransferase D Protein N-terminal acetyltransferase D (NatD, NAA40) that specifically acetylates the alpha-N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed an apparent Ki value of 1.0 nM. Biochemical studies indicated that bisubstrate inhibitor is competitive to the peptide substrate and noncompetitive to the cofactor, suggesting NatD undergoes an ordered Bi-Bi mechanism. We also demonstrated that these inhibitors are highly specific towards NatD, displaying about 1,000-fold selectivity over other closely related acetyltransferases. High-resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanism, providing a rational path for future inhibitor development.

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Clinically-relevant treatment of PDX models reveals patterns of neuroblastoma chemoresistance Chemotherapy resistance and relapses are common in high-risk neuroblastoma (NB), an aggressive pediatric solid tumor of the sympathetic nervous system. Here, we developed a clinically-relevant in vivo treatment protocol mimicking the first line five-chemotherapy treatment regimen of high-risk NB and applied this protocol to mice with MYCN-amplified NB patient-derived xenografts (PDXs). Genomic and transcriptomic analyses were used to reveal the genetic and non-genetic mechanisms involved in NB chemoresistance. We observed convergent and parallel evolution of key NB genetic aberrations over time. Intrinsic resistance to chemotherapy was associated with high genetic diversity and an embryonic phenotype. Relapsed NB PDX tumors with acquired resistance showed an immature mesenchymal-like phenotype resembling multipotent Schwann cell precursors that are found in the adrenal gland. NBs with a successful treatment response presented a lineage-committed adrenergic phenotype similar to normal neuroblasts, reduced cell cycle gene expression, and negative regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade. NB organoids established from relapsed PDX tumors retained drug resistance, tumorigenicity, and transcriptional cell states ex vivo. This work sheds light on mechanisms involved in NB chemotherapy response in vivo and ex vivo using a clinically-relevant protocol, and emphasizes the importance of transcriptional cell states in treatment response. Detailed characterization of resistance mechanisms is essential for the development of novel treatment strategies in non-responsive or relapsed high-risk NB.

One Sentence SummaryCOJEC chemotherapy treatment of neuroblastoma PDX models uncovers patterns of transcriptional plasticity and chemoresistance.

sentences: Leveraging a surrogate outcome to improve inference on a partially missing target outcome Sample sizes vary substantially across tissues in the Genotype-Tissue Expression (GTEx) project, where considerably fewer samples are available from certain inaccessible tissues, such as the substantia nigra (SSN), than from accessible tissues, such as blood. This severely limits power for identifying tissue-specific expression quantitative trait loci (eQTL) in undersampled tissues. Here we propose Surrogate Phenotype Regression Analysis (SO_SCPLOWPRAYC_SCPLOW) for leveraging information from a correlated surrogate outcome (e.g. expression in blood) to improve inference on a partially missing target outcome (e.g. expression in SSN). Rather than regarding the surrogate outcome as a proxy for the target outcome, SO_SCPLOWPRAYC_SCPLOW jointly models the target and surrogate outcomes within a bivariate regression framework. Unobserved values of either outcome are treated as missing data. We describe and implement an expectation conditional maximization algorithm for performing estimation in the presence of bilateral outcome missingness. SO_SCPLOWPRAYC_SCPLOW estimates the same association parameter estimated by standard eQTL mapping and controls the type I error even when the target and surrogate outcomes are truly uncorrelated. We demonstrate analytically and empirically, using simulations and GTEx data, that in comparison with marginally modeling the target outcome, jointly modeling the target and surrogate outcomes increases estimation precision and improves power.

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Assessment of deep learning algorithms for 3D instance segmentation of confocal image datasets Segmenting three dimensional microscopy images is essential for understanding phenomena like morphogenesis, cell division, cellular growth and genetic expression patterns. Recently, deep learning (DL) pipelines have been developed which claim to provide high accuracy segmentation of cellular images and are increasingly considered as the state-of-the-art for image segmentation problems. However, it remains difficult to define their relative performances as the concurrent diversity and lack of uniform evaluation strategies makes it difficult to know how their results compare. In this paper, we first made an inventory of the available DL methods for 3 dimensional (3D) cell segmentation. We next implemented and quantitatively compared a number of representative DL pipelines, alongside a highly efficient non-DL method named MARS. The DL methods were trained on a common dataset of 3D cellular confocal microscopy images. Their segmentation accuracies were also tested in the presence of different image artifacts. A specific method for segmentation quality evaluation was adopted which isolates segmentation errors due to under/over segmentation. This is complemented with a 3D visualization strategy for interactive exploration of segmentation quality. Our analysis shows that the DL pipelines have different levels of accuracy. Two of them, which are end to end 3D and were originally designed for cell boundary detection, show high performance, and offer clear advantages in terms of adaptability to new data.

Author summaryIn recent years a number of deep learning (DL) algorithms based on computational neural networks have been developed which claim to achieve high accuracy and automatic segmentation of 3D microscopy images. Although these algorithms have received considerable attention in the literature, it is difficult to evaluate their relative performances, while it remains unclear whether they really perform better than other, more classical segmentation methods.

To clarify these issues, we performed a detailed, quantitative analysis of a number of representative DL pipelines for cell instance segmentation from 3D confocal microscopy image datasets. We developed a protocol for benchmarking the performances of such DL based segmentation pipelines using common training and test datasets, evaluation metrics and visualizations. Using this protocol, we evaluated and compared four different DL pipelines to identify their strengths and limitations. A high performance non-DL method was also included in the evaluation. We show that DL pipelines may show significant differences in their performances depending on their model architecture and pipeline components but overall show excellent adaptability to unseen data. We also show that our benchmarking protocol could be extended to a variety of segmentation pipelines and datasets.

sentences: SIMBA: SIngle-cell eMBedding Along with features Recent advances in single-cell omics technologies enable the individual and joint profiling of cellular measurements. Currently, most single-cell analysis pipelines are cluster-centric and cannot explicitly model the interactions between different feature types. In addition, single-cell methods are generally designed for a particular task as distinct single-cell problems are formulated differently. To address these current shortcomings, we present SIMBA, a graph embedding method that jointly embeds single cells and their defining features, such as genes, chromatin accessible regions, and transcription factor binding sequences into a common latent space. By leveraging the co-embedding of cells and features, SIMBA allows for the study of cellular heterogeneity, clustering-free marker discovery, gene regulation inference, batch effect removal, and omics data integration. SIMBA has been extensively applied to scRNA-seq, scATAC-seq, and dual-omics data. We show that SIMBA provides a single framework that allows diverse single-cell analysis problems to be formulated in a unified way and thus simplifies the development of new analyses and integration of other single-cell modalities. SIMBA is implemented as an efficient, comprehensive, and extensible Python library (https://simba-bio.readthedocs.io) for the analysis of single-cell omics data using graph embedding.

sentences: Antibody escape and cryptic cross-domain stabilization in the SARS CoV-2 Omicron spike protein The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. The Omicron variant has two dominant sub-lineages, BA.1 and BA.2, each with unprecedented numbers of nonsynonymous and indel spike protein mutations: 33 and 29, respectively. Some of these mutations individually increase transmissibility and enhance immune evasion, but their interactions within the Omicron mutational background is unknown. We characterize the molecular effects of all Omicron spike mutations on expression, human ACE2 receptor affinity, and neutralizing antibody recognition. We show that key mutations enable escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein compensate for destabilizing mutations in the receptor binding domain, thereby enabling the record number of mutations in Omicron sub-lineages. Taken together, our results provide a comprehensive account of the mutational effects in the Omicron spike protein and illuminate previously unknown mechanisms of how the N-terminal domain can compensate for destabilizing mutations within the more evolutionarily constrained RBD.

sentences: Large-scale placenta DNA methylation mega-analysis reveals fetal sex-specific differentially methylated CpG sites and regions Although male-female differences in placental structure and function have been observed, little is understood about their molecular underpinnings. Here, we present a mega-analysis of 14 publicly available placenta DNA methylation (DNAm) microarray datasets to identify individual CpGs and regions associated with fetal sex. In the discovery dataset of placentas from full term pregnancies (N = 532 samples), 5,212 CpGs met genome-wide significance (p < 1E-8) and were enriched in pathways such as keratinization (FDR p-value = 7.37E-14), chemokine activity (FDR p-value = 1.56E-2), and eosinophil migration (FDR p-value = 1.83E-2). Nine differentially methylated regions were identified (fwerArea < 0.1) including a region in the promoter of ZNF300 that showed consistent differential DNAm in samples from earlier timepoints in pregnancy and appeared to be driven predominately by effects in the trophoblast cell type. We describe the largest study of fetal sex differences in placenta DNAm performed to date, revealing genes and pathways characterizing sex-specific placenta function and health outcomes later in life.

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A zebrafish model of COVID-19-associated cytokine storm syndrome reveals differential proinflammatory activities of Spike proteins of SARS-CoV-2 variants of concern. The sudden and unexpected appearance of the COVID-19 pandemic turned the whole world upside down in a very short time. One of the main challenges faced has been to understand COVID-19 patient heterogeneity, as a minority develop life-threatening hyperinflammation, the so-called cytokine storm syndrome (CSS). Using the unique advantages of the zebrafish model we report here the proinflammatory role of Spike (S) proteins from different SARS-CoV-2 variants of concern after injection into the hindbrain ventricle, a cavity filled with cerebrospinal fluid to which immune cells can be easily recruited and that mimics the alveolar environment of the human lung. We found that wild type/Wuhan variant S1 (S1WT) protein promoted neutrophil and macrophage recruitment, local and systemic hyperinflammation, emergency myelopoiesis, and hemorrhages. In addition, S1{gamma} protein was more proinflammatory and S1{delta} was less proinflammatory than S1WT and, strikingly, S1{beta} promoted delayed and long-lasting inflammation. Pharmacological inhibition of the canonical inflammasome robustly alleviated S1 protein-induced inflammation and emergency myelopoiesis. In contrast, genetic inhibition of angiotensin-converting enzyme 2 strengthened the proinflammatory activity of S1, and the administration of angiopoietin (1-7) fully rescued S1-induced hyperinflammation and hemorrhages. These results shed light into the mechanisms orchestrating the COVID-19-associated CSS and the host immune response to different SARS-CoV-2 S protein variants.

HighlightsO_LIS proteins of SARS-CoV-2 promote hyperinflammation, neutrophilia, monocytosis and hemorrhages in zebrafish.
C_LIO_LIS protein effects in zebrafish are mediated via the canonical inflammasome and the Ace2/Angiopoietin (1-7) axis.
C_LIO_LIDelta S1 is less proinflammatory than wild type S1 and fails to induce emergency myelopoiesis in zebrafish.
C_LIO_LINaive and primed human white blood cells are unable to respond to S proteins.
C_LI

sentences: In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage ([≥] 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.

sentences: Immediate neural network impact after the loss of a semantic hub The human brain extracts meaning from the world using an extensive neural system for semantic knowledge. Whether such broadly distributed systems1-3 crucially depend on or can compensate for the loss of one of their highly interconnected hubs4-6 is controversial4. The strongest level of causal evidence for the role of a brain hub is to evaluate its acute network-level impact following disconnection and any rapid functional compensation that ensues. We report rare neurophysiological data from two patients who underwent awake intracranial recordings during a speech prediction task immediately before and after neurosurgical treatment that required disconnection of the left anterior temporal lobe (ATL), a crucial hub for semantic knowledge4-6. Informed by a predictive coding framework, we tested three sets of hypotheses including diaschisis causing disruption in interconnected sites7 and incomplete or complete compensation by other language-critical and speech processing sites8-10. Immediately after ATL disconnection, we observed highly specific neurophysiological alterations in the recorded fronto-temporal network, including abnormally magnified high gamma responses to the speech sounds in auditory cortex. We also observed evidence for rapid compensation, seen as focal increases in effective connectivity involving language-critical sites in the inferior frontal gyrus and speech processing sites in auditory cortex. However, compensation was incomplete, in part because after ATL disconnection speech prediction signals were depleted in auditory cortex. This study provides direct causal evidence for a semantic hub in the human brain and shows striking neural impact and a rapid attempt at compensation in a neural network after the loss of one of its hubs.

sentences: A liquid +TIP-network drives microtubule dynamics through tubulin condensation Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here we show, that two conserved microtubule end-binding proteins, CLIP-170 and EB3, undergo phase separation and form dense liquid-networks. When CLIP-170 and EB3 act together the multivalency of the network increases, which synergistically increases the amount of protein in the dense phase. In vitro and in cells these liquid networks can condense tubulin. In vitro in the presence of microtubules, EB3/CLIP-170 phase separation can co-condense tubulin all along the microtubule. At this condition microtubule growth speed increases up to two-fold and depolymerization events are strongly reduced, compared to conditions with phase separation deficient networks. Our data show that phase separated EB3/CLIP-170 networks impact microtubule growth dynamics beyond direct protein-microtubule interactions.

sentences: Spatial redistribution of neurosecretory vesicles upon stimulation accelerates their directed transport to the plasma membrane Through the integration of results from an imaging analysis of intracellular trafficking of labelled neurosecretory vesicles in chromaffin cells, we develop a Markov state model to describe their transport and binding kinetics. Our simulation results indicate that a spatial redistribution of neurosecretory vesicles occurs upon secretagogue stimulation leading vesicles to the plasma membrane where they undergo fusion thereby releasing adrenaline and noradrenaline. Furthermore, we find that this redistribution alone can explain the observed up-regulation of vesicle transport upon stimulation and its directional bias towards the plasma membrane. Parameter fitting indicates that in the deeper compartment within the cell, vesicle transport is asymmetric and characterised by a bias towards the plasma membrane. We also find that crowding of neurosecretory vesicles undergoing directed transport explains the observed accelerated recruitment of freely diffusing vesicles into directed transport upon stimulation.

sentences: Creation of a single cell RNA seq meta-atlas to define human liver immune homeostasis The liver is unique in both its ability to maintain immune homeostasis and in its potential for immune tolerance following solid organ transplantation. Single-cell RNA sequencing (scRNA seq) is a powerful approach to generate highly dimensional transcriptome data to understand cellular phenotypes. However, when scRNA data is produced by different groups, with different data models, different standards, and samples processed in different ways, it can be challenging to draw meaningful conclusions from the aggregated data. The goal of this study was to establish a method to combine  human liver scRNA seq datasets by 1) characterizing the heterogeneity between studies and 2) using the meta-atlas to define the dominant phenotypes across immune cell subpopulations in healthy human liver. Publicly available scRNA seq data generated from liver samples obtained from a combined total of 17 patients and [~]32,000 cells were analyzed. Liver-specific immune cells (CD45+) were extracted from each dataset, and immune cell subpopulations (myeloid cells, NK and T cells, plasma cells, and B cells) were examined using dimensionality reduction (UMAP), differential gene expression, and ingenuity pathway analysis. All datasets co-clustered, but cell proportions differed between studies. Gene expression correlation demonstrated similarity across all studies, and canonical pathways that differed between datasets were related to cell stress and oxidative phosphorylation rather than immune-related function. Next, a meta-atlas was generated via data integration and compared against PBMC data to define gene signatures for each hepatic immune subpopulation. This analysis defined key features of hepatic immune homeostasis, with decreased expression across immunologic pathways and enhancement of pathways involved with cell death. This method for meta-analysis of scRNA seq data provides a novel approach to broadly define the features of human liver immune homeostasis. Specific pathways and cellular phenotypes described in this human liver immune meta-atlas provide a critical reference point for further study of immune mediated disease processes within the liver.

sentences: Computational study of the furin cleavage domain of SARS-CoV-2: delta binds strongest of extant variants We demonstrate that AlphaFold and AlphaFold Multimer, implemented within the ColabFold suite, can accurately predict the structures of the furin enzyme with known six residue inhibitory peptides. Noting the similarity of the peptide inhibitors to polybasic furin cleavage domain insertion region of the SARS-CoV-2, which begins at P681, we implement this approach to study the wild type furin cleavage domain for the virus and several mutants. We introduce mutations in silico for alpha, omicron, and delta variants, for several sequences which have been rarely observed, for sequences which have not yet been observed, for other coronaviruses (NL63, OC43, HUK1a, HUK1b, MERS, and 229E), and for the H5N1 flu. We show that interfacial hydrogen bonds between the furin cleavage domain and furin are a good measure of binding strength that correlate well with endpoint binding free energy estimates, and conclude that among all candidate viral sequences studied, delta is near the very top binding strength within statistical accuracy. However, the binding strength of several rare sequences match delta within statistical accuracy. We find that the furin S1 pocket is optimized for binding arginine as opposed to lysine. This residue, typically at sequence position five, contains the most hydrogen bonds to the furin, and hydrogen bond count for just this residue shows a strong positive correlation with the overall hydrogen bond count. We demonstrate that the root mean square backbone C-alpha fluctuation of the first residue in the furin cleavage domain has a strong negative correlation with the interfacial hydrogen bond count. We show by considering the variation with the number of basic residues that the maximum mean number of interfacial hydrogen bonds expected is 15.7 at 4 basic residues.

sentences: UniverSC: a flexible cross-platform single-cell data processing pipeline Single-cell RNA-sequencing analysis to quantify RNA molecules in individual cells has become popular owing to the large amount of information one can obtain from each experiment. We have developed UniverSC (https://github.com/minoda-lab/universc), a universal single-cell processing tool that supports any UMI-based platform. Our command-line tool enables consistent and comprehensive integration, comparison, and evaluation across data generated from a wide range of platforms.

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TAPAS: an open-source software package for Translational Neuromodeling and Computational Psychiatry Psychiatry faces fundamental challenges with regard to mechanistically guided differential diagnosis, as well as prediction of clinical trajectories and treatment response of individual patients. This has motivated the genesis of two closely intertwined fields: (i) Translational Neuromodeling (TN), which develops "computational assays" for inferring patient-specific disease processes from neuroimaging, electrophysiological, and behavioral data; and (ii) Computational Psychiatry (CP), with the goal of incorporating computational assays into clinical decision making in everyday practice. In order to serve as objective and reliable tools for clinical routine, computational assays require end-to-end pipelines from raw data (input) to clinically useful information (output). While these are yet to be established in clinical practice, individual components of this general end-to-end pipeline are being developed and made openly available for community use.

In this paper, we present the Translational Algorithms for Psychiatry-Advancing Science (TAPAS) software package, an open-source collection of building blocks for computational assays in psychiatry. Collectively, the tools in TAPAS presently cover several important aspects of the desired end-to-end pipeline, including: (i) tailored experimental designs and optimization of measurement strategy prior to data acquisition, (ii) quality control during data acquisition, and (iii) artifact correction, statistical inference, and clinical application after data acquisition. Here, we review the different tools within TAPAS and illustrate how these may help provide a deeper understanding of neural and cognitive mechanisms of disease, with the ultimate goal of establishing automatized pipelines for predictions about individual patients. We hope that the openly available tools in TAPAS will contribute to the further development of TN/CP and facilitate the translation of advances in computational neuroscience into clinically relevant computational assays.

sentences: Structural covariance of amygdala subregions is associated with trait aggression and endogenous testosterone in healthy individuals Many studies point toward volume reductions in the amygdala as a potential neurostructural marker for trait aggression. However, most of these findings stem from clinical samples, rendering unclear whether the findings generalize to non-clinical populations. Furthermore, the notion of neural networks suggests that interregional correlations in grey matter volume (i.e., structural covariance) can explain individual differences in aggressive behavior beyond local univariate associations. Here, we tested whether structural covariance between amygdala subregions and the rest of the brain is associated with self-reported aggression in a large sample of healthy young students (n=263; 51% women). Salivary testosterone concentrations were measured for a subset of n=76 participants (45% women), allowing us to investigate the influence of endogenous testosterone on structural covariance. Aggressive individuals showed enhanced covariance between superficial amygdala (SFA) and dorsal anterior insula (dAI), but lower covariance between laterobasal amygdala (LBA) and dorsolateral prefrontal cortex (dlPFC). These structural patterns overlap with functional networks involved in the genesis and regulation of aggressive behavior, respectively. With increasing endogenous testosterone, we observed stronger structural covariance between centromedial amygdala (CMA) and medial prefrontal cortex in men and between CMA and orbitofrontal cortex in women. These results speak for structural covariance of amygdala subregions as a robust correlate of trait aggression in healthy individuals. Moreover, regions that showed structural covariance with the amygdala modulated by either testosterone or aggression did not overlap, speaking for a more complex role of testosterone in human social behavior rather than the simple assumption that testosterone only increases aggressiveness.

sentences: Targeted degradation of the oncogenic phosphatase SHP2 SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras/MAPK pathway upon stimulation of receptor tyrosine kinases (RTKs), which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at sub-micromolar concentration, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.

sentences: Quantitative resistance loci to southern rust mapped in a temperate maize diversity panel Southern rust is a severe foliar disease of maize resulting from infection with the obligate biotrophic fungus, Puccinia polysora. The disease reduces photosynthetic productivity which reduces yields with the greatest yield losses (up to 50%) associated with earlier onset infections. Puccinia polysora urediniospores overwinter only in tropical and subtropical regions but cause outbreaks when environmental conditions favor initial infection. Increased temperatures and humidity during the growing season, combined with an increased frequency of moderate winters are likely to increase the frequency of severe southern rust outbreaks in the US corn belt. In summer 2020, a severe outbreak of Southern Rust was observed in eastern Nebraska (NE), USA. Disease incidence severity showed significant variation among maize genotypes. A replicated maize association panel planted in Lincoln, NE was scored for disease severity. Genome wide association studies identified four loci associated with significant quantitative variation in disease severity which were associated with candidate genes with plausible links to quantitative disease resistance and a transcriptome wide association study conducted identified additional associated genes. Together these results indicate substantial diversity in resistance to southern rust exists among current temperate adapted maize germplasm, including several candidate loci which may explain observed variation in resistance to southern rust.

sentences: Immunomodulatory Contribution of Mast Cells to the Regenerative Biomaterial Microenvironment Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials.

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Genome sequences of Aegilops species of section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for improvement of wheat. To understand their diversity and advance their utilization, we produced whole-genome assemblies of Ae. longissima and Ae. speltoides. Whole-genome comparative analysis, along with the recently sequenced Ae. sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides is the closest ancestor of the wheat B subgenome and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome-wide analysis of nucleotide-binding site leucine-rich repeat (NLR) genes revealed species-specific and lineage-specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.

TeaserGenome sequences of Aegilops species provides a key for efficient exploitation of this rich genetic resource in wheat improvement.

sentences: CRISPR-Cas Controls Cryptic Prophages The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12 and thought to be inactive against prophages. Instead, we demonstrate here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., 5 cryptic) prophages. Specifically, compared to the wild-type strain, reducing interfering RNAs (crRNA) from the spacer region due to a cas2 polar mutation, reduces growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA and subsequent cleavage of the double-stranded RNA, rather than through cleavage of cryptic prophage DNA; i.e., self-targeting. Consistently, seven of the 13 E. coli spacers contain partial matches to the cryptic prophages, and inactivation of CRISPR-Cas increases the level of cryptic prophage CP4-57 and DLP-12 lytic gene transcripts for lysis protein YdfD of Qin and lysis protein RzoD of DLP-12. Also, lysis is clearly seen via transmission electron microscopy when the CRISPR-Cas array is deleted. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages; i.e., unlike with active lysogens, CRISPR-Cas and cryptic prophages may stably exist.

sentences: Extrachromosomal DNA is associated with chromothripsis events and diverse prognoses in gastric cardia adenocarcinoma Extrachromosomal DNA plays an important role in oncogene amplification in tumour cells and poor outcomes across multiple cancers. However, the function of extrachromosomal DNA in gastric cardia adenocarcinoma (GCA) is very limited. Here, we investigated the availability and function of extrachromosomal DNA in GCA from a Chinese cohort of GCA using whole-genome sequencing (WGS), whole-exome sequencing (WES), and immunohistochemistry. For the first time, we identified the ecDNA amplicons present in most GCA patients, and found that some oncogenes are present as ecDNA amplicons in these patients. We found that oncogene ecDNA amplicons in the GCA cohort were associated with the chromothripsis process and may be induced by accumulated DNA damage due to local dietary habits in the geographic region. Strikingly, we observed diverse correlations between the presence of ecDNA oncogene amplicons and prognosis, where ERBB2 ecDNA amplicons correlated with good prognosis, EGFR ecDNA amplicons correlated with poor prognosis, and CCNE1 ecDNA amplicons did not correlate with prognosis. Large-scale ERBB2 immunohistochemistry results from 1668 GCA patients revealed that there was a positive correlation between the presence of ERBB2 and prognosis in 2-7-year survival; however, there was a negative correlation between the presence of ERBB2 and prognosis in 0-2-year survival. Our observations indicate that the presence of ERBB2 ecDNA in GCA patients may represent a good prognosis marker.

sentences: Suppression without inhibition: How retinal computation contributes to saccadic suppression Visual perception remains stable across saccadic eye movements, despite the concurrent strongly disruptive visual flow. This stability is partially associated with a reduction in visual sensitivity, known as saccadic suppression, which already starts in the retina with reduced ganglion cell sensitivity. However, the retinal circuit mechanisms giving rise to such suppression remain unknown. Here, we describe these mechanisms using electrophysiology in mouse, pig, and macaque retina, 2-photon calcium imaging, computational modeling, and human psychophysics. We find that sequential stimuli, such as those that naturally occur during saccades, trigger three independent suppressive mechanisms in the retina. The main suppressive mechanism is triggered by contrast-reversing sequential stimuli and originates within the receptive field center of ganglion cells. It does not involve inhibition or other known suppressive mechanisms such as saturation or adaptation. Instead, it relies on temporal filtering of the inherently slow response of cone photoreceptors coupled with downstream nonlinearities. Two further mechanisms of suppression are present predominantly in ON ganglion cells and originate in the receptive field surround, highlighting a novel disparity between ON and OFF ganglion cells. The mechanisms uncovered here likely play a role in shaping the retinal output following eye movements and other natural viewing conditions where sequential stimulation is ubiquitous.

sentences: A mechano-osmotic feedback couples cell volume to the rate of cell deformation Mechanics has been a central focus of physical biology in the past decade. In comparison, the osmotic and electric properties of cells are less understood. Here we show that a parameter central to both the physics and the physiology of the cell, its volume, depends on a mechano-osmotic coupling. We found that cells change their volume depending on the rate at which they change shape, when they spread, migrate or are externally deformed. Cells undergo slow deformation at constant volume, while fast deformation leads to volume loss. We propose a mechano-sensitive pump and leak model to explain this phenomenon. Our model and experiments suggest that volume modulation depends on the state of the actin cortex and the coupling of ion fluxes to membrane tension. This mechano-osmotic coupling defines a membrane tension homeostasis module constantly at work in cells, causing volume fluctuations associated with fast cell shape changes, with potential consequences on cellular physiology.

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Piezo2 mediates ultrasonic hearing via cochlear outer hair cells in mice Ultrasonic hearing is exploited for hunting and navigation as in echolocation by microbats and bottleneck dolphins, and for social communication like ultrasonic vocalization by mice and rats. However, the molecular and cellular basis for ultrasonic hearing is not known yet. Here we show that knockout of the mechanosensitive ion channel PIEZO2 in cochlea disrupts the ultrasonic hearing but not the low-frequency hearing in mice, as shown by audiometry and acoustically-associative freezing behavior. Deletion of Piezo2 in the outer hair cells specifically abolishes the associative learning of the mice upon hearing the ultrasonic frequency. Ex vivo cochlear Ca2+ imaging revealed that the ultrasonic transduction requires both PIEZO2 and the hair-cell mechanotransduction channel. Together, our study demonstrates that the outer hair cells are the effector cells with PIEZO2 as an essential molecule for ultrasonic hearing in mice.

Significance StatementSome animals have evolved an incredible ability for vocalizing and hearing ultrasonic frequencies that is inaudible for humans (> 20 kHz). For many years, it has been considered that animals hear ultrasonic frequencies with their cochlear hair cells, using the identical set of mechanotransduction molecules in the hair bundles for hearing audible frequencies. Here, we show that the mice lacking the mechanosensitive ion channel PIEZO2 hardly hear ultrasonic frequencies, while can still be sensitive to audible frequencies. Thus, animals may use a partially different mechanism for sensing physiological ultrasound.

sentences: Three-dimensional chromatin architecture of early-stage mouse embryos reconstructed via recurrence plots The chromatin conformation capture-related methods such as Hi-C have improved our understanding of nuclear architecture and organization in recent years. However, reconstruction of nuclear architecture of individual cells from single cell Hi-C (scHi-C) data has been challenging due to limited information of DNA contacts owing to the low efficiency of DNA recovery from a single cell. We have previously developed an algorithm named as "recurrence plot-based reconstruction (RPR) method" for reconstructing three dimensional (3D) genomic structure from Hi-C data of single haploid cells1 and diploid cells 2. This mathematical method is based on a recurrence plot, a tool of nonlinear time series analysis for visualizing temporal patterns within a time series 3,4, and enables the reconstruction of a unique 3D chromosome architecture even from sparse (low-coverage) DNA contact information. Here we applied the RPR method to analyzing published scHi-C data of diploid cells derived from early-stage F1 hybrid embryos 5. We found that paternal and maternal chromosomes become gradually intermingled from 1 cell to 64 cell stage and that discrete chromosome territories (CTs) are largely established between 8 cell and 64 cell stages. We also observed Rabl-like polarized distribution of chromosomes from 2 cell to 8 cell stage but this polarization becomes mostly dissolved by 64 cell stage. The formation of Rabl-like configuration precedes rod-like extension of the chromosomal shape and their parallel alignment, implicating a role of Rabl-like configuration in avoiding entanglement and promoting effective mixing of chromosomes before establishment of CTs. We also found a cell-to-cell variability in chromatin configuration. Combination of scHi-C and RPR analyses thus can characterize distinct 3D chromatin architecture of individual cells at different developmental stages during early embryogenesis.

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Preventing co-infection: a viral strategy with short-term benefits and long-term drawbacks Viral superinfection occurs when multiple viral particles subsequently infect the same host. In nature, several viral species are found to have evolved diverse mechanisms to prevent superinfection (superinfection exclusion) but how this strategic choice impacts the fate of mutations in the viral population remains unclear. Using stochastic simulations, we find that genetic drift is suppressed when superinfection occurs, thus facilitating the fixation of beneficial mutations and the removal of deleterious ones. Interestingly, we also find that the competitive (dis)advantage associated with variations in life history parameters is not necessarily captured by the viral growth rate for either infection strategy. Putting these together, we then show that a mutant with superinfection exclusion will easily overtake a superinfecting population even if the latter has a much higher growth rate. Our findings suggest that while superinfection exclusion can negatively impact the long-term adaptation of a viral population, in the short-term it is ultimately a winning strategy.

Author summaryViral social behaviour has recently been receiving increasing attention in the context of ecological and evolutionary dynamics of viral populations. One fascinating and still relatively poorly understood example is superinfection or co-infection, which occur when multiple viruses infect the same host. Among bacteriophages, a wide range of mechanisms have been discovered that enable phage to prevent superinfection (superinfection exclusion) even at the cost of using precious resources for this purpose. What is the evolutionary impact of this strategic choice and why do so many phages exhibit this behaviour? Here, we conduct an extensive simulation study of a phage population to address this question. In particular, we investigate the fate of viral mutations arising in an environment with a constant supply of bacterial hosts designed to mimic a "turbidostat," as these are increasingly being used in laboratory evolution experiments. Our results show that allowing superinfection in the long-term yields a population which is more capable of adapting to changes in the environment. However, when in direct competition, mutants capable of preventing superinfection experience a very large advantage over their superinfecting counterparts, even if this ability comes at a significant cost to their growth rate. This indicates that while preventing superinfection can negatively impact the long-term prospects of a viral population, in the short-term it is ultimately a winning strategy.

sentences: Detection of Rapidly Accumulating Stress-Induced SUMO in Prostate Cancer Cells by a Fluorescent SUMO Biosensor SUMO conjugates and SUMO chains form when SUMO, a small ubiquitin-like modifier protein, is covalently linked to other cellular proteins or itself. During unperturbed growth, cells maintain balanced levels of SUMO conjugates. In contrast, eukaryotic cells that are exposed to proteotoxic and genotoxic insults mount a cytoprotective SUMO-Stress Response (SSR). One hallmark of the SSR is a rapid and massive increase of SUMO conjugates in response to oxidative, thermal, and osmotic stress. Here, we use a recombinant fluorescent SUMO biosensor, KmUTAG-fl, to investigate differences in the SSR in a normal human prostate epithelial cell line immortalized with SV40 (PNT2) and two human prostate cancer cell lines that differ in aggressiveness and response to androgen (LNCaP and PC3). In cells that grow unperturbed, SUMO is enriched in the nuclei of all three cell lines. However, upon 30 minutes of exposure to ultraviolet radiation (UV) or oxidative stress, we detected significant cytosolic enrichment of SUMO as measured by KmUTAG-fl staining. This rapid enrichment in cytosolic SUMO levels was on average 5-fold higher in the LNCaP and PC3 prostate cancer cell lines compared to normal immortalized PNT2 cells. Additionally, this enhanced enrichment of cytosolic SUMO was reversible as cells recovered from stress exposure. Our study validates the use of the fluorescent KmUTAG-fl SUMO biosensor to detect differences of SUMO levels and localization between normal and cancer cells and provides new evidence that cancer cells may exhibit an enhanced SSR.

sentences: Unilateral corneal insult in Zebrafish results in a bilateral cell shape and identity modification, supporting wound closure Most of terrestrial and aquatic vertebrates are equipped with camera-type eyes, offering a focused and clear sight. This apparatus is rendered inefficient if its most superficial and transparent element, the cornea, is opaque. This structure, prone to environmental aggressions, bears excellent wound healing capabilities to preserve vision. Up to date, most of the corneal wound healing studies are made on mammals. Here, for the first time, zebrafish is used as model to study wound closure of corneal epithelium after abrasion. Our study demonstrates a swift wound closure after corneal insult. Interestingly, a unilateral wound induces a bilateral response. While cell proliferation is increased during wound closure, this parameter is not crucial, and cell rearrangements seems to be the driving force. Furthermore, we discovered a profound change in epithelial cell transcriptomic signature after abrasion, reflecting a modulation of cell identity and increase of phenotypic plasticity. The latter seems to unlock terminally differentiated cell capacities for wound healing, which could be the key for a speed up organ regeneration. Our results prove that zebrafish cornea is a powerful model to investigate, not only corneal wound healing, but ectodermal organ pathophysiology.

sentences: Homeostatic control of synaptic rewiring in recurrent networks induces the formation of stable memory engrams Brain networks store new memories using functional and structural synaptic plasticity. Memory formation is generally attributed to Hebbian plasticity, while homeostatic plasticity is thought to have an ancillary role in stabilizing network dynamics. Here we report that homeostatic plasticity alone can also lead to the formation of stable memories. We analyze this phenomenon using a new theory of network remodeling, combined with numerical simulations of recurrent spiking neural networks that exhibit structural plasticity based on firing rate homeostasis. These networks are able to store repeatedly presented patterns and recall them upon the presentation of incomplete cues. Storage is fast, governed by the homeostatic drift. In contrast, forgetting is slow, driven by a diffusion process. Joint stimulation of neurons induces the growth of associative connections between them, leading to the formation of memory engrams. These memories are stored in a distributed fashion throughout connectivity matrix, and individual synaptic connections have only a small influence. Although memory-specific connections are increased in number, the total number of inputs and outputs of neurons undergo only small changes during stimulation. We find that homeostatic structural plasticity induces a specific type of "silent memories", different from conventional attractor states.

sentences: Downregulation of RyR and NCX in the neonatal rat ventricular myocyte modulates cytosolic Calcium (Ca2+) is necessary for cardiac muscle contraction. RyR, NCX, and SERCA are key regulatory protein channels for cytosolic Ca2+ in cardiac myocytes. Expression levels of these proteins are a function of development, with protein expression shifting toward the adult phenotype over time. We investigated how downregulation by siRNAs of RyR and NCX affected expression levels of complimentary proteins and the corresponding intracellular Ca2+ transients. We compared experimentally observed Ca2+ transients to those predicted by mathematical models. Experiments show RyR downregulation decreased SERCA and increased NCX protein levels. The associated Ca2+ transient had a decreased amplitude, increased time-to-peak, 50%, and 90% Ca2+ removal with respect to the control cell. NCX downregulation increased SERCA production without significant changes in RyR expression levels. The corresponding [Ca2+] transient had increased amplitude, no change in time-to-peak and 50% Ca2+ removal, and increased 90% Ca2+ removal with respect to the control cell. Computational models that accurately predict the observed experimental data suggest compensatory changes occurring in the expression levels as well as biochemical activity of the regulatory proteins.

sentences: Visuo-vestibular gaze control - conserved subcortical processing Gaze stabilization compensates for movements of the head or external environment to minimize image blurring, which is critical for visually-guided behaviors. Multisensory information is used to stabilize the visual scene on the retina via the vestibulo-ocular (VOR) and optokinetic (OKR) reflexes. While the organization of neuronal circuits underlying VOR is well described across vertebrates, less is known about the contribution and evolutionary origin of the OKR circuits. Moreover, the integration of these two sensory modalities is still poorly understood. Here, we developed a novel experimental model, the isolated lamprey eye-brain-labyrinth preparation, to analyze the neuronal pathways underlying visuo-vestibular integration which allowed electrophysiological recordings while applying vestibular stimulation using a moving platform, coordinated with visual stimulation via two screens. We show that lampreys exhibit robust visuo-vestibular integration, with optokinetic information processed in the pretectum and integrated with vestibular inputs at several subcortical levels. The enhanced eye movement response to multimodal stimulation favored the vestibular response at increased velocities. The optokinetic signals can be downregulated from tectum. Additionally, saccades are present in the form of nystagmus. The lamprey represents the oldest living group of vertebrates, thus all basic components of the visuo-vestibular control of gaze were present already at the dawn of vertebrate evolution.

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Quantitative Restoration of Immune Defense in Old Animals Determined by Naive Antigen-Specific CD8 T cell Numbers Older humans and animals often exhibit reduced immune responses to infection and vaccination, and this often directly correlates to the numbers and frequency of naive T (Tn) cells. We found such a correlation between reduced numbers of blood CD8+ Tn cells and severe clinical outcomes of West Nile virus (WNV) in both humans naturally exposed to, and mice experimentally infected with, WNV.

To examine possible causality, we sought to increase the number of CD8 Tn cells by treating C57BL/6 mice with IL-7 complexes (IL-7C, anti-IL-7 mAb bound to IL-7), shown previously to efficiently increase peripheral T cell numbers by homeostatic proliferation. T cells underwent robust expansion following IL-7C administration to old mice increasing the number of total T cells (>four-fold) and NS4b:H-2Db-restricted antigen-specific CD8 T cells (two-fold). This improved the numbers of NS4b-specific CD8 T cells detected at the peak of the response against WNV, but not survival of WNV challenge. IL-7C treated old animals also showed no improvement in WNV-specific effector immunity (neutralizing antibody and in vivo T cell cytotoxicity). To test quantitative limits to which CD8 Tn cell restoration could improve protective immunity, we transferred graded doses of Ag-specific precursors into old mice and showed that injection of 5,400 (but not of 1,800 or 600) adult naive WNV-specific CD8 T cells significantly increased survival after WNV. These results set quantitative limits to the level of Tn reconstitution necessary to improve immune defense in older organisms and are discussed in light of targets of immune reconstitution.

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Maternal Western-Style Diet Impairs Bone Marrow Development and Drives a Hyperinflammatory Phenotype in Hematopoietic Stem and Progenitor Cells in Fetal Rhesus Macaques BackgroundMaternal obesity adversely impacts the in utero metabolic environment and offsprings health, but its effect on fetal hematopoiesis and immune cell development remains incompletely understood, particularly in models that resemble human development.

MethodsWe studied gestational day 130-135 fetuses derived from rhesus macaque dams chronically exposed to a high-fat Western-style diet (WSD) or a low-fat control diet. Fetal immune cell phenotypes and fetal bone marrow architecture and hematopoietic stem and progenitor cell (FBM HSPC) function were examined using bone computed tomography, histology, flow cytometry, single-cell RNA-sequencing, and HSPC transplantation assays.

FindingsMaternal WSD induced premature FBM cavity opening and a codominant increase in the number of FBM adipocytes. Furthermore, a maternal WSD induced a proinflammatory transcriptional response in FBM HSPCs. FBM macrophages from the WSD group exhibited heightened proinflammatory responses to toll-like receptor agonist stimulation. Maternal WSD exposure suppressed the expression of genes required for B-cell development and decreased the frequencies of FBM B-cells. Finally, maternal WSD led to poor engraftment of FBM HSPCs in nonlethally irradiated immunodeficient NOD/SCID/IL2r{gamma}-/- mice.

InterpretationsMaternal WSD impairs FBM development, drives a hyperinflammatory phenotype, and induces functional and differentiation impairment in FBM HSPCs in a translationally relevant nonhuman primate model.

FundingNational Institute of Health

RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSMaternal obesity is associated with increased risk of infections and proinflammatory disease in offspring. The translationally-relevant rhesus macaque model was utilized to address the effects of maternal obesogenic diet on fetal hematopoietic and immune cell development.

Added value of this studyWe assessed changes in fetal immune cell phenotypes and fetal hematopoietic stem and progenitor cell function using immunohistochemistry, flow cytometry, single-cell RNA sequencing, and transplantation assays. We determined that chronic consumption of a maternal obesogenic diet induced the development of adipogenic and proinflammatory environments in the fetal bone marrow. Additionally, we detected the impairment in B-cell differentiation program in fetal hematopoietic stem and progenitor cells.

Implications of all the available evidenceThese data demonstrate that maternal obesogenic diet modulates fetal hematopoietic development and could impact the offsprings immune system, including proinflammatory phenotype and a decline in B-cell function.

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A Method for Cost-Effective and Rapid Characterization of Genetic Parts Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a cost-effective and rapid method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house, as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR-amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components, or by titrating DNA concentrations rather than relying on limited plasmid copy numbers. Because this method retains a cell-like environment, the function of the genetic part will typically mimic its function in whole cells.

SUMMARYWell-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

sentences: Correlative imaging of spatio-angular dynamics of molecular assemblies and cells with multimodal instant polarization microscope Biological function depends on the spatio-angular architecture of macromolecules - for example, functions of lipid membrane and cytoskeletal polymers arise from both the spatial and the angular organization of the constituent molecules. Correlative imaging of cellular and molecular architecture is valuable across cell biology and pathology. However, current live imaging methods primarily focus on spatial component of the architecture. Imaging the dynamic angular architecture of cells and organelles requires fast polarization-, depth-, and wavelength-diverse measurement of intrinsic optical properties and fluorophore concentration, but remains challenging with current designs. We report a multimodal instant polarization microscope (miPolScope) that combines a broadband polarization-resolved detector, automation, and reconstruction algorithms to enable label-free imaging of phase, retardance, and orientation, multiplexed with fluorescence imaging of concentration, anisotropy, and orientation of molecules at diffraction-limited resolution and high speed. miPolScope enabled multimodal imaging of myofibril architecture and contractile activity of beating cardiomyocytes, cell and organelle architecture of live HEK293T and U2OS cells, and density and anisotropy of white and grey matter of mouse brain tissue across the visible spectrum. We anticipate these developments in joint quantitative imaging of density and anisotropy to enable new studies in tissue pathology, mechanobiology, and imaging-based screens.

sentences: ESCPE-1 Mediates Retrograde Endosomal Sorting of the SARS-CoV-2 Host Factor Neuropilin-1 Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed  cargoes) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify novel retrograde cargo proteins of the Endosomal SNX-BAR Sorting Complex Promoting Exit-1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterised host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalised with the NRP1-interacting peptide of the SARS-CoV-2 Spike protein. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

sentences: Performance comparison and in-silico harmonisation of commercial platforms for DNA methylome analysis by targeted bisulfite sequencing DNA methylation is a key epigenetic modification in the regulation of cell fate and differentiation, and its analysis is gaining increasing importance in both basic and clinical research. Targeted Bisulfite Sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. Here we benchmarked five commercially available TBS platforms, including three hybridization capture-based (Agilent, Roche, and Illumina) and two RRBS-based (Diagenode and NuGen), across 16 samples. A subset of these were also compared to whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed performance with respect to workflow complexity, on/off-target performance, coverage, accuracy and reproducibility. We find all platforms able to produce usable data but major differences for some performance criteria, especially in the number and identity of the CpG sites covered, which affects the interoperability of datasets generated on these different platforms. To overcome this limitation, we used imputation and show that it improves the interoperability from an average of 10.35% (0.8M CpG sites) to 97% (7.6M CpG sites). Our study provides cross-validated guidance on which TBS platform to use for different features of the methylome and offers an imputation-based harmonization solution for improved interoperability between platforms, allowing comparative and integrative analysis.

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Context-aware information selection and model recommendation with ACCORDION Querying new information from knowledge sources, in general, and published literature, in particular, aims to provide precise and quick answers to questions raised about a system under study. In this paper, we present ACCORDION (ACCelerating and Optimizing model RecommenDatIONs), a novel methodology and a tool to enable efficient addressing of biological questions, by automatically recommending models that recapitulate desired dynamic behavior. Our approach integrates information extraction from literature, clustering, simulation and formal analysis to allow for automated, consistent, and robust assembling, testing and selection of context-specific models. We used ACCORDION in nine benchmark case studies and compared its performance with other previously published tools. ACCORDION is comprehensive as it can capture all relevant knowledge from literature, obtained by automated literature search and machine reading. At the same time, as our results show, ACCORDION is selective, recommending only the most relevant and useful subset (15-20%) of candidate model extensions found in literature, while guided by baseline model context and goal properties. ACCORDION is very effective, also demonstrated by our results, as it can reduce the error of the initial baseline model by more than 80%, recommending models that closely recapitulate desired behavior, and outperforming previously published tools. In this process, ACCORDION can also suggest more than one highly scored model, thus providing alternative solutions to user questions and novel insights for treatment directions.

Contact{yaa38@pitt.edu, nmzivanov@pitt.edu}

Supplementary informationSupplementary data are available.

Availabilityhttp://www.biodesignlab.pitt.edu/accordion

sentences: Proton-pumping rhodopsins in marine diatoms Diatoms are a major phytoplankton group responsible for about 20% of Earths primary production. They carry out photosynthesis inside the plastid, an organelle obtained through eukaryote-eukaryote endosymbiosis. Recently, microbial rhodopsin, a photoreceptor distinct from chlorophyll-based photosystems, has been identified in certain diatoms. However, the physiological function of diatom rhodopsin is not well understood. Here we show that the diatom rhodopsin acts as a light-driven proton pump and localizes to the outermost membrane of the four membrane-bound complex plastids. Heterologous expression techniques were used to investigate the protein function and subcellular localization of diatom rhodopsin. Using model simulations, we further evaluated the physiological role of the acidic pool in the plastid produced by proton-transporting rhodopsin. Our results propose that the rhodopsin-derived acidic pool may be involved in a photosynthetic CO2-concentrating mechanism and assist CO2 fixation in diatom cells.

sentences: Gene conversion explains elevated diversity in the immunity modulating APL1 gene of the malaria vector Anopheles funestus The leucine rich repeat gene APL1 is a key component of immunity to Plasmodium and other microbial pathogens in Anopheles mosquitoes. In the malaria vector Anopheles funestus the APL1 gene has four paralogues which occur along the same chromosome arm. We show that APL1 has exceptional levels of non-synonymous polymorphism across the range of An. funestus with an average {pi}n of 0.027 versus a genome-wide average of 0.002, and {pi}n (and {pi}s) is consistently high in populations across Africa. The pattern of APL1 diversity was consistent between independent pooled-template and target-enrichment datasets, however no link between APL1 diversity and insecticide-resistance was observed with the phenotyped target-enrichment dataset. Two further innate immunity genes of the gambicin anti-microbial peptide family had {pi}n/{pi}s ratios greater than one, possibly driven by either positive or balancing selection. Cecropin antimicrobial peptides were expressed much more highly than other anti-microbial peptide genes, an observation discordant with current models of anti-microbial peptide activity. The observed APL1 diversity likely results from gene conversion between paralogs, as evidenced by shared polymorphisms, overlapping read mappings, and recombination events among paralogues. Gene conversion at APL1 versus alternative explanations is concordant with similarly elevated diversity in APL1 and TEP1 loci in An. gambiae. In contrast, the more closely related An. stephensi which also encodes a single-copy of APL1 does not show this elevated diversity. We hypothesise that a more open chromatin formation at the APL1 locus due to higher gene expression than its paralogues enhances gene conversion, and therefore increased polymorphism, at APL1.

sentences: Nonparametric Interrogation of Transcriptional Regulation in Single-Cell RNA and Chromatin Accessibility Multiomic Data Epigenetic control of gene expression is highly cell-type- and context-specific. Yet, despite its complexity, gene regulatory logic can be broken down into modular components consisting of a transcription factor (TF) activating or repressing the expression of a target gene through its binding to a cis-regulatory region. Recent advances in joint profiling of transcription and chromatin accessibility with single-cell resolution offer unprecedented opportunities to interrogate such regulatory logic. Here, we propose a nonparametric approach, TRIPOD, to detect and characterize three-way relationships between a TF, its target gene, and the accessibility of the TFs binding site, using single-cell RNA and ATAC multiomic data. We apply TRIPOD to interrogate cell-type-specific regulatory logic in peripheral blood mononuclear cells and contrast our results to detections from enhancer databases, cis-eQTL studies, ChIP-seq experiments, and TF knockdown/knockout studies. We then apply TRIPOD to mouse embryonic brain data during neurogenesis and gliogenesis and identified known and novel putative regulatory relationships, validated by ChIP-seq and PLAC-seq. Finally, we demonstrate TRIPOD on SHARE-seq data of differentiating mouse hair follicle cells and identify lineage-specific regulation supported by histone marks for gene activation and super-enhancer annotations.

sentences: Digenic traits under population admixture and inbreeding The population genetics of digenic genotypes in diploid populations, genotypes based on alleles at two loci, have been studied theoretically for decades with relevant digenic traits of medical interest being known for over 25 years. Given the effects of linkage and linkage disequilibrium on two locus genotypes, it should be expected that these factors can change the expected frequencies of digenic genotypes in many, sometimes unexpected, ways. In particular, the combination of linkage disequilibrium and inbreeding can combine to increase the frequencies of double homozygotes and double heterozygotes significantly over outbred comparisons. Given the prevalence of linkage disequilibrium in recently admixed populations, this can lead to large shifts in trait prevalence such that it can sometimes exceed that of either original pre-admixed population with the combined effects of linkage disequilibrium and inbreeding. Here we investigate the frequencies of digenic genotypes under the combined effects of linkage, linkage disequilibrium, and inbreeding to analyze how these interact to increase or decrease the frequency of the genotypes across two loci.

sentences: The recent range expansion of Great Cormorants (Phalacrocorax carbo hanedae) in Hokkaido, Japan. Though the Japanese subspecies of Great Cormorant (Phalacrocorax carbo hanedae) has increased its population in recent decades, information on its spread to the northern island of Hokkaido has not been reported outside of Japan. The purpose of this paper is to update the scientific community about the breeding and range ecology of P. carbo, and to provide comparative information on the abundant and similar looking resident Japanese Cormorant (P. capillatus). Several ornithological groups and researchers were contacted in order to gather information about the current distribution and breeding activities of P. carbo in the region. Here the findings of Japanese research groups, translated publications, and direct observations are made available.

sentences: Drug delivery via tattooing: Effect of needle and fluid properties Tattooing is a commonplace practice among the general populace in which ink is deposited within dermal tissue. Typically, an array of needles punctures the skin which facilitates the delivery of a fluid within the dermis. Although, a few studies in the past have investigated the potential of tattooing as an intradermal (ID) drug injection technique, an understanding of the fluid dynamics involved in the delivery of fluid into skin is still lacking. Herein, we sought to provide insight into the process via an in vitro study. We utilize a five needle flat array (5F) with a tattoo machine to inject fluids into gelatin gels. High-speed imaging was used to visualize the injection process and estimate the amount of fluid delive red after each injection upto the 50th injection. We investigate the role of reciprocating frequency (f) of the needle array and the physical properties of the fluids on the volume (Vo) and the percentage delivery ({eta}) after injection. In addition, we illustrate the physical mechanism of fluid infusion during tattooing, which has not been reported. An understanding of the injection process via tattooing can be useful in the development of ID tattoo injectors as drug delivery devices.

sentences: SARS-CoV-2 Nsp1 N-terminal and linker regions as a platform for host translational shutoff In the early stages of SARS-CoV-2 infection, non-structural protein 1 (Nsp1) inhibits the innate immune response by inserting its C-terminal helices into the mRNA entry channel of the ribosome and promoting mRNA degradation. Nevertheless, the mechanism by which Nsp1 achieves host translational shutoff while allowing for viral protein synthesis remains elusive. We set out to characterize the interactome of full-length Nsp1 and its topology by crosslinking mass spectrometry in order to investigate the role of the N-terminal domain and linker regions in host translational shutoff. We find that these regions are in contact with 40S proteins lining the mRNA entry channel and detect a novel interaction with the G subunit of the eIF3 complex. The crosslink-derived distance restraints allowed us to derive an integrative model of full-length Nsp1 on the 40S subunit, reporting on the dynamic interface between Nsp1, the ribosome and the eIF3 complex. The significance of the Nsp1-eIF3G interaction is supported by further evidence that Nsp1 predominantly binds to 40-43S complexes. Our results point towards a mechanism by which Nsp1 is preferentially recruited to canonical initiation complexes, leading to selective inhibition of host-translating ribosomes and subsequent mRNA degradation.

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Neuronal activity-driven oligodendrogenesis in selected brain regions is required for episodic memories The formation of long-term episodic memories requires the activation of molecular mechanisms in several regions of the medial temporal lobe, including the hippocampus and anterior cingulate cortex (ACC). The extent to which these regions engage distinct mechanisms and cell types to support memory formation is not well understood. Recent studies reported that oligodendrogenesis is essential for learning and long-term memory; however, whether these mechanisms are required only in selected brain regions is still unclear. Also still unknown are the temporal kinetics of engagement of learning-induced oligodendrogenesis and whether this oligodendrogenesis occurs in response to neuronal activity. Here we show that in rats and mice, episodic learning rapidly increases the oligodendrogenesis and myelin biogenesis transcripts olig2, myrf, mbp, and plp1, as well as oligodendrogenesis, in the ACC but not in the dorsal hippocampus (dHC). Region-specific knockdown and knockout of Myrf, a master regulator of oligodendrocyte maturation, revealed that oligodendrogenesis is required for memory formation in the ACC but not the dHC. Chemogenetic neuronal silencing in the ACC showed that neuronal activity is critical for learning-induced oligodendrogenesis. Hence, an activity-dependent increase in oligodendrogenesis in selected brain regions, specifically in the ACC but not dHC, is critical for the formation of episodic memories.

Impact statementOligodendrogenesis is required in the anterior cingulate cortex but not in the hippocampus for long-term memory consolidation.

sentences: Brief exposure of neuronal cells to levels of short chain fatty acids observed in human systemic circulation impair the cell lipid metabolism resulting in associated cell death by apoptosis Communication between gut microbiota and the brain is an enigma. Alterations in the gut microbial community affects enteric metabolite levels, such as SCFAs. SCFAs have been proposed as a possible mechanism through which the gut microbiome modulate brain health and function. This study analyzed for the first time the effects of SCFAs at levels reported in human systemic circulation on human neuronal cell energy metabolism, viability, survival and the brain lipidome. Cell and rat brain lipidomics was done using UHPLC-HESI-HRAMS/MS. Neuronal cells viability, survival and energy metabolism were analyzed via flow cytometer, immunofluorescence, and SeahorseXF platform. Lipidomics analysis demonstrated that SCFAs significantly remodeled the brain lipidome in vivo and in vitro. The most notable remodulation was observed in the metabolism of phosphatidylethanolamine plasmalogens, and mitochondrial lipids carnitine and cardiolipin. Increased mitochondrial mass, fragmentation, and hyperfusion occurred concomitant with the altered mitochondrial lipid metabolism resulting in decreased neuronal cell respiration, ATP production, and increased cell death. This suggests SCFAs at levels observed in human systemic circulation can adversely alter the brain lipidome and neuronal cell function potentially negatively impacting brain health outcomes.

sentences: Sustained morphine delivery suppresses bone formation and alters metabolic and circulating miRNA profiles in male C57BL/6J mice Opioid use is detrimental to bone health, causing both indirect and direct effects on bone turnover. While the mechanisms of these effects are not entirely clear, recent studies have linked chronic opioid use to alterations in circulating miRNAs. Our aim was to develop a model of opioid-induced bone loss to understand bone turnover and identify candidate miRNA-mediated regulatory mechanisms. We evaluated the effects of sustained morphine treatment on the skeleton, metabolism, and body composition of male and female C57BL/6J mice by treating with vehicle (0.9% saline) or morphine (18 mg/kg) using subcutaneous osmotic minipumps for 25 days. Morphine-treated mice had higher energy expenditure and respiratory quotient, indicating a shift toward carbohydrate metabolism. Microcomputed tomography ({micro}CT) analysis indicated that male mice treated with morphine had reduced trabecular bone volume fraction (Tb.BV/TV) (15%) and Tb. bone mineral density (BMD) (14%) in the distal femur compared to vehicle. Conversely, bone microarchitecture was not changed in females after morphine treatment. Histomorphometric analysis demonstrated that in males, morphine reduced bone formation rate compared to vehicle, but osteoclast parameters were not different. Furthermore, morphine reduced bone formation marker gene expression in the tibia of males (Bglap and Dmp1). Circulating miRNA profile changes were evident in males, with 14 differentially expressed miRNAs associated with morphine treatment. Target analysis indicated hypoxia inducible factor (HIF) signaling pathway was targeted by miR-223-3p and fatty acid metabolism by miR-484, - 223-3p, and -328-3p. In summary, we have established a model where morphine leads to a lower trabecular bone formation in males. Further, understanding the mechanisms of bone loss from opioid treatment will be important for improving management of the adverse effects of opioids on the skeleton.

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Deep phenotyping platform for microscopic plant-pathogen interactions 1O_LIThe initial phases of plant-pathogen interactions are critical since they are often decisive for the successful infection. However, these early stages of interaction are typically microscopic, making it challenging to study on a large scale.
C_LIO_LIFor this reason, using the powdery mildew fungi of cereals as a model, we have developed an automated microscopy pipeline coupled with deep learning-based image analysis for the high-throughput phenotyping of plant-pathogen interactions.
C_LIO_LIThe system can quantify fungal microcolony count and density, the precise area of the secondary hyphae of each colony, and different morphological parameters. Moreover, the high throughput and sensitivity allow quantifying rare microscopic phenotypes in a large sample size. One of these phenotypes is the cryptic infection of non-adapted pathogens, marking the hidden transition stages of pathogen adaptation and breaking the nonhost barrier. Thus, our tool opens the nonhost resistance phenomenon to genetics and genomics studies.
C_LIO_LIWe have developed an open-source high-throughput automated microscopy system for phenotyping the initial stages of plant-pathogen interactions, extendable to other microscopic phenotypes and hardware platforms. Furthermore, we have validated the systems performance in disease resistance screens of genetically diverse barley material and performed Genome-wide associations scans (GWAS), discovering several resistance-associated loci, including conferring nonhost resistance.
C_LI

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Protein-Ligand Interaction Graphs: Learning from Ligand-Shaped 3D Interaction Graphs to Improve Binding Affinity Prediction Graph Neural Networks (GNNs) have recently gained in popularity, challenging molecular fingerprints or SMILES-based representations as the predominant way to represent molecules for binding affinity prediction. Although simple ligand-based graphs alone are already useful for affinity prediction, better performance on multi-target datasets has been achieved with models that incorporate 3D structural information. Most recent advances utilize complex GNN architectures to capture 3D protein-ligand information by incorporating ligand-interacting protein atoms as additional nodes in the graphs; or by building a second protein-based graph in parallel. This expands the graph considerably while obfuscating the shape of the underlying ligand, diminishing the advantage that GNNs have when encoding molecular structures. There is therefore a need for a simple and elegant molecular graph representation that retains the topology of the ligand while simultaneously encoding 3D protein-ligand interactions.

We present Protein-Ligand Interaction Graphs (PLIGs): a simple way of representing atom-atom contacts of 3D protein-ligand complexes as node features for GNNs. PLIGs featurize an atom node in the molecular graph by describing each atoms properties as well as all atom-atom contacts made with protein atoms within a distance threshold. The edges of the graph are therefore identical to ligand-based graphs, but the nodes encode the 3D protein-ligand contacts. Since PLIGs are applicable to any GNN architecture, we have benchmarked their performance with six different GNN architectures, and compared them to conventional ligand-based graphs and fingerprint-based multi-layer perceptron (MLP) models using the CASF-2016 benchmark set where we found PLIG-based Graph Attention Networks (GATNet) to be the best performing model ({rho}=0.84, RMSE=1.22 pK). In summary, we created a novel graph-based representation that incorporates 3D structural information into the node features of ligand-shaped molecular graphs. The PLIG representation is simple, elegant, flexible and easily customizable, opening up many possibilities of incorporating other 2D and 3D properties into the graph.

AccessThe code and implementation for PLIGs and all models can be found at github.com/MarcMoesser/Protein-Ligand-Interaction-Graphs.

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Can cryptic female choice prevent invasive hybridization in external fertilizing fish? Polyandrous mating systems result in females mating with multiple males. This includes the potential for unintended matings and subsequent sperm competition with hybridizing species, especially in the presence of alternative reproductive tactics (sneaker males). Cryptic female choice allows females to bias paternity towards preferred males under sperm competition and may include conspecific sperm preference when under hybridization threat. The potential becomes particularly important in context of invasive species that can novelly hybridize with natives. We provide the first examination of conspecific sperm preference in a system of three species with potential to hybridize: North American native Atlantic salmon (Salmo salar) and brook char (Salvelinus fontinalis), and invasive brown trout (Salmo trutta) from Europe. Using naturalized populations on the island of Newfoundland, we measured changes in sperm swimming performance, a known predictor of paternity, to determine the degree of upregulation to female cues related to conspecific sperm preference. Compared to water alone, female ovarian fluid in general had a pronounced effect and upregulated sperm motility (mean 53%) and swimming velocity (mean 30%). However, patterns in the degree of upregulation suggest there is no conspecific sperm preference in the North American populations. Furthermore, female cues from both native species tended to boost the sperm of invasive males more than their own. We conclude that cryptic female choice is too weak in this system to prevent invasive hybridization and is likely insufficient to promote or maintain reproductive isolation between the native species.

Impact SummaryFemale mediated post-ejaculatory sexual selection, known as cryptic female choice, has only recently been researched in earnest, but has been documented across many taxa. This process allows females to bias paternity to favor a given male and can act as a filter to prevent fertilizations from unwanted males under sperm competition, including those of different species. In internal fertilizers like mammals, birds and insects, mechanisms of cryptic female choice can be very robust as the female can greatly modify the environment that sperm experience. In external fertilizers, females cannot control which males release sperm in close proximity to her eggs as she spawns with a chosen mate, but she can release reproductive fluids that act as a mechanism of cryptic female choice. In fishes, cryptic female choice is often mediated by ovarian fluid that is released with the eggs. This ovarian fluid alters sperm behavior, favoring certain males in situations of sperm competition. The mechanism is reportedly strong in native populations of European Atlantic salmon and brown trout, biasing paternity towards the females own species when eggs are under threat of hybridization under sperm competition. We examined cryptic female choice in three species of hybridizing salmonids on the North American island of Newfoundland, native Atlantic salmon and brook char, and invasive brown trout from Europe. Although the same species, salmon populations from both continents are quite distinct and our results suggest cryptic female choice is too weak in North American Atlantic salmon and brook char to prevent hybridization by invasive brown trout. We hope that this research inspires more work on cryptic female choice to better understand patterns across different species and locally adapted populations within species.

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Matrix topology guides collective cell migration in vivo Diverse modes of cell migration shape organisms in health and disease and much research has focused on the role of intracellular and extracellular components in different cell migration phenomena. What is less explored, however, is how the arrangement of the underlying extracellular matrix that many cells move upon in vivo influences migration.

Combining novel transgenic lines and image analysis pipelines, reveals that during zebrafish optic cup formation cells use cryptopodia-like protrusions to migrate collectively and actively over a topologically changing matrix. These changing topologies correspond to different cell-matrix interactions. Interference with matrix topology results in loss of cryptopodia and inefficient migration. Thus, matrix topology influences the efficiency of directed collective cell migration during eye morphogenesis, a concept likely conserved in other developmental and disease contexts.

One-Sentence SummaryDynamic cell-matrix interactions, crucial for successful collective rim cell migration, rely on extracellular matrix topologies during optic cup development in vivo.

sentences: Exploring the possible role of hybridization in the evolution of photosynthetic pathways in Flaveria (Asteraceae), the prime model of C4 photosynthesis evolution Flaveria (Asteraceae) is the prime model for the study of C4 photosynthesis evolution and seems to support a stepwise acquisition of the pathway through C3-C4 intermediate phenotypes, still existing in Flaveria today. Molecular phylogenies of Flaveria based on concatenated data matrices are currently used to reconstruct the complex sequence of trait shifts during C4 evolution. To assess the possible role of hybridization in C4 evolution in Flaveria, we re-analyzed transcriptome data of 17 Flaveria species to infer the extent of gene tree discordance and possible reticulation events. We found massive gene tree discordance as well as reticulation along the backbone and within clades containing C3-C4 intermediate and C4-like species. An early hybridization event between two C3 species might have triggered C4 evolution in the genus. The clade containing all C4 species plus the C4-like species F. vaginata and F. palmeri is robust but of hybrid origin involving F. angustifolia and F. sonorensis (both C3-C4 intermediate) as parental lineages. Hybridization seems to be a driver of C4 evolution in Flaveria and likely promoted the fast acquisition of C4 traits. This new insight can be used in further exploring C4 evolution and can inform C4 bioengineering efforts.

sentences: Homeostatic regulation of axonal Kv1.1 channels accounts for both synaptic and intrinsic modifications in CA3 circuit Homeostatic plasticity of intrinsic excitability goes hand-in-hand with homeostatic plasticity of synaptic transmission. However, the mechanisms linking the two forms of homeostatic regulation have not been identified so far. Using electrophysiological, imaging and immunohistochemical techniques, we show here that blockade of excitatory synaptic receptors for 2-3 days induces an up-regulation of synaptic strength at CA3-CA3 connexions and intrinsic excitability of CA3 pyramidal neurons. Activity-deprived connexions were found to express a high release probability, an insensitivity to dendrotoxin, and a lack of depolarization-induced presynaptic facilitation, indicating a loss of presynaptic Kv1.1 function. The down-regulation of Kv1.1 channels in activity-deprived neurons was confirmed by their broader action potentials measured in the axon that were insensitive to dendrotoxin. We conclude that regulation of axonal Kv1.1 channel constitutes a unique mechanism linking intrinsic excitability and synaptic strength that accounts for the functional synergy existing between homeostatic regulation of intrinsic excitability and synaptic transmission.

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Practical observations on the use of fluorescent reporter systems in C. difficile ObjectivesFluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherryOpt and phiLOV2.1, and the self-labelling tags SNAPCd and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells.

MethodsWe performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions.

ResultsWe show that the intrinsic fluorescence of C. difficile cells increases during growth, independent from sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced.

Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherryOpt were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype.

ConclusionsThe present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAPCd appears the most suitable candidate for live-cell imaging in C. difficile to date.

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sentences: The chromosome-scale genome assembly of Jasminum sambac var. unifoliatum provide insights into the formation of floral fragrance The high-quality genome assembly of the JSU-FSP provides valuable information on how jasmine produces the abundant VTs and VPBs that synergistically contribute to the pleasant aroma of jasmine flower.

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Monash DaCRA fPET-fMRI: A DAtaset for Comparison of Radiotracer Administration for high temporal resolution functional FDG-PET Background Functional [18F]-fluorodeoxyglucose positron emission tomography (FDG-fPET) is a new approach for measuring glucose uptake in the human brain. The goal of FDG-fPET is to maintain a constant plasma supply of radioactive FDG in order to track, with high temporal resolution, the dynamic uptake of glucose during neuronal activity that occurs in response to a task or at rest. FDG-fPET has most often been applied in simultaneous BOLD-fMRI/FDG-fPET (blood oxygenation level dependent functional MRI fluorodeoxyglucose functional positron emission tomography) imaging. BOLD-fMRI/FDG-fPET provides the capability to image the two primary sources of energetic dynamics in the brain, the cerebrovascular haemodynamic response and cerebral glucose uptake.

FindingsIn this Data Note, we describe an open access dataset, Monash DaCRA fPET-fMRI, which contrasts three radiotracer administration protocols for FDG-fPET: bolus, constant infusion, and hybrid bolus/infusion. Participants (n=5 in each group) were randomly assigned to each radiotracer administration protocol and underwent simultaneous BOLD-fMRI/FDG-fPET scanning while viewing a flickering checkerboard. The Bolus group received the full FDG dose in a standard bolus administration; the Infusion group received the full FDG dose as a slow infusion over the duration of the scan, and the Bolus-Infusion group received 50% of the FDG dose as bolus and 50% as constant infusion. We validate the dataset by contrasting plasma radioactivity, grey matter mean uptake, and task-related activity in the visual cortex.

ConclusionsThe Monash DaCRA fPET-fMRI dataset provides significant re-use value for researchers interested in the comparison of signal dynamics in fPET, and its relationship with fMRI task-evoked activity.

sentences: Microbiota links to neural dynamics supporting threat processing There is growing recognition that the composition of the gut microbiota influences behaviour, including responses to threat. The cognitive-interoceptive appraisal of threat-related stimuli relies on dynamic neural computations between the anterior insular (AIC) and the dorsal anterior cingulate (dACC) cortices. If, to what extent, and how microbial consortia influence the activity of this cortical threat processing circuitry is unclear. We addressed this question by combining a threat processing task, neuroimaging, 16S rRNA profiling, and computational modelling in healthy participants. Results showed interactions between high-level ecological indices with threat-related AIC-dACC neural dynamics. At finer taxonomic resolutions, the abundance of Ruminococcus was differentially linked to connectivity between, and activity within the AIC and dACC during threat updating. Functional inference analysis provides a strong rationale to motivate future investigations of microbiota-derived metabolites in the observed relationship with threat-related brain processes.

sentences: Structural modeling of the SARS-CoV-2 Spike/human ACE2 complex interface can identify high-affinity variants associated with increased transmissibility The COVID-19 pandemic has triggered concerns about the emergence of more infectious and pathogenic viral strains. As a public health measure, efficient screening methods are needed to determine the functional effects of new sequence variants. Here we show that structural modeling of SARS-CoV-2 Spike protein binding to the human ACE2 receptor, the first step in host-cell entry, predicts many novel variant combinations with enhanced binding affinities. By focusing on natural variants at the Spike-hACE2 interface and assessing over 700 mutant complexes, our analysis reveals that high-affinity Spike mutations (including N440K, S443A, G476S, E484R, G502P) tend to cluster near known human ACE2 recognition sites (K31 and K353). These Spike regions are conformationally flexible, allowing certain mutations to optimize interface interaction energies. Although most human ACE2 variants tend to weaken binding affinity, they can interact with Spike mutations to generate high-affinity double mutant complexes, suggesting variation in individual susceptibility to infection. Applying structural analysis to highly transmissible variants, we find that circulating point mutations S447N, E484K and N501Y form high-affinity complexes (~40% more than wild-type). By combining predicted affinities and available antibody escape data, we show that fast-spreading viral variants exploit combinatorial mutations possessing both enhanced affinity and antibody resistance, including S447N/E484K, E484K/N501Y and K417T/E484K/N501Y. Thus, three-dimensional modeling of the Spike/hACE2 complex predicts changes in structure and binding affinity that correlate with transmissibility and therefore can help inform future intervention strategies.

sentences: Biotext: Exploiting Biological-like format for text mining The large amount of existing textual data justifies the development of new text mining tools. Bioinformatics tools can be brought to Text Mining, increasing the arsenal of resources. Here, we present Biotext, a package of strategies for converting natural language text into biological-like information data, providing a general protocol with standardized functions, allowing to share, encode and decode textual data for amino acid data. The package was used to encode the arbitrary information present in the headings of the biological sequences found in a BLAST survey. The protocol implemented in this study consists of 12 steps, which can be easily executed and/ or changed by the user, depending on the study area. Biotext empowers user to perform text mining using bioinformatics tools. Biotext is Freely available at https://pypi.org/project/biotext/ (Python package) and https://sourceforge.net/projects/biotext-tools/files/AMINOcode_GUI/ (Standalone tool).

sentences: Geometric and Topological Approaches to Shape Variation in Ginkgo Leaves Leaf shape is a key plant trait that varies enormously. The diversity of leaf shape, and the range of applications for data on this trait, requires frequent methodological developments so that researchers have an up-to-date toolkit with which to quantify leaf shape. We generated a dataset of 468 leaves produced by Ginkgo biloba, and 24 fossil leaves produced by evolutionary relatives of extant Ginkgo. We quantified the shape of each leaf by developing a geometric method based on elastic curves and a topological method based on persistent homology. Our geometric method indicates that shape variation in our modern sample is dominated by leaf size, furrow depth, and the angle of the two lobes at the base of the leaf that is also related to leaf width. Our topological method indicates that shape variation in our modern sample is dominated by leaf size and furrow depth. We have applied both methods to modern and fossil material: the methods are complementary, identifying similar primary patterns of variation, but also revealing some different aspects of morphological variation. Our topological approach distinguishes long-shoot leaves from short-shoot leaves and both methods indicate that leaf shape influences or is at least related to leaf area.

sentences: Cellular signaling beyond the Wiener-Kolmogorov limit Accurate propagation of signals through stochastic biochemical networks involves significant expenditure of cellular resources. The same is true for regulatory mechanisms that suppress fluctuations in biomolecular populations. Wiener-Kolmogorov (WK) optimal noise filter theory, originally developed for engineering problems, has recently emerged as a valuable tool to estimate the maximum performance achievable in such biological systems for a given metabolic cost. However, WK theory has one assumption that potentially limits its applicability: it relies on a linear, continuum description of the reaction dynamics. Despite this, up to now no explicit test of the theory in nonlinear signaling systems with discrete molecular populations has ever seen performance beyond the WK bound. Here we report the first direct evidence the bound being broken. To accomplish this, we develop a theoretical framework for multi-level signaling cascades, including the possibility of feedback interactions between input and output. In the absence of feedback, we introduce an analytical approach that allows us to calculate exact moments of the stationary distribution for a nonlinear system. With feedback, we rely on numerical solutions of the systems master equation. The results show WK violations in two common network motifs: a two-level signaling cascade and a negative feedback loop. However the magnitude of the violation is biologically negligible, particularly in the parameter regime where signaling is most effective. The results demonstrate that while WK theory does not provide strict bounds, its predictions for performance limits are excellent approximations, even for nonlinear systems.

sentences: Changes in prefrontal GABA and glutamate through adolescence supports excitation/inhibition balance Animal and human postmortem studies provide evidence for changes in gamma-aminobutyric acid (GABA) and glutamate in prefrontal cortex (PFC) during adolescence, suggesting shifts in excitation and inhibition balance consistent with critical period plasticity. However, how GABA and glutamate change through adolescence and how the balance of these inhibitory and excitatory neurotransmitters changes is not well understood in vivo in humans. High field (7 Tesla) Magnetic Resonance Spectroscopic Imaging was used to investigate age-related changes in the balance of GABA/creatine (Cr) and glutamate/Cr in multiple developmentally-relevant regions of PFC in 144 10 to 30-year-olds. Results indicated a homogenous pattern of age-related Glu/Cr decreases across PFC regions, while age-related changes in GABA/Cr were heterogenous, with a mix of stable and decreasing age effects. Importantly, balance between glutamate/Cr and GABA/Cr in areas of prefrontal cortex increased through adolescence, suggesting the presence of critical period plasticity in PFC at this significant time of development when adult trajectories are established.

sentences: The Detection of Bergeyella zoohelcum in Young Children after Cat Bite Bergeyella zoohelcum is an uncommon zoonotic pathogen typically associated with cat or dog bites. Previously, only 14 cases of B. zoohelcum infection have been reported. We isolated the bacteria from the face of a 2-year-old girl who was bitten by a cat. The organism was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence, and phylogenetic analysise supported that this isolate was belong to B. zoohelcum. Due to the contradicts of culture characteristics of B. zoohelcum which described in reported literatures, we used different media to culture the bacteria. After 24 h incubation, Colombia blood agar (CBA), Mueller-Hinton agar plate with 5% sheep blood and chocolate agar (CA) grew well, but blood agar (BA) grew well until 72 h. The strain did not grow on McConkey agar (MAC), Mueller-Hinton agar (MHA) and chocolate agar (containing vancomycin). The low detection rate of this strain was related to its harsh growth conditions and the limitations of traditional identification techniques. With the popularization of MALDI-TOF MS and 16S rRNA gene sequence and our further understanding of the fastidious bacteria, we will quickly and accurately identify the B. zoohelcum, meanwhile the detection rate of this bacterium will also be significantly improved. So that clinicians can achieve precise anti-infective treatment according to antimicrobial susceptibility testing.

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Radiation-reprogrammed glioma stem cells generate vascular-like cells to build a trophic niche driving tumor recurrence Treatment-refractory glioma stem and tumor cells exhibit phenotypic plasticity driving recurrence, but the underlying molecular mechanisms remain to be elucidated. Here, we employed single-cell and whole transcriptomic analyses to discover that radiation induces a dynamic shift in functional states of glioma cells allowing for acquisition of vascular endothelial-like and pericyte-like cell phenotypes. These vascular-like cells provide a trophic niche to promote proliferation of irradiated glioma cells, and their selective depletion results in reduced tumor growth post-treatment in vivo. Mechanistically, the acquisition of vascular-like phenotype is driven by increased chromatin accessibility and H3K27 acetylation in specific vascular gene regions post-treatment. Blocking P300 histone acetyltransferase activity reverses the epigenetic changes induced by radiation, and inhibits the phenotypic transition and tumor growth. Our findings highlight an important role for P300 histone acetyltransferase in treatment-induced plasticity and opens a new therapeutic avenue for preventing glioma recurrence.

SignificanceOur study demonstrates that radiation therapy promotes glioma resistance by inducing vascular-like phenotypes in GSC that, in turn, aid in proliferation of the remaining tumor cells. This phenotype switch is mediated by P300 HAT, and inhibition of this enzyme is a potential therapeutic target for preventing glioma recurrence following radiation.

sentences: Plant genome response to incoming coding sequences: stochastic transcriptional activation independent of integration loci Horizontal gene transfer can occur between phylogenetically distant organisms, such as prokaryotes and eukaryotes. In these cases, how do the translocated genes acquire transcriptional competency in the alien eukaryotic genome? According to the conventional view, specific loci of the eukaryotic genome are thought to provide transcriptional competency to the incoming coding sequences. To examine this possibility, we randomly introduced the promoterless luciferase (LUC)-coding sequences into the genome of Arabidopsis thaliana cultured cells and performed a genome-wide "transgene location vs. expression" scan. We mapped 4,504 promoterless LUC inserts on the A. thaliana chromosomes, and found that about 30% of them were transcribed. Only a small portion of them were explained by the conventional transcriptional fusions with the annotated genes, and the remainder occurred in a quite different manner; (1) they occurred all over the chromosomal regions, (2) independently of the insertion sites relative to the annotated gene loci, inherent transcribed regions, or heterochromatic regions, and (3) with one magnitude lower transcriptional level than the conventional transcriptional fusions. This type of transcriptional activation occurred at about 30% of the inserts, raising a question as to what this 30% means. We tested two hypotheses: the activation occurred at 30% of the entire chromosomal regions, or stochastically at 30% of each insertion event. Our experimental analysis indicates that the latter model could explain this transcriptional activation, a new type of plant genome response to the incoming coding sequences. We discuss the possible mechanisms and evolutionary roles of this phenomenon in the plant genome.

sentences: Learning the rules of cell competition without prior scientific knowledge AO_SCPLOWBSTRACTC_SCPLOWDeep learning is now a powerful tool in microscopy data analysis, and is routinely used for image processing applications such as segmentation and denoising. However, it has rarely been used to directly learn mechanistic models of a biological system, owing to the complexity of the internal representations. Here, we develop an end-to-end machine learning model capable of learning the rules of a complex biological phenomenon, cell competition, directly from a large corpus of time-lapse microscopy data. Cell competition is a quality control mechanism that eliminates unfit cells from a tissue and during which cell fate is thought to be determined by the local cellular neighborhood over time. To investigate this, we developed a new approach ({tau}-VAE) by coupling a probabilistic encoder to a temporal convolution network to predict the fate of each cell in an epithelium. Using the{tau} -VAEs latent representation of the local tissue organization and the flow of information in the network, we decode the physical parameters responsible for correct prediction of fate in cell competition. Remarkably, the model autonomously learns that cell density is the single most important factor in predicting cell fate - a conclusion that is in agreement with our current understanding from over a decade of scientific research. Finally, to test the learned internal representation, we challenge the network with experiments performed in the presence of drugs that block signalling pathways involved in competition. We present a novel discriminator network that, using the predictions of the{tau} -VAE, can identify conditions which deviate from the normal behaviour, paving the way for automated, mechanism-aware drug screening.

sentences: Hippocampal sharp wave-ripples and the associated sequence replay emerge from structured synaptic interactions in a network model of area CA3 Hippocampal place cells are activated sequentially as an animal explores its environment. These activity sequences are internally recreated ("replayed"), either in the same or reversed order, during bursts of activity (sharp wave-ripples; SWRs) that occur in sleep and awake rest. SWR-associated replay is thought to be critical for the creation and maintenance of long-term memory. In order to identify the cellular and network mechanisms of SWRs and replay, we constructed and simulated a data-driven model of area CA3 of the hippocampus. Our results show that the chain-like structure of recurrent excitatory interactions established during learning not only determines the content of replay, but is essential for the generation of the SWRs as well. We find that bidirectional replay requires the interplay of the experimentally confirmed, temporally symmetric plasticity rule, and cellular adaptation. Our model provides a unifying framework for diverse phenomena involving hippocampal plasticity, representations, and dynamics, and suggests that the structured neural codes induced by learning may have greater influence over cortical network states than previously appreciated.

sentences: Astrovirus in Reunion Free-tailed Bat (Mormopterus francoismoutoui) Astroviruses (AstVs) are RNA viruses infecting of a large diversity of avian and mammalian species, including bats, livestock, and humans. We investigated AstV infection in a free-tailed bat species, Mormopterus francoismoutoui, endemic to Reunion Island. A total of 380 guano samples were collected in a maternity colony during 38 different sampling sessions, from June 21st 2016 to September 4th 2018. Each sample was tested for the presence of the AstV RNA-dependent RNA-polymerase (RdRp) gene using a pan-AstV semi-nested polymerase chain reaction assay. In total, 27 guano samples (7.1%) tested positive, with high genetic diversity of the partial RdRp gene sequences among positive samples. A phylogenetic analysis further revealed that the detected viruses were genetically related to AstVs reported in rats, reptiles, dogs, and pigs, but did not cluster with AstVs commonly found in bats. Although more investigations need to be conducted to assess the prevalence of infected bats in the studied population, our findings show that Reunion free-tailed bats are exposed to AstVs, and suggest that cross-species transmission may occur with other hosts sharing the same habitat.

sentences: Make Interactive Complex Heatmaps in R Heatmap is a powerful visualization method on two-dimensional data to reveal patterns shared by subsets of rows and columns. In R, there are many packages that make heatmaps. Among them, ComplexHeatmap provides rich tools for constructing highly customizable heatmaps. It can easily establish connections between information from multiple sources by automatically concatenating and adjusting multiple heatmaps as well as complex annotations, which makes it widely applied in data analysis in various fields, especially in Bioinformatics. Nevertheless, the limit of ComplexHeatmap still exists. It only generates static plots which restricts deeper inspections on complex heatmaps, e.g., to look into a subset of rows and columns when a specific pattern of interest is observed from the heatmap. In this work, we described a new R/Bioconductor package InteractiveComplexHeatmap that brings interactivity to ComplexHeatmap. InteractiveComplexHeatmap is designed with an easy-to-use interface where static complex heatmaps can be directly exported to an interactive Shiny web application only with one extra line of code. The interactive application contains comprehensive tools for manipulating heatmaps. Besides common tools as supported in other interactive heatmap packages, InteractiveComplexHeatmap additionally supports, e.g., selecting over multiple heatmaps and searching heatmaps via row or column labels. Also, InteractiveComplexHeatmap provides methods for exporting static heatmaps from other popular heatmap functions, e.g., heatmap.2() or pheatmap(), to interactive heatmap applications. Finally, InteractiveComplexHeatmap provides flexible functionalities for integrating interactive heatmap widgets into other Shiny applications. InteractiveComplexHeatmap provides a user interface for self-defining response to the selection events on heatmaps, which helps to implement more complex Shiny web applications.

sentences: Early life stress is associated with earlier emergence of permanent molars Exposure to adversity can accelerate biological aging. However, existing biomarkers of early aging are either difficult to detect in individuals at scale, like epigenetic signatures, or cannot be detected until late childhood, like pubertal onset. We evaluated the hypothesis that early adversity is associated with earlier molar eruption, an easily assessed measure that has been used to track the length of childhood across primates. In a pre-registered analysis (N = 117, ages 4-7), we demonstrate that lower family income and exposure to adverse childhood experiences (ACEs) are significantly associated with earlier eruption of the first permanent molars, as rated in T2-weighted magnetic resonance images (MRI). We replicate relationships between income and molar eruption in a population-representative dataset (NHANES; N = 1,973). These findings suggest that the impact of stress on the pace of biological development is evident in early childhood, and detectable in the timing of molar eruption.

sentences: Induction of neutralizing antibodies against SARS-CoV-2 variants by a multivalent mRNA-lipid nanoparticle vaccine encoding SARS-CoV-2/SARS-CoV Spike protein receptor-binding domains To address the need for multivalent vaccines against Coronaviridae that can be rapidly developed and manufactured, we compared antibody responses against SARS-CoV, SARS-CoV-2, and several variants of concern in mice immunized with mRNA-lipid nanoparticle vaccines encoding homodimers or heterodimers of SARS-CoV/SARS-CoV-2 receptor-binding domains. All vaccine constructs induced robust anti-viral antibody responses, and the heterodimeric vaccine elicited an IgG response capable of cross-neutralizing SARS-CoV, SARS-CoV-2 Wuhan-Hu-1, B.1.351 (beta), and B.1.617.2 (delta) variants.

sentences: Physiological and life history changes associated with seasonal adaptation in the cactophilic Drosophila mojavensis Many insects inhabiting temperate climates are faced with changing environmental conditions throughout the year. Depending on the species, these environmental fluctuations can be experienced within a single generation or across multiple generations. Strategies for dealing with these seasonal changes vary across populations. Drosophila mojavensis is a cactophilic Drosophila species endemic to the Sonoran Desert. The Sonoran Desert regularly reaches temperatures of 50{degrees}C in the summer months. As individuals of this population are rare to collect in the summer months, we therefore simulated in a temperature and light controlled chamber the cycling temperatures experienced by D. mojavensis in the Sonoran Desert from April to July (four generations) to understand the physiological and life history changes that allow this population to withstand these conditions. In contrast to our hypothesis of a summer aestivation, we found that D. mojavensis continue to reproduce during the summer months, albeit with lower viability, but the longevity of the population is highly reduced during this period. As expected, stress resistance increased during the summer months in both the adult and the larval stages. This study examines several strategies for withstanding the Sonoran Desert summer conditions which may be informative in the study of other desert endemic species.

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Generalizable EEG encoding models with naturalistic audiovisual stimuli In natural conversations, listeners must attend to what others are saying while ignoring extraneous background sounds. Recent studies have used encoding models to predict electroencephalography (EEG) responses to speech in noise-free listening situations, sometimes referred to as "speech tracking" in EEG. Researchers have analyzed how speech tracking changes with different types of background noise. It is unclear, however, whether neural responses from noisy and naturalistic environments can be generalized to more controlled stimuli. If encoding models for noisy, naturalistic stimuli are generalizable to other tasks, this could aid in data collection from populations who may not tolerate listening to more controlled, less-engaging stimuli for long periods of time. We recorded non-invasive scalp EEG while participants listened to speech without noise and audiovisual speech stimuli containing overlapping speakers and background sounds. We fit multivariate temporal receptive field (mTRF) encoding models to predict EEG responses to pitch, the acoustic envelope, phonological features, and visual cues in both noise-free and noisy stimulus conditions. Our results suggested that neural responses to naturalistic stimuli were generalizable to more controlled data sets. EEG responses to speech in isolation were predicted accurately using phonological features alone, while responses to noisy speech were more accurate when including both phonological and acoustic features. These findings may inform basic science research on speech-in-noise processing. Ultimately, they may also provide insight into auditory processing in people who are hard of hearing, who use a combination of audio and visual cues to understand speech in the presence of noise.

Significance StatementUnderstanding spoken language in natural environments requires listeners to parse acoustic and linguistic information in the presence of other distracting stimuli. However, most studies of auditory processing rely on highly controlled stimuli with no background noise, or with background noise inserted at specific times. Here, we compare models where EEG data are predicted based on a combination of acoustic, phonetic, and visual features in highly disparate stimuli - sentences from a speech corpus, and speech embedded within movie trailers. We show that modeling neural responses to highly noisy, audiovisual movies can uncover tuning for acoustic and phonetic information that generalizes to simpler stimuli typically used in sensory neuroscience experiments.

sentences: Salmonella Typhimurium outer membrane protein A (OmpA) renders protection against nitrosative stress by promoting SCV stability in murine macrophage Porins are highly conserved bacterial outer membrane proteins involved in the selective transport of charged molecules across the membrane. Despite their significant contributions to the pathogenesis of Gram-negative bacteria, their precise role in salmonellosis remains elusive. In this study, we investigated the role of porins (OmpA, OmpC, OmpD, and OmpF) in Salmonella Typhimurium (STM) pathogenesis. OmpA played a multifaceted role in STM pathogenesis, and a strain deleted for ompA (STM{Delta} ompA) showed enhanced proneness to phagocytosis and compromised proliferation in macrophages. However, in the epithelial cells, despite being invasion deficient, it was hyper-proliferative. The poor colocalization of STM{Delta} ompA with LAMP-1 confirmed impaired stability of SCV membrane around the intracellular bacteria, resulting in its (STM{Delta} ompA) release into the cytosol of macrophages where it is assaulted with reactive nitrogen intermediates (RNI). The cytosolic localization of STM{Delta} ompA was responsible for the downregulation of SPI-2 encoded virulence factor SpiC, which is required to suppress the activity of iNOS. The reduced recruitment of nitrotyrosine on STM in the macrophage cytosol upon ectopically expressing Listeriolysin O (LLO) explicitly supported the pro-bacterial role of OmpA against the host nitrosative stress. Further, we show that the generation of time-dependent redox burst could be responsible for the enhanced sensitivity of STM{Delta} ompA towards nitrosative stress. The absence of OmpA in STM{Delta} ompA resulted in the loss of integrity and enhanced porosity of the bacterial outer membrane, which was attributed to the upregulated expression of ompC, ompD, and ompF. We showed the involvement of OmpF in the entry of excess nitrite in STM{Delta} ompA, thus increasing the susceptibility of the bacteria towards in vitro and in vivo nitrosative stress. In conclusion, we illustrated a mechanism of strategic utilization of OmpA compared to other porins by wildtype Salmonella for combating the nitrosative stress in macrophages.

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Decoding Estimates of Curvilinear Self-Motion from Neural Signals in a Model of Primate MSTd Self-motion produces characteristic patterns of optic flow on the eye of the mobile observer. Movement along linear, straight paths without eye movements yields motion that radiates from the direction of travel (heading). The observer experiences more complex motion patterns while moving along more general curvilinear (e.g. circular) paths, the appearance of which depends on the radius of the curved path (path curvature) and the direction of gaze. Neurons in brain area MSTd of primate visual cortex exhibit tuning to radial motion patterns and have been linked with linear heading perception. MSTd also contains neurons that exhibit tuning to spirals, but their function is not well understood. We investigated in a computational model whether MSTd, through its diverse pattern tuning, could support estimation of a broader range of self-motion parameters from optic flow than has been previously demonstrated. We used deep learning to decode these parameters from signals produced by neurons tuned to radial expansion, spiral, ground flow, and other patterns in a mechanistic neural model of MSTd. Specifically, we found that we could accurately decode the clockwise/counterclockwise sign of curvilinear path and the gaze direction relative to the path tangent from spiral cells; heading from radial cells; and the curvature (radius) of the curvilinear path from activation produced by both radial and spiral populations. We demonstrate accurate decoding of these linear and curvilinear self-motion parameters in both synthetic and naturalistic videos of simulated self-motion. Estimates remained stable over time, while also rapidly adapting to dynamic changes in the observers curvilinear self-motion. Our findings suggest that specific populations of neurons in MSTd could effectively signal important aspects of the observers linear and curvilinear self-motion.

Author SummaryHow do we perceive our self-motion as we move through the world? Substantial evidence indicates that brain area MSTd contains neurons that signal the direction of travel during movement along straight paths. We wondered whether MSTd neurons could also estimate more general self-motion along curved paths. We tested this idea by using deep learning to decode signals produced by a neural model of MSTd. The system accurately decoded parameters that specify the observers self-motion along straight and curved paths in videos of synthetic and naturalistic scenes rendered in the Unreal game engine. Our findings suggest that MSTd could jointly signal self-motion along straight and curved paths.

sentences: Investigating the in vivo biodistribution of extracellular vesicles isolated from various human cell sources using positron emission tomography Noninvasive imaging is a powerful tool for understanding the in vivo behavior of drug delivery systems and successfully translating promising platforms into the clinic. Extracellular vesicles (EVs), nano-sized vesicles with a lipid bilayer produced by nearly all cell types, are emerging platforms for drug delivery. To date, the biodistribution of EVs has been insufficiently investigated, particularly using nuclear imaging-based modalities such as positron emission tomography (PET). Herein, we developed positron-emitting radiotracers to investigate the biodistribution of EVs isolated from various human cell sources using PET imaging. Chelator conjugation did not impact EVs size and subsequent radiolabeling was found to be highly efficient and stable with Zr-89 (t1/2 = 78.4 h). In vivo tracking of EVs isolated from bone marrow-derived mesenchymal stromal cells (BMSCs EVs), primary human macrophages (M{phi} EVs), and a melanoma cell line (A375 EVs) were performed in immunocompetent ICR mice. Imaging studies revealed excellent in vivo circulation for all EVs, with a half-life of approximately 12 h. Significantly higher liver uptake was observed for M{phi} EVs, evidencing the tissue tropism of EV and highlighting the importance of carefully choosing EVs cell sources for drug delivery applications. Conversely, the liver, spleen, and lung uptake of the BMSC EVs and A375 EVs was relatively low. We also investigated the impact of immunodeficiency on the biodistribution of BMSC EVs using NSG mice. The spleen uptake drastically increased in NSG mice, which could confound results of therapeutic studies employing this mouse models. Lastly, PET imaging studies in a melanoma tumor model demonstrated efficient tumor uptake of BMSC EVs following intravenous injection. Overall, these imaging studies evidenced the potential of EVs as carriers to treat a variety of diseases, such as cancer or in regenerative medicine applications, and the necessity to understand EVs tropism to optimize their therapeutic deployment.

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Public T-Cell Receptors (TCRs) Revisited by Analysis of the Magnitude of Identical and Highly-Similar TCRs in Virus-Specific T-Cell Repertoires of Healthy Individuals Since multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of T cells in a human body, the likelihood that many public identical (PUB-I) TCR-sequences frequently contribute to immune responses has been estimated to be low. Here, we quantitatively analyzed the TCR-repertoires of 190 purified virus-specific memory T-cell populations, directed against 21 antigens of Cytomegalovirus, Epstein-Barr virus and Adenovirus isolated from 29 healthy individuals, and determined the magnitude, defined as prevalence within the population and frequencies within individuals, of PUB-I TCR and of TCR-sequences that are highly-similar (PUB-HS) to these PUB-I TCR-sequences. We found that almost one third of all TCR nucleotide-sequences represented PUB-I TCR amino-acid (AA) sequences and found an additional 12% of PUB-HS TCRs differing by maximally 3 AAs. We illustrate that these PUB-I and PUB-HS TCRs were structurally related and contained shared core-sequences in their TCR-sequences. We found a prevalence of PUB-I and PUB-HS TCRs of up to 50% among individuals and showed frequencies of virus-specific PUB-I and PUB-HS TCRs making up more than 10% of each virus-specific T-cell population. These findings were confirmed by using an independent TCR-database of virus-specific TCRs. We therefore conclude that the magnitude of the contribution of PUB-I and PUB-HS TCRs to these virus-specific T-cell responses is high. Because the T cells from these virus-specific memory TCR-repertoires were the result of successful control of the virus in these healthy individuals, these PUB-HS TCRs and PUB-I TCRs may be attractive candidates for immunotherapy in immunocompromised patients that lack virus-specific T cells to control viral reactivation.

Significance statementPublic T-cell responses, in which T cells expressing the same T-cell receptor (TCR) are found in different individuals, have been described. However, the magnitude of the contribution of these TCRs to immune responses, defined as prevalence within the population and frequencies within individuals, is not known. In this study we characterized and quantified public T-cell responses within virus-specific memory T cells of healthy individuals by determining identical and highly-similar TCRs recognizing the same antigen and sharing conserved CDR3 motifs. The magnitude of public T-cell responses was surprisingly high and we argue that these dominant TCRs with shared core-sequences could be utilized for diagnostic purposes and may provide attractive TCRs to be used for immunotherapy in immunocompromised patients.

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Proteome, bioinformatic and functional analyses reveal a distinct and conserved metabolic pathway for bile salt degradation in the Sphingomonadaceae Bile salts are amphiphilic steroids with a C5 carboxylic side chain with digestive functions in vertebrates. Upon excretion, they are degraded by environmental bacteria. Degradation of the bile-salt steroid skeleton resembles the well-studied pathway for other steroids like testosterone, while specific differences occur during side-chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either {Delta}1,4- or {Delta}4,6-3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the {Delta}1,4- variant is well-known from many model organisms, the {Delta}4,6-variant involving a 7-hydroxysteroid dehydratase as key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic and functional analyses of the {Delta}4,6-variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to the {Delta}1,4-variant except for the elimination of the 7-OH as key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for side-chain degradation encoding a CoA-ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase, but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the {Delta}4,6-variant to be widespread among the Sphingomonadaceae, which was verified for three type strains which also have the predicted side-chain degradation cluster. A second amidase in the side-chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile-salt hydrolases. This study signifies members of the Sphingomonadaceae remarkably well-adapted to the utilization of bile salts via a partially distinct metabolic pathway.

ImportanceThis study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well-known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.

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EEG-based decoding and recognition of imagined music The authors have withdrawn their manuscript because they discovered an error in the analysis code after publication of the preprint, which turns out to have a major impact on the main results in the paper. The results on the imagination data become non-significant after correcting for the mistake. Significant results on the perception data are preserved, although the classification results are worse than what is reported.

Therefore, the authors do not wish this work to be cited as a reference.

If you have any questions, please contact the last author.

sentences: Pharmacokinetics of Orally Administered GS-441524 in Dogs Despite being FDA-approved for COVID-19, the clinical efficacy of remdesivir (Veklury(R)) remains contentious. We previously pointed out pharmacokinetic, pharmacodynamic and toxicology reasons for why its parent nucleoside GS-441524, is better suited for COVID-19 treatment. Here, we assess the oral bioavailability of GS-441524 in beagle dogs and show that plasma concentrations [~]24-fold higher than the EC50 against SARS-CoV-2 are easily and safely sustained. These data support translation of GS-441524 as an oral agent for COVID-19.

sentences: Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimer formation and enzymatic function Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways like DNA damage response and infection, making them attractive targets for therapeutic intervention. DTX3L, an E3 ubiquitin ligase, forms a heterodimer with PARP9. The complex has ubiquitin ligase activity and also ADP-ribosylates the C-terminus of ubiquitin on Gly76. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. By using individually produced proteins, we have studied the interaction between DTX3L and PARP9. We identify that the D3 domain (230 - 510) of DTX3L mediates interaction with PARP9 with nanomolar affinity and an apparent 1:1 stoichiometry. Our results also suggest the formation of a higher molecular weight oligomer mediated by the N-terminus of DTX3L (1-200). Furthermore, we show that ADP-ribosylation of ubiquitin at Gly76 is a reversible modification that can be removed by several macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination in an inter-regulatory manner.

sentences: Clostridium difficile Toxin B activates the NLRP3 inflammasome in human macrophages, demonstrating a novel regulatory mechanism for the Pyrin inflammasome Pyrin is a cytosolic immune sensor that forms an inflammasome when bacterial virulence factors inhibit RhoA, triggering the release of inflammatory cytokines, including IL-1{beta}. Gain of function mutations in the MEFV gene encoding Pyrin cause auto-inflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin associated auto-inflammation with Neutrophilic Dermatosis (PAAND). To precisely define the role of Pyrin in detecting pathogen virulence factors in relevant human immune cells, we investigated how the Pyrin inflammasome response was initiated and regulated in monocyte-derived macrophages (hMDM) compared to human monocytes. Unlike monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB). In contrast, TcdB activated the NLRP3 inflammasome in hMDM. Notably, we ascertained that the Pyrin inflammasome response could be re-enabled in hMDM by prolonged priming with either LPS or type I or II interferons, and required an increase in Pyrin expression. These data demonstrate an unexpected redundancy in detecting these toxins by inflammasome sensors.

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Amyloid beta oligomers modulate neuronal autophagy through the primary cilium The major neurodegenerative diseases, like Alzheimers disease (AD), accumulate neuropathogenic proteins that compromise autophagic function. In AD, autophagy contributes to intracellular APP processing and amyloid beta (A{beta}) generation by mutant presenilin-1 (PS1). However, how extracellular soluble A{beta} oligomers (A{beta}o) impact intracellular autophagy is not well understood. The primary cilium (PC), a signaling organelle on the surface of mature neurons and glia, is able to bind A{beta}. Since PC signaling pathways knowingly modify autophagy in non-brain cells, we here investigated the role of neuronal PC in the modulation of autophagy during acute extracellular A{beta}o overload. Our results show that, in vivo, recombinant A{beta}o require the presence of neuronal PC to modulate early autophagy and to induce the accumulation of autophagic vacuoles in an age-dependent manner. We show that activated Akt mediates these effects in an age-dependent manner, and that ciliary p75NTR receptor is required to block autophagy by A{beta}o. These findings demonstrate that neuronal PC in the adult brain participates in the deleterious effects mediated by soluble A{beta}o. The PC should therefore be considered as a target organelle to modulate autophagy for the treatment of neurodegenerative diseases.

HighlightsO_LIA{beta}o requires the neuronal PC to impair learning in young and old mice.
C_LIO_LIAutophagy in whole hippocampus differs from autophagy response in hippocampal neurons.
C_LIO_LIA{beta}o induce autophagolysosome accumulation through primary cilia- and age-dependent Akt phosphorylation.
C_LI

sentences: Live-Cell Imaging in Human Colonic Monolayers Reveals Erk Waves Limit the Stem Cell Compartment to Maintain Epithelial Homeostasis The establishment and maintenance of different cellular compartments in tissues is a universal requirement across all metazoans. Maintaining the correct ratio of cell types in time and space allows tissues to form patterned compartments and perform complex functions. Patterning is especially evident in the human colon, where tissue homeostasis is maintained by stem cells in crypt structures that balance proliferation and differentiation. Here we developed a 2D patient derived organoid (PDO) screening platform to study tissue patterning and kinase pathway dynamics in single cells across hundreds of conditions. Using this system, we discovered that waves of Erk signaling induced by apoptotic cells play a critical role in maintaining tissue patterning and homeostasis. If Erk is activated acutely across all cells instead of in wavelike patterns, then tissue patterning, and stem cell maintenance are lost. Conversely, if Erk activity is inhibited, then stem cells become unrestricted and expand dramatically. This work demonstrates that the colonic epithelium requires coordinated Erk signaling dynamics to maintain patterning and tissue homeostasis. Our work reveals how Erk can antagonize stem cells yet support cell replacement and the function of the gut.

sentences: Differential expression of striatal proteins in a mouse model of DOPA-responsive dystonia reveals shared mechanisms among dystonic disorders Dystonia is characterized by involuntary muscle contractions that cause debilitating twisting movements and postures. Although basal ganglia dysfunction is implicated in many forms of dystonia, the underlying mechanisms are unclear. Therefore, to reveal abnormal striatal cellular processes and pathways implicated in dystonia, we used an unbiased proteomic approach in a knockin mouse model of DOPA-responsive dystonia, a model in which the striatum is known to play a central role in the expression of dystonia. Fifty-seven of the 1805 proteins identified were differentially regulated in DOPA-responsive dystonia mice compared to control mice. Most differentially regulated proteins were associated with gene ontology terms that implicated either mitochondrial or synaptic dysfunction whereby proteins associated with mitochondrial function were generally over-represented whereas proteins associated with synaptic function were largely under-represented. Remarkably, nearly 20% of the differentially regulated proteins identified in our screen are associated with pathogenic variants that cause inherited dystonic disorders in humans suggesting shared mechanisms across many different forms of dystonia.

sentences: Engineering Protein Activity into Off-the-Shelf DNA Devices DNA-based devices are relatively straightforward to design by virtue of their predictable folding, but they lack biological activity. Conversely, protein-based devices offer a myriad of biological functions but are much more difficult to design due to their complex folding. This study bridges the fields of DNA engineering and protein engineering to generate a protein switch that is activated by a specific DNA sequence. A single protein switch, engineered from nanoluciferase using the alternate frame folding mechanism and herein called nLuc-AFF, is paired with different DNA technologies to create a biosensor for a DNA or RNA sequence of choice, sensors for serotonin and ATP, and a computational device that processes two DNA inputs. nLuc-AFF is a genetically-encoded, ratiometric, blue/green-luminescent biosensor whose output can be quantified by cell phone camera. nLuc-AFF is not falsely activated by decoy DNA and it retains full ratiometric readout in 100 % serum. The design approach can be applied to other proteins and enzymes to convert them into DNA-activated switches.

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Elexacaftor/Tezacaftor/Ivacaftor alters branching morphogenesis of the mouse embryonic lung IntroductionCFTR modulators triple combo-therapy Elexacaftor/Tezacaftor/Ivacaftor (ETI) has proven to clinically benefit homozygous and heterozygous F508del patients. As a result, an increasing number of pregnancies is expected. Studies of the potential impact of these modulators on the development of the foetus are mandatory.

MaterialsWe used the early mouse embryonic lung organ culture model to analyse ex vivo the lung branching process and the relative expression of Fgf10, Fgfr2IIIb, Shh, and Hhip development regulator genes in different conditions: standard culture medium, treatment with ETI or with Forskolin {+/-} Inh172. Development of lung branching and distal bud caliber were evaluated in lung explants from heterozygous F508del Cftrtm1Eur/+ and control Cftrtm1Eur+/+ (WT) mouse embryos at E12.5 during pseudo-glandular stage.

ResultsExposure to ETI of the Cftrtm1Eur/+ and WT lung explants induced a significant decrease in lung branching after 48h culture and the percentage of terminal bud dilations was significantly increased. These results were recapitulated by cAMP-dependent CFTR continuous activation by Forskolin and reversed by addition of Inh172.

ETI induced a significant decrease in Fgf10, Fgfr2IIIb, Shh and Hhip expression in lung explants of both E12.5 Cftrtm1Eur/+ and WT embryos treated with ETI for 72h.

ConclusionOur results provide evidence that the triple association Elexacaftor/Tezacaftor/Ivacaftor alters lung branching morphogenesis of WT and heterozygous F508del mouse embryos during the pseudo-glandular stage. Those results argue for a close monitoring of pregnancies in patients treated with these drugs.

Plain LanguageO_ST_ABSIntroductionC_ST_ABSThe triple combo-therapy Elexacaftor/Tezacaftor/Ivacaftor (ETI) improves homozygous and heterozygous F508del patients. As a result, an increasing number of pregnancies is expected. Studies of this treatment on the development of the foetus are lacking. We incubated lungs of murine foetus not carrying CFTR mutation or F508del heterozygous. We show that ETI induces significant defect of lung development and the formation of cysts. These results are at least partly due to CFTR activation. Those results argue for a close monitoring of pregnancies in patients treated with these drugs.

sentences: Distinct Mechanisms of Innate and Adaptive Immune Regulation Underlie Poor Oncologic Outcomes Associated with KRAS-TP53 Co-Alteration in Pancreatic Cancer Co-occurrent KRAS and TP53 mutations define a majority of patients with pancreatic ductal adenocarcinoma (PDAC) and define its pro-metastatic proclivity. Here, we demonstrate that KRAS-TP53 co-alteration is associated with worse survival compared with either KRAS-alone or TP53-alone altered PDAC in 245 patients with metastatic disease treated at a tertiary referral cancer center, and validate this observation in two independent molecularly annotated datasets. Compared with non-TP53 mutated KRAS-altered tumors, KRAS-TP53 co-alteration engenders disproportionately innate immune-enriched and CD8+ T-cell-excluded immune signatures. Leveraging in silico, in vitro, and in vivo models of human and murine PDAC, we discover a novel intersection between KRAS-TP53 co-altered transcriptomes, TP63-defined squamous trans-differentiation, and myeloid-cell migration into the tumor microenvironment. Comparison of single-cell transcriptomes between KRAS-TP53 co-altered and KRAS-altered/TP53WT tumors revealed cancer cell-autonomous transcriptional programs that orchestrate innate immune trafficking and function. Moreover, we uncover granulocyte-derived inflammasome activation and TNF signaling as putative paracrine mediators of innate immunoregulatory transcriptional programs in KRAS-TP53 co-altered PDAC. Immune subtyping of KRAS-TP53 co-altered PDAC reveals conflation of intratumor heterogeneity with progenitor-like stemness properties. Coalescing these distinct molecular characteristics into a KRAS-TP53 co-altered "immunoregulatory program" predicts chemoresistance in metastatic PDAC patients enrolled in the COMPASS trial, as well as worse overall survival.

sentences: Simultaneous Detection of Dopamine and Serotonin with carbon-based electrodes Graphite-based materials, like pyrolyzed carbon electrodes, are widely used as implantable electrochemical sensors, for the detection of neurotransmitters, neuromodulators, and gaseous species, thanks to their strong mechanical properties, superior electron-transfer kinetics, and in-vivo stability. Electrochemical properties of graphite can be improved by coating them with carbon nanotubes (CNTs) which improves sensitivity, selectivity, and resistance to biofouling. Although several types of electrodes have been developed to detect biologically relevant targets like monoamines, multiplexed sensing of dopamine and serotonin is not yet widely available. Herein we introduce pyrolyzed carbon electrodes coated with CNTs for fast scan cyclic voltammetry for simultaneous detection of dopamine and serotonin with a sensitivity of 52{+/-}8nA/M and 5{+/-}17nA/M, respectively. Serotonin shows a broad oxidation peak at 0.68V{+/-}0.12V. When dopamine and serotonin are probed simultaneously at 10 Hz, dopamine oxidizes at 0.1V{+/-} 0.1 and serotonin oxidizes at 0.78V{+/-}0.12 and dopamine reduces at -0.35V{+/-}0.1 and serotonin at 0.1V{+/-}0.2 V. Thus the sensors shows discrimination between dopamine and serotonin and are suitable for simultaneous detection of these monoamines.

sentences: Geometry of spiking activity in early visual cortex: a Topological Data Analytic approach In the brain, spiking patterns live in a high-dimensional space of neurons and time. Thus, determining the intrinsic structure of this state space for neural activity presents a theoretical and experimental challenge. To address this challenge, we introduce a new framework for applying topological data analysis (TDA) to spike train data to determine the geometry of neural activity space. Key to our approach is a parametrized family of distances based on the timing of spikes to determine the dissimilarity between neuronal responses. We applied TDA to visually driven single-unit and multiple single-unit spiking activity in macaque V1 and V2. TDA across timescales reveals a common geometry in V1 and V2 that is most consistent with a low-dimensional space endowed with Euclidean or hyperbolic geometry with modest curvature. Remarkably, the inferred geometry depends on timescale, and is most distinct for the timescales that are important for encoding contrast, orientation, and spatial correlations.

sentences: Biased credit assignment in motivational learning biases arises through prefrontal influences on striatal learning Actions are biased by the outcomes they can produce: Humans are more likely to show action under reward prospect, but hold back under punishment prospect. Such motivational biases derive not only from biased response selection, but also from biased learning: humans tend to attribute rewards to their own actions, but are reluctant to attribute punishments to having held back. The neural origin of these biases is unclear; in particular, it remains open whether motivational biases arise solely from an evolutionarily old, subcortical architecture or also due to younger, cortical influences. Simultaneous EEG-fMRI allowed us to track which regions encoded biased prediction errors in which order. Biased prediction errors occurred in cortical regions (ACC, vmPFC, PCC) before subcortical regions (striatum). These results highlight that biased learning is not a mere feature of the basal ganglia, but arises through prefrontal cortical contributions, revealing motivational biases to be a potentially flexible, sophisticated mechanism.

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Standardized genome-wide function prediction enables comparative functional genomics in plants: a new application area for Gene Ontologies BackgroundGenome-wide gene function annotations are useful for hypothesis generation and for prioritizing candidate genes potentially responsible for phenotypes of interest. We functionally annotated the genes of 18 crop plant genomes across 14 species using the GOMAP pipeline.

ResultsBy comparison to existing GO annotation datasets, GOMAP-generated datasets cover more genes, contain more GO terms, and produce datasets similar in quality (based on precision and recall metrics using existing gold standards as the basis for comparison). From there, we sought to determine whether the datasets across multiple species could be used together to carry out comparative functional genomics analyses in plants. To test the idea and as a proof of concept, we created dendrograms of functional relatedness based on terms assigned for all 18 genomes. These dendrograms were compared to well-established species-level evolutionary phylogenies to determine whether trees derived were in agreement with known evolutionary relationships, which they largely are. Where discrepancies were observed, we determined branch support based on jack-knifing then removed individual annotation sets by genome to identify the annotation sets causing unexpected relationships.

ConclusionsGOMAP-derived functional annotations used together across multiple species generally retain sufficient biological signal to recover known phylogenetic relationships based on genome-wide functional similarities, indicating that comparative functional genomics across species based on GO data hold promise for generating novel hypotheses about comparative gene function and traits.

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ColabFold - Making protein folding accessible to all ColabFold offers accelerated protein structure and complex predictions by combining the fast homology search of MMseqs2 with AlphaFold2 or RoseTTAFold. ColabFolds 40 - 60x faster search and optimized model use allows predicting close to a thousand structures per day on a server with one GPU. Coupled with Google Colaboratory, ColabFold becomes a free and accessible platform for protein folding. ColabFold is open-source software available at github.com/sokrypton/ColabFold. Its novel environmental databases are available at colabfold.mmseqs.com

Contactmilot.mirdita@mpibpc.mpg.de, so@fas.harvard.edu, martin.steinegger@snu.ac.kr

sentences: Ribosome Profiling Reveals a Dichotomy Between Ribosome Occupancy of Nuclear-Encoded and Mitochondrial-Encoded OXPHOS mRNA Transcripts in a Striatal Cell Model of Huntington Disease Huntington disease (HD) is caused by an expanded polyglutamine mutation in huntingtin (mHTT), which promotes a prominent atrophy in the striatum and subsequent psychiatric, cognitive, and choreiform movements. Multiple lines of evidence point to an association between HD and aberrant striatal mitochondrial functions. However, present knowledge about whether (or how) mitochondrial mRNA translation is differentially regulated in HD remains unclear. We have recently applied ribosome profiling (Ribo-Seq), a technique based on the high-throughput sequencing of ribosome-protected mRNA fragments, to analyze detailed snapshots of ribosome occupancy of the mitochondrial mRNA transcripts in control and HD striatal cells. Ribo-seq data revealed almost unaltered ribosome occupancy on the nuclear-encoded mitochondrial transcripts involved in oxidative phosphorylation (OXPHOS) and only a mild reduction in ribosome occupancy on a few selected transcripts (SHDA, Ndufv1, Timm23, Tomm5, and Mrps22) in HD cells. By contrast, ribosome occupancy of mitochondrially encoded OXPHOS mRNAs (mtNd-1, mtNd-2, mtNd-4, mtNd-4l, mtNd-5, mtNd-6, mt-Co1, mtCyt b, and mt-ATP8) was dramatically increased, implying widespread dichotomous effects on ribosome occupancy and OXPHOS mRNA translation in HD. Thus, mHTT may command signals that specifically regulate translation of the mitochondrial OXPHOS transcripts and influence HD pathogenesis.

sentences: Let-7b-5p, miR-184, and miR-22-3p: Multianalyte Plasma Biomarkers for NSCLC Diagnosis and for Predicting Resistance to Osimertinib Low-dose computed tomography (LDCT) Non-Small Cell Lung (NSCLC) screening is associated with high false-positive rates, leading to unnecessary expensive and invasive follow ups. There is a need for minimally invasive approaches to improve the accuracy of NSCLC diagnosis. Plasma extracellular vesicle (EV) and circulating microRNA (miRNA) have been proposed as cancer screening biomarkers. However, the identification of highly sensitive and broadly predictive core miRNA signatures remains a challenge. Also, how these systemic and diverse miRNAs impact cancer drug response is not well understood. Using an integrative approach, we examined plasma EV and circulating miRNA isolated from NSCLC patients versus screening controls with a similar risk profile. We found that combining EV (Hsa-miR-184, Let-7b-5p) and circulating (Hsa-miR-22-3p) miRNAs abundance robustly discriminates between NSCLC patients and high-risk cancer-free controls. Diagnosed NSCLC patients harboring sensitizing mutations in epidermal growth factor receptor EGFR (T790M, L578R) are treated with Osimertinib, a potent tyrosine kinase inhibitor (TKI). Nearly all patients develop TKI resistance via complex mechanisms and progress. We found that Hsa-miR-22-3p, Hsa-miR-184, and Let-7b-5p functionally converge on WNT/{beta}catenin and mTOR/AKT signaling axes, known cancer therapy resistance signals. Targeting Hsa-miR-22-3p and Hsa-miR-184 desensitized EGFR-mutated (T790M, L578R) NSCLC cells to Osimertinib. These findings suggest that the expression levels of circulating hsa-miR-22-3p combined with EV hsa-miR-184 and Let-7b-5p levels potentially define a core biomarker signature for improving the accuracy of NSCLC diagnosis. Importantly, these biomarkers have the potential to enable prospective identification of patients who are at risk of responding poorly to Osimertinib alone but likely to benefit from Osimertinib/AKT blockade combination treatments.

sentences: Transcriptional dynamics of colorectal cancer risk associated variation at 11q23.1 are correlated with tuft cell abundance and marker expression in silico Colorectal cancer (CRC) is characterised by heritable risk that is not well understood. Heritable, genetic variation at 11q23.1 is associated with increased colorectal cancer (CRC) risk, demonstrating eQTL effects on 3 cis- and 23 trans-eQTL targets. We sought to determine the relationship between 11q23.1 cis- and trans-eQTL target expression and test for potential cell-specificity. scRNAseq from 32,361 healthy colonic epithelial cells was aggregated and subject to weighted gene co-expression network analysis (WGCNA). One module (blue) included 19 trans-eQTL targets and was correlated with C11orf53 expression only. Following unsupervised clustering of single cells, the expression of 19 trans-eQTL targets was greatest and most variable in cluster number 11, which transcriptionally resembled tuft cells. 14 trans-eQTL targets were found to demarcate this cluster, 11 of which were corroborated in a second dataset. Intra-cluster WGCNA and module preservation analysis then identified twelve 11q23.1 trans-eQTL targets to comprise a network that was specific to cluster 11. Finally, linear modelling and differential abundance testing showed 11q23.1 trans-eQTL target expression was predictive of cluster 11 abundance. Our findings suggest 11q23.1 trans-eQTL targets comprise a C11orf53-related network that is likely tuft cell-specific and reduced expression of these genes correlates with reduced tuft cell abundance in silico.

sentences: Interrogation of genome-wide, experimentally dissected gene regulatory networks reveals mechanisms underlying dynamic cellular state control Pooled CRISPRi-mediated silencing of >1,000 transcriptional regulators expressed in single colorectal adenocarcinoma cells, followed by single-cell RNA-seq profiling at two timepoints, 1 day and 4 days, allowed reverse engineering the underlying tumor context-specific, causal regulatory network. Furthermore, the availability of experimentally derived, highly multiplexed gene reporter assays for each regulator, as identified by this analysis, allowed accurate assessment of differential protein activity following silencing of each regulator, thus providing proof-of-concept for generating comprehensive, tissue-specific networks of transcriptional and post-translational interactions. Analysis of this causal network allowed elucidation of complex autoregulatory mechanisms that have eluded previous computational approaches and supported systematic elucidation of cooperative mechanisms, where one regulatory protein can modulate the activity of another regulatory protein, as well as transcriptional mimicry, where one regulatory protein can phenocopy others.

sentences: SpinX: Time-resolved 3D Analysis of Mitotic Spindle Dynamics using Deep Learning Techniques and Mathematical Modelling Time-lapse microscopy movies have transformed the study of subcellular dynamics. However, manual analysis of movies can introduce bias and variability, obscuring important insights. While automation can overcome such limitations, spatial and temporal discontinuities in time-lapse movies render methods such as object segmentation and tracking difficult. Here we present SpinX, a framework for reconstructing gaps between successive frames by combining Deep Learning and mathematical object modelling. By incorporating expert feedback through selective annotations, SpinX identifies subcellular structures, despite confounding neighbour-cell information, non-uniform illumination and variable marker intensities. The automation and continuity introduced allows precise 3-Dimensional tracking and analysis of spindle movements with respect to the cell cortex for the first time. We demonstrate the utility of SpinX using distinct spindle markers and drug treatments. In summary, SpinX provides an exciting opportunity to study spindle dynamics in a sophisticated way, creating a framework for step changes in studies using time-lapse microscopy.

sentences: Rats show a preference for certain unfamiliar strains of rats Humans show distinct social behaviours when we recognise social similarity in opponents that are members of the same social group. However, little attention has been paid to the role of social similarity in non-human animals. In Wistar subject rats, the presence of an unfamiliar Wistar rat mitigated stress responses, suggesting the importance of social similarity in this phenomenon. We found that the presence of unfamiliar Sprague-Dawley (SD) or Long-Evans (LE) rats, but not an unfamiliar Fischer 344 (F344) rat, similarly mitigated stress in subject rats. It is therefore possible that the subject rats recognised social similarity to unfamiliar SD and LE rats. In this study, we demonstrated that Wistar subject rats were capable of categorizing unfamiliar rats based on their strain, and that Wistar subjects showed a preference for unfamiliar Wistar, SD, and LE rats over F344 rats. However, the subject rats did not show a preference among Wistar, SD, and LE rats. In addition, the results were not due to an aversion to F344 rats, and preference was not affected when anaesthetised rats were presented to subject rats. The findings suggested that rats recognise social similarity to certain unfamiliar strains of rats.

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Oxytocin reduces interoceptive influences on empathy-for-pain in the anterior insula Empathy-for-pain states are underpinned by interoception, i.e the central representation of internal states. Cardiac signals occur in a phasic manner; baroreceptor discharges at systole communicate the heartbeats strength. These signals modulate pain and emotion processing. We tested whether these phasic interoceptive signals modulate empathy-for-pain. As oxytocin (OT) enhances empathy and modulates interoceptive signals precision, we also tested if OT administration impacts empathy-for-pain via interoceptive mechanisms.

Male subjects (N=32) attended three sessions to perform psychometric tests and an fMRI empathy-for-pain task, after intranasal administration of OT or placebo (40IU). Pictures of hands in painful or non-painful context were presented at systole or diastole. Effects of drug, emotion and cardiac timing on behaviour and brain activity was tested using general and mixed-effects linear models.

Across conditions, activation was observed within regions implicated in pain and empathy-for-pain, with insula activation greater in the right than left hemisphere. OT administration, compared to placebo, attenuated the reactivity of some regions, including anterior cingulate cortex, but presentation of stimuli at systole blocked the OT attenuating effect.

Our data suggest that OT alters the processing of motivationally-salient social cues, interacting with interoceptive signals. Our findings may inform targeted use of OT in psychiatric conditions linked to aberrant interoceptive processing.

sentences: Genome binning of viral entities from bulk metagenomics data Despite the accelerating number of uncultivated virus sequences discovered in metagenomics and their apparent importance for health and disease, the human gut virome and its interactions with bacteria in the gastrointestinal are not well understood. In addition, a paucity of whole-virome datasets from subjects with gastrointestinal diseases is preventing a deeper understanding of the viromes role in disease and in gastrointestinal ecology as a whole. By combining a deep-learning based metagenomics binning algorithm with paired metagenome and metavirome datasets we developed the Phages from Metagenomics Binning (PHAMB) approach for binning thousands of viral genomes directly from bulk metagenomics data. Simultaneously our methodology enables clustering of viral genomes into accurate taxonomic viral populations. We applied this methodology on the Human Microbiome Project 2 (HMP2) cohort and recovered 6,077 HQ genomes from 1,024 viral populations and explored viral-host interactions. We show that binning can be advantageously applied to existing and future metagenomes to illuminate viral ecological dynamics with other microbiome constituents.

sentences: Generating heterokaryotic cells via bacterial cell-cell fusion Cell-cell fusion is fundamentally important for tissue repair, virus transmission, and genetic recombination, among other functions. Fusion has been mainly studied in eukaryotic cells and lipid vesicles, while cell-cell fusion in bacteria is less well characterized, due to the cell wall acting as a fusion-limiting barrier. Here we use cell wall-deficient bacteria to investigate the dynamics of cell fusion in bacteria that replicate without their cell wall. Stable, replicating cells containing differently labeled chromosomes were successfully obtained from fusion. We find that the rate of cell-cell fusion depends on the fluidity of cell membranes. Furthermore, we show that not only the efficiency but also the specificity of cell-cell fusion can be controlled via a pair of synthetic membrane-associated lipopeptides. Our results provide a molecular handle to understand and control cell-cell fusion to generate heterokaryotic cells, which was an important step in the evolution of protocells and of increasing importance for the design of synthetic cells.

sentences: Stomatal anatomy, leaf structure and nutrients of tropical rainforest tree species respond to altitude in a coordinated manner in accordance with the leaf economics spectrum Understanding taxon level responses of key plant traits to environmental variation across tropical rainforests (TRFs) is important to determine their response to climate change. We used an altitudinal gradient (from 150 to 2100 m above sea level, asl) across TRFs in Sri Lanka to answer the following questions: (a) Does the response to altitude by stomatal traits differ among plant taxa in TRFs?; (b) Are the altitudinal responses of key leaf structural traits (e.g. leaf mass per area, LMA) and major leaf nutrient (nitrogen, N, and phosphorus, P) concentrations linked to the corresponding variation in stomatal traits in a coordinated response across taxa?; (c) How strong is the influence of climatic variation on responses of leaf traits to altitude?. Leaf samples were collected in permanent sampling plots within rainforest reserves at Kanneliya (150 m asl), Sinharaja-Enasalwatta (1050 m), Hakgala (1800 m) and Piduruthalagala (2100 m) from 19 species in three plant genera (Calophyllum, Semecarpus and Syzygium). Stomatal density, guard cell length and epidermal density showed variation among taxa, but did not respond to altitude. Potential conductance index (PCI), a proxy for photosynthetic capacity, decreased with increasing altitude, in a common response across taxa. We found evidence that altitudinal responses of LMA, leaf N and P were linked to stomatal responses in a coordinated manner, where key features were the negative correlations between PCI and LMA and between proxy photosynthetic N- and P-use efficiencies ( PNUE and  PPUE) and LMA. We found strong responses to climatic variation across taxa and altitudes, where PCI,  PNUE and  PPUE increased and LMA decreased with increasing temperature, precipitation and solar irradiance. We conclude that stomatal traits of tree species in TRFs form part of a coordinated leaf trait response to environmental change which is in accordance with the leaf economics spectrum.

sentences: A T-cell independent universal cellular therapy strategy through antigen depletion CD19-targeting chimeric antigen receptor (CAR) T-cell therapeutics is a revolutionary, novel and successful treatment for B-cell malignancies. However, while CD19-CAR-T therapy can obtain high rates of complete responses in these patients, a significant fraction of patients may experience CD19-negative relapse. Moreover, the dependency on T-cell mediated cytotoxicity restricts CAR-T therapy as a patient-specific individualized therapy with severe side effects such as cytokine-release syndrome (CRS). Whether CAR-T therapy can be substituted by a non-T-cell based universal cellular therapy is largely unknown. Surprisingly, we have demonstrated here that T-lymphocytic cells, as well as non-lymphocytic cells, can cause CD19 internalization and subsequent depletion when they are armed with a CD19-recognizing moiety. This CD19 antigen depletion can efficiently induce T-cell independent apoptosis in target cancer cells whose survival is dependent upon CD19 expression, suggesting that CD19 antigen depletion constitutes a crucial tumor destroying mechanism for CD19-CAR-T, especially for its long-term efficacy. We therefore proposed a universal strategy for CRS-free cellular therapeutics, utilizing artificial antigen-recognizing cells (AARC), which can be manufactured universally and standardly as "off-the-shelf" mesenchymal stromal cells (MSCs) or other types of non-autologous cell expressing anergic CARs. Our results not only uncovered an unrecognized mechanism for CAR-T cytotoxicity and antigen loss, but also shed new insight into a shift in cellular therapeutics from unique patient-specific autologous therapeutics, to universal and standardized allogeneic treatment.

sentences: Transcriptome analysis of SARS-CoV-2 naive and recovered individuals vaccinated with inactivated vaccine The urgent approval of the use of the inactivated COVID-19 vaccine is essential to reduce the threat and burden of the epidemic on global public health, however, our current understanding of the host immune response to inactivated vaccine remains limited. Herein, we performed serum IgG antibody detection and transcriptomics analysis on 20 SARS-CoV-2 naive individuals who received multiple doses of inactivated vaccine and 5 SARS-CoV-2 recovered individuals who received single dose of inactivated vaccine. Our research revealed the important role of many innate immune pathways after vaccination, identified a significant correlation with the third dose of booster vaccine and proteasome-related genes, and found that SARS-CoV-2 recovered individuals can produces a strong immune response to a single dose of inactivated vaccine. These results help us understand the reaction mechanism of the hosts molecular immune system to the inactivated vaccine, and provide a basis for the choice of vaccination strategy.

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UPP2: Fast and Accurate Alignment Estimation of Datasets with Fragmentary Sequences MotivationMultiple sequence alignment (MSA) is a basic step in many bioinformatics pipelines. However, achieving highly accurate alignments on large datasets, especially those with sequence length heterogeneity, is a challenging task. UPP (Ultra-large multiple sequence alignment using Phylogeny-aware Profiles) is a method for MSA estimation that builds an ensemble of Hidden Markov Models (eHMM) to represent an estimated alignment on the full length sequences in the input, and then adds the remaining sequences into the alignment using selected HMMs in the ensemble. Although UPP provides good accuracy, it is computationally intensive on large datasets.

ResultsWe present UPP2, a direct improvement on UPP. The main advance is a fast technique for selecting HMMs in the ensemble that allows us to achieve the same accuracy as UPP but with greatly reduced runtime. We show UPP2 produces more accurate alignments compared to leading MSA methods on datasets exhibiting substantial sequence length heterogeneity, and is among the most accurate otherwise.

Availabilityhttps://github.com/gillichu/sepp

Contactwarnow@illinois.edu

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A CRISPR-based instant DNA repositioning system and the early intranuclear life of HSV-1 The intranuclear localization of viral DNA genomes in relation to the intranuclear environment plays critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of the infection, manipulative tools are needed. Instant repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery instantly (CRISPR-nuPin). Based on this system, investigations of herpes simplex virus 1 (HSV-1), a prototype member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: I) Viral genomes tethered to the nuclear periphery upon entry, compared with those in the nuclear interior, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent inhibition. II) Relocating HSV-1 genomes at 1 hour post infection significantly promoted transcription of viral {beta} and {gamma} genes, termed an "Escaping" effect. III) Early accumulation of ICP0 was a sufficient but not necessary condition mediating "Escaping". IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic should be widely applicable to many DNA viruses.

SummaryThe intranuclear localization of viral DNA genomes plays a critical role in determining virus fate. To elucidate the causal relationship between subnuclear localization and the pathogenic outcome of DNA viruses, manipulative tools are needed. Herein, we report an inducible CRISPR-based two-component platform that relocates DNA pieces (5 kb to 170 kb) to the nuclear periphery instantly (CRISPR-nuPin). Utilizing this tactic, we interrogated the early intranuclear life of herpes simplex virus 1 (HSV-1), a prototype of human herpesviruses, in space and time and revealed that I) viral genomes tethered to the nuclear edge upon entry were prone to suppressive histone packaging and severe inhibition. II) Relocating HSV-1 genomes to the nuclear fringe at 1 hour post infection promoted transcription of viral genes ("Escaping"). III) Early accumulation of ICP0 was a sufficient but not necessary condition mediating "Escaping". IV) Subnuclear localization was a critical factor only during early infection.

HighlightsO_LICRISPR-nuPin is an inducible two-component DNA repositioning system
C_LIO_LIIt mediates instant nuclear edging of viral DNA during infection
C_LIO_LIA powerful approach to interrogate DNA viruses in space and time
C_LIO_LIViral DNA at the nuclear periphery upon entry is strongly silenced
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In briefAn inducible two-component CRISPR-based platform that instantly repositions HSV-1 genomes to the nuclear edge unveils intranuclear space heterogeneity for the incoming viral genomes and dynamic stages of the host-virus interplay during early infection of the pathogen.

sentences: ATRIOVENTRICULAR NODAL FUNCTION, ATRIAL FIBRILLATION AND VENTRICULAR TACHYCARDIA IN GIRAFFES Unique cardiovascular adaptations in giraffes (Giraffa Camelopardalis reticulata) have been the focus of numerous investigations for almost a century. The vertical distance between the heart and brain impose high pressure on the giraffe left ventricle leading to thickening that exceeds heart in other mammals. Yet, cardiovascular function appears to be unimpacted by these morphologic differences. Physiologically adapted atrioventricular conduction may contribute to these unique cardiovascular characteristics. Atrioventricular (AV) function was assessed to determine whether physiologically adapted AV intervals might optimize the delay between atrial and ventricular contractions. Using ambulatory and intracardiac recordings, we report a longer PR interval in giraffes than predicted by allometric scaling. Slow ventricular response during atrial fibrillation further supports species-specific atrioventricular adaptations.

sentences: A scalp geometry parameter-space for optimization and implementation of conventional TMS coil placement Transcranial magnetic stimulation efficacy is largely dependent upon coil position and orientation. A good method for describing coil placement is required for both computational optimization (planning) and actual placement (implementation). In coordinate dependent parameter-spaces (CDPs), three-dimensional coordinates are used to represent coil position and three orthogonal unit vectors are used to represent coil orientation. A CDP can precisely describe arbitrary coil placement; therefore it offers great advantage in computational optimization which checks through all possible placements. However, a neuronavigation system is usually required to accurately implement the optimized CDP parameters on a participants head. Routine clinical practice, on the other hand, often uses the International 10-20 system to describe coil placement. Although the 10-20 system can only perform modeling and placement at limited scalp landmarks, it allows the synthesis of different individuals targeting effects to find group-optimal parameters; it also allows manual placement, which is important for commonly-seen use cases without individual MRI scans and navigation devices. This study proposes a new scalp geometry based parameter-space (SGP), integrating the advantages of CDP and 10-20 methods. Our SGP 1) can quantitatively specify all possible conventional coil positions and orientations on an individuals scalp, which is important for electrical modeling and optimization, 2) maintains inter-individual correspondence, which is important for synthesizing TMS effects from different individuals and studies. 3) enables fast and simple manual implementation. Demonstration experiments were conducted to illustrate the application of an SGP-based framework for both individual and group-based optimization. A measurement experiment was performed to evaluate speed, precision and reliability of SGP-based manual implementation; results show it surpasses previous manual placement methods.

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An Infant Sleep Electroencephalographic Marker of Thalamocortical Connectivity Predicts Behavioral Outcome in Late Infancy Infancy represents a critical period during which thalamocortical brain connections develop and mature. Deviations in the maturation of thalamocortical connectivity are linked to neurodevelopmental disorders. There is a lack of early biomarkers to detect and localize neuromaturational deviations, which can be overcome with mapping through high-density electroencephalography (hdEEG) assessed in sleep. Specifically, slow waves and spindles in non-rapid eye movement (NREM) sleep are generated by the thalamocortical system, and their characteristics, slow wave slope and spindle density, are closely related to neuroplasticity and learning. Recent studies further suggest that information processing during sleep underlying sleep-dependent learning is promoted by the temporal coupling of slow waves and spindles, yet slow wave-spindle coupling remains unexplored in infancy. Thus, we evaluated three potential biomarkers: 1) slow wave slope, 2) spindle density, and 3) the temporal coupling of slow waves with spindles. We use hdEEG to first examine the occurrence and spatial distribution of these three EEG features in healthy infants and second to evaluate a predictive relationship with later behavioral outcomes. We report four key findings: First, infants EEG features appear locally: slow wave slope is maximal in occipital and frontal areas, whereas spindle density is most pronounced frontocentrally. Second, slow waves and spindles are temporally coupled in infancy, with maximal coupling strength in the occipital areas of the brain. Third, slow wave slope, spindle density, and slow wave-spindle coupling are not associated with concurrent behavioral status (6 months). Fourth, spindle density in central and frontocentral regions at age 6 months predicts later behavioral outcomes at 12 and 24 months. Neither slow wave slope nor slow wave-spindle coupling predict behavioral development. Our results propose spindle density as an early EEG biomarker for identifying thalamocortical maturation, which can potentially be used for early diagnosis of neurodevelopmental disorders in infants. These findings are complemented by our companion paper that demonstrates the linkage of spindle density to infant nighttime movement, framing the possible role of spindles in sensorimotor microcircuitry development. Together, our studies suggest that early sleep habits, thalamocortical maturation, and behavioral outcome are closely interwoven. A crucial next step will be to evaluate whether early therapeutic interventions may be effective to reverse deviations in identified individuals at risk.

HighlightsO_LISlow waves and spindles occur in a temporally coupled manner in infancy
C_LIO_LISlow wave slope, spindle density, and slow wave-spindle coupling are not related to concurrent behavioral development
C_LIO_LISpindle density at 6 months predicts behavioral status at 12 and 24 months
C_LIO_LISlow wave slope and slow wave-spindle coupling are not predictive of behavioral development
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sentences: Computational Modeling of Aerosol Particle Transport through Lung Mucosa Delivery of aerosols to the lungs have great potential for the treatment of various lung diseases. However, the lungs are coated by a protective mucus layer whose complex properties make this form of delivery difficult. Mucus is non-Newtonian and cleared from the lungs over time by ciliated cells. Further, its gel-like structure hinders the diffusion of particles through it. Any aerosolized lung disease treatment must have certain properties to circumvent this barrier, and these properties may vary between diseases, drugs, and patients. Using Computational Fluid Dynamics (CFD) modeling, a model of this mucus layer was constructed such that the diffusion of an impacted aerosol might be studied. The model predicts what amount of a particle of a certain size might be expected to penetrate the mucus and reach the underlying tissue, as well as the distance downstream of the dosage site where concentration is maximized. Using this information, a personalized treatment plan may be designed. The model maintains modularity so that various lung regions and patient health states may be simulated.

sentences: Chimeric Inheritance and Crown-Group Acquisitions of Carbon Fixation Genes within Chlorobiales The geological record of microbial metabolisms and ecologies primarily consists of stable isotope fractionations and the diagenetic products of biogenic lipids. Carotenoid lipid biomarkers are particularly useful proxies for reconstructing this record, providing information on microbial phototroph primary productivity, redox couples, and oxygenation. The biomarkers okenane, chlorobactane, and isorenieratene are generally considered to be evidence of anoxygenic phototrophs, and provide a record that extends to [~]1.64 Ga. The utility of the carotenoid biomarker record may be enhanced by examining the carbon isotopic ratios in these products, which are diagnostic for specific pathways of biological carbon fixation found today within different microbial groups. However, this joint inference assumes that microbes have conserved these pathways across the duration of the preserved biomarker record. Testing this hypothesis, we performed phylogenetic analyses of the enzymes constituting the reductive tricarboxylic acid (rTCA) cycle in Chlorobiales, the group of anoxygenic phototrophic bacteria usually implicated in the deposition of chlorobactane and isorenieretane. We find phylogenetically incongruent patterns of inheritance across all enzymes, indicative of horizontal gene transfers to both stem and crown Chlorobiales from multiple potential donor lineages. This indicates that a complete rTCA cycle was independently acquired at least twice within Chlorobiales and was not present in the last common ancestor. When combined with recent molecular clock analyses, these results predict that the Mesoproterzoic lipid biomarker record diagnostic for Chlorobiales should not preserve isotopic fractionations indicative of a full rTCA cycle. Furthermore, we conclude that coupling isotopic and biomarker records is insufficient for reliably reconstructing microbial paleoecologies in the absence of a complementary and consistent phylogenomic narrative.

sentences: Topological supramolecular network enabled highly conductive and stretchable organic bioelectronics Intrinsically stretchable bioelectronic devices based on soft and conducting organic materials have been regarded as the ideal interface for seamless and biocompatible integration with the human body. However, the grand challenge remains for the conducting polymer to possess both high mechanical ductility and good electrical conduction at cellular level feature sizes. This longstanding material limitation in organic bioelectronics has impeded the full exploitation of its unique benefits. Here, we introduce a new molecular engineering strategy based on rationally designed topological supramolecular networks, which allows effective decoupling of competing effects from multiple molecular building blocks to meet complex requirements. We achieve two orders of magnitude improvement in the conductivity under 100% strain in physiological environment, along with the capability for direct photopatterning down to 2 m. These unprecedented capabilities allow us to realize previously inaccessible bioelectronic applications including high-resolution monitoring of  soft and malleable creatures, e.g., octopus, and localized neuromodulation down to single nucleus precision for controlling organ-specific activities through delicate tissues, e.g., brainstem.

sentences: Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis Derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome, impeding their use in biomedical research. Here, we developed a highly efficient chemically defined astrocyte differentiation strategy that overcomes current limitations. This approach largely bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro experiments. hPSCs were first differentiated into radial glial cells (RGCs) exhibiting in vivo-like radial glia signatures. Activation of NOTCH and JAK/STAT pathways in bona fide RGCs resulted in direct astrogliogenesis confirmed by expression of various glial markers (NFIA, NFIB, SOX9, CD44, S100B, GFAP). Transcriptomic and genome-wide epigenetic analyses confirmed RGC-to-astrocyte differentiation and absence of neurogenesis. The morphological and functional identity of hPSC-derived astrocytes was confirmed by using an array of methods (e.g. electron microscopy, calcium imaging, co-culture with neurons, grafting into mouse brains). Lastly, the scalable protocol was adapted to a robotic platform and used to model Alexander disease. In conclusion, our findings uncover remarkable plasticity in neural lineage progression that can be exploited to manufacture large numbers of human hPSC-derived astrocytes for drug development and regenerative medicine.

sentences: Cylindromatosis Drives Synapse Pruning and Weakening by Promoting Macroautophagy through Akt-mTOR Signaling The lysine-63 deubiquitinase cylindromatosis (CYLD) is long recognized as a tumor suppressor in immunity and inflammation and its loss-of-function mutations lead to familial cylindromatosis. However, recent studies reveal that CYLD is enriched in mammalian brain postsynaptic densities, and a gain-of-function mutation causes frontotemporal dementia (FTD), suggesting critical roles at excitatory synapses. Here we report that CYLD drives synapse elimination and weakening by acting on the Akt-mTOR-autophagy axis. Mice lacking CYLD display abnormal sociability, anxiety- and depression-like behaviors, and cognitive inflexibility. These behavioral impairments are accompanied by excessive synapse numbers, increased postsynaptic efficacy, augmented synaptic summation, and impaired NMDA receptor-dependent hippocampal long-term depression (LTD). Exogenous expression of CYLD results in removal of established dendritic spines from mature neurons in a deubiquitinase activity-dependent manner. In search of underlying molecular mechanisms, we find that CYLD knockout mice display marked overactivation of Akt and mTOR and reduced autophagic flux and, conversely, CYLD overexpression potently suppresses Akt and mTOR activity and promotes autophagy. Consequently, abrogating the Akt-mTOR-autophagy signaling pathway abolishes CYLD-induced spine loss, whereas enhancing autophagy in vivo by the mTOR inhibitor rapamycin rescues the synaptic pruning and LTD deficits in mutant mice. Our findings establish CYLD, via Akt-mTOR signaling, as a synaptic autophagy activator that exerts critical modulations on synapse maintenance, function, and plasticity.

sentences: Citrus photosynthesis and morphology acclimate to phloem-affecting huanglongbing disease at the leaf and shoot levels Huanglongbing (HLB) is a phloem-affecting disease of citrus that reduces growth and has impacted global citrus production. HLB is caused by a phloem-limited bacterium (Candidatus Liberibacter asiaticus; CLas). By inhibiting phloem function, HLB stunts sink growth, including reducing production of new shoots and leaves, and induces hyperaccumulation of foliar starch. HLB induces feedback inhibition of photosynthesis by reducing foliar carbohydrate export. In this work we assessed the relationship of bacterial distribution within the foliage, foliar starch accumulation, and net CO2 assimilation (Anet). Because HLB impacts canopy morphology, we developed a chamber to measure whole-shoot Anet to test the effects of HLB at both leaf and shoot levels. Whole-shoot-level Anet saturated at high irradiance, and green stems had high photosynthetic rates compared to leaves. Starch accumulation was correlated with bacterial population, and starch was negatively correlated with Anet at the leaf level but not at the shoot level. Starch increased initially after infection, then decreased progressively with increasing length of infection. HLB infection reduced Anet at the leaf level, but increased it at the whole shoot level, in association with reduced leaf size and greater relative contribution of stems to photosynthetic surface area. Although HLB-increased photosynthetic efficiency, total carbon fixed per shoot decreased because photosynthetic surface area was reduced. We conclude that the localized effects of infection on photosynthesis are mitigated by whole shoot morphological acclimation over time. Stems contribute important proportions of whole shoot Anet, and these contributions are likely increased by the morphological acclimation induced by HLB.

sentences: Phylogenetic and population structure analyses uncover pervasive misclassification and help assessing the biosafety of Pseudomonas putida for biotechnological applications The Pseudomonas putida group comprises strains with biotechnological and clinical relevance. P. alloputida was proposed as a new species and highlighted the misclassification of P. putida. Nevertheless, the population structure of P. alloputida remained unexplored. We retrieved 11,025 Pseudomonas genomes and used P. alloputida Kh7T to delineate the species. The P. alloputida population structure comprises at least 7 clonal complexes (CCs). Clinical isolates are mainly found in CC4 and acquired resistance genes are present at low frequency in plasmids. Virulence profiles support the potential of CC7 members to outcompete other plant or human pathogens through a type VI secretion system. Finally, we found that horizontal gene transfer had an important role in shaping the ability of P. alloputida to bioremediate aromatic compounds such as toluene. Our results provide the grounds to understand P. alloputida genetic diversity and safety for environmental applications.

sentences: PhycoMine: A Microalgae Data Warehouse PhycoMine is data warehouse system created to fostering the analysis of complex and integrated data from microalgae species in a single computational environment. The PhycoMine was developed on top of the InterMine software system, and it has implemented an extended database model, containing a series of tools that help the users in the analysis and mining of individual data and group data. The platform has widgets created to facilitate simultaneous data mining of different datasets. Among the widgets implemented in PhycoMine, there are options for mining chromosome distribution, gene expression variation via transcriptomics, proteomics sets, Gene Onthology enrichment, KEGG enrichment, publication enrichment, EggNOG, Transcription factors and transcriptional regulators enrichment and phenotypical data. These widgets were created to facilitate data visualization of the gene expression levels in different experimental setups, for which RNA-seq experimental data is available in data repositories. For comparative purposes, we have reanalyzed 200 RNA-seq datasets from Chlamydomonas reinhardtii, a model unicellular microalga, for optimizing the performance and accuracy of data comparisons. We have also implemented widgets for metabolic pathway analysis of selected genes and proteins and options for biological network analysis. The option for analysis of orthologue genes was also included. With this platform, the users can perform data mining for a list of genes or proteins of interest in an integrated way through accessing the data from different sources and visualizing them in graphics and by exporting the data into table formats. The PhycoMine platform is freely available and can be visited through the URL https://PhycoMine.iq.usp.br.

sentences: Annotation of Putative Circadian Rhythm-Associated Genes in Diaphorina citri (Hemiptera : Liviidae) The circadian rhythm is a process involving multiple genes that generates an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the earths rotation on its axis. This report presents the results of the manual curation of twenty-seven genes likely associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect acts as the vector of the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease (Huanglongbing). This disease is the most severe detriment to citrus industries and has drastically decreased crop yields worldwide. Based on the genes identified in the psyllid genome, namely cry1 and cry2, D. citri likely possesses a circadian model similar to that of the lepidopteran butterfly, Danaus plexippus. Manual annotation of these genes will allow future molecular therapeutics to be developed that can disrupt the psyllid biology.

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The Effect of COVID-19 on the Postdoctoral Experience: a comparison of pre-pandemic and pandemic surveys. In the interest of advocating for the postdoctoral community in the United States, we present results from survey data collected before and during the COVID-19 pandemic on the same population of postdocs. In 2019, 5,929 postdocs in the US completed a comprehensive survey, and in 2020, a subset completed a follow-up survey several months into the pandemic. The results show that the pandemic has substantially impacted postdocs mental health and wellness irrespective of gender, race, citizenship, or other identities. Postdocs also reported a significant impact on their career trajectories and progression, reduced confidence in achieving career goals, and negative perceptions of the job market compared to pre-COVID-19. International postdocs also reported experiencing distinct stressors due to the changes in immigration policy. Notably, having access to Postdoctoral Associations and Postdoctoral Offices positively impacted postdocs overall well-being and helped mitigate the personal and professional stresses and career uncertainties caused by the pandemic.

Graphical Abstract

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org.highwire.dtl.DTLVardef@174d7e3org.highwire.dtl.DTLVardef@9893dorg.highwire.dtl.DTLVardef@114470org.highwire.dtl.DTLVardef@1a4278f_HPS_FORMAT_FIGEXP  M_FIG Graphical Abstract of survey responses to: Why or how has your research been disrupted or not disrupted due to the pandemic? Overall, postdocs responded with feelings of loss of control as the pandemic was acting upon them and taking away their ability to complete their work.

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sentences: The Myogenesis Program Drives Clonal Selection and Drug Resistance in Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is a pediatric cancer with features of skeletal muscle; patients with unresectable or metastatic RMS fare poorly due to high rates of disease recurrence. Here, we use single cell and single nucleus RNA-sequencing to show that RMS tumors recapitulate the spectrum of embryonal myogenesis. Using matched patient samples from a clinical trial and orthotopic patient-derived xenografts (O-PDXs), we show chemotherapy eliminates the most proliferative component with features of myoblasts; after treatment, the quiescent immature population with features of paraxial mesoderm expands to reconstitute the developmental hierarchy of the original tumor. We discovered that this paraxial mesoderm population is dependent on EGFR signaling and is sensitive to EGFR inhibitors. Taken together, this data serves as a proof-of-concept that targeting each developmental state in RMS is an effective strategy for improving outcomes by preventing disease recurrence.

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Plasticity in female timing may explain current shifts in breeding phenology of a North American songbird O_LIClimate change has driven changes in breeding phenology. Identifying the magnitude of phenological shifts and whether selection in response to climate change drives these shifts is key for determining species reproductive success and persistence in a changing world.
C_LIO_LIWe investigated reproductive timing in a primarily sedentary population of the dark-eyed junco (Junco hyemalis) over 32 years. We predicted that juncos would breed earlier in warmer springs in response to selection favouring earlier breeding.
C_LIO_LITo test this prediction, we compared the annual median date for reproductive onset (i.e., egg one date) to monthly spring temperatures and examined evidence for selection favouring earlier breeding and for plasticity in timing.
C_LIO_LIEgg one dates occurred earlier over time, with the timing of breeding advancing up to 24 days over the 32-year period. Breeding timing also strongly covaried with maximum April temperature. We found significant overall selection favouring earlier breeding (i.e., higher relative fitness with earlier egg one dates) that became stronger over time, but strength of selection was not predicted by temperature. Lastly, individual females exhibited plastic responses to temperature across years.
C_LIO_LIOur findings provide further evidence that phenotypic plasticity plays a crucial role in driving phenological shifts in response to climate change. For multi-brooded bird populations, a warming climate might extend the breeding season and provide more opportunities to re-nest rather than drive earlier breeding in response to potential phenological mismatches. However, as plasticity will likely be insufficient for long-term survival in the face of climate change, further research in understanding the mechanisms of female reproductive timing will be essential for forecasting the effects of climate change on population persistence.
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sentences: Pellaea zygophylla, a new combination for a distinctive & well-known but neglected fern. Pellaea ovata is a widespread species, sexual diploid in Texas &northeastern Mexico but an apogamous triploid in northwestern Mexico, south to northern Argentina, &on Hispaniola. The type belongs to the southern, apogamous triploid form. Although these two forms have been discussed repeatedly in the literature, morphological distinctions between them have been overlooked and they have not been recognized taxonomically. However, they are distinct. Pellaea ovata s.s. has puberulent rachides & costae; pinnae usually 2-pinnate with a well-defined main axis &pinnules borne singly; fertile pinnules ovate, cordate basally &rounded apically. The sexual diploid form has rachides &costae glabrous; pinnae pseudo-dichotomously branched &pinnules usually paired; fertile pinnules narrowly rounded-trapeziform, obliquely truncate to cordate basally &truncate apically. Riddell named the sexual diploid form Pteris zygophylla, from which I give it the new combination Pellaea zygophylla.

sentences: Multifunctionality of belowground food webs: resource, size and spatial energy channels The belowground compartment of terrestrial ecosystems drives nutrient cycling, the decomposition and stabilisation of organic matter, and supports aboveground life. Belowground consumers create complex food webs that regulate functioning, ensure stability and support biodiversity both below and above ground. However, existing soil food-web reconstructions do not match recently accumulated empirical evidence and there is no comprehensive reproducible approach that accounts for the complex resource, size and spatial structure of food webs in soil. Here I build on generic food-web organization principles and use multifunctional classification of soil protists, invertebrates and vertebrates, to reconstruct  multichannel food-web across size classes of soil-associated consumers. This reconstruction is based on overlying feeding preference, prey protection, size spectrum and spatial distribution matrices combined with biomasses of trophic guilds to infer weighted trophic interactions. I then use food-web reconstruction, together with assimilation efficiencies, to calculate energy fluxes assuming a steady-state energetic system. Based on energy fluxes, I describe a number of indicators, related to stability, biodiversity and multiple ecosystem-level functions such as herbivory, top-down control, translocation and transformation of organic matter. I illustrate the approach with an empirical example, comparing it with traditional resource-focused soil food-web reconstruction. The multichannel reconstruction can be used to assess trophic multifunctionality (analogous to ecosystem multifunctionality), i.e. simultaneous support of multiple trophic functions by the food-web, and compare it across communities and ecosystems spanning beyond the soil. With further validation and parametrization, my multichannel reconstruction approach provides an effective tool for understanding and analysing soil food webs. I believe that having this tool will inspire more people to comprehensively describe soil communities and belowground-aboveground interactions. Such studies will provide informative indicators for including consumers as active agents in biogeochemical models, not only locally but also on regional and global scales.

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Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms.

Author SummaryPseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

sentences: Loss of the DYRK1A protein kinase results in reduction of ribosomal protein genes expression, ribosome mass and translation defects Ribosomal proteins (RPs) are evolutionary conserved proteins that are essential for protein translation. RP expression must be tightly regulated to ensure the appropriate assembly of ribosomes and to respond to the growth demands of cells. The elements regulating the transcription of RP genes (RPGs) have been characterized in yeast and Drosophila, yet how cells regulate the production of RPs in mammals is less well understood. Here, we show that a subset of RPG promoters is characterized by the presence of the palindromic TCTCGCGAGA motif and marked by the recruitment of the protein kinase DYRK1A. The presence of DYRK1A at these promoters is associated with enhanced binding of the TATA-binding protein, TBP, and it is negatively correlated with the binding of the GABP transcription factor, establishing at least two clusters of RPGs that could be coordinately regulated. However, DYRK1A silencing leads to a global reduction of RPGs, mRNA pointing at DYRK1A activities beyond those dependent on its chromatin association. Significantly, cells in which DYRK1A is depleted have reduced RP levels, fewer ribosomes, reduced global protein synthesis and smaller size. We therefore propose a novel role for DYRK1A in coordinating the expression of genes encoding RPs, thereby controlling cell growth in mammals.

sentences: Biological integrity enhances the qualitative effectiveness of conditional mice-oak mutualisms Scatter-hoarding decisions by rodents are key for the long-term maintenance of scattered tree populations. Decisions are determined by seed value, competition and predation risk, so that they can be influenced by the integrity of the biological system composed by trees, rodents, ungulate competitors, and rodent predators. We manipulate and model the oak-mice interaction in a Spanish dehesa, an anthropogenic savanna system suffering chronic tree regeneration failure, and quantify the joint effect of intrinsic and extrinsic factors on acorn dispersal effectiveness. First, we conducted a large-scale cafeteria field experiment, where we modified ungulate presence and predation risk, and followed mouse scatter-hoarding decisions under contrasting levels of moonlight and acorn availability. Then, we estimated the net effects of competition and risk by means of transition probability models that simulated mouse scatter-hoarding decisions according to the environmental context. Our results show that suboptimal conditions for mice balance the interaction towards the mutualism as they force mice to forage less efficiently. Under stressful conditions (predation risks and presence of ungulates), lack of antipredatory cover around dehesa trees limited transportation of acorns, but also precluded mice activities outside tree canopies. As a result, post-dispersal predation rates were reduced and large acorns had a higher probability to survive. Our work shows that inter-specific interactions preventing efficient foraging by scatter-hoarders benefitted seed dispersal. Therefore, the maintenance of the full set of producers, consumers, dispersers and predators in ecosystems is key for promoting seed dispersal effectiveness in conditional mutualisms.

sentences: Sexually dimorphic placental responses to maternal SARS-CoV-2 infection There is a persistent male bias in the prevalence and severity of COVID-19 disease. Underlying mechanisms accounting for this sex difference remain incompletely understood. Interferon responses have been implicated as a modulator of disease in adults, and play a key role in the placental anti-viral response. Moreover, the interferon response has been shown to alter Fc-receptor expression, and therefore may impact placental antibody transfer. Here we examined the intersection of viral-induced placental interferon responses, maternal-fetal antibody transfer, and fetal sex. Placental interferon stimulated genes (ISGs), Fc-receptor expression, and SARS-CoV-2 antibody transfer were interrogated in 68 pregnancies. Sexually dimorphic placental expression of ISGs, interleukin-10, and Fc receptors was observed following maternal SARS-CoV-2 infection, with upregulation in males. Reduced maternal SARS-CoV-2-specific antibody titers and impaired placental antibody transfer were noted in pregnancies with a male fetus. These results demonstrate fetal sex-specific maternal and placental adaptive and innate immune responses to SARS-CoV-2.

sentences: ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19. Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchioalveolar lavage fluid from critically ill COVID-19 patients was associated with reduced ICU and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.

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Expression of CD49f defines subsets of human regulatory T cells with divergent transcriptional landscape and function that correlate with ulcerative colitis disease activity ObjectiveAdoptive regulatory T cell (Treg) therapy is being trialled for treatment of different autoimmune disorders, including inflammatory bowel diseases (IBD). In-depth understanding of the biological variability of Treg in the human blood may be required to improve IBD immune monitoring and treatment strategies.

MethodsThrough a combination of quantitative proteomic, multiparametric flow cytometry, RNA-sequencing data analysis and functional assays on Treg enriched from the blood of ulcerative colitis (UC) patients and healthy controls, we investigated the association between CD49f expression, Treg phenotype and function, and UC disease activity.

ResultsHigh-dimensional analysis and filtering defined two distinct subsets of human Treg based on the presence or absence of CD49f with divergent transcriptional landscape and functional activities. CD49f negative Treg are enriched for functional Treg markers and present significantly increased suppressive capacity. In contrast, CD49f high Treg display a pro-inflammatory Th17-like phenotype and accumulate in the blood of UC patients. Dysregulation on CD49f Treg subsets in UC patients correlate with disease activity.

ConclusionOverall our findings uncover the importance of CD49f expression on Treg in physiological immunity and in pathological autoimmunity.

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Genomes contain relics of a triplet code connecting the origins of primordial RNA synthesis to the origins of genetically coded protein synthesis Life on earth relies on three types of information polymers-DNA, RNA and proteins. In all organisms and viruses, these molecules are synthesized by the copying of pre-existing templates. A triplet-based code known as the genetic code guides the synthesis of proteins by complex enzymatic machines that decode genetic information in RNA sequences. The origin of the genetic code is one of the most fundamental questions in biology. In this study, computational analysis of about 5,000 species level metagenomes using techniques for the analysis of human language suggests that the genomes of extant organisms contain relics of a distinct triplet code that potentially predates the genetic code. This code defines the relationship between adjacent triplets in DNA/RNA sequences, whereby these triplets predominantly differ by a single base. Furthermore, adjacent triplets encode amino acids that are thought to have emerged around the same period in the earths early history. The results suggest that the order of triplets in primordial RNA sequences was associated with the availability of specific amino acids, perhaps due to a coupling of a triplet-based primordial RNA synthesis mechanism to a primitive mechanism of peptide bond formation. Together, this coupling could have given rise to early nucleic acid sequences and a system for encoding amino acid sequences in RNA, i.e. the genetic code. Thus, the central role of triplets in biology potentially extends to the primordial world, contributing to both the origins of genomes and the origins of genetically coded protein synthesis.

SignificanceOne of the most intriguing discoveries in biology is that the order of amino acids in each protein is determined by the order of nucleotides (commonly represented by the letters A, U, G, C) in a biological molecule known as RNA. The genetic code serves as a dictionary that maps each of the 64 triplets  words in RNA to the 20 amino acids, thereby specifying how information encoded in RNA is decoded into sequences of amino acids (i.e., proteins). The deciphering of the genetic code was one of the greatest discoveries of the 20th century (1968 Nobel Prize in Medicine and Physiology) and is central to modern molecular biology. Yet, how it came to be that the order of triplets in RNA encodes the sequence of the protein synthesized remains one of the most important enigmas of biology. Paradoxically, in all life forms proteins cannot be synthesized without RNA and RNA itself cannot also be synthesized without proteins, presenting a chicken and egg dilemma. By analyzing thousands of microbial genomes using approaches drawn from the field of natural language processing, this study finds that the order of triplets across genomes contains relics of an ancient triplet code, distinct from but closely connected to the genetic code. Unlike the genetic code which specifies the relationship between information in RNA and the sequence of proteins, this ancient code describes the relationship between adjacent triplets in extant genome sequences, whereby such triplets are often different from each other by a single letter. Triplets that are closely related by this ancient code encode amino acids that are thought to have emerged around the same period in the earths early history. In other words, a fossil record of the chronological order of appearance of amino acids on early earth appears written in genome sequences. This potentially demonstrates that the process by which RNA sequences were synthesized in the primordial world relied on triplets and was coupled to amino acids available at the time. Hence, the connections between primordial RNA synthesis and a primitive mechanism for linking amino acids to form peptides could have enabled one type of molecule (RNA) to code for the other (protein), facilitating the emergence of the genetic code.

sentences: Humans trade-off energetic cost with fatigue avoidance while walking Metabolic cost minimization is widely regarded as the principal optimality criterion that governs walking. Minimizing muscle activation has, nevertheless, outperformed energy optimization in simulating human gait and predicting certain gait behaviors. The highly coupled nature of metabolic and muscle activation costs makes it difficult to empirically discern the interrelationship between these objectives. We implemented a unique experimental design that pits metabolic cost against muscle activation costs estimated from electromyography of seven lower limb muscles. Healthy adults (N=10) selected between walking on a treadmill incline versus walking in a crouched posture (that disproportionately affected activation cost), forcing a choice between minimizing metabolic cost or activation cost. When experiencing these Competing-Cost-Pairs, participants systematically protected their activation cost at the expense of high metabolic power ([Formula]; 19% penalty, p<0.05). This held true when activation cost was expressed as the sum of the muscle activations squared ([Formula]; 66% saving, p<0.05) and as the maximal activation across muscles ([Formula]; 44% saving, p<0.05), both of which penalize overburdening any individual muscle and thus indicate fatigue avoidance. Activation cost, expressed as the sum of muscle volume-normalized activation [Formula], more closely models energy use and was also protected by the participants decision (23% saving, p<0.05) demonstrating that activation was, at best, an inaccurate proxy signal for metabolic energy. Energy minimization was only observed when there was no adverse effect on muscle activation. By decoupling metabolic and activation costs, we provide the first empirical evidence of humans embracing non-energetic optimality in favor of a clearly defined alternate neuromuscular objective.

sentences: Direct neural evidence for the contrastive roles of the complementary learning systems in adult acquisition of native vocabulary The Complementary Learning Systems (CLS) theory provides a powerful framework for considering the acquisition, consolidation and generalisation of new knowledge. We tested this proposed neural division of labour in adults through an investigation of the consolidation and long-term retention of newly-learned native vocabulary with post-learning functional neuroimaging. Newly-learned items were compared to two conditions: (i) previously known items to highlight the similarities and differences with established vocabulary; and (ii) unknown/untrained items to provide a control for non-specific perceptual and motor-speech output. Consistent with the CLS, retrieval of newly-learned items was supported by a combination of regions associated with episodic memory (including left hippocampus) and the language-semantic areas that support established vocabulary (left inferior frontal gyrus and left anterior temporal lobe). Furthermore, there was a shifting division of labour across these two networks in line with the items consolidation status; faster naming was associated with more activation of language-semantic areas and lesser activation of episodic memory regions. Hippocampal activity during naming predicted more than half the variation in naming retention six months later.

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Benchmarking 50 classification algorithms on 50 gene-expression datasets By classifying patients into subgroups, clinicians can provide more effective care than using a uniform approach for all patients. Such subgroups might include patients with a particular disease subtype, patients with a good (or poor) prognosis, or patients most (or least) likely to respond to a particular therapy. Diverse types of biomarkers have been proposed for assigning patients to subgroups. For example, DNA variants in tumors show promise as biomarkers; however, tumors exhibit considerable genomic heterogeneity. As an alternative, transcriptomic measurements reflect the downstream effects of genomic and epigenomic variations. However, high-throughput technologies generate thousands of measurements per patient, and complex dependencies exist among genes, so it may be infeasible to classify patients using traditional statistical models. Machine-learning classification algorithms can help with this problem. However, hundreds of classification algorithms exist--and most support diverse hyperparameters--so it is difficult for researchers to know which are optimal for gene-expression biomarkers. We performed a benchmark comparison, applying 50 classification algorithms to 50 gene-expression datasets (143 class variables). We evaluated algorithms that represent diverse machine-learning methodologies and have been implemented in general-purpose, open-source, machine-learning libraries. When available, we combined clinical predictors with gene-expression data. Additionally, we evaluated the effects of performing hyperparameter optimization and feature selection in nested cross-validation folds. Kernel- and ensemble-based algorithms consistently outperformed other types of classification algorithms; however, even the top-performing algorithms performed poorly in some cases. Hyperparameter optimization and feature selection typically improved predictive performance, and univariate feature-selection algorithms outperformed more sophisticated methods. Together, our findings illustrate that algorithm performance varies considerably when other factors are held constant and thus that algorithm selection is a critical step in biomarker studies.

Author Summary

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Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation In neurodegenerative diseases including Alzheimers and ALS, proteins that bind RNA are found in aggregated forms in autopsied brains. Evidence suggests that RNA aids nucleation of these pathological aggregates; however, the mechanism has not been investigated at the level of atomic structure. Here we present the 3.4 [A] resolution structure of fibrils of full-length recombinant tau protein in the presence of RNA, determined by electron cryo-microscopy (cryoEM). The structure reveals the familiar in-register cross-{beta} amyloid scaffold, but with a small fibril core spanning residues Glu391 to Ala426, a region disordered in the fuzzy coat in all previously studied tau polymorphs. RNA is bound on the fibril surface to the positively charged residues Arg406 and His407 and runs parallel to the fibril axis. The fibrils dissolve when RNAse is added, showing that RNA is necessary for fibril integrity. While this structure cannot exist simultaneously with the tau fibril structures extracted from patients brains, it could conceivably account for the nucleating effects of RNA cofactors followed by remodeling as fibrils mature.

Significance statementApplication of cryoEM has greatly expanded our understanding of atomic structures of mature pathological amyloid fibrils, but little is known at the molecular level of the initiation of fibril formation. RNA has been shown to be one cofactor for formation of fibrils of tau protein, and is known also to bind to other proteins, including TDP-43, FUS, and HNRNPA2, which form pathological inclusions. Our cryoEM structure of recombinant tau protein with RNA reveals a 36 residue, C-terminal fibril core bound to RNA which runs parallel to the fibril axis. We speculate that this structure could represent an early step in the formation of tau fibrils.

sentences: SURFMAP: a software for mapping in two dimensions protein surface features Molecular cartography using two-dimensional (2D) representation of protein surfaces has been shown to be very promising for protein surface analysis. Here, we present SURFMAP, a free standalone and easy-to-use software that enables the fast and automated 2D projection of either predefined features of protein surface (i.e., electrostatic potential, Kyte-Doolittle hydrophobicity, stickiness, and surface relief) or any descriptor encoded in the temperature factor column of a PDB file. SURFMAP uses a pseudo-cylindrical sinusoidal "equal-area" projection that has the advantage of preserving the area measures. It provides the user with (i) 2D maps that enable the easy and visual analysis of protein surface features of interest and (ii) maps in a text file format allowing the fast and straightforward quantitative comparison of 2D maps of homologous proteins.

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The chemokine receptor CXCR3 promotes CD8+ T cell-dependent lung pathology during influenza pathogenesis While the role of CD8+ T cells in influenza clearance is established, their contribution to pathological lung injury is increasingly appreciated. To explore if protective versus pathological functions can be linked to CD8+ T cell subpopulations, we dissected their responses in influenza-infected murine lungs. Our single-cell RNASeq (scRNAseq) analysis revealed significant diversity in CD8+ T cell subpopulations during peak viral load vs. infection-resolved state. While enrichment of Cxcr3hi CD8+ T effector (Teff) subset was associated with a more robust cytotoxic response, both CD8+ Teff and CD8+ T central memory (TCM) exhibited equally potent effector potential. The scRNAseq analysis identified unique regulons regulating the cytotoxic response in CD8+ T cells. The neutralization of CXCR3 mitigated lung injury without affecting viral clearance. IFN-{gamma} was dispensable to regulate the cytotoxic response of Cxcr3hi CD8+ T cells. Collectively, our data imply that CXCR3 interception could have a therapeutic effect in preventing influenza-linked lung injury.

TEASERThe CXCR3 expressing CD8+ T cell subset causes severe lung pathology and exacerbates disease severity without affecting viral clearance during influenza infection

sentences: SHAPR - An AI approach to predict 3D cell shapes from 2D microscopic images Reconstruction of shapes, forms, and sizes of three-dimensional (3D) objects from two-dimensional (2D) information is one of the most complex functions of the human brain. It also poses an algorithmic challenge and at present is a widely studied subject in computer vision. We here focus on the single cell level and present a neural network-based SHApe PRediction autoencoder SHAPR that accurately reconstructs 3D cellular and nuclear shapes from 2D microscopic images and may have great potential for application in the biomedical sciences.

sentences: A high-resolution comparative atlas across 74 fish genomes illuminates teleost evolution after whole-genome duplication Teleost fishes are ancient tetraploids stemming from an ancestral whole-genome duplication that may have contributed to the impressive diversification of this clade. Whole-genome duplications can occur via self-doubling (autopolyploidy) or via hybridization between different species (allopolyploidy). The mode of tetraploidization conditions subsequent evolutionary processes by which descendant genomes return to a diploid state through gene losses and functional divergence. How teleosts became tetraploid remains however unresolved, leaving a fundamental gap to interpret their functional evolution. As legacy of the whole genome duplication, identifying orthologous and paralogous genomic regions across teleosts is challenging, hindering genome-wide investigations into their polyploid history. Here, we combine tailored gene phylogeny methodology together with the state-of-the-art ancestral karyotype reconstruction to establish the first high-resolution comparative atlas of paleopolyploid regions across 74 teleost genomes. We then leverage this atlas to investigate the genome-wide mechanisms of rediploidization after tetraploidization in teleosts. We uncover that meiotic recombination persisted between duplicated chromosomes for over 60 million years, with entire chromosomes only functionally diverging after the separation of major teleost families. This evidence suggests that the teleost ancestor was an autopolyploid. Further, ancient duplicated chromosomes have evolved asymmetrically in teleosts, disputing unbalanced evolution as a hallmark of polyploidization through hybridization.

sentences: Oxylipin metabolism is controlled by mitochondrial b-oxidation during bacterial inflammation. Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial {beta}-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin {beta}-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-{gamma}. Last, dampening oxylipin levels by {beta}-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial {beta}-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

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Transmission distortion and genetic incompatibilities between alleles in a multigenerational mouse advanced intercross line Background/ObjectivesWhile direct additive and dominance effects on complex traits have been mapped repeatedly, additional genetic factors contributing to the heterogeneity of complex traits have been scarcely investigated. To assess genetic background effects, we investigated transmission ratio distortions (TRDs) of alleles from parent to offspring using an advanced intercross line (AIL) of an initial cross between the mouse inbred strains C57BL/6NCrl (B6N) and BFMI860-12 (BFMI).

Subjects/Methods341 males of generation 28 and their respective 61 parents and 66 grandparents were genotyped using Mega Mouse Universal Genotyping Arrays (MegaMUGA). TRDs were investigated using allele transmission asymmetry tests, and pathway overrepresentation analysis was performed. Sequencing data was used to test for overrepresentation of non-synonymous SNPs in TRD regions. Genetic incompatibilities were tested using the Bateson-Dobzhansky-Muller two-locus model.

Results62 TRD regions were detected, many in close proximity to the telocentric centromere. TRD regions contained 44.5% more non-synonymous SNPs than randomly selected regions (182 vs. 125.9 {+/-} 17.0, P < 1x10-4). Testing for genetic incompatibilities between TRD regions identified 29 genome-wide significant incompatibilities between TRD regions (P(BF) < 0.05). Pathway overrepresentation analysis of genes in TRD regions showed that DNA methylation, epigenetic regulation of RNA, and meiotic/meiosis regulation pathways were affected independent of the parental origin of the TRD. Paternal BFMI TRD regions showed overrepresentation in the small interfering RNA (siRNA) biogenesis and in the metabolism of lipids and lipoproteins. Maternal B6N TRD regions harbored genes involved in meiotic recombination, cell death, and apoptosis pathways. The analysis of genes in TRD regions suggests the potential distortion of protein-protein interactions influencing obesity and diabetic retinopathy as a result of disadvantageous combinations of allelic variants in Aass, Pgx6 and Nme8.

ConclusionsUsing an AIL significantly improves the resolution at which we can investigate TRD. Our analysis implicates distortion of protein-protein interactions as well as meiotic drive as the underlying mechanisms leading to the observed TRD in our AIL. Furthermore, genes with large amounts of non-synonymous SNPs located in TRD regions are more likely to be involved in pathways that are related to the phenotypic differences between the parental strains. Genes in these TRD regions provide new targets for investigating genetic adaptation, protein-protein interactions, and determinants of complex traits such as obesity.

sentences: Shaping coral traits: plasticity more than filtering The structure of ecosystems is usually determined by the shape of the organisms that build it, commonly known as ecosystem engineers. Understanding to what extent plasticity and environmental filtering determine variation in ecosystem engineer physical structure is necessary to predict how ecosystem structure may change. Here, we explored coral survival and the plasticity of morphological traits that are critical for habitat provision in coral reefs. We conducted a reciprocal clonal transplant experiment in which branching corals from the genus Porites and Acropora were moved to and from a deep and a shallow site within a lagoon in the Maldives. Survival and trait analyses showed that transplant destination consistently induced the strongest changes, particularly among Acropora spp. The origin of the corals only marginally affected some of the traits. We also detected variation in the way individuals from the same species and site differentiate their shape, showing that traits linked to habitat provision are phenotypically plastic. The results suggest coral phenotypic plasticity plays a stronger role than environmental filtering, in determining zonation of coral morphologies, and consequently the habitats they provide for other taxa.

sentences: Deciphering the potential niche of novel black yeast fungal isolates in a biological soil crust based on genomes, phenotyping, and melanin regulation Black yeasts are polyextremotolerant fungi that contain high amounts of melanin in their cell wall and maintain a primarily yeast form. These fungi grow in xeric, nutrient deplete environments which implies that they require highly flexible metabolisms and the ability to form lichen-like mutualisms with nearby algae and bacteria. However, the exact ecological niche and interactions between these fungi and their surrounding community is not well understood. We have isolated two novel black yeast fungi of the genus Exophiala: E. viscosium and E. limosus, which are from dryland biological soil crusts. A combination of whole genome sequencing and various phenotyping experiments have been performed on these isolates to determine their fundamental niches within the biological soil crust consortium. Our results reveal that these Exophiala spp. are capable of utilizing a wide variety of carbon and nitrogen sources potentially from symbiotic microbes, they can withstand many abiotic stresses, and can potentially provide UV resistance to the crust community in the form of secreted melanin. Besides the identification of two novel species within the genus Exophiala, our study also provides new insight into the production and regulation of melanin in extremotolerant fungi.

sentences: Th2 single-cell heterogeneity and clonal interorgan distribution in helminth-infected mice Th2 cells provide effector functions in type 2 immune responses to helminths and allergens. Despite knowledge about molecular mechanisms of Th2 cell differentiation, there is little information on Th2 cell heterogeneity and clonal distribution between organs. To address this, we performed combined single-cell transcriptome and TCR clonotype analysis on murine Th2 cells in mesenteric lymph nodes (MLN) and lung after infection with Nippostrongylus brasiliensis (Nb) as a human hookworm infection model. We find organ-specific expression profiles, but also populations with conserved migration or effector/resident memory signatures that unexpectedly cluster with potentially regulatory Il10posFoxp3neg cells. A substantial MLN subpopulation with an interferon response signature suggests a role for interferon-signaling in Th2 differentiation or diversification. Further RNA-inferred developmental directions indicate proliferation as a hub for differentiation decisions. We also link long noted Cxcr3 expression in the Th2 compartment to a population of Il4pos NKT cells. Although the TCR repertoire is highly heterogeneous, we identified expanded clones and CDR3 motifs. Clonal relatedness between distant organs confirmed effective exchange of Th2 effector cells, although locally expanded clones dominated the response. These results provide new insights in Th2 cell subset diversity and clonal relatedness in distant organs.

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Limited Evidence for a Triple Dissociation in the Medial Temporal Lobe: an fMRI Recognition Memory Replication Study Common research practices in neuroimaging studies using functional magnetic resonance imaging may produce outcomes that are difficult to replicate. Results that cannot be replicated have contributed to a replication crisis in psychology, neuroscience, and other disciplines over the years. Here we replicate two previous papers in which the authors present two analysis paths for a dataset in which participants underwent fMRI while performing a recognition memory test for old and new words. Both studies found activation in the hippocampus, with the first demonstrating a distinction in hippocampal activation corresponding to true and perceived oldness of stimuli and the second demonstrating that hippocampal activation reflects the subjective experience of the participant. We replicated the behavioral and MRI acquisition parameters reported in the two target articles (Daselaar, Fleck, & Cabeza, 2006; Daselaar, Fleck, Prince, et al., 2006) with N=53 participants. We focused fMRI analyses on regions of interest reported in the target articles examining fMRI activation for differences corresponding with true and perceived oldness and those associated with the subjective memory experiences of recollection, familiarity, and novelty. Comparisons between true and perceived oldness revealed main effects not only for true, but also perceived oldness along with significant interaction effects. We replicate the findings of recollection and familiarity signals in the hippocampus and medial temporal lobe cortex, respectively, but failed to replicate a novelty signal in the anterior medial temporal lobe. These results remained when we analyzed only correct trials, indicating that the effects were not due to selectively averaging correct and incorrect trials. Taken together, we replicate the previous finding that activation in the hippocampus corresponds to the subjective experience associated with correct recognition memory retrieval.

HighlightsO_LIWe replicated the methods of two influential papers using fMRI to examine recognition memory.
C_LIO_LIThe hippocampus consistently signals the subjective experience of remembering but not the objective old/new status of stimuli.
C_LIO_LIHippocampal response was consistent with recollection while the medial temporal lobe (MTL) cortex response was consistent with familiarity.
C_LIO_LIWe did not observe any regions in the MTL that signaled novelty, indicating recognition memory does not involve a triple dissociation of recollection, familiarity, and novelty in the MTL.
C_LI

sentences: Impact of Starmerella bacillaris and Zygosaccharomyces bailii on ethanol reduction and Saccharomyces cerevisiae metabolism during mixed wine fermentations The bulk of grape juice fermentation is carried out by the yeast Saccharomyces cerevisiae, but non-Saccharomyces yeasts can modulate many sensorial aspects of the final products in ways not well understood. In this study, some of such non-conventional yeasts were screened as mixed starter cultures in a fermentation defined medium in both simultaneous and sequential inoculations. One strain of Starmerella bacillaris and another of Zygosaccharomyces bailii were chosen by their distinct phenotypic footprint and their ability to reduce ethanol levels at the end of fermentation, particularly during simultaneous vinification. S. bacillaris losses viability strongly at the end of mixed fermentation, while Z. bailii remains viable until the end of vinification. Interestingly, for most non-Saccharomyces yeasts, simultaneous inoculation helps for survival at the end of fermentation compared to sequential inoculation. S. cerevisiae viability was unchanged by the presence of the either yeast. Characterization of both strains indicates that S. bacillaris behavior is overall more different from S. cerevisiae than Z. bailii. S. bacillaris has a less strict glucose repression mechanism and molecular markers like catabolite repression kinase Snf1 is quite different in size. Besides, S. cerevisiae transcriptome changes to a bigger degree in the presence of S. bacillaris than when inoculated with Z. bailii. S. bacillaris induces the translation machinery and repress vesicular transport. Both non-Saccharomyces yeast induce S. cerevisiae glycolytic genes, and that may be related to ethanol lowering, but there are specific aspects of carbon-related mechanisms between strains: Z. bailii presence increases the stress-related polysaccharides trehalose and glycogen while S. bacillaris induces gluconeogenesis genes.

sentences: Find me if you can: Pre- and Post-hurricane Densities of the Red-bellied Racer (Alsophis rufiventris) on St. Eustatius, and a review of the genus in the Caribbean We estimated population densities of the red-bellied racer (Alsophis rufiventris) on the Caribbean island of St. Eustatius in 2011, 2018 and 2019 to determine the likely influence of hurricanes Irma and Maria (September 2017), in addition to evaluating abiotic parameters which may be correlated with its presence. Surveys were conducted at seven sites in 2011 prior to the hurricanes, and at 81 and 108 sites in 2018 and 2019 respectively posterior to the hurricanes. A total of 8.2 ha was surveyed in 2011, and 11.42 ha in 2018/2019. The pre-hurricane (2011) racer density estimate was 9.2/ha (min 7.3 - max 11.6); post-hurricane estimates were 4.6/ha (min 3.4 - max 6.0) in 2018 and 5.0/ha (min 3.8 - max 6.5) in 2019. The pre-hurricane encounter rate of individual racers was 16.0 snakes/hour compared to 0.34 snakes/hour in 2018 and 0.41 snakes/hour in 2019 (post-hurricane). The decrease in encounter rates between 2011 and 2019 implies a negative impact of the hurricanes on racer abundance. Based on calculations of detection probability (0.02 in 2018 and 0.03 in 2019), post-hurricane lambda estimates were 1.82 (95% CI 0.66 - 5.01) in 2018 and 1.60 (95% CI 0.39 - 6.65) snakes/ha in 2019. Given the current small size of the remaining population and the presence of invasive species across the snakes range, this species could be at risk of local extirpation. We suggest conservation actions such as invasive species management and habitat restoration to enable further recovery.

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Evolutionary coexistence in a fluctuating environment by specialization on resource level Microbial communities in fluctuating environments can contain many species and diversity within species, both in natural environments such as the human gut, and in laboratory settings when communities are propagated for a long time. Whether this diversity is at the species level, within the species level, or a combination of both, the question remains: what processes lead to the origination and maintenance of this diversity?

When nutrient levels fluctuate over time, one possibly relevant process is that different types specialize on low and high nutrient levels. The relevance of this process is supported by observations of types co-existing through this mechanism when put together in the laboratory, and by simple models, that show that negative frequency dependence of two types, specialized on low and high resource level, can stabilize coexistence. However, when microbial populations are in an environment for a long time, they evolve. In this article we determine what happens when species can evolve; whether branching can occur to create diversity and whether evolution will destabilize coexistence.

We find that for the trade-off data between growth at low and high substrate concentrations, available for the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae on glucose, there is only a small portion of the trait-space that allows for coexistence. Moreover, this coexistence is destabilized by evolution, and the only evolutionary stable outcome is a single type. When we combine two species that are well-adapted on their own, we do find that they can form an evolutionary singular coalition, i.e. a coexistence that is evolutionarily stable. We conclude that although specialization on resource level can support diversity within a species, it is likely not a cause by itself. In contrast, for species consortia this specialization can lead to evolutionarily stable coexistence.

sentences: An improved assembly of the Albugo candida Ac2V genome reveals the expansion of the "CCG" class of effectors Albugo candida is an obligate oomycete pathogen that infects many plants in the Brassicaceae family. We re-sequenced the genome of isolate Ac2V using PacBio long reads and constructed an assembly augmented by Illumina reads. The Ac2VPB genome assembly is 10% larger and more contiguous compared to a previous version. Our annotation of the new assembly, aided by RNASeq information, revealed a dramatic 250% expansion (40 to 110) in the CHxC effector class, which we redefined as "CCG" based on motif analysis. This class of effectors consist of arrays of phylogenetically related paralogs residing in gene sparse regions, and shows signatures of positive selection and presence/absence polymorphism. This work provides a resource that allows the dissection of the genomic components underlying A. candida adaptation and particularly the role of CCG effectors in virulence and avirulence on different hosts.

sentences: SARS-CoV-2 variant B.1.1.7 is susceptible to neutralizing antibodies elicited by ancestral Spike vaccines The SARS-CoV-2 Spike glycoprotein mediates virus entry and is a major target for neutralizing antibodies. All current vaccines are based on the ancestral Spike with the goal of generating a protective neutralizing antibody response. Several novel SARS-CoV-2 variants with multiple Spike mutations have emerged, and their rapid spread and potential for immune escape have raised concerns. One of these variants, first identified in the United Kingdom, B.1.1.7 (also called VUI202012/01), contains eight Spike mutations with potential to impact antibody therapy, vaccine efficacy and risk of reinfection. Here we employed a lentivirus-based pseudovirus assay to show that variant B.1.1.7 remains sensitive to neutralization, albeit at moderately reduced levels (~2-fold), by serum samples from convalescent individuals and recipients of two different vaccines based on ancestral Spike: mRNA-1273 (Moderna), and protein nanoparticle NVX-CoV2373 (Novavax). Some monoclonal antibodies to the receptor binding domain (RBD) of Spike were less effective against the variant while others were largely unaffected. These findings indicate that B.1.1.7 is not a neutralization escape variant that would be a major concern for current vaccines, or for an increased risk of reinfection.

sentences: Identification of Kinases Activated by Multiple Pro-Angiogenic Growth Factors Antiangiogenic therapy began as an effort to inhibit VEGF signaling, which was thought to be the sole factor driving tumor angiogenesis. It has become clear that there are more pro-angiogenic growth factors that can participate in and can substitute for VEGF during tumor vascularization. This has led to the development of multi-kinase inhibitors that simultaneously target multiple growth factor receptors. These inhibitors perform better than monotherapies yet to date no multi-kinase inhibitor targets all receptors known to be involved in pro-angiogenic signaling and resistance inevitably occurs. Given the large number of pro-angiogenic growth factors identified, it may be impossible to simultaneously target all pro-angiogenic growth factor receptors with a single agent. Here we search downstream of the receptors for kinase targets that would be critical to endothelial cell proliferation, irrespective of the growth factor signal. To do this systematically we develop a quantitative endothelial cell proliferation assay and combine it with "kinome regression" or KIR, a recently developed computational and kinase screening method capable of identifying kinases that influence any measurable and quantitative phenotype, such as proliferation. We report the result of this analysis. The kinases implicated by KIR are both expected and unexpected intracellular kinases. Using RNA interference we provide orthogonal evidence of their importance in endothelial cell proliferation. This approach points to a new strategy that can be employed to develop a more complete anti-angiogenic blockade.

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Specific plasticity loci and their synergism mediate operant	conditioning Despite numerous studies examining the mechanisms of operant conditioning (OC), the diversity of plasticity loci and their synergism have not been examined sufficiently. In the well-characterized feeding neural circuit of Aplysia, appetitive OC increases neuronal excitability and electrical coupling among several neurons. Here we found OC decreased the intrinsic excitability of B4 and the strength of its inhibitory connection to a key decision-making neuron, B51. The OC-induced changes were specific without affecting the B4-to-B8 inhibitory connection or excitability of another neuron critical for feeding behavior, B8. A conductance-based circuit model indicated certain sites of plasticity mediated the OC phenotype more effectively and that plasticity loci acted synergistically. This synergy was specific in that only certain combinations of loci synergistically enhanced feeding. Taken together, these results suggest modifications of diverse loci work synergistically to mediate OC.

Significance StatementThe diversity and synergism of plasticity loci mediating operant conditioning (OC) is poorly understood. Here we found that OC decreased the intrinsic excitability of a critical neuron mediating Aplysia feeding behavior and specifically reduced the strength of one of its inhibitory connections to a key decision-making neuron. A conductance-based computational model indicated that the known plasticity loci showed a surprising level of synergism to mediate the behavioral changes associated with OC. These results highlight the importance of understanding the diversity, specificity and synergy among different types of plasticity that encode memory. Also, because OC in Aplysia is mediated by dopamine (DA), the present study provides insights into specific and synergistic mechanisms of DA-mediated reinforcement of behaviors.

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An Off-the-Shelf Bioadhesive Patch for Sutureless Repair of Gastrointestinal Defects Surgical sealing and repair of injured and resected gastrointestinal (GI) organs are critical requirements for successful treatment and tissue healing. Despite being the standard of care, hand-sewn closure of GI defects using sutures faces various limitations and challenges. The process remains technically complicated and time-consuming. The needle-piercing and pointwise closure also inflict tissue damage and stress concentration, raising the risk of local failure and subsequent anastomotic leaks. To address these limitations and challenges, we introduce an off-the-shelf bioadhesive GI patch capable of atraumatic, rapid, robust, and sutureless repair of GI defects. The GI patch synergistically integrates a non-adhesive top layer and a dry bioadhesive bottom layer, resulting in a thin, flexible, transparent, and ready to use dressing with tissue-matching mechanical properties. Rapid, robust, and sutureless sealing capability of the GI patch is systematically characterized based on various standard tests in ex vivo porcine GI organ models. In vitro and in vivo rat models are utilized to validate biocompatibility and biodegradability of the GI patch including comprehensive cytotoxicity, histopathology, immunofluorescence, and blood analyses. To validate the GI patchs efficacy in a clinically relevant setting, we demonstrate successful sutureless in vivo sealing and healing of GI defects; namely in rat stomach and colon, and porcine colon injury models. The proposed GI patch not only provides a promising alternative to suture for repair of GI defects but also offers potential clinical opportunities in the treatment and repair of other organs.

One Sentence SummaryAn off-the-shelf bioadhesive patch is introduced for facile sutureless repair of gastrointestinal defects, addressing various limitations of conventional suture-based treatments.

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VirB4- and VirD4-like ATPases, components of a putative type 4C secretion system in Clostridioides difficile The type 4 secretion system (T4SS) represents a bacterial nanomachine capable of trans-cell wall transportation of proteins and DNA and which has attracted intense interest due to its roles in the pathogenesis of infectious diseases. During the current investigation we uncovered three distinct gene clusters in Clostridioides difficile strain 630 coding for proteins structurally related to components of the VirB4/D4 type 4C secretion system from Streptococcus suis strain 05ZYH33 and located within sequences of conjugative transposons (CTn). Phylogenic analysis shows that VirB4- and VirD4-like proteins of CTn4 locus, on one hand, and those of CTn2 and CTn5 loci, on the other hand, fit into separate clades, suggesting specific roles of identified secretion system variants in physiology of C. difficile. Our further study on VirB4- and VirD4-like products coded by CTn4 revealed that both proteins possess Mg2+-dependent ATPase activity, form oligomers (most probably, hexamers) in water solutions, and rely on potassium but not sodium ions for the highest catalytic rate. VirD4 binds nonspecifically to DNA and RNA. Its DNA binding activity strongly decreased with the W241A variant. Mutations in the nucleotide sequences coding for presumable Walker A and Walker B motifs decreased stability of the oligomers and significantly but not completely attenuated enzymatic activity of VirB4. In VirD4, substitutions of amino acid residues in the peptides reminiscent of Walker structural motifs resulted neither in attenuation of enzymatic activity of the protein nor influenced the oligomerization state of the ATPase.

ImportanceC. difficile is a Gram-positive, anaerobic, spore-forming bacterium that causes life-threatening colitis in humans. Major virulence factors of the microorganism include toxins TcdA, TcdB and CDT. However, other bacterial products, including a type 4C secretion system, have been hypothesized to contribute to the pathogenesis of the infection and are considered as possible virulence factors of C. difficile. In the current paper we describe structural organization of putative T4SS machinery in C. difficile and characterize its VirB4- and VirD4-like components. Our studies, in addition to significance for basic science, can potentially aid development of anti-virulence drugs suitable for treatment of C. difficile infection.

sentences: Correlation analysis among single nucleotide polymorphisms in thirteen language genes and culture/education parameters from twenty-six countries Language is a vital feature of any human culture, but whether language gene polymorphisms have meaningful correlations with some cultural characteristics during the long-run evolution of human languages largely remains obscure (uninvestigated). This study would be an endeavor example to find evidences for the above questions answer. In this study, the collected basic data include 13 language genes and their randomly selected 111 single nucleotide polymorphisms (SNPs), SNP profiles, 29 culture/education parameters, and estimated cultural context values for 26 representative countries. In order to undertake principal component analysis (PCA) for correlation search, SNP genotypes, cultural context and all other culture/education parameters have to be quantitatively represented into numerical values. Based on the above conditions, this study obtained its preliminary results, the main points of which contain: (1) The 111 SNPs contain several clusters of correlational groups with positive and negative correlations with each other; (2) Low cultural context level significantly influences the correlational patterns among 111 SNPs in the principal component analysis diagram; and (3) Among 29 culture/education parameters, several basic characteristics of a language (the numbers of alphabet, vowel, consonant and dialect) demonstrate least correlations with 111 SNPs of 13 language genes.

sentences: Towards a structurally resolved cancer interactome Protein-protein interactions (PPIs) are involved in almost all essential cellular processes. Perturbation of PPI networks plays critical roles in tumorigenesis, cancer progression and metastasis. While numerous high-throughput experiments have produced a vast amount of data for PPIs, these datasets suffer from high false positive rates and exhibit a high degree of discrepancy. Coevolution of amino acid positions between protein pairs has proven to be useful in identifying interacting proteins and providing structural details of the interaction interfaces with the help of deep learning methods like AlphaFold (AF). In this study, we applied AF to investigate the cancer protein-protein interactome. We predicted 1,798 PPIs for cancer driver proteins involved in diverse cellular processes such as transcription regulation, signal transduction, DNA repair and cell cycle. We modeled the spatial structure for the predicted binary protein complexes, 1,087 of which lacked previous 3D structure information. Our predictions offer novel structural insight into many cancer-related processes such as the MAP kinase cascade and Fanconi anemia pathway. We further investigated the cancer mutation landscape by mapping somatic missense mutations (SMMs) in cancer to the predicted PPI interfaces and performing enrichment and depletion analyses. Interfaces enriched or depleted with SMMs exhibit different preferences for functional categories. Interfaces enriched in mutations tend to function in pathways that are deregulated in cancers and they may help explain the molecular mechanisms of cancers in patients; interfaces lacking mutations appear to be essential for the survival of cancer cells and thus may be future targets for PPI modulating drugs.

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Multiple mechanisms impact fluconazole resistance of mutant Erg11 proteins in Candida glabrata Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol -14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. These azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance alleles from C. albicans ERG11. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. Crucially, the presence of the DM ERG11 allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in ERG11 activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.

ImportanceAzole drugs target the Erg11 enzyme and lead to a reduction in fungal ergosterol, a vital sterol in yeast. Mutations in Erg11 are common among azole resistant Candida albicans clinical isolates, but not in C. glabrata, a major human pathogen. In this study, we showed that ERG11 mutations were tolerated in C. glabrata, and these mutations could confer azole resistance. We found that the strongest azole-resistant allele of ERG11 led to induction of the Pdr1 transcription factor and Cdr1 ATP-binding cassette transporter protein in the absence of drug. ERG11 mutations can cause azole resistance via altered enzymatic properties but also by triggering induction of other resistance systems owing to impacts on ergosterol biosynthesis. These data illustrate the deep connections between ergosterol biosynthesis and regulation of membrane transporter proteins via Pdr1 and the ergosterol-responsive transcription factor Upc2A.

sentences: Systematic assessment of the accuracy of subunit counting in biomolecular complexes using automated single molecule brightness analysis Analysis of single molecule brightness allows subunit counting of high-order oligomeric biomolecular complexes. Although the theory behind the method has been extensively assessed, systematic analysis of the experimental conditions required to accurately quantify the stoichiometry of biological complexes remains challenging. In this work, we develop a high-throughput, automated computational pipeline for single molecule brightness analysis that requires minimal human input. We use this strategy to systematically quantify the accuracy of counting under a wide range of experimental conditions in simulated ground-truth data and then validate its use on experimentally obtained data. Our approach defines a set of conditions under which subunit counting by brightness analysis is designed to work optimally and helps establishing the experimental limits in quantifying the number of subunits in a complex of interest. Finally, we combine these features into a powerful, yet simple, software that can be easily used for the stoichiometry analysis of such complexes.

sentences: Daily orexinergic modulation of the rat superficial layers of the superior colliculus - implications for intrinsic clock activities in the visual system The orexinergic system delivers excitation for multiple brain centres to facilitate behavioural arousal, with its malfunction resulting in narcolepsy, somnolence, and notably, visual hallucinations. Since the circadian clock underlies the daily arousal, a timed coordination is expected between the orexin system and its target subcortical visual system, including the superior colliculus (SC). Here, we use a combination of electrophysiological, immunohistochemical, and molecular approaches across 24 h, together with the neuronal tract tracing methods in rodents to elucidate the daily coordination between the orexin system and the superficial layers of the SC. We find the daily orexinergic innervation onto the SC, coinciding with the daily silencing of spontaneous firing in this visual brain area. We identify autonomous daily and circadian expression of clock genes in the SC, which may underlie these day-night changes. Additionally, we establish the lateral hypothalamic origin of orexin innervation to the SC and that the SC neurons robustly respond to orexin A via OX2 receptor in both excitatory and GABAA receptor-dependent inhibitory manners. Together, our evidence supports that the clock coordination between the orexinergic input and its response in the SC provides arousal-related excitation needed to detect sparse visual information during the behaviourally active phase.

sentences: G4-Selective Ligand Induced Autophagy G-quadruplex (G4) structures have emerged as singular therapeutic targets for cancer and neurodegeneration. Autophagy is a housekeeping cellular homeostatic mechanism and deregulation of autophagy is common in cancer and in neurodegenerative diseases. In this study, we identified the presence of 46 putative G4 sequences in the MTOR gene by use of QGRS mapper tool. We sought to connect these putative G4 sequences to a functional context by leveraging G4-targeting ligands. A G4-selective dimeric carbocyanine dye Bis-4,3 and the porphyrin TMPyP4 were used to affect the replication, transcription and translation of the MTOR gene. The ligand-induced induction of autophagic pathway via MTOR gene regulation was monitored upon treatment of HeLa and SHSY-5Y cells with G4-targeting ligands. The use of Bis-4,3 was compared with the known G4-stabilizing activity of TMPyP4. Our results show that treatment with G4-selective ligands downregulates mTOR activity and leads to the induction of excessive autophagy. This is first report on effect of G4-selective ligands on MTOR regulation and mTOR expression. mTOR being the key negative regulator of autophagy, the current work suggests potential of G4 stabilizing ligands towards induction of autophagy through the downregulation of mTOR.

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Dynamics of peripheral blood flow across sleep stages Study ObjectivesPulse rate variability (PRV) derived from peripheral blood flow has been reported as a surrogate parameter for heart rate variability (HRV). However, there are currently no studies reporting systematic comparisons of PRV with HRV in a normal sleep state. Whether PRV can provide similar information regarding sleep stages remains unclear. Peripheral blood flow may also be modulated differently across sleep stages. Thus, we aimed to investigate blood flow dynamics and compare PRV with HRV across sleep stages to see if blood flow can provide further information about sleep stages.

MethodsWe performed electrocardiography and simultaneously measured blood flow from the right index finger and ear concha of 45 healthy participants (13 women; mean age, 22.5 {+/-} 3.4 years) during one night of sleep. Time-domain, frequency-domain, and non-linear indices of PRV/HRV, and time- and frequencydomain blood flow parameters were calculated.

ResultsFinger-PRV results showed similar patterns to HRV results for most parameters. Finger-blood flow parameters in the time and frequency domains also showed information about the different sleep stages. Further, both finger- and ear-blood flow results showed 0.2-0.3 Hz oscillations that varied with sleep stages, with a significant increase in N3, suggesting a modulation (of respiration) within this frequency band.

ConclusionsThese results suggest that PRV can provide as much information as HRV for different sleep stages. Furthermore, the results show that blood flow + PRV could be more advantageous than HRV alone in the assessment of the sleep state and related autonomic nervous activity.

Statement of SignificanceThe research provides comprehensive information on peripheral blood flow (BF) activity across sleep stages for the first time, as the major novelty of our work. The second contribution is a systematic study comparing BF-derived pulse rate variability (PRV) with heart rate variability across sleep stages in a normal sleep state. We believe that our work makes a significant contribution to the literature because it provides comprehensive information on the potential of BF+PRV as a new biomarker for assessing the sleep state. Further, this study contributes to developing a more convenient method of assessing the sleep state in the clinical and home/work setting.

sentences: Degradation intermediates as an indicator of mRNA and cell metabolism Traditional mRNA degradation rate measurements involves complex experimental design with RNA labeling or transcription blocking together with sampling at multiple timepoints1,2. These experimental requirements limit the application of transcriptome-wide mRNA degradation rate analysis mainly in cultured cells, but rarely in in vivo samples. Therefore, a direct and simple strategy needs to be developed to study mRNA degradation rate. Here, we defined mRNA degradation intermediates as transcripts where decay is about to occur or has partially occurred in the 3-untranslated regions after poly(A) tail deadenylation, and found that the proportion of mRNA degradation intermediates is a very simple and convenient indicator for evaluating the degradation rate of mRNA in mouse and human cell lines. In addition, we showed that a higher proportion of mRNA degradation intermediates is correlated with faster cell cycle and higher turnover rate of mouse tissues. Further, we validated that in mouse maturing oocytes where transcription is silent3,4, the proportion of mRNA degradation intermediates is positively correlated with the mRNA degradation rate. Together, these results demonstrate that degradation intermediates can function as a good indicator of mRNA, cell, and tissue metabolism, and can be easily assayed by total RNA 3-end sequencing from a single bulk cell sample without the need for drug treatment or multi-timepoint sampling. This finding is of great potential for studies on mRNA degradation rate at the molecular, cellular, or organic level, including samples or systems that cannot be assayed with previous methods. In addition, further application of the findings into single cells will likely greatly aid the identification and study of rare cells with unique cellular metabolism dynamics such as tissue stem cells and tumor cells.

sentences: Identification of CRF66_BF, a new HIV-1 circulating recombinant form of South American origin Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Among 108 reported in the literature, 17 are BF1 intersubtype recombinant, most of which are of South American origin. Among these, all 5 identified in the Southern Cone and neighboring countries, except Brazil, derive from a common recombinant ancestor related to CRF12_BF, which circulates widely in Argentina, as deduced from coincident breakpoints and clustering in phylogenetic trees. In a HIV-1 molecular epidemiological study in Spain, we identified a phylogenetic cluster of 20 samples from 3 separate regions which were of F1 subsubtype, related to the Brazilian strain, in protease-reverse transcriptase (Pr-RT) and of subtype B in integrase. Remarkably, 14 individuals from this cluster (designated BF9) were Paraguayans and only 4 were native Spaniards. HIV-1 transmission was predominantly heterosexual, except for a subcluster of 6 individuals, 5 of which were men who have sex with men. Ten additional database sequences, from Argentina (n=4), Spain (n=3), Paraguay (n=1), Brazil (n=1), and Italy (n=1), branched within the BF9 cluster. To determine whether it represents a new CRF, near full-length genome (NFLG) sequences were obtained for 6 viruses from 3 Spanish regions. Bootscan analyses showed a coincident BF1 recombinant structure, with 5 breakpoints, located in p17gag, integrase, gp120, gp41-rev overlap, and nef, which was identical to that of two BF1 recombinant viruses from Paraguay previously sequenced in NFLGs. Interestingly, none of the breakpoints coincided with those of CRF12_BF. In a maximum likelihood phylogenetic tree, all 8 NFLG sequences grouped in a strongly supported clade segregating from previously identified CRFs and from the CRF12_BF  family clade. These results allow us to identify a new HIV-1 CRF, designated CRF66_BF. Through a Bayesian coalescent analysis, the most recent common ancestor of CRF66_BF was estimated around 1984 in South America, either in Paraguay or Argentina. Among Pr-RT sequences obtained by us from HIV-1-infected Paraguayans living in Spain, 14 (20.9%) of 67 were of CRF66_BF, suggesting that CRF66_BF may be one of the major HIV-1 genetic forms circulating in Paraguay. CRF66_BF is the first reported non-Brazilian South American HIV-1 CRF_BF unrelated to CRF12_BF.

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To See, Not to See, or to See Poorly: Perceptual Quality and Guess Rate as a Function of Electroencephalography (EEG) Brain Activity in an Orientation Perception Task Detection of visual stimuli fluctuates over time, and these fluctuations have been shown to correlate with time-domain evoked activity and frequency-domain periodic activity. However, it is unclear if these fluctuations are related to a change in guess rate, perceptual quality, or both. Here we determined whether the quality of perception randomly varies across trials or is fixed so that the variability is the same. Then we estimated how perceptual quality and guess rate on an orientation perception task relates to EEG activity. Response errors were fitted to variable precision models and the standard mixture model to determine whether perceptual quality is from a varying or fixed distribution. Overall, the best fit was the standard mixture model that assumes response variability can be defined by a fixed distribution.

The power and phase of 2-7 Hz post-target activities were found to vary along with task performance in that more accurate trials had greater power, and the preferred phase differed significantly between accurate and guess trials. Guess rate and {sigma} were significantly lower on trials with high 2-3 Hz power than low and the difference started around 250 ms post-target.

These effects coincide with changes in the P3 ERP: there was a more positive deflection in the accurate trials vs guesses. These results suggest that the spread of errors (perceptual quality) can be characterized by a fixed range of values. Where the errors fall within that range is modulated by the post-target power in the lower frequency bands and their analogous ERPs.

sentences: Unsupervised physiological noise correction of fMRI data using phase and magnitude information (PREPAIR) Of the sources of noise which affect BOLD fMRI, respiration and cardiac fluctuations are responsible for the largest part of the variance, particularly at high and ultra-high field. Existing approaches to removing physiological noise either use external recordings, which can be unwieldy and unreliable, or attempt to identify physiological noise from the magnitude fMRI data. Data-driven approaches are limited by sensitivity, temporal aliasing and the need for user interaction. In the light of the sensitivity of the phase of the MR signal to local changes in the field stemming from physiological processes, we have developed an unsupervised physiological noise correction method which uses the information carried in both the phase and the magnitude of EPI data. Our technique, Physiological Regressor Estimation from Phase and mAgnItude, sub-tR (PREPAIR) derives time series signals which are sampled at the slice TR from both phase and magnitude images. It allows physiological noise to be captured without aliasing, and efficiently removes other sources of signal fluctuations which are not related to physiology, prior to regressor estimation. We demonstrate that the physiological signal time courses identified with PREPAIR not only agree well with those from external devices, but also retrieve challenging cardiac dynamics. The removal of physiological noise was as effective as that achieved with the most commonly used approach based on external recordings, RETROICOR. In comparison with widely used physiological noise correction tools which do not use external signals, PESTICA and FIX, PREPAIR removed more respiratory and cardiac noise and achieved a larger increase in tSNR at both 3 T and 7 T.

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Spatiotemporally Mapping Thermodynamics of Lysosomes and Mitochondria using Cascade Organelle-Targeting Upconversion Nanoparticles The intracellular metabolism of organelles, like lysosomes and mitochondria, are highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of ATP generation during active metabolism. Here, we develop temperature-sensitive LysoDots and MitoDots to monitor the in situ thermodynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K-1 and accuracy of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the intracellular ion concentrations- and pH values. With Ca2+ ion shock, the temperatures of both lysosomes and mitochondria increased by 2[~]4 {degrees}C. Intriguingly, with Chloroquine treatment, the lysosomal temperature was observed to decrease by up to [~]3 {degrees}C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed a 3[~]7 {degrees}C thermal increase and transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multi-modality functional imaging of subcellular organelles and interactions with high spatial, temporal and thermal dynamics resolutions.

Graphical AbstractCascade organelle-targeted nano-thermometers based on upconversion LysoDots and MitoDots.

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sentences: Effects of arousal and movement on secondary somatosensory and visual thalamus All neocortical sensory areas have an associated primary and secondary thalamic nucleus. While the primary nuclei encode sensory information for transmission to cortex, the nature of information encoded in secondary nuclei is poorly understood. We recorded juxtasomally from neurons in secondary somatosensory (POm) and visual (LP) thalamic nuclei of awake head-fixed mice with simultaneous whisker tracking and pupilometry. POm activity correlated with whether or not a mouse was whisking, but not precise whisking kinematics. This coarse movement modulation persisted after unilateral paralysis of the whisker pad and thus was not due to sensory reafference. POm continued to track whisking even during optogenetic silencing of primary somatosensory and motor cortex and after lesion of superior colliculus, indicating that motor efference copy cannot explain the correlation between movement and POm activity. Whisking and pupil dilation were strongly correlated, raising the possibility that POm may track arousal rather than movement. LP, being part of the visual system, is not expected to encode whisker movement. We discovered, however, that LP and POm track whisking equally well, suggesting a global effect of arousal on both nuclei. We conclude that secondary thalamus is a monitor of behavioral state, rather than movement, and may exist to alter cortical activity accordingly.

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Stability of DNA methylation and chromatin accessibility in structurally diverse maize genomes Accessible chromatin and unmethylated DNA are associated with many genes and cis-regulatory elements. Attempts to understand natural variation for accessible chromatin regions (ACRs) and unmethylated regions (UMRs) often rely upon alignments to a single reference genome. This limits the ability to assess regions that are absent in the reference genome assembly and monitor how nearby structural variants influence variation in chromatin state. In this study, de novo genome assemblies for four maize inbreds (B73, Mo17, Oh43 and W22) are utilized to assess chromatin accessibility and DNA methylation patterns in a pan-genome context. The number of UMRs and ACRs that can be identified is more accurate when chromatin data is aligned to the matched genome rather than a single reference genome. While there are UMRs and ACRs present within genomic regions that are not shared between genotypes, these features are substantially enriched within shared regions, as determined by chromosomal alignments. Characterization of UMRs present within shared genomic regions reveals that most UMRs maintain the unmethylated state in other genotypes with only a small number being polymorphic between genotypes. However, the majority of UMRs between genotypes only exhibit partial overlaps suggesting that the boundaries between methylated and unmethylated DNA are dynamic. This instability is not solely due to sequence variation as these partially overlapping UMRs are frequently found within genomic regions that lack sequence variation. The ability to compare chromatin properties among individuals with structural variation enables pan-epigenome analyses to study the sources of variation for accessible chromatin and unmethylated DNA.

Article summaryRegions of the genome that have accessible chromatin or unmethylated DNA are often associated with cis-regulatory elements. We assessed chromatin accessibility and DNA methylation in four structurally diverse maize genomes. There are accessible or unmethylated regions within the non-shared portions of the genomes but these features are depleted within these regions. Evaluating the dynamics of methylation and accessibility between genotypes reveals conservation of features, albeit with variable boundaries suggesting some instability of the precise edges of unmethylated regions.

sentences: Finding structure in time: Humans, machines, and language The emergence of artificial neural networks (ANNs) that seem capable of emulating the remarkable human capacity for language has raised fundamental questions about complex cognition in humans and machines. This debate has taken place, however, in the context of limited empirical evidence about how the internal operations of ANNs relate to dynamic processes in the human brain as listeners understand language. Using Representational Similarity Analysis, we conducted a set of in-depth comparisons between these two types of systems, focusing on a core aspect of human language - the building of a structured and meaningful interpretation as each word in a sentence is heard in sequence. We extracted incremental structural measures of unfolding sentences from a deep language ANN and tested these against the spatiotemporally resolved brain activity recorded by electro/magnetoencephalography when human participants were listening to the same sentences. These uniquely neurocomputationally specific comparisons revealed strong behavioral and neural alignments between humans and ANNs in the use of multi-dimensional probabilistic constraints to build word-by-word structural interpretations, suggesting important commonalities in basic computational strategies for finding structure in time.

sentences: αS oligomers generated from polyunsaturated fatty acid and dopamine metabolite differentially interact with Aβ to enhance neurotoxicity It is increasingly becoming clear that neurodegenerative diseases are not as discrete as originally thought to be but rather display significant overlap in histopathological and clinical presentations. For example, nearly half of the patients with Alzheimer disease (AD) and synucleinopathies such as Parkinson disease (PD) show symptoms and pathological features of one another. Yet, the molecular events and features that underlie such comorbidities in neurodegenerative diseases remain poorly understood. Here, inspired to uncover the molecular underpinnings of the overlap between AD and PD, we investigated the interactions between amyloid-{beta} (A{beta}) and -synuclein (S), aggregates of which form the major components of amyloid plaques and Lewy bodies, respectively. Specifically, we focused on S oligomers generated from the dopamine metabolite called dihydroxyphenylacetaldehyde (DOPAL), and a polyunsaturated fatty acid docosahexaenoic acid (DHA). Both S oligomers showed structural and conformational differences confirmed by their disparity in size, secondary structure, susceptibility to proteinase K digestion and cytotoxicity. More importantly, the two oligomers differentially modulated A{beta} aggregation. While both oligomers inhibited A{beta} aggregation to varying extents, they induced structurally different A{beta} assemblies. Furthermore, A{beta} seeded with DHA-derived S oligomers showed greater toxicity than DOPAL-derived S oligomers in SH-SY5Y neuroblastoma cells. These results provide insights into the interactions between two amyloid proteins with empirically distinctive biophysical and cellular manifestations, enunciating a basis for potentially ubiquitous cross-amyloid interactions across many neurodegenerative diseases.

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Oscillatory calcium release and sustained store-operated oscillatory calcium signaling prevents differentiation of human oligodendrocyte progenitor cells Endogenous remyelination in demyelinating diseases such as multiple sclerosis is contingent upon the successful differentiation of oligodendrocyte progenitor cells (OPCs). Signaling via the Gq-coupled muscarinic receptor (M1/3R) inhibits human OPC differentiation and impairs endogenous remyelination in experimental models. We hypothesized that calcium release following Gq-coupled receptor (GqR) activation directly regulates human OPC (hOPC) cell fate. In this study, we show that specific GqR agonists activating muscarinic and metabotropic glutamate receptors induce characteristic oscillatory calcium release in hOPCs and that these agonists similarly block hOPC maturation in vitro. Both agonists induce calcium release from endoplasmic reticulum (ER) stores and store operated calcium entry (SOCE) likely via STIM/ORAI-based channels. siRNA mediated knockdown (KD) of obligate calcium sensors STIM1 and STIM2 decreased the magnitude of muscarinic agonist induced oscillatory calcium release and attenuated SOCE in hOPCs. In addition, STIM2 expression was necessary to maintain the frequency of calcium oscillations and STIM2 KD reduced spontaneous OPC differentiation. Furthermore, STIM2 siRNA prevented the effects of muscarinic agonist treatment on OPC differentiation suggesting that SOCE is necessary for the anti-differentiative action of muscarinic receptor-dependent signaling. Finally, using a gain-of-function approach with an optogenetic STIM lentivirus, we demonstrate that independent activation of SOCE was sufficient to significantly block hOPC differentiation and this occurred in a frequency dependent manner while increasing hOPC proliferation. These findings suggest that intracellular calcium oscillations directly regulate hOPC fate and that modulation of calcium oscillation frequency may overcome inhibitory Gq-coupled signaling that impairs myelin repair.

Significance StatementIn this study, Seidman et al. show that SOCE is a common component of ligand-based Gq-coupled signaling in hOPCs and that SOCE alone is sufficient to block hOPC differentiation and drive proliferation. Therefore, SOCE blocks differentiation and pathological SOCE could contribute to myelin disease.

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Kidins220 regulates the development of B cells bearing the {lambda} light chain The ratio between Ig{kappa} and Ig{lambda} light chain (LC)-expressing B cells varies considerably between species. We recently identified Kinase D-interacting substrate of 220 kDa (Kidins220) as an interaction partner of the BCR. In vivo ablation of Kidins220 in B cells resulted in a marked reduction of {lambda}LC-expressing B cells. Kidins220 knockout B cells fail to open and recombine the genes of the{lambda} LC locus, even in genetic scenarios where the{kappa} LC genes cannot be rearranged or where the {kappa}LC confers autoreactivity.{kappa} LC gene recombination and expression in Kidins220-deficient B cells is normal. Kidins220 regulates the development of {lambda}LC B cells by enhancing the survival of developing B cells and thereby extending the time-window in which the{lambda} LC locus opens and the genes are rearranged and transcribed. Further, our data suggest that Kidins220 guarantees optimal pre-BCR and BCR signaling to induce{lambda} LC locus opening and gene recombination during B cell development and receptor editing.

One Sentence SummaryWe demonstrate that the scaffold protein Kidins220 regulates the development of {lambda}LC B cells by supporting B cell precursor survival and optimizing pre-BCR and BCR signaling to open, recombine, and transcribe the genes of the{lambda} LC locus.

sentences: Triad interaction stabilizes the voltage sensor domains in a constitutively open KCNQ1-KCNE3 channel Tetrameric voltage-gated K+ channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K+ channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the recently determined KCNQ1-KCNE3-calmodulin complex structure, we identified amino acid residues on KCNE3 facing the S1 segment of KCNQ1 that are required for constitutive activity. In addition, we found that the interaction of these amino acid residues of KCNE3 and the S1 segment affects the voltage sensor movement via M238 and V241 residues of the S4 segment. This triad interaction shifts the voltage sensor domains equilibrium, leading to stabilization of the channels open state.

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Nonclassical monocytes are prone to migrate into tumor in diffuse large B-cell lymphoma Absolute count of circulating monocytes has been proposed as an independent prognostic factor in diffuse large B-cell lymphoma (DLBCL). However, monocyte nomenclature includes various subsets with pro-, anti-inflammatory, or suppressive functions, and their clinical relevance in DLBCL has been poorly explored. Herein, we broadly assessed circulating monocyte heterogeneity in 91 DLBCL patients. Classical- (cMO, CD14pos CD16neg) and intermediate- (iMO, CD14pos CD16pos) monocytes accumulated in DLBCL peripheral blood and exhibited an inflammatory phenotype. On the opposite, nonclassical monocytes (ncMO, CD14low CD16pos) were decreased in peripheral blood. Tumor-conditioned monocytes presented similarities with ncMO phenotype from DLBCL and were prone to migrate in response to CCL3, CCL5, and CXCL12, and presented similarities with DLBCL-infiltrated myeloid cells, as defined by mass cytometry. Finally, we demonstrated the adverse value of an accumulation of nonclassical monocytes in 2 independent cohorts of DLBCL.

Key pointsO_LINonclassical monocytes are prone to migrate to DLBCL tumor
C_LIO_LIHigh count of circulating nonclassical monocytes is an independent adverse event in DLBCL
C_LI

sentences: Arginine 65 methylation of Neurogenin 3 by PRMT1 is a prerequisite for development of hESCs into pancreatic endocrine cells In the developing pancreas, Neurogenin 3 (NGN3) is a key transcription factor in the cell fate determination of endocrine progenitors (EPs). Although the activation and stability of NGN3 are regulated by phosphorylation, the role of arginine methylation of NGN3 is poorly understood. Here, we report arginine 65 methylation of NGN3 is absolutely required for the pancreatic lineage development of human embryonic stem cells (hESCs) in vitro. First, we found inducible protein arginine methyltransferase 1 (PRMT1)-knockout (P-iKO) hESCs did not differentiate from EPs to endocrine cells (ECs) in the presence of doxycycline. Loss of PRMT1 caused an accumulation of NGN3 in the cytoplasm of EPs and blocked NGN3s transcriptional activity in PRMT1-KO EPs. We also found that PRMT1 specifically methylates NGN3 arginine 65, and this modification is a prerequisite for ubiquitin-mediated NGN3 degradation. Our findings indicate arginine 65 methylation of NGN3 is a key molecular switch in hESCs in vitro permitting the differentiation into pancreatic endocrine lineages.

sentences: Predicted pH-dependent stability of SARS-CoV-2 spike protein trimer from interfacial acidic groups Transition between receptor binding domain (RBD) up and down forms of the SARS-CoV-2 spike protein trimer is coupled to receptor binding and is one route by which variants can alter viral properties. It is becoming apparent that key roles in the transition are played by pH and a more compact closed form, termed locked. Calculations of pH-dependence are made for a large set of spike trimers, including locked form trimer structures that have recently become available. Several acidic sidechains become sufficiently buried in the locked form to give a predicted pH-dependence in the mild acidic range, with stabilisation of the locked form as pH reduces from 7.5 to 5, consistent with emerging characterisation by cryo-electron microscopy. The calculated pH effects in pre-fusion spike trimers are modulated mainly by aspartic acid residues, rather than the more familiar histidine role at mild acidic pH. These acidic sidechains are generally surface located and weakly interacting when not in a locked conformation. In this model, their replacement (perhaps with asparagine) would remove the pH-dependent destabilisation of locked spike trimer conformations, and increase their recovery at neutral pH. This would provide an alternative or supplement to the insertion of disulphide linkages for stabilising spike protein trimers, with potential relevance for vaccine design.

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Persistent horizontal and vertical, MR-induced nystagmus in resting state Human Connectome Project data ObjectiveStrong magnetic fields from magnetic resonance (MR) scanners induce a Lorentz force that contributes to vertigo and persistent nystagmus. Prior studies have reported a predominantly horizontal direction for healthy subjects in a 7 Tesla (T) MR scanner, with slow phase velocity (SPV) dependent on head orientation. Less is known about vestibular signal behavior for subjects in a weaker, 3T magnetic field, the standard strength used in the Human Connectome Project (HCP). The purpose of this study is to characterize the form and magnitude of nystagmus induced at 3T.

MethodsForty-two subjects were studied after being introduced head-first, supine into a Siemens Prisma 3T scanner. Eye movements were recorded in four separate acquisitions over 20 minutes. A biometric eye model was fit to the recordings to derive rotational eye position and then SPV. An anatomical template of the semi-circular canals was fit to the T2 anatomical image from each subject, and used to derive the angle of the B0 magnetic field with respect to the vestibular apparatus.

ResultsRecordings from 37 subjects yielded valid measures of eye movements. The population-mean SPV {+/-} SD for the horizontal component was -1.38 {+/-} 1.27 deg/sec, and vertical component was -0.93 {+/-} 1.44 deg/sec, corresponding to drift movement in the rightward and downward direction. Although there was substantial inter-subject variability, persistent nystagmus was present in half of subjects with no significant adaptation over the 20 minute scanning period. The amplitude of vertical drift was correlated with the roll angle of the vestibular system, with a non-zero vertical SPV present at a 0 degree roll.

InterpretationNon-habituating vestibular signals of varying amplitude are present in resting state data collected at 3T.

sentences: Fragmented habitat compensates for the adverse effects of genetic bottleneck According to the population genetics theory, population subdivision helps organisms retain genetic diversity, crucial for adaptation in a changing environment. Habitat shape is thought to be important for generating and maintaining population subdivision, but empirical cases are needed to test this assumption. We studied Saimaa ringed seals, land-locked in a labyrinthine lake and recovering from a drastic bottleneck, by whole-genome sequencing 105 individuals and additional individuals from three other ringed seal subspecies. We analyzed the distribution of variation and the genetic relatedness among the individuals in relation to the habitat shape. Despite a severe history of a genetic bottleneck with prevalent homozygosity in Saimaa ringed seals, we found evidence for population structure mirroring the subregions of the lake. Highlighting the significance of habitat connectivity in conservation biology and the power of genomic tools in understanding its impact, the genome wide analyses showed the subpopulations to have retained unique variation and largely complementary patterns of homozygosity, giving hope to this endangered species.

sentences: Improved Predictions Of MHC-Peptide Binding Using Protein Language Models AO_SCPLOWBSTRACTC_SCPLOWMajor histocompatibility complex (MHC) molecules bind to peptides from exogenous antigens, and present them on the surface of cells, allowing the immune system (T cells) to detect them. Elucidating the process of this presentation is essential for regulation and potential manipulation of the cellular immune system [1]. Predicting whether a given peptide will bind to the MHC is an important step in the above process, motivating the introduction of many computational approaches. NetMHCPan [2], a pan-specific model predicting binding of peptides to any MHC molecule, is one of the most widely used methods which focuses on solving this binary classification problem using a shallow neural network. The successful results of AI methods, especially Natural Language Processing (NLP-based) pretrained models in various applications including protein structure determination, motivated us to explore their use in this problem as well. Specifically, we considered fine-tuning these large deep learning models using as dataset the peptide-MHC sequences. Using standard metrics in this area, and the same training and test sets, we show that our model outperforms NetMHCpan4.1 which has been shown to outperform all other earlier methods [2].

sentences: Evolutionary history of mammalian UDP-glucuronosyltransferase (UGT)1 and UGT2 families: the emergence of UGT2B subfamily in eutherians after the diversification of flowering plants The UDP-glucuronosyltransferase (UGT) gene family is responsible for the transfer of glucuronic acid to exogenous and endogenous chemicals. Based on the highly diversified number of genes, the mammalian UGT1A and UGT2B subfamily genes are believed to be involved in the conjugation reactions of xenobiotic metabolism. However, it is speculated that the UGT2 family genes are not involved in the xenobiotic metabolism of avian species due to the less diverse number of genes. In this study, we aimed to investigate the evolutionary history of mammalian UGT1 and UGT2 family genes and determine when the diversification of UGT2B genes occurred. We also attempted to identify the main factors responsible for the diversification of UGT genes. By examining the genomic information and feeding habits of 67 species representing each mammalian family, we discovered that the UGT2B genes emerged in the Eutheria on or after Cretaceous period and that their number were higher in plant-eating mammals (herbivore or omnivore) than in carnivorous mammals. We also found that the UGT2B genes in some herbivorous mammals underwent positive selection. In contrast, the diversity of the UGT1 family genes was inherited from the common ancestor of birds and mammals. Thus, our findings suggest that the emergence of angiosperms (flowering plants) and the occurrence of "animal-plant warfare" influenced the evolution of this gene family involved in the xenobiotic metabolism of eutherians. Furthermore, future research investigating the marsupials and birds that do not possess UGT2B genes is required to elucidate the mechanisms underlying the metabolism of chemical substances in these species.

sentences: Synchronized Neural Activity Indexes Engagement with Spoken Stories Despite Acoustic Masking Older people with hearing problems often experience difficulties understanding speech in the presence of background sound. As a result, they may disengage in social situations, which has been associated with negative psychosocial health outcomes. Measuring listening (dis-)engagement during challenging listening situations has received little attention thus far. We recruit young, normal-hearing human adults (both sexes) and investigate how speech intelligibility and engagement during naturalistic story listening is affected by the level of acoustic masking (12-talker babble) at different signal-to-noise ratios (SNRs). In Experiment 1, we observed that word-report scores were above 80% for all but the lowest SNR (-3 dB SNR) we tested, at which performance dropped to 54%. In Experiment 2, we calculated inter-subject correlation (ISC) using electroencephalography (EEG) data to identify dynamic spatial patterns of shared neural activity evoked by the stories. ISC has previously been used as a neural measure of participants engagement with naturalistic materials. Our results show that ISC was stable across all but the lowest SNRs, despite reduced speech intelligibility. Comparing ISC and intelligibility demonstrated that word-report performance declined more strongly with decreasing SNR compared to ISC. Our measure of neural engagement suggests that individuals remain engaged in story listening despite missing words due to background noise. Our work provides a potentially fruitful approach to investigate listener engagement with naturalistic, spoken stories that may be used to investigate (dis)engagement in older adults with hearing impairment.

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Myopia alters the structural organization of the retinal astrocyte template, associated vasculature and ganglion layer thickness. PurposeTo describe the effect of myopic eye growth on the structure and distribution of astrocytes, vasculature and ganglion cell thickness, critical for inner retinal tissue homeostasis and survival.

MethodsAstrocyte and capillary distribution, retinal nerve fiber (RNFL) and ganglion cell layer (GCL) thicknesses were assessed using immunochemistry and spectral domain optical coherence tomography on eleven retinas of juvenile common marmosets (Callithrix Jacchus), six of which were induced with lens-induced myopia (refraction, Rx: -7.01{+/-}1.8D). Five untreated age-matched juvenile marmoset retinas were used as controls (Rx: -0.74{+/-}0.4D).

ResultsAs control marmoset eyes grew normally, there was an age-related increase in astrocyte numbers associated with RNFL thickening. Marmosets with induced myopia did not show this trend and, on the contrary, had reduced astrocyte numbers, increased positive GFAP immunopositive staining, thinner RNFL, lower peripheral capillary branching, and increased numbers of string vessels.

ConclusionThe myopic changes in retinal astrocytes, vasculature, and ganglion cell layer thickness suggest a reorganization of the astrocyte and vascular templates during myopia development and progression. Whether these adaptations are beneficial or harmful to the retina remains to be investigated.

Summary StatementThis article provides new information on how progressive myopia affects key elements of the retinal neurovascular unit.

sentences: The lipid raft-associated protein stomatin is required for accumulation of dectin-1 in the phagosomal membrane and for full activity of macrophages against Aspergillus fumigatus Alveolar macrophages are among the first cells to come into contact with inhaled fungal conidia of the human pathogenic fungus Aspergillus fumigatus. In lung alveoli, they contribute to phagocytosis and elimination of conidia. As a counter defense, conidia contain a grey-green pigment allowing them to survive in phagosomes of macrophages for some time. Previously, we showed that this conidial pigment interferes with the formation of flotillin-dependent lipid-rafts in the phagosomal membrane thereby preventing the formation of functional phagolysosomes. In this study, the role of the lipid raft-associated protein stomatin in macrophages during antifungal defense was investigated. To determine the function of this integral membrane protein, a stomatin-deficient macrophage cell line was generated by CRISPR/Cas9 gene editing. Immunofluorescence and flow cytometry revealed that stomatin contributes to the phagocytosis of conidia and is important for recruitment of both the {beta}-glucan receptor dectin-1 and the vATPase to the membrane of phagosomes. In the stomatin knockout cell line, fusion of phagosomes and lysosomes was reduced when infected with pigmentless pksP conidia. Thus, our data suggest that stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes.

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Increased risk of thrombocytopenia and death in patients with bacteremia caused by high alpha toxin-producing methicillin-resistant Staphylococcus aureus. BackgroundAlpha toxin (Hla) is a major virulence factor of Staphylococcus aureus that targets platelets but clinical data on Hla pathogenesis in bacteremia (SAB) is limited.

ObjectiveWe examined the link between in vitro Hla activity and outcome.

MethodsStudy isolates obtained from 100 patients with SAB (50 survivors; 50 non-survivors) were assessed for in vitro Hla production and activity by Western immunoblotting and hemolysis assay, respectively. Relevant demographics, laboratory and clinical data were extracted from patients medical records to correlate Hla activity of the infecting isolates with outcome.

ResultsHla production strongly correlated with hemolytic activity (rs=0.93) in vitro. A trend towards higher hemolytic activity was observed for MRSA compared to MSSA and with high-risk source infection. Significantly higher hemolytic activity was noted for MRSA strains isolated from patients who developed thrombocytopenia (median 52.48 vs 16.55 HU/ml in normal platelet count, p=0.012) and from non survivors (median 30.96 vs 14.87 HU/ml in survivors, p= 0.014) but hemolytic activity of MSSA strains did not differ between patient groups.

ConclusionsIn vitro Hla activity of S. aureus strains obtained from patients with bacteremia may be used to predict risk for thrombocytopenia and death which supports bedside phenotyping and therapeutic targeting in the future.

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Langerhans cells and cDC1s play redundant roles in mRNA-LNP induced protective anti-influenza and anti-SARS-CoV-2 responses Nucleoside modified mRNA combined with Acuitas Therapeutics lipid nanoparticles (LNP) have been shown to support robust humoral immune responses in many preclinical animal vaccine studies and later in humans with the SARS-CoV-2 vaccination. We recently showed that this platform is highly inflammatory due to the LNPs ionizable lipid component. The inflammatory property is key to support the development of potent humoral immune responses. However, the mechanism by which this platform drives T follicular helper cells (Tfh) and humoral immune responses remains unknown. Here we show that lack of Langerhans cells or cDC1s neither significantly affected the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cells and humoral immune responses, nor susceptibility towards the lethal challenge of influenza and SARS-CoV-2. However, the combined deletion of these two DC subsets led to a significant decrease in the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cell and humoral immune responses. Despite these observed defects, the still high antibody titers were sufficient to confer protection towards lethal viral challenges. We further found that IL-6, but not neutrophils, was required to generate Tfh cells and antibody responses.

In summary, here we bring evidence that the mRNA-LNP platform can support protective adaptive immune responses in the absence of specific DC subsets through an IL-6 dependent and neutrophil independent mechanism.

sentences: Thermodynamics and kinetics of aggregation of flexible peripheral membrane proteins Biomembrane remodeling is essential for cellular trafficking, with membrane-binding peripheral proteins playing a key role in it. Significant membrane remodeling as in endo- and exocytosis is often due to aggregates of many proteins with direct or membrane-mediated interactions. Understanding this process via computer simulations is extremely challenging: protein-membrane systems involve time- and lengthscales that make atomistic simulations impractical, while most coarse-grained models fall short in resolving dynamics and physical effects of protein and membrane flexibility. Here, we develop a coarse-grained model of the bilayer membrane bestrewed with rotationally-symmetric flexible proteins, parametrized to reflect local curvatures and lateral dynamics of proteins. We investigate the kinetics, equilibrium distributions, and the free energy landscape governing the formation and break-up of protein clusters on the surface of the membrane. We demonstrate how the flexibility of the proteins as well as their surface concentration play deciding roles in highly selective macroscopic aggregation behavior.

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Age-associated gut microbiota impairs hippocampus-dependent memory in a vagus dependent manner Aging is known to be associated with hippocampus-dependent memory decline, but the underlying causes of this age-related memory impairment are not yet elucidated. Here we show that the colonization of mice with the gut microbiota from aged, but not young animals is sufficient to trigger profound hippocampal alterations including astrogliosis, decreased adult neurogenesis, decreased novelty-induced neuronal activation and impairment in hippocampal-dependent memory. Similar alterations were reported in mice following the transfer of microbiota from aged human healthy donors. To decipher the mechanisms involved in mediating these microbiota-induced effects on brain functioning, we mapped the neuronal activity patterns and report that aged-microbiota transplantation reduced neuronal activity upstream to the vagus nerve. Targeted pharmacogenetic manipulation of the ascending branch of the vagus nerve demonstrated that the mere decrease in vagal activity was also detrimental to hippocampal functions. In contrast, increasing vagal activity alleviated the adverse effects of age-associated microbiota transfer on hippocampal functions and reinstated normal hippocampal memory in aged mice. We conclude that vagus nerve stimulation is a potential therapeutic strategy to lessen microbiota-dependent age-associated impairments in hippocampal functions.

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Systematic Elucidation and Pharmacological Targeting of Tumor-Infiltrating Regulatory T Cell Master Regulators Due to their immunosuppressive role, tumor-infiltrating regulatory T cells (TI-Tregs) represent attractive therapeutic targets. Analysis of TI vs. peripheral Tregs (P-Tregs) from 36 patients, across four malignancies, identified 17 candidate Master Regulators (MRs), predicted to mechanistically regulate TI-Tregs transcriptional state. In vivo, pooled CRISPR-KO screening, using a hematopoietic stem cell transplant model, confirmed essentiality of 7 of 17 MRs in TI-Treg recruitment and/or retention to the TME, without affecting other T cell subtypes, while individual knockout of the most significant MR (TRPS1) significantly reduced tumor allograft growth. TI-Treg drug perturbation profile analysis identified drugs capable of inverting the TI-Treg-specific MR activity signature at low concentration. Low dose treatment with gemcitabine (top prediction) inhibited tumor growth in immunocompetent but not immunocompromised allografts, increased PD-1 inhibitor efficacy, and depleted TI-Tregs in vivo. The study provides key insight into Treg infiltration mechanism and a gene reporter assay to identify additional small molecule inhibitors.

Graphical Abstract

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sentences: Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.

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Emergence of compensatory mutations reveal the importance of electrostatic interactions between HIV-1 integrase and genomic RNA ABSTRACTHIV-1 integrase (IN) has a non-catalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit IN-gRNA binding and result in non-infectious viruses marked by mislocalization of the gRNA within virions. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA. However, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R and D279R substitutions was sufficient to restore IN-RNA binding and infectivity for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding. Finally, HIV-1 IN R269A/K273A, but not IN R262A/R263A, bearing compensatory mutations was more sensitive to ALLINIs providing key genetic evidence that specific IN residues required for RNA binding also influence ALLINI activity. Taken together, our findings highlight the essential role of CTD in gRNA binding and ALLINI sensitivity, and reveal the importance of pliable electrostatic interactions between the IN- CTD and the gRNA.

IMPORTANCEIn addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues within its C-terminal domain (CTD) and regulates proper virion maturation. Here we show that compensatory mutations in nearby acidic residues (i.e. D256N and D270N) restore the ability to bind gRNA for IN variants bearing substitutions in these positively charged CTD residues. Similarly, charge reversals through individual D-to-R and D-to-K substitutions at these positions enabled the respective IN mutants to bind gRNA and restore virion infectivity. Further, we show that specific residues within the IN-CTD required for RNA binding also influence sensitivity to allosteric integrase inhibitors, a class of novel IN- targeting compounds that target the non-catalytic function of IN. Taken together, our findings reveal the importance of electrostatic interactions in IN-gRNA binding and provide key evidence for a crucial role of the IN-CTD in allosteric integrase inhibitor mechanism of action.

sentences: Analysis of paralogs in target enrichment data pinpoints multiple ancient polyploidy events in Alchemilla s.l. (Rosaceae) Target enrichment is becoming increasingly popular for phylogenomic studies. Although baits for enrichment are typically designed to target single-copy genes, paralogs are often recovered with increased sequencing depth, sometimes from a significant proportion of loci, especially in groups experiencing whole-genome duplication (WGD) events. Common approaches for processing paralogs in target enrichment datasets include random selection, manual pruning, and mainly, the removal of entire genes that show any evidence of paralogy. These approaches are prone to errors in orthology inference or removing large numbers of genes. By removing entire genes, valuable information that could be used to detect and place WGD events is discarded. Here we use an automated approach for orthology inference in a target enrichment dataset of 68 species of Alchemilla s.l. (Rosaceae), a widely distributed clade of plants primarily from temperate climate regions. Previous molecular phylogenetic studies and chromosome numbers both suggested ancient WGDs in the group. However, both the phylogenetic location and putative parental lineages of these WGD events remain unknown. By taking paralogs into consideration, we identified four nodes in the backbone of Alchemilla s.l. with an elevated proportion of gene duplication. Furthermore, using a gene-tree reconciliation approach we established the autopolyploid origin of the entire Alchemilla s.l. and the nested allopolyploid origin of four major clades within the group. Here we showed the utility of automated tree-based orthology inference methods, previously designed for genomic or transcriptomic datasets, to study complex scenarios of polyploidy and reticulate evolution from target enrichment datasets.

sentences: Retinoid-X Receptor Agonists Increase Thyroid Hormone Competence in Lower Jaw Remodeling of Pre-Metamorphic Xenopus laevis tadpoles Thyroid hormone (TH) signaling plays critical roles during vertebrate development, including regulation of skeletal and cartilage growth. TH acts through its receptors (TRs), nuclear hormone receptors (NRs) that heterodimerize with Retinoid-X receptors (RXRs), to regulate gene expression. A defining difference between NR signaling during development compared to in adult tissues, is competence, the ability of the organism to respond to an endocrine signal. Amphibian metamorphosis, especially in Xenopus laevis, the African clawed frog, is a well-established in vivo model for studying the mechanisms of TH action during development. Previously, weve used one-week post-fertilization X. laevis tadpoles, which are only partially competent to TH, to show that in the tail, which is naturally refractive to exogenous T3 at this stage, RXR agonists increase TH competence, and that RXR antagonism inhibits the TH response. Here, we focused on the jaw that undergoes dramatic TH-mediated remodeling during metamorphosis in order to support new feeding and breathing styles. We used a battery of approaches in one-week-old tadpoles, including quantitative morphology, differential gene expression and whole mount cell proliferation assays, to show that both pharmacologic (bexarotene) and environmental (tributyltin) RXR agonists potentiated TH-induced responses but were inactive in the absence of TH; and the RXR antagonist UVI 3003 inhibited TH action. At this young age, the lower jaw has not developed to the point that T3-induced changes produce an adult-like jaw morphology, and we found that increasing TH competence with RXR agonists did not give us a more natural-metamorphic phenotype, even though Bex and TBT significantly potentiated cellular proliferation and the TH induction of runx2, a transcription factor critical for developing cartilage and bone. Prominent targets of RXR-mediated TH potentiation were members of the matrix metalloprotease family, suggesting that RXR potentiation may emphasize pathways responsible for rapid changes during development.

sentences: Spontaneous formation of prebiotic compartment colonies on Hadean Earth and pre-Noachian Mars The primitive cells that emerged at the origin of life are commonly viewed as spherical biosurfactant shells, freely suspended in aqueous media1-3. This model explains initial, but not subsequent events in the development process towards structured protocells. Taking into consideration the involvement of naturally occurring surfaces, which were abundant on the early Earth4, we report feasible and productive pathways for the development of primitive cells. Surfaces intrinsically possess energy, easily utilized by the interfacing amphiphiles, such as lipids, to attain self-organization and spontaneous transformations5-7. We show that the physical interaction of phospholipid pools with 20 Hadean Earth analogue materials as well as a Martian meteorite composed of fused regolith representing the ancient crust of Mars, consistently lead to the shape transformation and autonomous formation of surfactant compartment assemblies. Dense, colony-like protocell populations grow from these lipid deposits, predominantly at the grain boundaries or cleavages of the investigated natural surfaces, and remain there for several days. The model protocells in our study are able to autonomously develop, transform and pseudo-divide, and encapsulate RNA as well as DNA. We also demonstrate that they can accommodate non-enzymatic, DNA strand displacement reactions. Our findings suggest a feasible route towards the transformation from nonliving to living entities, and provide fresh support for the  Lipid World hypothesis8.

sentences: DyNeuMo Mk-1: Design and Pilot Validation of an Investigational Motion-Adaptive Neurostimulator with Integrated Chronotherapy There is growing interest in using adaptive neuro-modulation to provide a more personalized therapy experience that might improve patient outcomes. Current implant technology, however, can be limited in its adaptive algorithm capability. To enable exploration of adaptive algorithms with chronic implants, we designed and validated the  Picostim DyNeuMo Mk-1 (DyNeuMo Mk-1 for short), a fully-implantable, adaptive research stimulator that titrates stimulation based on circadian rhythms (e.g. sleep, wake) and the patients movement state (e.g. posture, activity, shock, free-fall). The design leverages off-the-shelf consumer technology that provides inertial sensing with low-power, high reliability, and relatively modest cost. The DyNeuMo Mk-1 system was designed, manufactured and verified using ISO 13485 design controls, including ISO 14971 risk management techniques to ensure patient safety, while enabling novel algorithms. The system was validated for an intended use case in movement disorders under an emergency-device authorization from the Medicines and Healthcare Products Regulatory Agency (MHRA). The algorithm configurability and expanded stimulation parameter space allows for a number of applications to be explored in both central and peripheral applications. Intended applications include adaptive stimulation for movement disorders, synchronizing stimulation with circadian patterns, and reacting to transient inertial events such as posture changes, general activity, and walking. With appropriate design controls in place, first-in-human research trials are now being prepared to explore the utility of automated motion-adaptive algorithms.

sentences: Time-Encoding Migrates from Prefrontal Cortex to Dorsal Striatum During Learning of a Self-Timed Response Duration Task Although time is a fundamental dimension of life, we do not know how the brain encodes the temporal information. Several brain areas underlie the temporal information, such as the hippocampus, prefrontal cortex, and striatum, but evidence of how they cooperate to process temporal information is scarce. Notably, the analysis of neural activity during learning are rare, mainly because timing tasks usually take a long time to train. Here we investigated how the time encoding evolves when animals learn to time a 1.5 s interval. We designed a novel training protocol where rats go from naive- to proficient-level timing performance within a single session, allowing us to investigate neuronal activity from very early learning stages. We used pharmacological experiments and machine-learning algorithms to evaluate the level of time encoding in the medial prefrontal cortex and the dorsal striatum. Our results show a double dissociation between the roles of the medial prefrontal cortex and the dorsal striatum during temporal learning, where the former commits to early learning stages while the latter become more engaged as animals become more proficient in the task.

sentences: Decoding regulatory specificity of human ribosomal proteins Human ribosomes, made of around 80 ribosomal proteins (RPs) and four ribosomal RNAs, have long been thought as uniform passive protein-making factories with little regulatory function. Recently, accumulating evidence showed heterogeneity of RP composition in ribosomes responsible for regulating gene expression in development and tumorigenesis. However, a comprehensive understanding of regulatory spectrum of RPs is still lacking. In this study, we conducted a systematic survey of regulatory specificity of human RPs on global gene expression. We quantified translational and transcriptional changes of gene expression upon deficiency of 75 RPs, including 44 from the large subunit (60S) and 31 from the small subunit (40S), by ribosomal profiling and RNA sequencing analysis. We showed that RP deficiency induced diverse expression changes, particularly at the translational level. RPs were subjected to co-translational regulation under ribosomal stress where deficiency of the 60S and the 40S RPs had opposite effects on the two subunits. RP deficiency perturbed expression of genes related to cell cycle, cellular metabolism, signal transduction and development. Deficiency of RPs from the 60S led to a greater repression effect on cell growth than that from the 40S by perturbing P53 signaling and cell cycle pathways. To demonstrate functional specificity of RPs, we showed that RPS8 deficiency stimulated cellular apoptosis and RPL13 or RPL18 deficiency promoted cellular senescence. We also showed that RPL11 and RPL15 played important roles in retina development and angiogenesis, respectively. Overall, our study demonstrated a widespread regulatory role of RPs in controlling cellular activity, providing an important resource which can offer novel insights into ribosome regulation in human diseases and cancer.

sentences: Epigenetic reprogramming ameliorates type 1 diabetes by decreasing the generation of Th1 and Th17 subsets and restoring self-tolerance in CD4+ T cells The histone modifier Trichostatin A (TSA) ameliorated diabetes and repressed IFN-{gamma} and IL-17A expression in prediabetic female NOD mice. Purified CD4+ cells could be polarized ex vivo into Th1 and Th17 subsets, which comparably transferred diabetes into NOD.scid mice. Polarized Th1 cells were devoid of IL-17A-producing cells and did not transdifferentiate into Th17 cells in an immunodeficient environment. However, Th17 cells had contaminant Th1 cells, which expressed IFN-{gamma} upon adoptive transfer into lymphopenic recipients. Notably, TSA treatment abrogated the transfer of diabetes by CD4+ T-cells cultured under Th1 or Th17 polarizing conditions accompanied by the absence of Ifng and Il17a expression in NOD.scid recipients. Significantly, the histone modifier restored the ability of CD4+ but not CD8+ T-cells to undergo CD3-mediated apoptosis ex vivo in a caspase-dependent manner. Thus, the histone modifier afforded protection against autoimmune diabetes by negative regulation of signature lymphokines and restitution of self-tolerance in CD4+ T cells.

sentences: Mechanistic insights into dideoxygenation in gentamicin biosynthesis Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathway of gentamicin has been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are converted to C1a and C2a by concerted actions of two PLP (pyridoxal 5-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then re-aminated by GenB3 using an amino donor. Crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed new light on the already unrivalled catalytic versatility of PLP-dependent enzymes.

sentences: Patterned endogenous activity controls migration, morphogenesis and survival of adult-born neurons in the mouse olfactory bulb The development and survival of adult-born neurons is believed to be driven by sensory signaling. By genetically manipulating excitability of adult-born cells (via cell-specific overexpression of either Kv1.2 or Kir2.1 K+ channels), longitudinal in vivo monitoring of their Ca2+ signaling and transcriptome analyses, we show that endogenous but not sensory-driven activity governs migration, morphogenesis, survival, and functional integration of adult-born juxtaglomerular neurons in the mouse olfactory bulb. The proper development of these cells required fluctuations of cytosolic Ca2+ levels, phosphorylation of CREB, and pCREB-mediated gene expression. Attenuating Ca2+ fluctuations via K+ channel overexpression strongly downregulated genes involved in neuronal migration, differentiation, and morphogenesis and upregulated apoptosis-related genes, thus locking adult-born cells in the vulnerable and immature state. Together, the data reveal signaling pathways connecting the endogenous intermittent neuronal activity/Ca2+ fluctuations as well as proper Kv1.2/Kir2.1 K+ channel function to migration, maturation, and survival of adult-born neurons.

sentences:
Risk of Bias Assessment in Preclinical Literature using Natural Language Processing ObjectiveWe sought to apply natural language processing to the task of automatic risk of bias assessment in preclinical literature, which could speed the process of systematic review, provide information to guide research improvement activity, and support translation from preclinical to clinical research.

Materials and MethodsWe use 7,840 full-text publications describing animal experiments with yes/no annotations for five risk of bias items. We implement a series of models including baselines (support vector machine, logistic regression, random forest), neural models (convolutional neural network, recurrent neural network with attention, hierarchical neural network) and models using BERT with two strategies (document chunk pooling and sentence extraction). We tune hyperparameters to obtain the highest F1 scores for each risk of bias item on the validation set and compare evaluation results on the test set to our previous regular expression approach.

ResultsThe F1 scores of best models on test set are 82.0% for random allocation, 81.6% for blinded assessment of outcome, 82.6% for conflict of interests, 91.4% for compliance with animal welfare regulations and 46.6% for reporting animals excluded from analysis. Our models significantly outperform regular expressions for four risk of bias items.

ConclusionFor random allocation, blinded assessment of outcome, conflict of interests and animal exclusions, neural models achieve good performance, and for animal welfare regulations, BERT model with sentence extraction strategy works better.

sentences: A target capture approach for phylogenomic analyses at multiple evolutionary timescales in rosewoods (Dalbergia spp.) and the legume family (Fabaceae) Understanding the genetic changes associated with the evolution of biological diversity is of fundamental interest to molecular ecologists. The assessment of genetic variation at hundreds or thousands of unlinked genetic loci forms a sound basis to address questions ranging from micro-to macro-evolutionary timescales, and is now possible thanks to advances in sequencing technology. Major difficulties are associated with i) the lack of genomic resources for many taxa, especially from tropical biodiversity hotspots, ii) scaling the numbers of individuals analyzed and loci sequenced, and iii) building tools for reproducible bioinformatic analyses of such datasets. To address these challenges, we developed a set of target capture probes for phylogenomic studies of the highly diverse, pantropically distributed and economically significant rosewoods (Dalbergia spp.), explored the performance of an overlapping probe set for target capture across the legume family (Fabaceae), and built a general-purpose bioinformatics pipeline. Phylogenomic analyses of Dalbergia species from Madagascar yielded highly resolved and well supported hypotheses of evolutionary relationships. Population genomic analyses identified differences between closely related species and revealed the existence of a potentially new species, suggesting that the diversity of Malagasy Dalbergia species has been underestimated. Analyses at the family level corroborated previous findings by the recovery of monophyletic subfamilies and many well-known clades, as well as high levels of gene tree discordance, especially near the root of the family. The new genomic and bioinformatics resources will hopefully advance systematics and ecological genetics research in legumes, and promote conservation of the highly diverse and endangered Dalbergia rosewoods.

sentences: Live-Cell Imaging of (p)ppGpp with RNA-based Fluorescent Sensors Guanosine tetra- and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to directly image and measure cellular (p)ppGpp. Here, we report a type of RNA-based fluorescent sensors for live-cell imaging of (p)ppGpp. Our sensor is engineered by conjugating a recently identified (p)ppGpp-specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell-to-cell variation and cellular dynamics of (p)ppGpp levels can now be obtained under different nutritional conditions. We predict that these RNA-based sensors will be broadly adapted to study bacterial stringent response.

sentences: Culture and genomic analysis of Arsenophonus apicola sp. nov. isolated from the honeybee 	Apis mellifera The genus Arsenophonus has been traditionally considered to be comprised of heritable bacterial symbionts of arthropods. Recent work has reported a microbe related to the type species A. nasoniae as infecting the honey bee, Apis mellifera. The association was unusual for members of the genus in that the microbe-host interaction arose through environmental and social exposure rather than vertical transmission. In this study, we describe the in vitro culture of ArsBeeUS, a strain of this microbe isolated from A. mellifera in the USA. The 16S rRNA sequence of the isolated strain clearly indicates it falls within the genus Arsenophonus. Biolog analysis indicates the bacterium has a restricted range of nutrients that support growth. In vivo experiments demonstrate the strain proliferates rapidly on injection into A. mellifera hosts. We further report the closed genome sequence for the strain. The genome is 3.3 MB and 37.6% GC, which is smaller than A. nasoniae but larger than the genomes reported for non-culturable Arsenophonus symbionts. The genome is complex, with 6 plasmids and 11 predicted intact phage elements, but notably less complex than A. nasoniae. With 92% average nucleotide identity (ANI) to the type strain A. nasoniae, the ArsBeeUS strain isolated is clearly distinct from the type species, and we propose the name Arsenophonus apicola sp. nov. (CECT 30499; LMG 32504).

sentences: Transferability of Geometric Patterns from Protein Self-Interactions to Protein-Ligand Interactions There is significant interest in developing machine learning methods to model protein-ligand interactions but a scarcity of experimentally resolved protein-ligand structures to learn from. Protein self-contacts are a much larger source of structural data that could be leveraged, but currently it is not well understood how this data source differs from the target domain. Here, we characterize the 3D geometric patterns of protein self-contacts as probability distributions. We then present a flexible statistical framework to assess the transferability of these patterns to protein-ligand contacts. We observe that the level of transferability from protein self-contacts to protein-ligand contacts depends on contact type, with many contact types exhibiting high transferability. We then demonstrate the potential of leveraging information from these geometric patterns to aid in ligand pose-selection problems in protein-ligand docking. We publicly release our extracted data on geometric interaction patterns to enable further exploration of this problem.

sentences: A functional logic for neurotransmitter co-release in the cholinergic forebrain pathway The forebrain cholinergic system has recently been shown to co-release both acetylcholine and GABA. We have discovered that such co-release by cholinergic inputs to the claustrum differentially affects neurons that project to cortical versus subcortical targets. The resulting changes in neuronal gain toggles network efficiency and discriminability of output between two different projection subcircuits. Our results provide a potential logic for neurotransmitter co-release in cholinergic systems.

sentences: Rare long-range cortical connections enhance information processing What are the key topological features of connectivity critically relevant for generating the dynamics underlying efficient cortical function? A candidate feature that has recently emerged is that the connectivity of the mammalian cortex follows an exponential distance rule, which includes a small proportion of long-range high-weight anatomical exceptions to this rule. Whole-brain modelling of large-scale human neuroimaging data in 1003 participants offers the unique opportunity to create two models with and without long-range exceptions and explicitly study their functional consequences. We found that rare long-range exceptions are crucial for significantly improving information processing. Furthermore, modelling in a simplified ring architecture shows that this improvement is greatly enhanced by the turbulent regime found in empirical neuroimaging data. Overall, the results provide strong empirical evidence for the immense functional benefits of long-range exceptions combined with turbulence for information processing.

sentences: Accurate MHC Motif Deconvolution of Immunopeptidomics Data Reveals a Significant Contribution of DRB3, 4 and 5 to the Total DR Immunopeptidome Mass spectrometry (MS) based immunopeptidomics is used in several biomedical applications including neo-epitope discovery in oncology and next-generation vaccine development. Immunopeptidome data are highly complex given the expression of multiple HLA alleles on the cell membrane and presence of co-immunoprecipitated contaminants. The absence of tools that accurately deal with these challenges is currently a major bottleneck for the large-scale application of this technique. Here, we present the MHCMotifDecon that benefits from state-of-the-art HLA class-I and class-II predictions to accurately deconvolute immunopeptidome datasets and assign individual ligands to the most likely HLA allele while discarding co-purified contaminants. We have benchmarked the tool against other state-of-the-art methods and illustrated its application on experimental datasets for HLA-DR demonstrating a previously underappreciated role for HLA-DRB3/4/5 molecules in defining HLA class II immune repertoires. With its ease of use MHCMotifDecon can efficiently guide interpretation of immunopeptidome datasets, serving the discovery of novel T cell targets.

sentences: Transcription factor lineages in plant-pathogenic fungi, connecting diversity with fungal virulence Plant-pathogenic fungi span diverse taxonomic lineages. Their host-infection strategies are often specialised and require the coordinated regulation of molecular virulence factors. Transcription factors (TFs) are fundamental regulators of gene expression, controlling development and virulence in plant pathogenic fungi. Recent research has established regulatory roles for several taxonomically conserved fungal TFs, but the evolution of specific virulence regulators is not well understood. This study sought to explore the representation of TFs across a taxonomically-diverse range of fungi, with a focus on plant pathogens. A significant trend was observed among the obligate, host-associated pathogens, which possess a reduced overall TF repertoire, alluding to a lack of pressure for maintaining diversity. A novel orthology-based analysis is then presented that refined TF classifications, traditionally based on the nature of the DNA-binding domains. Using this analysis, cases of TF over/underrepresentation across fungal pathogen lineages are systematically highlighted. Specific examples are then explored and discussed that included the TF orthologues of Ste12, Pf2 and EBR1, plus phytotoxic secondary-metabolite cluster regulators, which all presented novel and distinct evolutionary insights. Ultimately, as the examples presented demonstrate, this resource can be interrogated to guide functional studies that seek to characterise virulence-specific regulators and shed light on the factors underpinning plant pathogenicity.

sentences:
Metformin Confers Cardiac and Renal Protection in Sudden Cardiac Arrest via AMPK Activation Sudden cardiac arrest (SCA) affects over 600,000 individuals annually in the United States and is associated with substantial mortality. After resuscitation, multi-system organ damage is common and largely attributable to ischemia-reperfusion injury. The anti-diabetic drug metformin improves cardiac outcomes in models of myocardial ischemia and ischemia-reperfusion. In this study, we evaluated the role of metformin pretreatment in a mouse model of SCA. We found that two weeks of metformin pretreatment protects cardiac ejection fraction and reduces acute kidney injury post-SCA in non-diabetic mice. Further, metformin pretreatment prior to SCA activates AMPK signaling and is associated with altered mitochondrial dynamics and markers of autophagy following arrest. Direct AMPK activation and inhibition studies demonstrate that activation is necessary and sufficient for metformin-mediated protection of cardiac and renal tissues in this model. We were unable to demonstrate cardiac improvement with a single-dose metformin rescue therapy. Importantly, the protective findings translate into patients. We retrospectively evaluated the extent of cardiac and kidney damage in diabetic patients resuscitated from SCA. Metformin-treated patients have less evidence of heart and kidney damage after arrest than diabetics who have not received metformin. Together, these data support AMPK activation as a preventive mechanism in ischemia-reperfusion injury.

Graphical Abstract

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sentences: High-throughput phenotyping of leaf discs infected withgrapevine downy mildew using shallow convolutionalneural networks Objective and standardized recording of disease severity in mapping crosses and breeding lines is a crucial step in characterizing resistance traits utilized in breeding programs and to conduct QTL or GWAS studies. Here we report a system for automated high-throughput scoring of disease severity on inoculated leaf discs. As proof of concept, we used leaf discs inoculated with Plasmopara viticola causing grapevine downy mildew (DM). This oomycete is one of the major grapevine pathogens and has the potential to reduce grape yield dramatically if environmental conditions are favorable. Breeding of DM resistant grapevine cultivars is an approach for a novel and more sustainable viticulture. This involves the evaluation of several thousand inoculated leaf discs from mapping crosses and breeding lines every year. Therefore, we trained a shallow convolutional neural-network (SCNN) for efficient detection of leaf disc segments showing P. viticola sporangiophores. We could illustrate a high and significant correlation with manually scored disease severity used as ground truth data for evaluation of the SCNN performance. Combined with an automated imaging system, this leaf disc-scoring pipeline has the potential to reduce the amount of time during leaf disc phenotyping considerably. The pipeline with all necessary documentation for adaptation to other pathogens is freely available.

sentences: Aging and white matter microstructure and macrostructure: a longitudinal multi-site diffusion MRI study of 1,184 participants Quantifying the microstructural and macrostructural geometrical features of the human brains connections is necessary for understanding normal aging and disease. Here, we examine brain white matter diffusion magnetic resonance imaging data from one cross-sectional and two longitudinal datasets totaling in 1184 subjects and 2236 sessions of people aged 50-97 years. Data was drawn from well-established cohorts, including the Baltimore Longitudinal Study of Aging dataset, Cambridge Centre for Ageing Neuroscience dataset, and the Vanderbilt Memory & Aging Project. Quantifying 4 microstructural features and, for the first time, 11 macrostructure-based features of volume, area, and length across 120 white matter pathways, we apply linear mixed effect modeling to investigate changes in pathway-specific features over time, and document large age associations within white matter. Conventional diffusion tensor microstructure indices are the most age-sensitive measures, with positive age associations for diffusivities and negative age associations with anisotropies, with similar patterns observed across all pathways. Similarly, pathway shape measures also change with age, with negative age associations for most length, surface area, and volume-based features. A particularly novel finding of this study is that while trends were homogeneous throughout the brain for microstructure features, macrostructural features demonstrated heterogeneity across pathways, whereby several projection, thalamic, and commissural tracts exhibited more decline with age compared to association and limbic tracts. The findings from this large-scale study provide a comprehensive overview of the age-related decline in white matter and demonstrate that macrostructural features may be more sensitive to heterogeneous white matter decline. Therefore, leveraging macrostructural features may be useful for studying aging and could have widespread implications for a variety of neurodegenerative disorders.

sentences: First record of a mermithid nematode (Nematoda: Mermithidae) parasitizing winged females of gall-forming aphids (Hemiptera: Aphididae: Eriosomatinae) Juvenile mermithid nematodes were found to parasitize winged females (sexuparae) of Erisoma auratum and Tetraneura radicicola. The morphological characteristics of mermithid nematodes are briefly described. The 18S rDNA and 28S rDNA extracted from one nematode were sequenced and used to construct a Bayesian phylogenetic tree, on which the host ranges of mermithid nematodes were represented. Our study indicated that mermithid parasitism of sexuparae led to fewer and smaller sexual female embryos. This is the first record of a mermithid in relation to eriosomatine aphids and the fourth record with respect to Aphididae.

sentences: Functional and structural deficiencies of Gemin5 variants associated with neurological disease Dysfunction of RNA-binding proteins are often linked to a wide range of human disease in general, and particularly with neurological conditions. Gemin5 is a member of the survival of motor neuron (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 protein have been reported but the functional consequences of these variants remain elusive. Here we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)-like dimerization module and the non-canonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization while the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, abolishing the cap-dependent and selective translation control of Gemin5, and establishing a functional difference with the wild type protein. Our study provides a molecular basis of disease associated with malfunction of Gemin5 protein.

sentences: Teosinte introgression modulates phosphatidylcholine levels and induces early maize flowering time Native Americans domesticated maize (Zea mays ssp. mays) from lowland teosinte parviglumis (Zea mays ssp.parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability, and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 (HPC1), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern US, Canada and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.

sentences: Using the UniFrac metric on Whole Genome Shotgun data The UniFrac metric has proven useful in revealing diversity across metagenomic communities. Due to the phylogeny-based nature of this measurement, UniFrac has historically only been applied to 16S rRNA data. Simultaneously, Whole Genome Shotgun (WGS) metagenomics has been increasingly widely employed and proven to provide more information than 16S data, but a UniFrac-like diversity metric suitable for WGS data has not previously been developed. The main obstacle for UniFrac to be applied directly to WGS data is the absence of phylogenetic distances in the taxonomic relationship derived from WGS data. In this study, we demonstrate a method to overcome this intrinsic difference and compute the UniFrac metric on WGS data by assigning branch lengths to the taxonomic tree obtained from input taxonomic profiles. We conduct a series of experiments to demonstrate that this WGSUniFrac method is comparably robust to traditional 16S UniFrac and is not highly sensitive to branch lengths assignments, be they data-derived or model-prescribed. Code implementing a prototype of WGSUniFrac along with paper reproducible are available at https://github.com/KoslickiLab/WGSUniFrac.

sentences: Characterising the Photoplethysmography Pulse Waveform for Use in Human Neuroscience: The Hybrid Excess and Decay (HED) Model Photoplethysmography (PPG) offers a widely-used, convenient and non-invasive approach to monitoring basic indices of cardiovascular function such as heart rate and blood oxygenation. However, while the pulse waveform, generated by PPG comprises features that are shaped by physiological and psychological factors, it is frequently overlooked in analyses of such data. We suggest that studies could be enriched by exploiting the possibilities afforded by a systematic analysis of PPG waveforms. To do this we initially require a robust and automated means of characterising it, thereby allowing us to examine variations across individuals and between different physiological and psychological contexts. We present a psychophysiologically-relevant model, the Hybrid Excess and Decay (HED) Model, which characterises pulse wave morphology in terms of three underlying pressure waves and a decay function. We show that these parameters capture PPG data with a high degree of precision and, moreover, are sensitive to specific, physiologically-relevant changes within individuals. We present the theoretical and practical basis for the model and demonstrate its performance when applied to a pharmacological dataset of 105 participants receiving intravenous administrations of the sympathomimetic drug isoproterenol (Isoprenaline). We conclude by discussing the possible value in using the HED model to complement standard measures of PPG outputs.

sentences: Visual exposure enhances stimulus encoding and persistence in primary cortex The brain adapts to the sensory environment. For example, simple sensory exposure can modify the response properties of early sensory neurons. How these changes affect the overall encoding and maintenance of stimulus information across neuronal populations remains unclear. We perform parallel recordings in the primary visual cortex of anesthetized cats and find that brief, repetitive exposure to structured visual stimuli enhances stimulus encoding by decreasing the selectivity and increasing the range of the neuronal responses that persist after stimulus presentation. Low-dimensional projection methods and simple classifiers demonstrate that visual exposure increases the segregation of persistent neuronal population responses into stimulus-specific clusters. These observed refinements preserve the representational details required for stimulus reconstruction and are detectable in post-exposure spontaneous activity. Assuming response facilitation and recurrent network interactions as the core mechanisms underlying stimulus persistence, we show that the exposure-driven segregation of stimulus responses can arise through strictly local plasticity mechanisms, also in the absence of firing rate changes. Our findings provide evidence for the existence of an automatic, unguided optimization process that enhances the encoding power of neuronal populations in early visual cortex, thus potentially benefiting simple readouts at higher stages of visual processing.

sentences: Contribution of the Golgi apparatus in morphogenesis of a virus induced cytopathic vacuolar system The Golgi apparatus (GA) in mammalian cells is pericentrosomally anchored and exhibits a stacked architecture. During infections by members of the alphavirus genus, the host cell GA is thought to give rise to distinct mobile pleomorphic vacuoles known as CPV-II (cytopathic vesicle-II) via unknown morphological steps. To dissect this, we adopted a phased electron tomography approach to image multiple overlapping volumes of a cell infected with Venezuelan equine encephalitis virus (VEEV) and complemented it with localization of peroxidase tagged Golgi marker. Analysis of the tomograms revealed a pattern of progressive cisternal bending into double-lamellar vesicles as a central process underpinning the biogenesis and the morphological complexity of this vacuolar system. Here we propose a model for the conversion of GA to CPV-II that reveals a unique pathway of intracellular virus envelopment. Our result has implications for alphavirus virus induced displacement of Golgi cisternae to the plasma membrane to aid viral egress operating late in the infection cycle.

sentences: HistoClean: Open-source Software for Histological Image Pre-processing and Augmentation to Improve Development of Robust Convolutional Neural Networks The growth of digital pathology over the past decade has opened new research pathways and insights in cancer prediction and prognosis. In particular, there has been a surge in deep learning and computer vision techniques to analyse digital images. Common practice in this area is to use image pre-processing and augmentation to prevent bias and overfitting, creating a more robust deep learning model. Herein we introduce HistoClean; user-friendly, graphical user interface that brings together multiple image processing modules into one easy to use toolkit. In this study, we utilise HistoClean to pre-process images for a simple convolutional neural network used to detect stromal maturity, improving the accuracy of the model at a tile, region of interest, and patient level. HistoClean is free and open-source and can be downloaded from the Github repository here: https://github.com/HistoCleanQUB/HistoClean.

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Regulation of DNA (de)methylation positively impacts seed germination during seed development under heat stress Seed development needs the coordination of multiple molecular mechanisms to promote correct tissue development, seed filling and the acquisition of germination capacity, desiccation tolerance, longevity and dormancy. Heat stress can negatively impact these processes and upon the increasing of global mean temperatures, global food security is threatened. Here, we explored the impact of heat stress on seed physiology, morphology, gene expression and methylation on three stages of seed development. Notably, Arabidopsis Col-0 plants under heat stress presented a decrease in germination capacity and also a decrease in longevity. We observed that upon mild stress, gene expression and DNA methylation were moderately affected. Nevertheless, upon severe heat stress during seed development, gene expression was intensively modified, promoting heat stress response mechanisms, including the activation of ABA pathway. By analyzing candidate epigenetic marks using mutants physiological assays, we observed that the lack of DNA demethylation by ROS1 gene impaired seed germination by affecting germination-related genes expression. On the other hand, we also observed that upon severe stress, a large proportion of differentially methylated regions (DMRs) were located in promoters and gene sequences of germination-related genes. To conclude, our results indicate that DNA (de)methylation could be a key regulatory process to ensure proper seed germination of seeds produced under heat stress.

Graphic Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: The invasive land flatworm Obama nungara in La Reunion, a French island in the Indian Ocean, the first report of the species for Africa The land flatworm Obama nungara, a species originating from South America and already invasive in many European countries, is recorded from La Reunion, a French island in the Indian Ocean. This is the first record of O. nungara from this locality and also the first record of the species for Africa. Two specimens were collected, one from Petite France (commune of Saint Paul) and one from La Plaine des Gregues (commune of Saint Joseph); the two localities are widely separated, one in the Western part and one in the South-eastern part of the island. This suggests that the species is already present in several locations in La Reunion. The sightings were communicated to us in 2021, but it is likely that the species is already present since 2020. A molecular analysis of the specimen from Petite France showed that it had the same cox1 haplotype as specimens previously recorded from several countries of Europe; it is hypothesized that the species was imported from Europe, probably from France. We mapped climatic suitability of the species in La Reunion and found that O. nungara could potentially invade a large part of the island. One record was apparently associated with the transport of plates of travertine, a construction material which has numerous cavities, suitable for the transport and survival of adult or cocoons of land flatworms.

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No One Is Leaving This Time: Appraisal of Awareness and Knowledge of Port Health Officers on International Health Regulation IHR (2005) BackgroundHistorically, ships have played an important role in transmitting infectious diseases around the world. The spread of cholera pandemics in the 19th century was thought to be linked to trade routes and facilitated by merchant shipping. The international maritime traffic of people and goods has often contributed to the spread of pathogens affecting public health.

ObjectivesTo assess level of awareness and knowledge of international Health regulation (IHR 2005) content among port health officer

MethodsThe study design was descriptive cross-sectional evaluation, questionnaires were used to capture the respondents knowledge, awareness and sanitary condition of ship in accordance with (IHR 2005)

ResultsOn awareness and knowledge, Majority of the respondent (77.1 %) demonstrate good awareness of the IHR (2005), while 22.9% had not and some even testified of hearing the said document for the first time. Despite the fact that majority of respondent were aware but only 24.6% of them can actually demonstrate good knowledge of IHR (2005) and its intent to protect and prevent spread of disease along the international route.

ConclusionThere is need to improve the knowledge of port health officers by expand training and guidance on application of the IHRs to frontline officer at point of entries. Also ensure more thorough inspection and avoid influence of ship agent during inspection of ship.

sentences: An activity-based labelling method for the detection of ammonia and methane-oxidizing bacteria. The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia- and methane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. However, the in situ detection and phylogenetic identification of novel ammonia- and methane-oxidizing bacteria remains a challenge. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and methane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labelling of cells harboring active ammonia and methane monooxygenases. The biotinylation of these enzymes in combination with immunogold labelling reveals the subcellular localization of the tagged proteins, while the fluorescent labelling of cells harboring active ammonia or methane monooxygenases provides a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labelling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and methanotrophs from complex environmental samples, facilitating the retrieval of their high quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and methane-oxidizing bacteria present in complex microbial communities.

sentences: Systematic and quantitative view of the antiviral arsenal of prokaryotes Facing the abundance and diversity of phages, bacteria have developed multiple anti-phage mechanisms. In the past three years, the number of known anti-phage mechanisms has been expanded by at least 5-fold rendering our view of prokaryotic immunity obsolete. Most anti-phage systems have been studied as standalone mechanisms, however many examples demonstrate strains encode not one but several anti-viral mechanisms. How these different systems integrate into an anti-viral arsenal at the strain level remains to be elucidated. Much could be learned from establishing fundamental description of features such as the number and diversity of anti-phage systems encoded in a given genome. To address this question, we developed DefenseFinder, a tool that automatically detects known anti-phage systems in prokaryotic genomes. We applied DefenseFinder to >20 000 fully sequenced genomes, generating a systematic and quantitative view of the anti-viral arsenal of prokaryotes. We show prokaryotic genomes encode on average five anti-phage systems from three different families of systems. This number varies drastically from one strain to another and is influenced by the genome size and the number of prophages encoded. Distributions of different systems are also very heterogenous with some systems being enriched in prophages and in specific clades. Finally, we provide a detailed comparison of the anti-viral arsenal of 15 common bacterial species, revealing drastic differences in anti-viral strategies. Overall, our work provides a free and open-source software, available as a command line tool or, on a webserver. It allows the rapid detection of anti-phage systems, enables a comprehensive description of the anti-viral arsenal of prokaryotes and paves the way for large scale genomics study in the field of anti-phage defense.

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Foldseek: fast and accurate protein structure search Highly accurate structure prediction methods are generating an avalanche of publicly available protein structures. Searching through these structures is becoming the main bottleneck in their analysis. Foldseek enables fast and sensitive comparisons of large structure sets. It reaches sensitivities similar to state-of-the-art structural aligners while being four orders of magnitude faster. Foldseek is free open-source software available at foldseek.com and as a webserver at search.foldseek.com.

Contactsoeding@mpinat.mpg.de, martin.steinegger@snu.ac.kr

sentences: Antibiotic action revealed by real-time imaging of the mycobacterial membrane The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to understanding such time-dependent processes. We employed a fluorogenic chemical probe to image how antibiotics perturb mycobacterial cell envelope assembly in real-time. Time-lapse microscopy revealed that differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for simultaneously monitoring cell envelope construction and remodeling responses. Our data show that rifampicin, which does not directly inhibit cell wall biosynthesis, affords a readily detected mycomembrane phenotype. The fluorogenic probe revealed the production of extracellular vesicles in response to antibiotics, and analyses of these vesicles indicate that antibiotic treatment elicits the release of agents that attenuate macrophage activation.

sentences: PTT-quant - a new method for direct identification and absolute quantification of premature transcription termination events, following the example of bacterial riboswitches. Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism is hampered by lack of highly sensitive and accurate experimental methods. Here we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of SAM riboswitch induction, which turned to be [~]23% higher in comparison the results obtained without cDNA synthesis normalization.

sentences: Dichotomous Feedback: A Signal Sequestration-based Feedback Mechanism for Biocontroller Design We introduce a new design framework for implementing negative feedback regulation in Synthetic Biology, which we term  dichotomous feedback. Our approach is different from current methods, in that it sequesters existing fluxes in the process to be controlled, and in this way takes advantage of the processs architecture to design the control law. This signal sequestration mechanism appears in many natural biological systems and can potentially be easier to realise than  molecular sequestration and other comparison motifs that are nowadays common in biomolecular feedback control design. The loop is closed by linking the strength of signal sequestration to the process output. Our feedback regulation mechanism is motivated by two-component signalling systems, where we introduce a second response regulator competing with the natural response regulator thus sequestering kinase activity. Here, dichotomous feedback is established by increasing the concentration of the second response regulator as the level of the output of the natural process increases. Extensive analysis demonstrates how this type of feedback shapes the signal response, attenuates intrinsic noise while increasing robustness and reducing crosstalk.

sentences: CRISPR metabolic screen identifies ATM and KEAP1 as targetable genetic vulnerabilities in solid tumors. Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase ATM using a metabolism-centered CRISPR/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both in vitro and in vivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In the TCGA pan-cancer datasets, we found that ATM levels strongly correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.

sentences: YAP1 Activation by Human Papillomavirus E7 Promotes Basal Cell Identity in Squamous Epithelia Persistent human papillomavirus (HPV) infection of stratified squamous epithelial cells causes nearly five percent of cancer cases worldwide. HPV-positive oropharyngeal cancers harbor few mutations in the Hippo signaling pathway compared to HPV-negative cancers at the same anatomical site, prompting the hypothesis that an HPV-encoded protein inactivates the Hippo pathway and activates the Hippo effector YAP1. The HPV E7 oncoprotein is required for HPV infection and for HPV-mediated oncogenic transformation. We investigated the effects of HPV oncoproteins on YAP1 and found that E7 activates YAP1, promoting YAP1 nuclear localization in basal epithelial cells. YAP1 activation by HPV E7 required that E7 bind and degrade the tumor suppressor PTPN14. E7 required YAP1 transcriptional activity to extend the lifespan of primary keratinocytes, indicating that YAP1 activation contributes to E7 carcinogenic activity. Maintaining infection in basal cells is critical for HPV persistence, and here we demonstrate that YAP1 activation causes HPV E7 expressing cells to be retained in the basal compartment of stratified epithelia. We propose that YAP1 activation resulting from PTPN14 inactivation is an essential, targetable activity of the HPV E7 oncoprotein relevant to HPV infection and carcinogenesis.

sentences: Rhes protein transits from neuron to neuron and facilitates mutant huntingtin spreading in the brain. Rhes (RASD2) is a thyroid hormone-induced gene that regulates striatal motor activity and promotes neurodegeneration in Huntington disease (HD) and tauopathy. Previously, we showed that Rhes moves between cultured striatal neurons and transports the HD protein, polyglutamine-expanded huntingtin (mHTT) via tunneling nanotube (TNT)-like membranous protrusions. However, similar intercellular Rhes transport has not yet been demonstrated in the intact brain. Here, we report that Rhes induces TNT-like protrusions in the striatal medium spiny neurons (MSNs) and transported between dopamine-1 receptor (D1R)-MSNs and D2R-MSNs of intact striatum and organotypic brain slices. Notably, mHTT is robustly transported within the striatum and from the striatum to the cortical areas in the brain, and Rhes deletion diminishes such transport. Moreover, we also found transport of Rhes to the cortical regions following restricted expression in the MSNs of the striatum. Thus, Rhes is a first striatum-enriched protein demonstrated to move and transport mHTT between neurons and brain regions, providing new insights on interneuronal protein transport in the brain.

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Joint actions of diverse transcription factor families establish neuron-type identities and promote enhancer selectivity To systematically investigate the complexity of neuron-specification regulatory networks we performed an RNA interference (RNAi) screen against all 875 transcription factors (TFs) encoded in Caenorhabditis elegans genome and searched for defects in nine different neuron types of the monoaminergic (MA) superclass and two cholinergic motoneurons.

We identified 91 TF candidates to be required for correct generation of these neuron types of which 28 were confirmed by mutant analysis. We found that correct reporter expression in each individual neuron type requires at least nine different TFs. Individual neuron types do not usually share TFs involved in their specification but share a common pattern of TFs belonging to the five most common TF families: Homeodomain (HD), basic Helix Loop Helix (bHLH), Zinc Finger (ZF), Basic Leucine Zipper Domain (bZIP) and Nuclear Hormone Receptors (NHR). HD TF members are over-represented, supporting a key role for this family in the establishment of neuronal identities. These five TF families area also prevalent when considering mutant alleles with previously reported neuronal phenotypes in C. elegans, Drosophila or mouse. In addition, we studied terminal differentiation complexity focusing on the dopaminergic terminal regulatory program. We found two HD TFs (UNC-62 and VAB-3) that work together with known dopaminergic terminal selectors (AST-1, CEH-43, CEH-20). Combined TF binding sites for these five TFs constitute a cis-regulatory signature enriched in the regulatory regions of dopaminergic effector genes. Our results provide new insights on neuron-type regulatory programs in C. elegans that could help better understand neuron specification and evolution of neuron types.

sentences: The F-box protein AFF1 regulates ARF protein accumulation to regulate auxin response Auxin critically regulates nearly every aspect of plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. Although ARF protein stability is regulated via the 26S proteasome, molecular mechanisms underlying ARF stability and turnover are unknown. Here, we report the identification and functional characterization of an F-box E3 ubiquitin ligase, which we have named AUXIN RESPONSE FACTOR F-BOX1 (AFF1). AFF1 directly interacts with ARF19 and regulates its accumulation. Mutants defective in AFF1 display ARF19 protein hyperaccumulation, attenuated auxin responsiveness, and developmental defects. Together, our data suggest a new mechanism, namely control of ARF protein stability, in regulating auxin response.

sentences: Ultrastructure expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a microtubule organisation centre coordinating mitosis with axoneme assembly The transmission of malaria-causing parasites to mosquitoes relies on the production of the gametocyte stages and their development into gametes upon a blood feed. These stages display various microtubule cytoskeletons and the architecture of the corresponding microtubule organisation centres (MTOC) remains elusive. Combining ultrastructure expansion microscopy (U-ExM) with bulk proteome labelling, we first reconstructed in 3D the subpellicular microtubule network and its associated actin cytoskeleton, which confer cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it simultaneously assembles axonemes to form eight flagellated microgametes. Here, U-ExM combined with Pan-ExM revealed the molecular architecture of a single bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking acentriolar mitotic plaques to cytoplasmic basal bodies by proteinaceous filaments. The eight basal bodies are concomitantly de novo assembled from a deuterosome-like structure, where centrin, {gamma}-tubulin, SAS4/CPAP and SAS6 form distinct subdomains. Once assembled, the basal bodies show a fusion of the proximal and central cores where colocalised centrin and SAS6 are surrounded by a SAS4/CPAP-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of {gamma}-tubulin from the basal bodies to the acentriolar plaques. We finally show that this atypical MTOC architecture relies on two non-canonical MTOC regulators, the calcium-dependent protein kinase 4 and the serine/arginine-protein kinase 1. Altogether, these results provide insights into the molecular organisation of a bipartite MTOC that may reflect a functional transition of a basal body to coordinate axoneme formation with mitosis.

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Cost efficiency of institutional incentives in finite populations Institutions can provide incentives to increase cooperation behaviour in a population where this behaviour is infrequent. This process is costly, and it is thus important to optimize the overall spending. This problem can be mathematically formulated as a multi-objective optimization problem where one wishes to minimize the cost of providing incentives while ensuring a desired level of cooperation within the population. In this paper, we provide a rigorous analysis for this problem. We study cooperation dilemmas in both the pairwise (the Donation game) and multi-player (the Public Goods game) settings. We prove the regularity of the (total incentive) cost function, characterize its asymptotic limits (infinite population, weak selection and large selection) and show exactly when reward or punishment is more efficient. We prove that the cost function exhibits a phase transition phenomena when the intensity of selection varies. We calculate the critical threshold in regards to the phase transition and study the optimization problem when the intensity of selection is under and above the critical value. It allows us to provide an exact calculation for the optimal cost of incentive, for a given intensity of selection. Finally, we provide numerical simulations to demonstrate the analytical results. Overall, our analysis provides for the first time a selection-dependent calculation of the optimal cost of institutional incentives (for both reward and punishment) that guarantees a minimum amount of cooperation. It is of crucial importance for real-world applications of institutional incentives since intensity of selection is specific to a given population and the underlying game payoff structure.

In this Supplementary Information, we provide detailed calculations and proofs as well as illustrative figures for the analytical results in the main text of the present paper.

sentences: FACT-mediated maintenance of chromatin integrity during transcription is essential for viability of mammalian stem cells Preservation of nucleosomes during replication has been extensively studied, while the maintenance of nucleosomes during transcription has gotten less attention. The histone chaperone FACT is involved in transcription elongation, although whether it disassembles or assembles nucleosomes during this process is still unclear. We deleted the FACT subunit in adult mice to clarify the function of FACT in mammals. FACT loss was lethal due to the loss of the earliest progenitors in bone marrow and intestine, while mor differentiated cells were not affected. Using cells isolated from several tissues, we showed that FACT loss was lethal only for stem cells but not cells differentiated in vitro. FACT depletion led to increased chromatin accessibility in a transcription-dependent manner, suggesting that nucleosomes are lost during transcription in the absence of FACT. The most prominent response to the loss of nucleosomes was the activation of interferon signaling and the accumulation of immunocytes in sensitive organs. FACT maintained chromatin integrity during transcription in mammalian adult stem cells, suggesting that chromatin transcription in these cells is different from more differentiated cells.

sentences: Dark kinase annotation, mining and visualization using the Protein Kinase Ontology The Protein Kinase Ontology (ProKinO) is an integrated knowledge graph that conceptualizes the complex relationships connecting protein kinase sequence, structure, function, and disease in a human and machine-readable format. Here we extend the scope of ProKinO as a discovery tool by including new classes and relationships capturing information on kinase ligand binding sites, expression patterns, and functional features, and demonstrate its application in uncovering new knowledge regarding understudied members of the protein kinase family. Specifically, through graph mining and aggregate SPARQL queries, we identify the p21-activated protein kinase 5 (PAK5) as one of the most frequently mutated dark kinase in human cancers with abnormal expression in multiple cancers, including an unappreciated role in acute myeloid leukemia. We identify recurrent oncogenic mutations in the PAK5 activation loop predicted to alter substrate binding and phosphorylation and identify common ligand/drug binding residues in PAK family kinases, highlighting the potential application of ProKinO in drug discovery. The updated ontology browser and a web component, ProtVista, which allows interactive mining of kinase sequence annotations in 3D structures and Alphafold models, provide a valuable resource for the signaling community. The updated ProKinO database is accessible at http://prokino.uga.edu/browser/.

sentences: Low photorespiratory capacity is sufficient to block photosynthetic capacity acclimation in high light The induction of high photosynthetic capacity in high light (HL) is a common response among many herbaceous dicot plants, however, the signals that control this response remain largely unknown. Here, multiple independent lines of evidence are presented in support of the conclusion that low photorespiratory capacity acts a negative signal to limit photosynthetic capacity acclimation in HL in Arabidopsis thaliana. Using a panel of natural accessions, primary nitrogen (N) assimilation and photorespiration rates early after a shift to growth in HL, as well as activities for key enzymes in these pathways, were shown to positively correlate with the magnitude of the subsequent induction of photosynthetic capacity, which occurred several days later. Time-resolved metabolomic data during acclimation to HL were collected using a strongly acclimating ecotype and a weakly acclimating ecotype, revealing in greater detail the differences in N assimilation, photorespiration, and triose-phosphate utilization pathways underlying efficient photosynthetic capacity acclimation. When shifted into HL growth conditions under non-photorespiratory conditions, weakly acclimating ecotypes and even photorespiratory mutants gained the ability to strongly induce high photosynthetic capacity in HL. Thus, a negative, photorespiration-dependent signal early in the HL shift appears to block photosynthetic capacity acclimation in accessions with low photorespiratory capacity, whereas accessions with high photorespiratory capacity are licensed to increase photosynthetic capacity.

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Human and wildlife use of mountain glacier habitat in western North America ContextThe global recession of glaciers and perennial snowfields is reshaping mountain ecosystems. Beyond physical changes to the landscape and altered downstream hydrology, the implications of glacier decline for biodiversity are poorly known. Before predictions can be made about how climate change will affect wildlife in glacier-associated ecosystems, a more thorough accounting of the role that glaciers play in species life histories is needed. However, typical approaches for documenting wildlife presence and behavior--remote camera traps--are difficult to use in glaciated terrain due to limited options for securing them (e.g., no trees) and dramatic seasonal changes in snowpack.

AimsIn this study, we sought to test a novel camera trap designed for glaciated mountain ecosystems. We also aimed to use this approach to gain insight into wildlife and human usage of a mountain glacier in western North America.

MethodsWe deployed an elevational transect of uniquely designed camera traps along the western margin of the Paradise Glacier, a rapidly receding mountain glacier on the south side of Mount Rainier, WA, USA. Our simple camera trap design consisted of a wildlife camera attached to a camouflaged cylindrical cooler filled with snow and rocks.

Key resultsOur camera design proved ideal for a mountain glacier ecosystem and from June to September 2021, we detected at least 16 vertebrate species (seven birds, nine mammals) over 770 trap nights using glacier-associated habitats. Humans, primarily skiers, were the most common species detected, but we also recorded 99 observations of wildlife (birds and mammals). These included three species of conservation concern in Washington: wolverine (Gulo gulo), Cascade red fox (Vulpes vulpes cascadensis), and white-tailed ptarmigan (Lagopus leucura).

ConclusionsCollectively, our results provide proof-of-concept for a novel camera trap design that is ideal for treeless, perennially snow-covered landscapes and revealed a rich diversity of wildlife using mountain glacier habitat in the Pacific Northwest. We highlight the global need for similar studies to better understand the true scale of biodiversity that will be impacted by glacier recession in mountain ecosystems.

sentences: Photons guided by axons may enable backpropagation-based learning in the brain Despite great advances in explaining synaptic plasticity and neuron function, a complete understanding of the brains learning algorithms is still missing. Artificial neural networks provide a powerful learning paradigm through the backpropagation algorithm which modifies synaptic weights by using feedback connections. Backpropagation requires extensive communication of information back through the layers of a network. This has been argued to be biologically implausible and it is not clear whether backpropagation can be realized in the brain. Here we suggest that biophotons guided by axons provide a potential channel for backward transmission of information in the brain. Biophotons have been experimentally shown to be produced in the brain, yet their purpose is not understood. We propose that biophotons can propagate from each post-synaptic neuron to its pre-synaptic one to carry the required information backward. To reflect the stochastic character of biophoton emissions, our model includes the stochastic backward transmission of teaching signals. We demonstrate that a three-layered network of neurons can learn the MNIST handwritten digit classification task using our proposed backpropagation-like algorithm with stochastic photonic feedback. We model realistic restrictions and show that our system still learns the task for low rates of biophoton emission, information-limited (one bit per photon) backward transmission, and in the presence of noise photons. Our results suggest a new functionality for biophotons and provide an alternate mechanism for backward transmission in the brain.

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Single cell transcriptome analysis defines heterogeneity of the murine pancreatic ductal tree Lineage tracing using genetically engineered mouse models is an essential tool for investigating cell-fate decisions of progenitor cells and biology of mature cell types, with relevance to physiology and disease progression. To study disease development, an inventory of an organs cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify SPP1 as a regulator of this fate decision as well as human duct cell de-differentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for Geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.

SIGNIFICANCEMurine models are extensively used for pancreatic lineage tracing experiments and investigation of pancreatic disease progression. Here, we describe the transcriptome of murine pancreatic duct cells, intrapancreatic bile duct cells, and pancreatobiliary cells at single cell resolution. Our analysis defines novel heterogeneity within the pancreatic ductal tree and supports the paradigm that more than one population of pancreatic duct cells harbors progenitor capacity. We identify and validate unique functional properties of subpopulations of pancreatic duct cells including an epithelial-mesenchymal transcriptomic axis and roles in chronic pancreatic inflammation.

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Anchorage by seed mucilage prevents seed dislodgement in high surface flow: a mechanistic investigation O_LIBackground and Aims: Seed mucilage is a common and highly diverse trait shared among thousands of angiosperm species. While long recognized that mucilage allows seeds to anchor to substrates (antitelechory), resisting abiotic and biotic dislodgement, we still lack a mechanistic understanding of this process.
C_LIO_LIMethods: We propose a mechanistic model of how mucilage affects substrate anchorage and fluid resistance, ultimately contributing to dislodgement resistance. To test this model, we subjected mucilaginous seeds of 52 species, varying in eight measured seed traits, to seven days of continuous water flow at a range of dislodgement potentials.
C_LIO_LIKey Results: Supporting our model, mucilage mass increased force necessary to dislodge both dry and wet seeds; our measurement of the dislodgement force of dry mucilage explained time to dislodgement well. The effect size was remarkably large; increasing the standardized mucilage mass by one standard deviation resulted in a 280-fold increase in the time to dislodgement. Fluid resistance was largely dependent on speed of water flow and the seeds modified drag coefficient, but not seed traits. Neither mucilage expansion speed nor mucilage decay rate explained dislodgement potential well.
C_LIO_LIConclusions: Our results suggest that the degree of anchorage to substrate, measured with a simple dislodgement force assay, is highly predictive of mucilaginous seed retention in highly erosive environments. In contrast, we found that other seed and mucilage traits are of lesser importance to anchorage.
C_LI

sentences: DNA Methylation Dynamics and Dysregulation Delineated by High-Throughput Profiling in the Mouse We have developed a mouse Infinium DNA methylation array that contains 297,415 probes to capture the diversity of mouse DNA methylation biology. We present a mouse DNA methylation atlas as a rich reference resource of 1,239 DNA samples encompassing distinct tissues, strains, age, sex, and pathologies. We describe applications for comparative epigenomics, genomic imprinting, epigenetic inhibitors, PDX assessment, backcross tracing, and epigenetic clocks. We dissect DNA methylation processes associated with differentiation, aging and tumorigenesis. Notably, we find that tissue-specific methylation signatures localize to binding sites for transcription factors controlling the corresponding tissue development. Age-associated hypermethylation is enriched at regions of Polycomb repression, while hypomethylation is enhanced at regions bound by cohesin complex members. ApcMin/+ polyp-associated hypermethylation affects enhancers regulating intestinal differentiation, while hypomethylation targets AP-1 binding sites. This MM285 mouse array is widely accessible to the research community, and will accelerate future high sample-throughput studies in this important model organism.

sentences: Biofilms preserve transmissibility of a multi-drug resistance plasmid Self-transmissible multidrug resistance (MDR) plasmids are a major health concern because they can spread antibiotic resistance to pathogens. Even though most pathogens form biofilms, little is known about how MDR plasmids persist and evolve in biofilms. We hypothesize that (i) biofilms act as refugia of MDR plasmids by retaining them in the absence of antibiotics longer than well-mixed planktonic populations, and that (ii) the evolutionary trajectories that account for the improvement of plasmid persistence over time differ between biofilms and planktonic populations. In this study, we evolved Acinetobacter baumannii with an MDR plasmid in biofilm and planktonic populations with and without antibiotic selection. In the absence of selection biofilm populations were better able to maintain the MDR plasmid than planktonic populations. In planktonic populations plasmid persistence improved rapidly but was accompanied by a loss of genes required for the horizonal transfer of plasmids. In contrast, in biofilms most plasmids retained their transfer genes, but on average plasmid persistence improved less over time. Our results showed that biofilms can act as refugia of MDR plasmids and favor the horizontal mode of plasmid transfer, which has important implications for the spread of MDR.

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Molecular diversity maintained by long-term balancing selection in mating loci defines multiple mating types in fungi Balancing selection, an evolutionary force that retains genetic diversity, has been detected in multiple genes and organisms, such as the sexual mating loci in fungi. However, to quantify the strength of balancing selection and define the mating-related genes require a large number of specimens. In tetrapolar basidiomycete fungi, sexual type is determined by two unlinked loci, MATA and MATB. Genes in both loci defines mating type identity, control successful mating and completion of the life cycle. These loci are usually highly diverse. Previous studies have speculated, based on culture crosses, that species of the non-model genus Trichaptum (Hymenochaetales, Basidiomycota) possess a tetrapolar mating system, with multiple alleles. Here, we sequenced a hundred and eighty specimens of three Trichaptum species. We characterized the chromosomal location of MATA and MATB, the molecular structure of MAT regions and their allelic richness. Our sequencing effort was sufficient to molecularly characterize multiple MAT alleles segregating before the speciation event of Trichaptum species. Our analyses suggested that long-term balancing selection has generated trans-species polymorphisms. Mating sequences were classified in different allelic classes based on an amino acid identity (AAI) threshold supported by phylogenetics. The inferred allelic information mirrored the outcome of in vitro crosses, thus allowing us to support the degree of allelic divergence needed for successful mating. Even with the high amount of divergence, key amino acids in functional domains are conserved. The observed allelic classes could potentially generate 14,560 different mating types. We conclude that the genetic diversity of mating in Trichaptum loci is due to long-term balancing selection, with limited recombination and duplication activity. Our large number of sequenced specimens highlighted the importance of sequencing multiple individuals from different species to detect the mating-related genes, the mechanisms generating diversity and the evolutionary forces maintaining them.

Author summaryFungi have complex mating systems, and basidiomycete fungi can encode thousands of mating types. Individuals with the same mating type cannot mate. This sexual system has evolved to facilitate sexual mating, increasing the chances to recombine into advantageous allelic combination and prune deleterious alleles. We explored the genomes of hundred and eighty specimens, combined with experimental mating studies of selected specimens, from a non-model organism (Trichaptum). We characterized the genomic regions controlling sex. The mating ability of the specimens confirmed the role of the mating alleles observed in the genomic data. The detailed analyses of many specimens allowed us to observe gene duplication and rearrangements within the mating loci, increasing the diversity within these loci. We supported previous suggestions of balancing selection in this region, an evolutionary force that maintains genomic diversity. These results supports that our fungal specimens are prone to outcross, which might facilitate the adaptation to new conditions.

sentences: Structures of full-length glycoprotein hormone receptor signaling complexes Luteinizing hormone (LH) and chorionic gonadotropin (CG) are members of the glycoprotein hormone family essential to human reproduction and are important therapeutic drugs. They activate the same G protein-coupled receptor, LHCGR, by binding to the large extracellular domain (ECD). Here we report four cryo-EM structures of LHCGR, two wildtype receptor structures in the inactive and active states, and two constitutively active mutated receptor structures. The active structures are bound to CG and Gs heterotrimer, with one of the structure also containing the allosteric agonist, Org43553. The structures reveal a distinct  push and pull" mechanism of receptor activation, in which the ECD is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain (TMD). A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the ECD-TMD interface functions as a tethered agonist to induce conformational changes in TMD and G-protein coupling. Org43553 binds to a TMD pocket and interacts directly with P10 that further stabilizes the receptor in the active conformation. Together, these structures provide a common model for understanding glycoprotein hormone signal transduction and dysfunction, and inspire the search for clinically suitable small molecular compounds to treat endocrine diseases.

sentences: Organic stabilization of extracellular elemental sulfur in a Sulfurovum-rich biofilm: a new role for EPS? This work shines light on the role of extracellular polymeric substances (EPS) in the formation and preservation of elemental sulfur biominerals produced by sulfur-oxidizing bacteria. We characterized elemental sulfur particles produced within a Sulfurovum-rich biofilm in the Frasassi Cave System (Italy). The particles adopt spherical and bipyramidal morphologies, and display both stable (-S8) and metastable ({beta}-S8) crystal structures. Elemental sulfur is embedded within a dense matrix of EPS and the particles are surrounded by organic envelopes rich in amide and carboxylic groups. Organic encapsulation and the presence of metastable crystal structures are consistent with elemental sulfur organomineralization, i.e. the formation and stabilization of elemental sulfur in the presence of organics, a mechanism that has previously been observed in laboratory studies. This research provides new evidence for the important role of microbial EPS in mineral formation in the environment. We hypothesize that extracellular organics are used by sulfur-oxidizing bacteria for the stabilization of elemental sulfur minerals outside of the cell wall as a store of chemical energy. The stabilization of energy sources (in the form of a solid electron acceptor) in biofilms is a potential new role for microbial EPS that requires further investigation.

sentences: Choose carefully, act quickly: Efficient decision making with selective inhibition in attractor neural networks The ability to decide swiftly and accurately in an urgent scenario is crucial for an organisms survival. The neural mechanisms underlying the perceptual decision and trade-off between speed and accuracy have been extensively studied in the past few decades. Among several theoretical models, the attractor neural network model has successfully captured both behavioral and neuronal data observed in many decision experiments. However, a recent experimental study revealed additional details that were not considered in the original attractor model. In particular, the study shows that the inhibitory neurons in the posterior parietal cortex of mice are as selective to decision making results as the excitatory neurons, whereas the previous attractor model assumes the inhibitory neurons to be unselective. In this study, we investigate the attractor model with selective inhibition (selective model), which can be considered as a general case of the previous attractor model (unselective model). Our model reproduces several behavioral and neurophysiological observations. To analyze the dynamics of the selective model, we reduce it and show that selectivity adds a time-varying component to the energy landscape. This time dependence of the energy landscape allows the selective model to integrate information carefully in initial stages, then quickly converge to an attractor once the choice is clear. This results in the selective model having a more efficient speed-accuracy trade-off that is unreachable by unselective models.

sentences: Error quantification in multi-parameter mapping facilitates robust estimation and enhanced group level sensitivity Multi-Parameter Mapping (MPM) is a comprehensive quantitative neuroimaging protocol that enables estimation of four physical parameters (longitudinal and effective transverse relaxation rates R1 and [Formula], proton density PD, and magnetization transfer saturation MTsat) that are sensitive to microstructural tissue properties such as iron and myelin content. Their capability to reveal microstructural brain differences, however, is tightly bound to controlling random noise and artefacts (e.g. caused by head motion) in the signal. Here, we introduced a method to estimate the local error of PD, R1 and MTsat maps that captures both noise and artefacts on a routine basis without requiring additional data. To investigate the methods sensitivity to random noise, we calculated the model-based signal-to-noise ratio (mSNR) and showed in measurements and simulations that it correlated linearly with an experimental raw-image-based SNR map. We found that the mSNR varied with MPM protocols, magnetic field strength (3T vs. 7T) and MPM parameters: it halved from PD to R1 and decreased from PD to MTsat by a factor of 3-4. Exploring the artefact-sensitivity of the error maps, we generated robust MPM parameters using two successive acquisitions of each contrast and the acquisition-specific errors to down-weight erroneous regions. The resulting robust MPM parameters showed reduced variability at the group level as compared to their single-repeat or averaged counterparts. The error and mSNR maps may better inform power-calculations by accounting for local data quality variations across measurements. Code to compute the mSNR maps and robustly combined MPM maps is available in the open-source hMRI toolbox.

sentences: Virulence and antimicrobial resistance profile of non-typhoidal Salmonella enterica serovars recovered from poultry processing environments at wet markets in Dhaka, Bangladesh The rapid emergence of virulent and multidrug-resistant (MDR) non-typhoidal Salmonella (NTS) enterica serovars are a growing public health concern globally. The present study focused on the assessment of the pathogenicity and antimicrobial resistance (AMR) profiling of NTS enterica serovars isolated from chicken processing environments at wet markets in Dhaka, Bangladesh. A total number of 870 samples consisting of carcass dressing water (CDW), chopping board swabs (CBS), and knife swabs (KS) were collected from 29 wet markets. The prevalence of Salmonella was found to be 20% in CDW, 19.31% in CBS and 17.58% in KS, respectively. Meanwhile, the MDR Salmonella was found to be 72.41%, 73.21% and 68.62% in CDW, CBS, and KS, respectively. All isolates were screened by polymerase chain reaction (PCR) for eight virulence genes, namely invA, agfA, IpfA, hilA, sivH, sefA, sopE, and spvC. The S. Enteritidis and untyped Salmonella isolate harbored all virulence genes while S. Typhimurium isolates carried six virulence genes except sefA and spvC. Phenotypic resistance revealed decreased susceptibility to ciprofloxacin, streptomycin, ampicillin, tetracycline, gentamycin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid and azithromycin. Genotypic resistance showed higher prevalence of plasmid mediated blaTEM followed by tetA, sul1, sul2, sul3, and strA/B genes. Harmonic and symmetrical trend was observed among the phenotypic and genotypic resistance patterns of the isolates. The research findings anticipate that MDR and virulent NTS enterica serovars are prevailing in the wet market environments which can easily enter into the human food chain. There was a resilient and significant correlation existent among the phenotypic and genotypic resistance patterns and virulence genes of Salmonella isolate recovered from carcass dressing water, chopping board swabs, and knife swabs (p < 0.05), respectively.

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Epigenetic regulator genes direct lineage switching in MLL-AF4 leukaemia The fusion gene MLL-AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukaemia. However, relapse can be associated with a switch from acute lymphoblastic to acute myeloid leukaemia. Here we show that these myeloid relapses share oncogene fusion breakpoints with their matched lymphoid presentations and can originate in either early, multipotent progenitors or committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programmes indicating that the execution and maintenance of lymphoid lineage differentiation is impaired. We show that this subversion is recurrently associated with the dysregulation of repressive chromatin modifiers, notably the nucleosome remodelling and deacetylation complex, NuRD. In addition to mutations, we show differential expression or alternative splicing of NuRD members and other genes is able to reprogram the B lymphoid into a myeloid gene regulatory network. Lineage switching in MLL-AF4 leukaemia is therefore driven and maintained by defunct epigenetic regulation.

Statement of SignificanceWe demonstrate diverse cellular origins of lineage switched relapse within MLL-AF4 pro-B acute leukaemia. Irrespective of the developmental origin of relapse, dysregulation of NuRD and/or other epigenetic machinery underpins fundamental lineage reprogramming with profound implications for the increasing use of epitope directed therapies in this high-risk leukaemia.

sentences: Aging-regulated TUG1 is dispensable for endothelial cell function The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function.

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Annotation of glycolysis, gluconeogenesis, and trehaloneogenesis pathways provide insight into carbohydrate metabolism in the Asian citrus psyllid Citrus greening disease is caused by the pathogen Candidatus Liberibacter asiaticus, which is transmitted by the Asian citrus psyllid, Diaphorina citri. There is no curative treatment or significant prevention mechanism for this detrimental disease that causes continued economic losses from reduced citrus production. A high quality genome of D. citri is being manually annotated to provide accurate gene models required to identify novel control targets and increase understanding of this pest. Here, we annotated genes involved in glycolysis, gluconeogenesis, and trehaloneogenesis in the D. citri genome, as these are core metabolic pathways and suppression could reduce this pest. Specifically, twenty-five genes were identified and annotated in the glycolysis and gluconeogenesis pathways and seven genes for the trehaloneogenesis pathway. Comparative analysis showed that the glycolysis genes in D. citri are highly conserved compared to orthologs in other insect systems, but copy numbers vary in D. citri.

Expression levels of the annotated gene models were analyzed and several enzymes in the glycolysis pathway showed high expression in the thorax. This is consistent with the primary use of glucose by flight muscles located in the thorax. A few of the genes annotated in D. citri have been targeted for gene knockdown as a proof of concept, for RNAi therapeutics. Thus, manual annotation of these core metabolic pathways provides accurate genomic foundations for developing gene-targeting therapeutics to reduce D. citri.

sentences: An astrocytic signaling loop for frequency-dependent control of dendritic integration and spatial learning Dendrites of hippocampal CA1 pyramidal cells amplify clustered glutamatergic input by activation of voltage-gated sodium channels and N-methyl-D-aspartate receptors (NMDARs). NMDAR activity depends on the presence of NMDAR co-agonists such as D-serine, but how co-agonists influence dendritic integration is not well understood. Using combinations of whole-cell patch clamp, iontophoretic glutamate application, two-photon excitation fluorescence microscopy and glutamate uncaging we found that exogenous D-serine reduces the threshold of dendritic spikes and increases their amplitude. Triggering an astrocytic mechanism controlling endogenous D-serine supply via endocannabinoid receptors (CBRs) also increased dendritic spiking. Unexpectedly, this pathway was activated by pyramidal cell activity primarily in the theta range, which required HCN channels and astrocytic CB1Rs. Therefore, astrocytes close a positive and frequency-dependent feedback loop between pyramidal cell activity and their integration of dendritic input. Its disruption led to an impairment of spatial memory, which demonstrates its behavioral relevance.

sentences: The transcription factor Roc1 is a regulator of cellulose degradation in the wood-decaying mushroom Schizophyllum commune Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in the nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly up-regulated during growth on cellulose, when compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase activity was reduced in the growth medium of the {Delta}roc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A GC-rich motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota.

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The openCARP Simulation Environment for Cardiac Electrophysiology Background and ObjectiveCardiac electrophysiology is a medical specialty with a long and rich tradition of computational modeling. Nevertheless, no community standard for cardiac electrophysiology simulation software has evolved yet. Here, we present the openCARP simulation environment as one solution that could foster the needs of large parts of this community.

Methods and ResultsopenCARP and the Python-based carputils framework allow developing and sharing simulation pipelines which automate in silico experiments including all modeling and simulation steps to increase reproducibility and productivity. The continuously expanding openCARP user community is supported by tailored infrastructure. Documentation and training material facilitate access to this complementary research tool for new users. After a brief historic review, this paper summarizes requirements for a high-usability electrophysiology simulator and describes how openCARP fulfills them. We introduce the openCARP modeling workflow in a multi-scale example of atrial fibrillation simulations on single cell, tissue, organ and body level and finally outline future development potential.

ConclusionAs an open simulator, openCARP can advance the computational cardiac electrophysiology field by making state-of-the-art simulations accessible. In combination with the carputils framework, it offers a tailored software solution for the scientific community and contributes towards increasing use, transparency, standardization and reproducibility of in silico experiments.

sentences: STOmicsDB: a database of Spatial Transcriptomic data Recent technological development in spatial transcriptomics allows researchers to measure gene expression of cells and their spatial locations at the almost single-cell level, which generates detailed biological insight into biological processes. However, specialized spatial transcriptomics databases are rare. Here, we present the Spatial TranscriptOmics DataBase (STOmicsDB), a user-friendly database with multifunctions including search of relevant publications and tools, public dataset visualization, customized specialized databases, new data archive, and online analysis. The current version of STOmicsDB consists of 141 curated spatial transcript datasets covering 12 species, and includes 5,618 spatial multi-omics publications and 674 tools. STOmicsDB is freely accessible at https://db.cngb.org/stomics/.

sentences: Sequential functional differentiation of luminal epithelial cells regulated by maternal and embryonic signaling in the mouse endometrium Embryo implantation requires temporospatial maternal-embryo dialog to regulate the interactions between an activated blastocyst and a receptive endometrium; however, the details of the related cell-specific coordination is largely unknown because of the cellular complexity and dynamic developmental processes of both the embryo and endometrium during peri-implantation. By comparing single-cell RNA (scRNA) data obtained from entire uteri of pregnant and pseudo-pregnant mice with embryos that were in the oviduct (dpc2.5 days post-coitum [dpc]) or had entered the uterus (after 3.5 dpc), we found a maternal estrogen and progesterone signaling-dependent functional differentiation process in which progesterone induced estrogen-responsive luminal epithelial cells to differentiate into two new types of epithelial cells: adhesion epithelial cells (AECs) and supporting epithelial cells (SECs) on 2.5 dpc. In addition to maternal signaling data, the uterine scRNA and corresponding embryo bulk sequencing data were obtained, and analyses revealed that embryonic Pdgfa and Efna3/4 signaling activated AECs and SECs, enhancing the attachment of embryos to the endometrium after implantation. Specifically, embryonic signaling induced additional transformation of AECs by preventing adjacent AECs, but not AECs away from the embryo in the implantation chamber, from undergoing apoptosis. Furthermore, we demonstrated that epithelial cell differentiation and related regulatory signaling were largely conserved in humans and mice. In the human endometrium, developmental defects of SOX9-positive epithelial cells similar to luminal epithelial cells were related to thin endometrium. Our work provides comprehensive and systematic information on endometrial luminal epithelial cell development directed by maternal and embryonic signaling, which is important for endometrial receptivity and embryo implantation.

sentences: Discovering molecular features of intrinsically disordered regions by using evolution for contrastive learning A major challenge to the characterization of intrinsically disordered regions (IDRs), which are widespread in the proteome, but relatively poorly understood, is the identification of molecular features that mediate functions of these regions, such as short motifs, amino acid repeats and physicochemical properties. Here, we introduce a proteome-scale feature discovery approach for IDRs. Our approach, which we call "reverse homology", exploits the principle that important functional features are conserved over evolution. We use this as a contrastive learning signal for deep learning: given a set of homologous IDRs, the neural network has to correctly choose a held-out homologue from another set of IDRs sampled randomly from the proteome. We pair reverse homology with a simple architecture and standard interpretation techniques, and show that the network learns conserved features of IDRs that can be interpreted as motifs, repeats, or bulk features like charge or amino acid propensities. We also show that our model can be used to produce visualizations of what residues and regions are most important to IDR function, generating hypotheses for uncharacterized IDRs. Our results suggest that feature discovery using unsupervised neural networks is a promising avenue to gain systematic insight into poorly understood protein sequences.

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Glycine targets NINJ1-mediated plasma membrane rupture to provide cytoprotection First recognized 35 years ago, glycine is known to protect cells against plasma membrane rupture from diverse types of tissue injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine cytoprotection, however, remains entirely elusive. We hypothesized that glycine targets ninjurin-1 (NINJ1), a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptotic cell death. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. Here, we first demonstrate that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in mouse and human macrophages stimulated to undergo lytic cell death. Next, we show that glycine treatment prevents NINJ1 clustering thereby preserving cellular integrity. By identifying NINJ1 as a glycine target, our data help resolve a long-standing mechanism of glycine cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death pathways.

Graphical abstract

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org.highwire.dtl.DTLVardef@b9c3d9org.highwire.dtl.DTLVardef@1e903f0org.highwire.dtl.DTLVardef@1018498org.highwire.dtl.DTLVardef@a819b9_HPS_FORMAT_FIGEXP  M_FIG C_FIG SummaryGlycine is known to protect cells against plasma membrane rupture from diverse types of tissue injury by an unknown mechanism. The authors demonstrate that NINJ1, a newly identified executioner of plasma membrane rupture across lytic cell death pathways, is a glycine target and resolve a longstanding mechanism of glycine cytoprotection.

sentences: Transcriptional control of nucleus accumbens neuronal excitability by Retinoid X Receptor Alpha tunes sensitivity to drug rewards The complex nature of the transcriptional networks underlying addictive behaviors suggests intricate cooperation between diverse gene regulation mechanisms that go beyond canonical activity-dependent pathways. Here we implicate in this process a novel nuclear receptor transcription factor, Retinoid X Receptor Alpha (RXR), which we identified bioinformatically as associated with addiction-like behaviors. In the nucleus accumbens (NAc) of male and female mice, we show that, while its own expression remains unaltered after cocaine exposure, RXR controls plasticity- and addiction-relevant transcriptional programs in both dopamine receptor D1- and D2-expressing medium spiny neurons, which in turn modulate intrinsic excitability and synaptic activity of these NAc cell types. Behaviorally, bidirectional viral and pharmacological manipulation of RXR regulates drug reward sensitivity in both non-operant and operant paradigms. Together, this study demonstrates a key role for NAc RXR in promoting drug addiction, and paves the way for future studies of rexinoid signaling in psychiatric disease states.

sentences: Dynamic expression of smRNA from fecal exosome in disease progression of an inflammatory bowel disorder mouse model. Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract affecting over 3 million adults in the United States. Despite being widespread, reliable early diagnostic tests are not available. We examined exosomal small RNA (smRNA), specifically targeting microRNA (miRNA) and piRNA from the stool samples of IBD model mice, interleukin 10 knockout mice (IL-10 KO), as a potential diagnostic marker. Stool samples were specifically chosen because they are readily available, and collection is noninvasive. At the end of the experimental period, the gastrointestinal (GI) tract was collected, and disease severity was scored. Histopathology showed a significant increase in inflammation and proliferation within the proximal and distal large intestines. smRNA profiles were examined upon conventional housing (start-point) which is a determinant factor of spontaneous IBD progression in the IL-10 KO mice, terminal illness (end-point), and 6 weeks before the end-point (mid-point), when the mice were still phenotypically healthy. We found 504 smRNA that were significantly differentially expressed between before symptom onset and terminal sedation. These changes were not detected in wild-type samples. Moreover, clustering analysis of expression changes over the disease progression identified a unique set of smRNAs that primarily target pro-inflammatory or anti-inflammatory genes. The expression of smRNAs that suppresses pro-inflammatory genes was higher at 6 weeks before terminal sedation, suggesting the downregulation of the pro-inflammatory genes advances the terminal illness of the IBD. In summary, our study proposes that fecal exosomal smRNA profiling offers a new opportunity to monitor the inflammatory status of the gut with a capability of detecting its pro-inflammatory (asymptomatic) status. Our next step is to understand the spatiotemporal interplay of these exosomes and the host cells in the gut as well as the complete biochemical makeup of fecal exosomes, such as mRNA, DNA, protein, and lipids. This will lead to an exciting development of reengineered exosomes that can be utilized to treat or even prevent the pro-inflammatory colonic lesion while the host is still clinically asymptomatic.

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Thermodynamic constraints on the diversity of microbial ecosystems Non-equilibrium thermodynamics has long been an area of substantial interest to ecologists because most fundamental biological processes, such as protein synthesis and respiration, are inherently energy-consuming. However, most of this interest has focused on developing coarse ecosystem-level maximisation principles, providing little insight into underlying mechanisms that lead to such emergent constraints. Microbial communities are a natural system to decipher this mechanistic basis because their interactions in the form of substrate consumption, metabolite production, and cross-feeding can be described explicitly in thermodynamic terms. Previous work has considered how thermodynamic constraints impact competition between pairs of species, but restrained from analysing how this manifests in complex dynamical systems. To address this gap, we develop a thermodynamic microbial community model with fully reversible reaction kinetics, which allows direct consideration of free-energy dissipation. This also allows species to interact via products rather than just substrates, increasing the dynamical complexity, and allowing a more nuanced classification of interaction types to emerge. Using this model, we find that community diversity increases with substrate lability, because greater free-energy availability allows for faster generation of niches. Thus, more niches are generated in the time frame of community establishment, leading to higher final species diversity. We also find that allowing species to make use of near-to-equilibrium reactions increases diversity in a low free-energy regime. In such a regime, two new thermodynamic interaction types that we identify here reach comparable strengths to the conventional (competition and facilitation) types, emphasising the key role that thermodynamics plays in community dynamics. Our results suggest that accounting for realistic thermodynamic constraints is vital for understanding the dynamics of real-world microbial communities.

Author summaryThere is a growing interest in microbial communities due to their important role in biogeochemical cycling as well as plant and animal health. Although our understanding of thermodynamic constraints on individual cells is rapidly improving, the impact of these constraints on complex microbial communities remains largely unexplored theoretically and empirically. Here, we develop a new microbial community model which allows thermodynamic efficiency and entropy production to be calculated directly. We find that availability of substrates with greater free-energy allows for a faster rate of niche generation, leading to higher final species diversity. We also show that when the free-energy availability is low, species with reactions close to thermodynamic equilibrium are favoured, leading to more diverse and efficient communities. In addition to the conventional interaction types (competition and facilitation), our model reveals the existence of two novel interaction types mediated by products rather than substrates. Though the conventional interactions are generally the strongest, the novel interaction types are significant when free-energy availability is low. Our results suggest that non-equilibrium thermodynamics need to be considered when studying microbial community dynamics.

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G-Protein Coupled Receptor-Ligand Dissociation Rates and Mechanisms from {tau}RAMD Simulations There is a growing appreciation of the importance of drug-target binding kinetics for lead optimization. For G protein-coupled receptors (GPCRs), which mediate signaling over a wide range of timescales, the drug dissociation rate is often a better predictor of in vivo efficacy than binding affinity, although it is more challenging to compute. Here, we assess the ability of the {tau}-Random Acceleration Molecular Dynamics ({tau}RAMD) approach to reproduce relative residence times and reveal dissociation mechanisms and the effects of allosteric modulation for two important membrane-embedded drug targets: the {beta}2-adrenergic receptor and the muscarinic acetylcholine receptor M2. The dissociation mechanisms observed in the relatively short RAMD simulations (in which molecular dynamics (MD) simulations are performed using an additional force with an adaptively assigned random orientation applied to the ligand) are in general agreement with much more computationally intensive conventional MD and metadynamics simulations. Remarkably, although decreasing the magnitude of the random force generally reduces the number of egress routes observed, the ranking of ligands by dissociation rate is hardly affected and agrees well with experiment. The simulations also reproduce changes in residence time due to allosteric modulation and reveal associated changes in ligand dissociation pathways.

TABLE OF CONTENTS GRAPHIC

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sentences: Exploring the interaction network of a synthetic gut bacterial community A key challenge in microbiome research is to predict functionality from microbial community composition. As central microbiota functions are determined by bacterial community networks it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments, as well as in community batch culture. Metabolomics analysis of spent culture supernatant of individual strains in combination with genome-informed pathway reconstruction provided insights into the metabolic potential of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both, exploitative and interference competition in vitro. In particular, Enterococcus faecalis KB1 was identified as important driver of community composition by affecting the abundance of several other consortium members. Together, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network, which serves as knowledge base for future mechanistic studies.

sentences: Transcriptional activation of Il36A by C/EBPβ via a half-CRE*C/EBP element in murine macrophages is independent of its CpG methylation level Interleukin-36(Il-36) is a member of the novel Il-1-like proin-flammatory cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types. We have recently shown that CCAAT enhancer binding protein{beta} (C/EBP{beta}) binds specifically to an essential half cAMP response element (half-CRE)*C/EBP motif in the Il36A promoter to induce Il36A expression upon LPS stimulation. C/EBPs are transcription factors belonging to the basic leucine zipper (bZIP) family of transcriptional regulators. C/EBP proteins can form homo- and heterodimers and regulate gene expression by binding to C/EBP specific recognition sequences and composite sites that can contain 5'-cytosine-phosphate-guanine-3'dinucleotides (CpG). CpG methylation of such elements has been shown to influence transcription factor binding and gene expression. Here we show that the half-CREoC/EBP element in the Il36A promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. By using electrophoretic mobility gel shift and fluorescence polarization assays we demonstrate that C/EBP{beta} binding to the half-CRE*C/EBP element in the Il36A promoter following LPS stimulation is insensitive to CpG methylation. Transfection assays also show that methylation of the CpG in the half-CRE*C/EBP element does not alter LPS-induced Il36A promoter activity. A direct comparison of Il36A mRNA copy numbers as well as the pro-Il-36 protein level in RAW264.7 and primary macrophages revealed similar amounts in both cell types. Taken together, our data suggest that C/EBP{beta} binding to the half-CRE*C/EBP element and C/EBP{beta} mediated gene activation occurs independently of the CpG methylation status of the target DNA sequence and underline the potential of C/EBP{beta} to recognize methylated as well as unmethylated binding sites.

sentences: Predictability of drug sensitivity in multi-species environments Microbes associate in nature forming complex communities, but they are often studied in purified form. Here I show that differences in antibiotic sensitivity between co-existing bacterial species can modify the diffusion of antibiotic molecules, and alter the drug efficacy measured in conventional in vitro assays. I developed a model, validated experimentally using an artificial two-species community, showing antibiotic molecules re-distribute evenly across all species if they have similar drug sensitivity. The result is lower drug efficacy with respect to conventional in vitro assays. However, when species have different sensitivities, the re-distribution of antibiotic is uneven and most sensitive species accumulate more drug. The result is increased drug efficacy against these species. Despite this discrepancy with conventional laboratory assays, my data suggests the changes in drug efficacy are predictable and provides insight into how communities can affect the diffusion of other molecules in the environment.

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Chronic exercise protects against the progression of renal cyst growth and dysfunction in rats with polycystic kidney disease IntroductionPolycystic kidney disease (PKD) is a genetic disorder characterized by the progressive enlargement of renal epithelial cysts and renal dysfunction. Previous studies have reported the beneficial effects of chronic exercise on chronic kidney disease. However, the effects of chronic exercise have not been fully examined in PKD patients or models. The effects of chronic exercise on the progression of PKD were investigated in a polycystic kidney (PCK) rat model.

MethodsSix-week-old male PCK rats were divided into a sedentary group and an exercise group. The exercise group underwent forced treadmill exercise for 12 weeks (28 m/min, 60 min/day, 5 days/week). After 12 weeks, kidney function and histology were examined, protein expressions were analyzed, and signaling cascades of PKD were examined.

ResultsChronic exercise reduced the excretion of urinary protein, liver-type fatty acid-binding protein, plasma creatinine, urea nitrogen, and increased plasma irisin and urinary arginine vasopressin (AVP) excretion. Chronic exercise also slowed renal cyst growth, glomerular damage, and interstitial fibrosis, and led to reduced Ki-67 expression. Chronic exercise had no effect on cAMP content but decreased the renal expression of B-Raf and reduced the phosphorylation of extracellular signal-regulated kinase (ERK), mammalian target of rapamycin (mTOR), and S6.

ConclusionChronic exercise slows renal cyst growth and damage in PCK rats, despite increasing AVP, with down-regulation of the cAMP/B-Raf/ERK and mTOR/S6 pathways in the kidney of PCK rats.

sentences: Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth To maintain stable DNA concentrations, proliferating cells need to coordinate DNA replication with cell growth. For nuclear DNA, eukaryotic cells achieve this by coupling DNA replication to cell cycle progression, ensuring that DNA is doubled exactly once per cell cycle. By contrast, mitochondrial DNA replication is typically not strictly coupled to the cell cycle, leaving the open question of how cells maintain the correct amount of mitochondrial DNA during cell growth. Here, we show that in budding yeast, mitochondrial DNA copy number increases with cell volume, both in asynchronously cycling populations and during G1 arrest. Our findings suggest that cell-volume-dependent mitochondrial DNA maintenance is achieved through nuclear encoded limiting factors, including the mitochondrial DNA polymerase Mip1 and the packaging factor Abf2, whose amount increases in proportion to cell volume. By directly linking mitochondrial DNA maintenance to nuclear protein synthesis, and thus cell growth, constant mitochondrial DNA concentrations can be robustly maintained without a need for cell-cycle-dependent regulation.

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Classification of the plant-associated phenotype of Pseudomonas strains using genome properties and machine learning The rhizosphere, the region of soil surrounding roots of plants, is colonized by a unique population of Plant Growth Promoting Rhizobacteria (PGPR). By enhancing nutrient uptake from the soil and through modulation of plant phytohormone status and metabolism, PGPR can increase the stress tolerance, growth and yield of crop plants. Many important PGPR as well as plant pathogens belong to the genus Pseudomonas. There is, however, uncertainty on the divide between phytobeneficial and phytopathogenic strains as previously thought to be signifying genomic features have limited power to separate these strains. Here the Genome properties (GP) common biological pathways annotation system was applied to establish the relationship between the genome wide GP composition and the plant-associated phenotype of 91 Pseudomonas strains representing both phenotypes. GP enrichment analysis, Random Forest model fitting and feature selection revealed 28 discriminating features. A validation dataset of 67 new strains confirmed the importance of the selected features for classification. A number of unexpected discriminating features were found, suggesting involvement of novel molecular mechanisms. The results suggest that GP annotations provide a promising computational tool to better classify the plant-associated phenotype.

Author summaryWith a growing population the need to double the agricultural food production is specified. Simultaneously, there is an urgent need to implement sustainable and climate change resilient agricultural practices that preserve natural ecosystems. Cooperative microbiomes play important positive roles in plant growth development and fitness. Properly tuned, these microbiomes can significantly reduce the need for synthetic fertilizers and can replace chemicals in crop pest control. To select beneficial candidates, their traits need to be described and likewise, potential detrimental traits should be avoided. Here we applied GP-based comparative functional genomics, enrichment analysis and Random Forest model fitting to compare known phytobeneficial and phytopathogenic Pseudomonas strains. A number of unexpected discriminating features were found suggesting the involvement of novel molecular mechanisms.

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Disrupted development and altered hormone signaling in male Padi2/Padi4 double knockout mice BackgroundPeptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo.

MethodsPreliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice for a range of reproductive defects.

ResultsControlled breeding trials found that DKO breeding pairs, particularly males, appeared to take longer to have their first litter than wild-type FVB controls (WT), and that pups and DKO male weanlings weighed significantly less than their WT counterparts. Additionally, DKO males took significantly longer than WT males to reach puberty and had lower serum testosterone levels. Furthermore, DKO males had smaller testes than WT males with increased rates of germ cell apoptosis.

ConclusionsThe Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling.

sentences: Principles governing the topological organization of object selectivities in ventral temporal cortex To achieve the computational goal of rapidly recognizing miscellaneous objects in the environment despite large variations in their appearance, our mind represents objects in a high-dimensional object space to provide separable category information and enable the extraction of different kinds of information necessary for various levels of the visual processing. To implement this abstract and complex object space, the ventral temporal cortex (VTC) develops different object-selective regions with certain topological organization as the physical substrate. However, the principle that governs the topological organization of object selectivities in the VTC remains unclear. Here, equipped with the wiring cost minimization principle constrained by the wiring length of neurons in human temporal lobe, we constructed a hybrid self-organizing map (SOM) model as an artificial VTC (VTC-SOM) to explain how the abstract and complex object space is faithfully implemented in the brain. In two in silico experiments with the empirical brain imaging and single-unit data, our VTC-SOM predicted the topological structure of fine-scale functional regions (face-, object-, body-, and place-selective regions) and the boundary (i.e., middle Fusiform Sulcus) in large-scale abstract functional maps (animate vs. inanimate, real-word large-size vs. small-size, central vs. peripheral), with no significant loss in functionality (e.g., categorical selectivity, a hierarchy of view-invariant representations). These findings illustrated that the simple principle utilized in our model, rather than multiple hypotheses such as temporal associations, conceptual knowledge, and computational demands together, was apparently sufficient to determine the topological organization of object-selectivities in the VTC. In this way, the high-dimensional object space is implemented in a two-dimensional cortical surface of the brain faithfully.

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A comprehensive transcriptome signature of murine hematopoietic stem cell aging We surveyed 16 published and unpublished data sets to determine whether a consistent pattern of transcriptional deregulation in aging murine hematopoietic stem cells (HSC) exists. Despite substantial heterogeneity between individual studies, we uncovered a core and robust HSC aging signature. We detected increased transcriptional activation in aged HSCs, further confirmed by chromatin accessibility analysis. Unexpectedly, using two independent computational approaches, we established that deregulated aging genes consist largely of membrane-associated transcripts, including many cell surface molecules previously not associated with HSC biology. We show that Selp, the most consistent deregulated gene, is not merely a marker for aged HSCs but is associated with HSC functional decline. Additionally, single-cell transcriptomics analysis revealed increased heterogeneity of the aged HSC pool. We identify the presence of transcriptionally "young-like" HSCs in aged bone marrow. We share our results as an online resource and demonstrate its utility by confirming that exposure to sympathomimetics, and deletion of Dnmt3a/b, molecularly resembles HSC rejuvenation or aging, respectively.

Key PointsO_LIA comprehensive transcriptome analysis of aged murine hematopoietic stem cells identifies an aging signature;
C_LIO_LIThe HSC aging signature is highly enriched for cell membrane-related transcripts and identifies age-associated heterogeneity.
C_LI

sentences: Sequence-based modeling of genome 3D architecture from kilobase to chromosome-scale The structural organization of the genome plays an important role in multiple aspects of genome function. Understanding how genomic sequence influences 3D organization can help elucidate their roles in various processes in healthy and disease states. However, the sequence determinants of genome structure across multiple spatial scales are still not well understood. To learn the complex sequence dependencies of multiscale genome architecture, here we developed a sequence-based deep learning approach, Orca, that predicts genome 3D architecture from kilobase to whole-chromosome scale, covering structures including chromatin compartments and topologically associating domains. Orca also makes both intrachromosomal and interchromosomal predictions and captures the sequence dependencies of diverse types of interactions, from CTCF-mediated to enhancer-promoter interactions and Polycomb-mediated interactions. Orca enables the interpretation of the effects of any structural variant at any size on multiscale genome organization and provides an in silico model to help study the sequence-dependent mechanistic basis of genome architecture. We show that the models accurately recapitulate effects of experimentally studied structural variants at varying sizes (300bp-80Mb) using only sequence. Furthermore, these sequence models enable in silico virtual screen assays to probe the sequence-basis of genome 3D organization at different scales. At the submegabase scale, the models predicted specific transcription factor motifs underlying cell-type-specific genome interactions. At the compartment scale, based on virtual screens of sequence activities, we propose a new model for the sequence basis of chromatin compartments: sequences at active transcription start sites are primarily responsible for establishing the expression-active compartment A, while the inactive compartment B typically requires extended stretches of AT-rich sequences (at least 6-12kb) and can form  passively without depending on any particular sequence pattern. Orca thus effectively provides an "in silico genome observatory" to predict variant effects on genome structure and probe the sequence-based mechanisms of genome organization.

sentences: Neuro-molecular characterization of fish cleaning interactions Coral reef fish exhibit a large variety of behaviours crucial for fitness and survival. The cleaner wrasse Labroides dimidiatus displays cognitive abilities during interspecific interactions by providing services of ectoparasite cleaning, thus serving as a good example to understand the processes of complex social behaviour. However, little is known about the molecular underpinnings of cooperative behaviour between L. dimidiatus and a potential client fish (Acanthurus leucosternon). Therefore, we investigated the molecular mechanisms in three regions of the brain (fore-, mid-, and hindbrain) during the interaction of these fishes. Here we show, using transcriptomics, that most of the transcriptional response in both species was regulated in the hindbrain and forebrain regions and that the interacting behaviour responses of L. dimidiatus involved immediate early gene alteration, dopaminergic and glutamatergic pathways, the expression of neurohormones (such as isotocin) and steroids (e.g. progesterone and estrogen). In contrast, in the client, fewer molecular alterations were found, mostly involving pituitary hormone responses. The particular pathways found suggested learning and memory processes in the cleaner wrasse, while the client indicated stress relief.

sentences: Chemoproteomic profiling reveals cellular targets of nitro-fatty acids Nitro-fatty acids are a class of endogenous electrophilic lipid mediators with anti-inflammatory and cytoprotective effects in a wide range of inflammatory and fibrotic disease models. While these beneficial biological effects of nitro-fatty acids are mainly attributed to their ability to form covalent adducts with proteins, only a small number of proteins are known to be nitro-alkylated and the scope of protein nitro-alkylation remains undetermined. Here we describe the synthesis and application of a clickable nitro-fatty acid probe for the detection and first global identification of mammalian proteins that are susceptible to nitro-alkylation. 184 high confidence nitro-alkylated proteins were identified in human macrophages, majority of which are novel targets of nitro-fatty acids, including Extended synaptotagmin 2 (ESYT2), Signal transducer and activator of transcription 3 (STAT3), Toll-like receptor 2 (TLR2), Retinoid X receptor alpha (RXR) and Glucocorticoid receptor (NR3C1). In particular, we showed that 9-nitro-oleate covalently modified and inhibited dexamethasone binding to NR3C1. Bioinformatic analyses revealed that nitro-alkylated proteins are highly enriched in endoplasmic reticulum and transmembrane proteins, and are overrepresented in lipid metabolism and transport pathways. This study significantly expands the scope of protein substrates targeted by nitro-fatty acids in living cells and provides a useful resource towards understanding the pleiotropic biological roles of nitro-fatty acids as signaling molecules or as multi-target therapeutic agents.

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Low ozone concentration and negative ions for rapid SARS-CoV-2 inactivation Ozone is a powerful anti-bacterial, anti-fungal and anti-viral agent, yet exposure to high levels of ozone can pose risks to human/animal health and, in the long term, corrode certain objects. In order to overcome these risks, we evaluated the potential of using a relatively short exposure of a low concentration of ozone to disinfect an indoor environment in the absence of individuals and animals. ICON3 by O3ZONO/M2L, a new disinfection device generating both ozone and negative ions, was selected to assess the potential of this strategy to inactivate different viral isolates of SARS-CoV-2.

Tests under controlled laboratory conditions were performed in a system consisting of an ozone-proof airtight plastic box inside a biological safety cabinet, where suspensions of two strains of SARS-CoV-2 were exposed to ozone and negative ions and virucidal activity was measured by means of two complementary methodologies: viral replication capacity and viral titer determination.

These studies revealed that low concentration ozone (average 3.18 ppm after the peak) inactivated up to >99% of SARS-CoV-2 within 20 minutes of exposure. Under controlled conditions, similar ozone exposure was recreated with ICON3 in different volume rooms (15, 30, 60 m3) where a linear relationship was observed between the room volume and the time of continuous ozone/ions flow required to reach and maintain the desired ozone levels used in the laboratory studies.

These studies suggest that ICON3 may have the potential for use in the disinfection of SARS-CoV-2 in indoor environments in the absence of individuals and animals, under properly controlled and monitored safety conditions.

sentences: Comparative proteomic analysis of Fusarium oxysporum f. sp. cubense strains (Foc R1 and Foc TR4) provides better insights into mechanisms of their virulence, habitat adaptation and pathogenesis Fusarium oxysporum f. sp. cubense (Foc), a devastative soil-borne fungal pathogen causing vascular wilt (i.e. Panama disease) which leads to severe crop losses in most of the banana-growing regions of the world. As there is no single source of effective management practices available so far, understand the pathogenicity of the organism may help in designing effective control measures through molecular approaches. The study aims to compare the proteome of the two pathogenic Foc virulent strains, Race 1 (Foc R1) and tropical race 4 (Foc TR4) that are capable of infecting the Cavendish group of bananas using 2-dimensional (2-D) gel electrophoresis, MALDI-TOF/MS and MS/MS analysis. The results of the study revealed that the proteins, peroxiredoxins, NAD-aldehyde dehydrogenase (NAD-ALDH), MAPK protein, pH-response regulator protein palA/rim-20 and isotrichodermin C15 hydroxylase have shared homology with the fungal proteins, which regulate the osmotic stress response, signal transduction, root colonization and toxin biosynthesis. These are the important functions for the pathogen survival in an unfavourable environment, and successful establishment and infection of the banana host. The present study also identified several putative pathogenicity related proteins in both Foc R1 and Foc TR4. Specifically, certain Foc TR4 specific putative pathogenicity related proteins, phytotoxins biosynthesis gene, fructose 1,6-bisphosphate aldolase class II, Synembryn-like proteins found to contribute strong virulence. Overexpression or knockout of the elective genes could help in devising better control measures for the devastative pathogens in the future. To the best of our knowledge, this is the first report on the proteomics of Foc R1 and Foc TR4 strains of Indian origin that infect Cavendish bananas.

sentences: Single-Cell Analysis of Human Testis Aging, and Impact of Elevated Body Mass Index Aging human males display reduced reproductive health, however testis aging is poorly understood at the molecular and genomic level. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from both young and older men (>60 years old) - and examined age-related changes in germline development and in the somatic niche. Interestingly, age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and the somatic niche ranged from moderate to severe. Altered pathways included signaling and inflammation in multiple cell types, metabolic signaling in Sertoli cells, hedgehog signaling and testosterone production in Leydig cells, cell death and growth in testicular peritubular cells, and possible developmental regression in both Leydig and peritubular cells. Remarkably, the extent of dysregulation correlated with body mass index in older, but not younger men. Taken together, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging, and their exacerbation by concurrent chronic conditions such as obesity.

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Identifying potential natural inhibitors of Brucella melitensis Methionyl-tRNA synthetase through an in-silico approach BackgroundBrucellosis is an infectious disease caused by bacteria of the genus Brucella. Although it is the most common zoonosis worldwide, there are increasing reports of drug resistance and cases of relapse after long term treatment with the existing drugs of choice. This study therefore aims at identifying possible natural inhibitors of Brucella melitensis Methionyl-tRNA synthetase through an in-silico approach.

MethodsUsing PyRx 0.8 virtual screening software, the target was docked against a library of natural compounds obtained from edible African plants. The compound, 2-({3-[(3,5-dichlorobenzyl) amino] propyl} amino) quinolin-4(1H)-one (OOU) which is a co-crystallized ligand with the target was used as the reference compound. Screening of the molecular descriptors of the compounds for bioavailability, pharmacokinetic properties, and bioactivity was performed using the SWISSADME, pkCSM, and Molinspiration web servers respectively. The Fpocket and PLIP webservers were used to perform the analyses of the binding pockets and the protein ligand interactions. Analysis of the time-resolved trajectories of the Apo and Holo forms of the target was performed using the Galaxy and MDWeb servers. The lead compounds, Strophanthidin and Isopteropodin are present in Corchorus olitorius and Uncaria tomentosa (cat-claw) plants respectively.

ResultsIsopteropodin had a binding affinity score of -8.9 kcal / ml with the target and had 17 anti-correlating residues in pocket 1 after molecular dynamics simulation. The complex formed by Isopteropodin and the target had a total RMSD of 4.408 and a total RMSF of 9.8067. However, Strophanthidin formed 3 hydrogen bonds with the target at ILE21, GLY262 and LEU294, and induced a total RMSF of 5.4541 at Pocket 1.

ConclusionOverall, Isopteropodin and Strophanthidin were found to be better drug candidates than OOU and they showed potentials to inhibit the Brucella melitensis Methionyl-tRNA synthetase at Pocket 1, hence abilities to treat brucellosis. In vivo and in vitro investigations are needed to further evaluate the efficacy and toxicity of the lead compounds.

Author SummaryO_LIStrophanthidin and Isopteropodin showed potentials to inhibit the Brucella melitensis Methionyl-tRNA synthetase at Pocket 1
C_LIO_LIBoth compounds can be used to treat brucellosis.
C_LIO_LIBoth compounds showed potentials of being safe to use in humans.
C_LI

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In vitro inhibition of H. pylori in a preferential manner using bioengineered L. lactis releasing guided Antimicrobial peptides ObjectivesTargeted therapies seek to selectively eliminate a pathogen without disrupting the resident microbial community. This is even more important when a pathogen like H. pylori resides in stomach, a sensitive microbial ecosystem. Using a probiotic like Lactococcus lactis and bioengineering it to release a guided Antimicrobial Peptide (AMP) targeted towards the pathogen offers a pathway to specifically knock-out the deleterious species and not disturbing the stomach microbiome.

ResultsThree AMPs, Alyteserin, CRAMP and Laterosporulin, were genetically fused to a guiding peptide MM1, which selectively binds to Vacuolating Toxin A (VacA) of H. pylori and cloned into an excretory vector pTKR inside L. lactis. When cultured together in vitro, the L. lactis bioengineered with guided AMPs selectively killed H. pylori when compared to E. coli or Lactobacillus plantarum, as determined by qPCR. Chemically synthesized Alyteserin and MM1-Alyteserin showed similar preferential inhibition of H. pylori when compared against E. coli, with the MIC of MM1-Alyteserin becoming significantly higher for E. coli than Alytserin whereas no such effet was observed against H. pylori.

ConclusionsProbiotics bioengineered to excrete guided AMPs can be a novel and useful approach for combating pathogens without endangering the natural microbial flora. Given the wealth of AMPs and guiding ligands, both natural and synthetic, this approach can be adapted to develop a diverse array of chimeric guided AMPs and can be cloned into probiotics to create a safe and effective alternative to conventional chemical antibiotics.

sentences: The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.

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Non-invasive stimulation with Temporal Interference: Optimization of the electric field deep in the brain with the use of a genetic algorithm ObjectiveSince the introduction of transcranial temporal interference stimulation (tTIS), there has been an ever-growing interest in this novel method, as it theoretically allows non-invasive stimulation of deep brain target regions. To date, attempts have been made to optimize the electrode montages and injected current to achieve personalized area targeting using two electrode pairs. Most of these methods use exhaustive search to find the best match, but faster and, at the same time, reliable solutions are required. In this study, the aforementioned stimulation settings were optimized using a genetic algorithm.

MethodsSimulations were performed on head models from the Population Head Model (PHM) repository. First, each model was fitted with an electrode array based on the 10-10 international EEG electrode placement system. Following electrode placement, the models were meshed and solved for all single-pair electrode combinations using an electrode on the left mastoid as a reference (ground). At the optimization stage, different electrode pairs and injection currents were tested using a genetic algorithm to obtain the optimal combination for each model.

ResultsGreater focality was achieved with optimization, specifically for the right hippocampus (region of interest), with a significant decrease in the surrounding electric field intensity. In the non-optimized case, the brain volumes stimulated above 0.2V/m were 99.9% in the region of interest (ROI), and 76.4% in the rest of the gray matter. In contrast, the stimulated volumes were 91.4% and 29.6% for the best optimization case. Additionally, the maximum electric field intensity inside the region of interest was consistently higher than that outside the ROI for all optimized cases.

SignificanceGiven that the accomplishment of a globally optimal solution requires a brute-force approach, the use of a genetic algorithm can significantly decrease the optimization time and provide usable results. Although the method is promising and the indications are becoming more robust, such that optimal solutions can be achieved at the individual level, there are limitations on what can be achieved using only two electrode pairs.

sentences: Genetic lineage of the Amami islanders inferred from classical genetic markers The genetic structure of the people of mainland Japan and Okinawa has been gradually unveiled in recent years. However, previous anthropological studies dealing with people in the Amami islands, located between mainland Japan and Okinawa, were less informative because of the lack of genetic data. In this study, we collected DNAs from 104 subjects in two of the Amami islands, Amami-Oshima island and Kikai island. We analyzed the D-loop region of mtDNA, four Y-STRs, and four autosomal nonsynonymous SNPs to clarify the Amami islanders genetic structure compared with peoples in Okinawa, mainland Japan, and other regions of East Asia. We found that the Amami islanders showed a genetically intermediate position between mainland Japan and Okinawa in mtDNA and Y-STR. However, the frequencies of several autosomal SNPs in the Amami islanders indicated a significant difference from mainland Japanese, which may be because of the gene flow from Okinawa but not natural selection. Moreover, extremely high or low frequencies of several alleles implied a founder effect in Kikai islanders. Note that there is room for the interpretation of the results because of the small sample size and number of alleles in the present study. Geographically broad and detailed samplings and genome-wide analyses are awaited.

sentences: Adenylate kinase 1 overexpression increases locomotor activity in medaka fish Maintenance of the energy balance is indispensable for cell survival and function. Adenylate kinase (Ak) is a ubiquitous enzyme highly conserved among many organisms. Ak plays an essential role in energy regulation by maintaining adenine nucleotide homeostasis in cells. However, its role at the whole organism level, especially in animal behavior, remains unclear. Here, we established a model using medaka fish (Oryzias latipes) to examine the function of Ak in environmental adaptation. Medaka overexpressing the major Ak isoform Ak1 exhibited increased locomotor activity compared to that of the wild type. Interestingly, this increase was temperature dependent. Our findings suggest that cellular energy balance can modulate locomotor activity.

sentences: Genetic characterization of ABC pesticide transporters establishes Drosophila as an in vivo model for toxicogenetics Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge in mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect D. melanogaster. An RNAi screen was performed, which knocked down individual ABCs in targeted epithelial tissue, examining the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This simultaneously implicated a novel ABC drug transporter CG4562 but also highlighted a predominant role for the P-glycoprotein orthologue Mdr65. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, heterologous expression of the Mdr65 orthologue from the major malaria vector Anopheles gambiae was used to establish an in vivo characterization system for the characterization of P-glycoproteins from non-model insects in D. melanogaster. This study provides the basis for establishing Drosophila as a model for toxicology research regarding drug transporters.

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Effectiveness of the protected areas on the Mornington Peninsula for the common resident shorebird species using citizen science data In the absence of comprehensive survey data this study used citizen science bird counts, extracted from the Atlas of Living Australia, to assess which species benefit most from protected areas. This was done by fitting temporal models using the Integrated Laplace Approximation (INLA) method.

The trends for five resident shorebird species were compared to the Australian Pied Oystercatcher, with significantly steeper upward trends identified for the Black-fronted Dotterel, Red-capped Dotterel and Red-kneed Dotterel. Steeper upward trends were observed in protected than unprotected areas for the Black-fronted Dotterel, Masked Lapwing and Red-kneed Dotterel.

This work suggests that, with some limitations, statistical models can be used with citizen science data for monitoring the persistence of resident shorebirds and for investigating factors that are impacting these data. The results for the Dotterel species in protected areas are particularly encouraging.

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Bromodomain and Extraterminal Protein Inhibitor, Apabetalone (RVX-208), Reduces ACE2 Expression and Attenuates SARS-CoV-2 Infection in vitro Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.

Graphical Abstract

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sentences: Manipulation of carotenoid metabolism stimulates biomass and stress tolerance in tomato Improving yield, nutritional value and tolerance to abiotic stress are major targets of current breeding and biotechnological approaches that aim at increasing crop production and ensuring food security. Metabolic engineering of carotenoids, the precursor of Vitamin-A and plant hormones that regulate plant growth and response to adverse growth conditions, has been mainly focusing on provitamin A biofortification or the production of high-value carotenoids. Here, we show that the introduction of a single gene of the carotenoid biosynthetic pathway in different tomato cultivars simultaneously improved photosynthetic capacity and tolerance to various abiotic stresses (e.g., high light, salt, and drought), caused an up to 77% fruit yield increase and enhanced fruits provitamin A content and shelf life. Our findings pave the way for developing a new generation of crops that combine high productivity and increased nutritional value with the capability to cope with climate change-related environmental challenges.

sentences: Low-Toxin Clostridioides difficile RT027 Strains Exhibit Robust Virulence Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. Currently, there is lack of consensus on a single optimal diagnostic method for C. difficile infection (CDI). Nucleic acid amplification tests (NAAT) that detect toxin genes are highly sensitive, but their specificity limitations could inflate CDI rates. Alternate multi-step diagnostic algorithms emphasize the detection of C. difficile toxins TcdA/TcdB, and are premised on the rationale that stool toxin-negative (Tox-) CDI patients have less severe disease, shorter diarrhea duration, and fewer disease complications. There have been no systematic assessments, however, of the virulence of C. difficile strains from Tox-/NAAT+ (discrepant) specimens. In our prospective analysis of 1243 C. difficile-positive patient stool specimens from three Southern Arizona hospitals, 31% were discrepant. Ribotype 027 (RT027) strains were recovered from 221 specimens; of these, 23% were discrepant. Post-culture, RT027 strains produced a range of toxin amounts including levels lower than that of the non-epidemic strain CD630. These low-toxin RT027 (LT-027) strains harbored both tcdA and tcdB genes, and their culture supernatants were cytotoxic to cultured fibroblasts. We confirmed robust colonization and virulence of a subset of LT-027 strains using multiple rodent models; lethality in animals infected with LT-027 strains was comparable to that potentiated by a high-toxin RT027 strain. Comparative genomics and proteomics analyses of several LT-027 strains identified unique genes and altered protein abundances relative to closely-related high-toxin strains. Collectively our data highlight the robust virulence and clinical relevance of low-toxin-producing, RT027 C. difficile isolates.

sentences: Cationic inhalable particles for enhanced drug delivery to M. tuberculosis infected macrophages Inhalable microparticle-based drug delivery platforms are being investigated extensively for Tuberculosis (TB) treatment as they offer efficient deposition in lungs and improved pharmacokinetics of the encapsulated cargo. However, the effect of physical parameters of microcarriers on interaction with Mycobacterium tuberculosis (Mtb) infected mammalian cells is underexplored. In this study, we report that Mtb-infected macrophages are highly phagocytic and microparticle surface charge plays a major role in particle internalization by infected cells. Microparticles of different sizes (0.5 - 2 m) were internalized in large numbers by Mtb-infected THP-1 macrophages and murine primary Bone Marrow Derived Macrophages in vitro. Drastic improvement in particle uptake was observed with cationic particles in vitro and in mice lungs. Rapid uptake of rifampicin-loaded cationic microparticles allowed high intracellular accumulation of the drug and lead to enhanced anti-bacterial function when compared to non-modified rifampicin-loaded microparticles. Cytocompatibility assay and histological analysis in vivo confirmed that the formulations were safe and did not elicit any adverse reaction. Additionally, pulmonary delivery of cationic particles in mice resulted in two-fold higher uptake in resident alveolar macrophages compared to non-modified particles. This study provides a framework for future design of drug carriers to improve delivery of anti-TB drugs inside Mtb-infected cells.

sentences: High vitamin B2 production by the Lactiplantibacillus plantarum B2 strain carrying a mutation in the aptamer P1 helix of the FMN-riboswitch that regulates expression of the bacterial rib operon Manufacturing of probiotics and functional foods using lactic acid bacteria that overproduce vitamin B2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analyzed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for lactic acid bacteria, exceeding by ~25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~6-fold higher in B2. Mutation B2 could decrease the stability of the aptamers regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavine, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

sentences: Sexual selection for males with beneficial mutations Sexual selection is the process by which traits providing a mating advantage are favoured. Theoretical treatments of the evolution of sex by sexual selection propose that it operates by reducing the load of deleterious mutations. Here, we postulate instead that sexual selection primarily acts through females preferentially mating with males carrying beneficial mutations. We used simulation and analytical modelling to investigate the evolutionary dynamics of beneficial mutations in the presence of sexual selection. We found that female choice for males with beneficial mutations had a much greater impact on genetic quality than choice for males with low mutational load. We also relaxed the typical assumption of a fixed mutation rate. For deleterious mutations, mutation rate should always be minimized, but when rare beneficial mutations can occur, female choice for males with those rare beneficial mutations could overcome a decline in average fitness and allow an increase in mutation rate. We propose that sexual selection for beneficial mutations could overcome the  two-fold cost of sex much more readily than choice for males with low mutational load and may therefore be a more powerful explanation for the prevalence of sexual reproduction than the existing theory. If sexual selection results in higher fitness at higher mutation rates and if the variability produced by mutation itself promotes sexual selection, then a feedback loop between these two factors could have had a decisive role in driving adaptation.

sentences: Publication practices during the COVID-19 pandemic: Biomedical preprints and peer-reviewed literature The coronavirus pandemic introduced many changes to our society, and deeply affected the established in biomedical sciences publication practices. In this article, we present a comprehensive study of the changes in scholarly publication landscape for biomedical sciences during the COVID-19 pandemic, with special emphasis on preprints posted on bioRxiv and medRxiv servers. We observe the emergence of a new category of preprint authors working in the fields of immunology, microbiology, infectious diseases, and epidemiology, who extensively used preprint platforms during the pandemic for sharing their immediate findings. The majority of these findings were works-in-progress unfitting for a prompt acceptance by refereed journals. The COVID-19 preprints that became peer-reviewed journal articles were often submitted to journals concurrently with the posting on a preprint server, and the entire publication cycle, from preprint to the online journal article, took on average 63 days. This included an expedited peer-review process of 43 days and journals production stage of 15 days, however there was a wide variation in publication delays between journals. Only one third of COVID-19 preprints posted during the first nine months of the pandemic appeared as peer-reviewed journal articles. These journal articles display high Altmetric Attention Scores further emphasizing a significance of COVID-19 research during 2020. This article will be relevant to editors, publishers, open science enthusiasts, and anyone interested in changes that the 2020 crisis transpired to publication practices and a culture of preprints in life sciences.

sentences: Mediator-RNA Polymerase II Interactions Critical for Transcriptional Activation Are Mediated by the N-terminal Half of MED14 and C-terminal Domain of RPB1 Mediator is a large and evolutionarily conserved coactivator complex essential for RNA polymerase II (Pol II)-mediated gene regulation at multiple steps of the transcription process, including preinitiation complex (PIC) assembly and function. Here, we used the MultiBac baculovirus expression system to generate recombinant human core Mediator subcomplexes and subsequent biochemical approaches to dissect the mechanism by which Mediator facilitates direct recruitment of Pol II to core promoters. Our results highlight a pivotal role in this process for the N-terminal half (NTD) of the MED14 subunit. We show that a reconstituted 15-subunit human core Mediator complex that contains only the MED14-NTD is fully functional in facilitating both basal and activated (p53) transcription. This complex directly interacts with the C-terminal domain (CTD) of the RPB1 subunit of Pol II (RPB1 CTD) and is required for recruiting Pol II to core promoters. Moreover, recombinant RPB1 can completely reverse the human core Mediator-Pol II interaction. Notably, the human MED14-NTD region has secondary structure conservation with Schizosaccharomyces pombe. In addition, reanalysis of published cryo-EM structures of yeast Mediator-Pol II complexes strongly supports our conclusion. Thus, our analyses provide critical new insights into how Mediator binds to Pol II and recruits it to the promoters to facilitate transcription.

sentences: Neuron-radial glial cell communication via BMP/Id1 signaling maintains the regenerative capacity of the adult zebrafish telencephalon The mechanisms of the management of the neural stem cell (NSC) pool underlying the regenerative capacity of the adult zebrafish brain are not understood. We show that Bone Morphogenetic Proteins (BMPs) which are exclusively expressed by neurons in the adult telencephalon and the helix-loop-helix (HLH) transcription co-regulator, Inhibitor of differentiation 1 (Id1), control quiescence of NSCs. Upon injury, lack of id1 function leads to an initial over-proliferation and subsequent loss of NSCs and the regenerative capacity. BMP/Id1 signaling up-regulates the transcription factor her4.1 which is also a target of Notch signaling mediating short-range control of NSC quiescence. Hence, the two signaling systems converge onto Her4.1. Our data show that neurons feedback on NSC proliferation. BMP1/Id1 signaling appears as the predominant safeguard of the NSC pool under regenerative conditions while Notch signaling is sufficient to maintain NSCs under homeostatic baseline neurogenesis in the uninjured animal.

sentences: Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is driven by differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network by defining cell-type-specific enhancers.

sentences: Single Tube qPCR detection and quantitation of hotspot mutations down to 0.01% VAF Clinically and biologically, rare DNA sequence variants are significant and informative. However, existing detection technologies are either complex in workflow, or restricted in the limit of detection (LoD), or do not allow for multiplexing. Blocker displacement amplification (BDA) method can stably and effectively detect and enrich multiple rare variants with LoD around 0.1% variant allele fraction (VAF). Nonetheless, the detailed mutation information has to be identified by additional sequencing technologies. Here, we present allele-specific BDA (As-BDA), a method combining BDA with allele-specific TaqMan (As-TaqMan) probes for effective variant enrichment and simultaneous SNV profiling. We demonstrated that As-BDA could detect mutations down to 0.01% VAF. Further, As-BDA could detect up to four mutations with low to 0.1% VAF per reaction using only 15 ng DNA input. The median error of As-BDA in VAF determination is approximately 9.1%. Comparison experiments using As-BDA and droplet digital PCR (ddPCR) on peripheral blood mononuclear cell (PBMC) clinical samples showed 100% concordance for samples with mutations at [≥] 0.1% VAF. Hence, we have shown that As-BDA can achieve simultaneous enrichment and identification of multiple targeted mutations within the same reaction with high clinical sensitivity and specificity, thus helpful for clinical diagnosis.

sentences: Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations Naturally occurring structural variations (SVs) are a considerable source of genomic variation and can reshape chromosomes 3D architecture. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system has been proved to generate random SVs to impact phenotypes and thus constitutes powerful drivers of directed genome evolution. However, how to reveal the molecular mechanism insights into the interactions between phenotypes and complex SVs, especially inversions and translocations, has so far remained challenging. In this study, we develop a SV-prone yeast strain by using SCRaMbLE with two synthetic chromosomes, synV and synX. An heterologous biosynthesis pathway allowing a high throughput screen for increased yield of astaxanthin is used as readout and a proof of concept for the application of SV in industry. We report here that complex SVs, including a pericentric inversion and a trans-chromosomes translocation between synV and synX, result in two neochromosomes and a 2.7-fold yield of astaxanthin. We demonstrated that inversion and inversion reshaped chromosomes 3D architecture and led to large reorganization of the genetic information nearby the breakpoint of the SVs along the chromosomes. Specifically, the pericentric inversion increased the expression of STE18 and the trans-chromosomic translocation increased the expression of RPS5 and MCM22, which contributed to higher astaxanthin yield. We also used the model learned from the aforementioned random screen and successfully harnessed the precise introduction of trans-chromosomes translocation and pericentric inversions by rational design. Overall, our work provides an effective tool to not only accelerate the directed genome evolution but also reveal mechanistic insight of complex SVs for altering phenotypes.

sentences: Induction of selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work we demonstrate the effect of DEAD-box polypeptide 3, X-Linked (DDX3) inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). RNA sequencing analysis revealed that while overall gene expression was minimally dysregulated, DDX3 inhibition in independent donor CD4+ T cells led to significant downregulation of BIRC5 and HSPB1A, genes critical to cell survival during HIV-1 infection. We used single-cell FISH-Flow technology to characterise latency reversal and the contribution of viral RNA to inducing cell death; pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3, upregulation of IFN{beta} and selective induction of apoptosis in viral RNA-expressing CD4+ T cells from PLWHIV but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV in an in vitro culture model over five days resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir as determined by quantitation of CA HIV-1 RNA, by TILDA, as well as by FISH-Flow technology. Our data support the translation of DDX3 inhibitor class compounds into HIV-1 curative strategies and provide proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards elimination of the inducible reservoir.

sentences: Commensal Pseudomonas fluorescens protect Arabidopsis from closely-related Pseudomonas pathogens in a colonization-dependent manner Plants form commensal associations with soil microorganisms, creating a root microbiome that provides benefits to the host including protection against pathogens. While bacteria can inhibit pathogens through production of antimicrobial compounds in vitro, it is largely unknown how microbiota contribute to pathogen protection in planta. We developed a gnotobiotic model system consisting of Arabidopsis thaliana, and an opportunistic pathogen Pseudomonas sp. N2C3, to identify mechanisms that determine the outcome of plant-pathogen-microbiome interactions in the rhizosphere. We screened 25 phylogenetically diverse Pseudomonas strains for their ability to protect against N2C3 and found that commensal strains closely related to N2C3 were more likely to protect against pathogenesis. We used a comparative genomics approach to identify unique genes in the protective strains that revealed no genes that correlate with protection, suggesting that variable regulation of components of the core Pseudomonas genome may contribute to pathogen protection. We found that commensal colonization level was highly predictive of protection and so tested deletions in genes previously shown to be required for Arabidopsis rhizosphere colonization. We identified a response regulator colR that is required for Pseudomonas protection from N2C3 and fitness in competition with N2C3 indicating that competitive exclusion may contribute to pathogen protection. We found that Pseudomonas WCS365 also protects against the agricultural pathogen Pseudomonas fuscovaginae SE-1, the causal agent of bacterial sheath brown rot of rice. This work establishes a gnotobiotic model to uncover mechanisms by which members of the microbiome can protect hosts from pathogens and informs our understanding of the use of beneficial strains for microbiome engineering in dysbiotic soil systems.

sentences: SIA: Selection Inference Using the Ancestral Recombination Graph Detecting signals of selection from genomic data is a central problem in population genetics. Coupling the rich information in the ancestral recombination graph (ARG) with a powerful and scalable deep learning framework, we developed a novel method to detect and quantify positive selection: Selection Inference using the Ancestral recombination graph (SIA). Built on a Long Short-Term Memory (LSTM) architecture, a particular type of a Recurrent Neural Network (RNN), SIA can be trained to explicitly infer a full range of selection coefficients, as well as the allele frequency trajectory and time of selection onset. We benchmarked SIA extensively on simulations under a European human demographic model, and found that it performs as well or better as some of the best available methods, including state-of-the-art machine-learning and ARG-based methods. In addition, we used SIA to estimate selection coefficients at several loci associated with human phenotypes of interest. SIA detected novel signals of selection particular to the European (CEU) population at the MC1R and ABCC11 loci. In addition, it recapitulated signals of selection at the LCT locus and several pigmentation-related genes. Finally, we reanalyzed polymorphism data of a collection of recently radiated southern capuchino seedeater taxa in the genus Sporophila to quantify the strength of selection and improved the power of our previous methods to detect partial soft sweeps. Overall, SIA uses deep learning to leverage the ARG and thereby provides new insight into how selective sweeps shape genomic diversity.

sentences: A waveform-independent measure of recurrent neural activity Rhythmic neural activity, so called oscillations, play a key role for neural information transmission, processing and storage. Neural oscillations in distinct frequency bands are central to physiological brain function and alterations thereof have been associated with several neurological and psychiatric disorders. The most common methods to analyse neural oscillations, e.g. short-term Fourier transform or wavelet analysis, assume that measured neural activity is composed of a series of symmetric prototypical waveforms, e.g. sinusoids. However, usually the models generating the signal, including waveform shapes of experimentally measured neural activity are unknown. Decomposing asymmetric waveforms of nonlinear origin using these classic methods may result in spurious harmonics visible in the estimated frequency spectra. Here, we introduce a new method for capturing rhythmic brain activity based on recurrences of similar states in phase-space. This method allows for a time-resolved estimation of amplitude fluctuations of recurrent activity irrespective of or specific to waveform-shapes. The algorithm is derived from the well-established field of recurrence analysis, which has rarely been adopted in neuroscience. In this paper, we show its advantages and limitations in comparison to short-time Fourier transform and wavelet convolution using periodic signals of different waveform shapes. Further, we demonstrate its application using experimental data, i.e. intracranial electrophysiological recordings from the human motor cortex of one epilepsy patient.

sentences: Gene name errors: lessons not learned Erroneous conversion of gene names into other dates and other data types has been a frustration for computational biologists for years. We hypothesized that such errors in supplementary files might diminish after a report in 2016 highlighting the extent of the problem. To assess this, we performed a scan of supplementary files published in PubMed Central from 2014 to 2020. Overall, gene name errors continued to accumulate unabated in the period after 2016. An improved scanning software we developed identified gene name errors in 30.9% of articles with supplementary Excel gene lists; a figure significantly higher than previously estimated. This is due to gene names being converted not just to dates and floating-point numbers, but also to internal date format (five-digit numbers). These findings further reinforce that spreadsheets are ill-suited to use with large genomic data.

sentences: Glycosaminoglycans and glycoproteins influence the elastic response of synovial fluid nanofilms on model oxide surfaces Synovial fluid (SF) is the natural lubricant found in articulated joints, providing unique cartilage surface protecting films under confinement and relative motion. While it is known that the synergistic interactions of the macromolecular constituents provide its unique load-bearing and tribological performance, it is not fully understood how two of the main constituents, glycosaminoglycans (GAGs) and glycoproteins, regulate the formation and mechanics of robust load-bearing films. Here, we present evidence that the load-bearing capabilities, rather than the tribological performance, of the formed SF films depend strongly on its components integrity. For this purpose, we used a combination of enzymatic treatments, quartz crystal microbalance with dissipation (QCM-D) and the surface forces apparatus (SFA) to characterize the formation and load-bearing capabilities of SF films on model oxide (i.e., silicates) surfaces. We find that, upon cleavage of proteins, the elasticity of the films is reduced and that cleaving GAGs results in irreversible (plastic) molecular re-arrangements of the film constituents when subjected to confinement. Understanding thin film mechanics of SF can provide insight into the progression of diseases, such as arthritis, but may also be applicable to the development of new implant surface treatments or new biomimetic lubricants.

sentences: Highly efficient multiplex editing: One-shot generation of 8x Nicotiana benthamiana and 12x Arabidopsis mutants Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well-documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 sgRNAs. We used this toolkit to explore the limits of multiplexing in two major model species, and report on isolation of transgene-free octuple Nicotiana benthamiana and duodecuple (12x) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on production of toxic 5-fluorouracyl in presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (~112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher order multiplexing applications. We identify a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.

sentences: Identification of 370 genetic loci for age at first sex and birth linked to externalising behaviour Age at first sexual intercourse (AFS) and age at first birth (AFB) have implications for health and evolutionary fitness. In the largest genome-wide association study to date (AFS, N=387,338; AFB, N=542,901), we identify 370 independent signals, 11 sex-specific, with a 5-6% polygenic score (PGS) prediction. Heritability of AFB shifted from 9% [CI=4-14] for women born in 1940 to 22% [CI=19-25] in 1965. Signals are driven by the genetics of reproductive biology and externalising behaviour, with key genes related to follicle stimulating hormone (FSHB), implantation (ESR1), infertility, and spermatid differentiation. Polycystic Ovarian Syndrome leads to later AFB, linking with infertility. Late AFB is protective against later-life disease and associated with parental longevity. Higher childhood socioeconomic circumstances and those in the highest PGS decile (90%+) experience markedly later reproductive onset. Results are relevant for improving teenage and late-life health, for understanding longevity, and guiding experimentation into mechanisms of infertility.

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A unique mineralizing pool of Gli1+ stem cells builds the tendon enthesis and demonstrates therapeutic potential The enthesis, a fibrocartilaginous transition between tendon and bone, is necessary for the transfer of force from muscle to bone to produce joint motion. The enthesis is prone to injury due to mechanical demands, and it cannot regenerate. A better understanding of how the enthesis develops will lead to more effective therapies to prevent pathology and promote regeneration. Here, we used single-cell RNA sequencing to define the development transcriptome of the entheses over postnatal stages. Six resident cell types, including enthesis progenitors and mineralizing chondrocytes, were identified along with their transcription factor regulons and temporal regulation. Following our prior discovery of the necessity of Gli1-lineage cells for enthesis development and healing, we then examined their transcriptomes at single cell resolution and demonstrated their clonogenicity and multipotency. Transplantation of these Gli1- lineage cells to enthesis injuries improved healing, demonstrating their therapeutic potential for enthesis regeneration.

HighlightsO_LIThe transcriptome and differentiation trajectory of enthesis stem cells during postnatal development are defined at single cell resolution.
C_LIO_LITranscription factor regulons drive enthesis stem cell differentiation.
C_LIO_LIGli1-lineage enthesis stem cells demonstrate in vivo and in vitro clonogenicity and multipotency.
C_LIO_LITransplantation of Gli1-lineage enthesis stem cells to enthesis injuries improves healing.
C_LI

sentences: DataJoint Elements: Data Workflows for Neurophysiology A new resource--DataJoint Elements--provides modular designs for assembling complete workflow solutions to organize data and computations for common neurophysiology experiments. The designs are derived from working solutions developed in leading research groups using the open-source DataJoint framework to integrate data collection and analysis in collaborative workflows.

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Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, Magainin 2 and Melittin. This small genetic modification, capable of efficient non-autonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei. It delays the release of infectious sporozoites while it simultaneously reduces the lifespan of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of endemic settings.

One sentence summaryWe developed a gene drive effector that retards Plasmodium development in transgenic Anopheles gambiae mosquitoes via the expression of antimicrobial peptides in the midgut and which is predicted to eliminate malaria under a range of transmission scenarios.

sentences: Inferring brain-wide interactions using data-constrained recurrent neural network models Behavior arises from the coordinated activity of numerous anatomically and functionally distinct brain regions. Modern experimental tools allow unprecedented access to large neural populations spanning many interacting regions brain-wide. Yet, understanding such large-scale datasets necessitates both scalable computational models to extract meaningful features of inter-region communication and principled theories to interpret those features. Here, we introduce Current-Based Decomposition (CURBD), an approach for inferring brain-wide interactions using data-constrained recurrent neural network models that directly reproduce experimentally-obtained neural data. CURBD leverages the functional interactions inferred by such models to reveal directional currents between multiple brain regions. We first show that CURBD accurately isolates inter-region currents in simulated networks with known dynamics. We then apply CURBD to multi-region neural recordings obtained from mice during running, macaques during Pavlovian conditioning, and humans during memory retrieval to demonstrate the widespread applicability of CURBD to untangle brain-wide interactions underlying behavior from a variety of neural datasets.

sentences: Production of Volatile Moth Sex Pheromones in Transgenic Nicotiana benthamiana Plants Plant-based bio-production of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here we describe the engineering of transgenic plants producing (Z)-11-hexadecen-1-ol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them in Nicotiana benthamiana plants. The constructs comprised the Amyelois transitella Atr{Delta}11 desaturase gene, the Helicoverpa armigera farnesyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, whose severity correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered harbouring an intact pheromone pathway and producing moderate levels of Z11-16OAc (11.8 {micro}g g-1 FW), next to high levels of Z11-16OH (111.4 {micro}g g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone bio-dispenser strategies and establish a roadmap for biotechnological improvements.

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Hunting the plant surrender signal activating apoplexy in grapevines after Neofusicoccum parvum infection O_LIApoplectic breakdown from Grapevines Trunk Diseases (GTDs) has become a serious challenge to viticulture in consequence to drought stress. We hypothesise that fungal aggressiveness is controlled by a chemical communication between host and colonising fungus.
C_LIO_LIWe introduce the new concept of a "plant surrender signal" accumulating in host plants under stress and triggering aggressive behaviour of the strain Neofusicoccum parvum (Bt-67) causing Botryosphaeriaceae-related dieback in grapevines.
C_LIO_LIUsing a cell-based experimental system (Vitis cells) and bioactivity-guided fractionation, we identify trans-ferulic acid, a monolignol precursor, as "surrender signal". We show that this signal specifically activates secretion of the fungal phytotoxin Fusicoccin A. We show further that this phytotoxin, mediated by 14-3-3 proteins, activates programmed cell death in Vitis cells.
C_LIO_LIWe arrive at a model pinpointing the chemical communication driving apoplexy in Botryosphaeriaceae-Vitis interaction and define the channelling of phenylpropanoid pathway from the lignin precursor, trans-ferulic acid to the phytoalexin transresveratrol as target for future therapy.
C_LI

sentences: Interfacing Spinal Motor Units in Non-Human Primates Identifies a Principal Neural Component for Force Control Constrained by the Size Principle Motor units convert the last neural code of movement into muscle forces. The classic view of motor unit control is that the central nervous system sends common synaptic inputs to motoneuron pools and that motoneurons respond in an orderly fashion dictated by the size principle. This view however is in contrast with the large number of dimensions observed in motor cortex which may allow individual and flexible control of motor units. Evidence for flexible control of motor units may be obtained by tracking motor units longitudinally during the performance of tasks with some level of behavioural variability. Here we identified and tracked populations of motor units in the brachioradialis muscle of two macaque monkeys during ten sessions spanning over one month during high force isometric contractions with a broad range of rate of force development (1.8 - 38.6 N{middle dot}m{middle dot}s-1). During the same sessions we recorded intramuscular EMG signals from 16 arm muscles of both limbs and elicited the full recruitment through neural stimulation of the median and deep radial nerves. We found a very stable recruitment order and discharge characteristics of the motor units over sessions and contraction trials. The small deviations from orderly recruitment were observed between motor units with close recruitment thresholds, and only during high rate of force development. Moreover, we also found that one component explained more than ~50% of the motor unit discharge rate variance, and that the remaining components could be described as a time-shifted version of the first, as it could be predicted from the interplay between the size principle of recruitment and one common input. In conclusion, our results show that motoneurons recruitment is determined by the interplay of the size principle and common input and that this recruitment scheme is not violated over time nor by the speed of the contractions.

sentences: CandyCollect: At-home saliva sampling for respiratory pathogen capture Streptococcus pyogenes is a major human-specific bacterial pathogen and a common cause of a wide range of symptoms from mild infection such as pharyngitis (commonly called strep throat) to lifethreatening invasive infection. Traditional methods for diagnosis include collecting a sample using a pharyngeal swab, which can cause discomfort and even discourage adults and children from seeking proper testing and treatment in the clinic. Saliva samples are an alternative to pharyngeal swabs. To improve the testing experience for strep throat, we developed a novel lollipop-inspired sampling platform (called CandyCollect) to capture bacteria in saliva. The device can be used in clinics or in the home and shipped back to a lab for analysis, integrating with telemedicine. CandyCollect is designed to capture bacteria on an oxygen plasma treated polystyrene surface embedded with flavoring substances to enhance the experience for children and inform the required time to complete the sampling process. In addition, the open channel structure prevents the tongue from scraping and removing the captured bacteria. Flavoring substances did not affect bacterial capture and the device has a shelf life of at least 2 months (with experiments ongoing to extend the shelf life). We performed a usability study with 17 participants who provided feedback on the device design and the dissolving time of the candy. This technology and advanced processing techniques, including polymerase chain reaction (PCR), will enable user-friendly and effective diagnosis of streptococcal pharyngitis.

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Independent and Interacting Value Systems for Reward and Information in the Human Brain Theories of Prefrontal Cortex (PFC) as optimizing reward value have been widely deployed to explain its activity in a diverse range of contexts, with substantial empirical support in neuroeconomics and decision neuroscience. Theoretical frameworks of brain function, however, suggest the existence of a second, independent value system for optimizing information during decision-making. To date, however, there has been little direct empirical evidence in favor of such frameworks. Here, by using computational modeling, model-based fMRI analysis, and a novel experimental paradigm, we aim at establishing whether independent value systems exist in human PFC. We identify two regions in the human PFC which independently encode distinct value signals. These value signals are then combined in subcortical regions in order to implement choices. Our results provide empirical evidence for PFC as an optimizer of independent value signals during decision-making under realistic scenarios, with potential implications for the interpretation of PFC activity in both healthy and clinical population.

One Sentence SummaryReward and information value are independently optimized in the Human PFC and are then combined in subcortical regions in order to implement choices.

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Genome-wide signatures of geographic expansion and breeding process in soybean The clarification of genomic signatures left during evolutionary histories of crops is crucial for breeding varieties adapting to changing climate. Soybean, a leguminous crop, provides both plant oil and protein. Here, we analyzed genome sequences of 2,214 soybeans and proposed its evolutionary route, which includes four geographic paths, expansion of annual wild soybean (Glycine soja Sieb. & Zucc.) from Southern China, domestication in Central China, expansion of landrace (G. max (L.) Merr.), and local breeding. We observed that local adaptation of the wild and cultivated soybeans was largely independent, and that genetic introgression was mostly derived from sympatric rather than allopatric wild populations during the range expansion of soybean landraces. Range expansion and breeding processes were accompanied with positive selection of flowering-time genes including GmSPA3c as validated by knock-out mutants. Our study shed lights on the evolutionary history of soybean and provides valuable genetic resources for future breeding.

TeaserThe expansion and selection history of soybean

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Mechanically induced nuclear shuttling of β-catenin requires co-transfer of actin Mesenchymal stem cells (MSC) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, {beta}-catenin and Yes-Associated Protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles x 2%) and static (1 x 2% hold) strain on nuclear translocation of {beta}-catenin and YAP1 at 3h after force application. Dynamic strain induced nuclear accumulation of {beta}-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced {beta}-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid (LPA) did not increase nuclear content of {beta}-catenin or actin, but induced robust nuclear increase in YAP1. As actin binds {beta}-catenin, we considered whether {beta}-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both {beta}-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of {beta}-catenin via mechanisms utilized for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, {beta}-catenin and YAP1.

Significance statementCells integrate both static and dynamic mechanical signals through the actin cytoskeleton which is attached to the nuclear envelope, affecting nuclear transport of {beta}-catenin and YAP1. Dynamic strain induces nuclear translocation of {beta}-catenin, but not YAP1, while static strain causes nuclear translocation of YAP1, but not {beta}-catenin. Importantly, nuclear transport of actin is induced by dynamic but not static force. Furthermore, nuclear import of {beta}-catenin depends on cofilin/importin-9 dependent actin transport mechanisms. Thus the presence of {beta}-catenin and YAP1 in the nucleus represent specific responses to regulatory mechanical signals.

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Vimentin intermediate filament assembly is a reversible process Networks of intermediate filaments (IF) need to constantly reorganize to fulfill their functions at different locations within the cell. IF assembly results from end-to-end annealing, which is commonly assumed to be irreversible. By contrast, the mechanisms involved in IF disassembly are far less understood. IF fragmentation has however been observed in many cell types, and it has been suggested that it could be associated with post-translational modifications. In this article, we investigate the contribution of filament fragmentation in the assembly dynamics of type III vimentin IF using a combination of in vitro reconstitution, fluorescence imaging, and theoretical modeling. We first show that vimentin assembly at low concentrations results in an equilibrium between filament annealing and fragmentation at time [≥] 24 h. At higher concentration, entanglements kinetically trap the system out of equilibrium, and we show that this trapping is reversible upon dilution. Taking into account both fragmentation and entanglement, we estimated that the mean bond breaking time was [~]18 hours Finally, we provide direct evidence through dual color imaging that filament fragmentation and annealing coexist during assembly. By showing that IF fragmentation can occur without cofactors or post-translational-modifications, our study provides a physical understanding of the IF length regulation.

Significance StatementVimentin intermediate filaments are a key component of the cytoskeleton and are involved in many cellular functions, such as the regulation of cell shape, migration and division. These functions require cytoskeletal filaments to simultaneously assemble and disassemble throughout the life of the cell. While the mechanisms of intermediate filament assembly have been widely studied, the minimal ingredients underpinning their disassembly are not understood. Here, we demonstrate that vimentin constantly disassembles through filament breakage without the assistance of any other protein or post-translational modification, contrary to common wisdom. Our findings suggest that the dynamic cytoskeletal steady-states observed in cells could be largely shaped by simple physical effects linked to the reversible association of vimentin subunits, combined with dramatic kinetic trapping effects that hinder network reorganization as soon as the filaments become too dense and too long.

sentences: Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro Development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are urgently needed to combat the coronavirus disease 2019 (COVID-19). Oxysterols, defined as oxidized derivatives of cholesterol, include endogenous (naturally occurring) cholesterol metabolites as well as semi-synthetic oxysterol derivatives. We have previously studied the use of semi-synthetic oxysterol derivatives as drug candidates for inhibition of cancer, fibrosis, and bone regeneration. In this study, we have screened a panel of naturally occurring and semi-synthetic oxysterol derivatives for anti-SARS-CoV-2 activity, using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy186 displayed antiviral activity comparable to natural oxysterols. In addition, related oxysterol analogues Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 M and 99% at 15 M, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fall into a therapeutically relevant range (19 M), based on the dose-dependent curve for antiviral activity in our cell culture infection assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 with disrupting the formation of double membrane vesicles (DMVs), intracellular membrane compartments associated with viral replication. Oxy210 also inhibited the replication of hepatitis C virus, another RNA virus whose replication is associated with DMVs, but not the replication of the DMV-independent hepatitis D virus. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk developing COVID-19.

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Ground tissue circuitry regulates organ complexity in cereal roots Most plant roots have multiple cortex layers that make up the bulk of the organ and play key roles in physiology, such as flood tolerance and symbiosis. However, little is known about the formation of cortical layers outside of the highly reduced anatomy of the model Arabidopsis. Here we use single-cell RNAseq to rapidly generate a cell resolution map of the maize root, revealing an alternative configuration of the tissue formative SHORT-ROOT (SHR) signaling pathway adjacent to an expanded cortex. We show that maize SHR protein is hypermobile, moving at least eight cell layers into the cortex. Higher-order SHR mutants in both maize and Setaria have reduced numbers of cortical layers, showing that the SHR pathway controls expansion of cortical tissue in grasses that sets up anatomical complexity and a host of key traits.

One sentence summarySingle-cell RNA-seq maps the maize root transcriptome uncovering a mechanism that regulates cortex layer number.

sentences: Neuroinflammation plays a critical role in cerebral cavernous malformation disease Background: Cerebral Cavernous Malformations (CCMs) are neurovascular lesions caused by loss-of-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect ~1/200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown. Methods: We use genetic, RNA-seq, histology, flow cytometry, and imaging techniques to report the interaction between CCM-endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CALMN interaction) during the pathogenesis of CCMs in the brain tissue. Results: Expression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP-tagged ribosomes (Aldh1l1-EGFP/Rpl10a) in the presence or absence of CCM lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) identifies a novel gene signature for neuroinflammatory astrogliosis. CCM reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-seq analysis on RNA isolated from brain endothelial cells (BECs) in chronic Pdcd10BECKO mice (CCM-endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM-endothelium was associated with increased expression of Nlrp3 and Il1b. Pharmacological inhibition of NLRP3 significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA caspase-1) in BECs from Pdcd10BECKO in the chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium can trigger the recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-kB activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in an elevated number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-kB inhibition may contribute to detrimental side effects. Conclusions: Our study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-kB activity may contribute to detrimental side effects.

sentences: Frequency Effects on Spelling Error Recognition: An ERP Study Spelling errors are ubiquitous in all writing systems. Most studies exploring spelling errors focused on the phonological plausibility of errors. However, unlike typical pseudohomophones, spelling errors occur in naturally produced written language with variable frequencies. We investigated the time course of recognition of the most frequent orthographic errors in Russian (error in an unstressed vowel at the root) and the effect of word frequency on this process. During ERP recording, 26 native Russian speakers silently read high-frequency correctly spelled words, low-frequency correctly spelled words, high-frequency words with errors, and low-frequency words with errors. The amplitude of P200 was more positive for correctly spelled words than for misspelled words and did not depend on the frequency of the words. Word frequency affected spelling recognition in the later stages of word processing (350-700 ms): high-frequency misspelled words elicited a greater P300 than high-frequency correctly spelled words, and low-frequency misspelled words elicited a greater N400 than low-frequency correctly spelled words. We observe spelling effects in the same time window for both the P300 and N400, which may reflect temporal overlap between mainly categorization processes based on orthographic properties for high-frequency words and phonological processes for low-frequency words. We concluded that two independent pathways can be active simultaneously during spelling recognition: one reflects mainly orthographic processing of high-frequency words and the other is the phonological processing of low-frequency words. Our findings suggest that these pathways are associated with different ERP components. Therefore, our results complement existing reading models and demonstrate that the neuronal underpinnings of spelling error recognition during reading depend on word frequency.

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Emergence and maintenance of different sized actin filaments in a common pool of building blocks Actin is one of the key structural components of the eukaryotic cytoskeleton that regulates cellular architecture and mechanical properties. Dynamic regulation of actin filament length and organization is essential for the control of many physiological processes including cell adhesion, motility and division. While previous studies have mostly focused on the mechanisms controlling the mean length of individual actin filaments, it remains poorly understood how distinct actin filament populations in cells maintain different size using the same set of molecular building blocks. Here we develop a theoretical model for the length regulation of multiple actin filaments by nucleation and growth rate modulation by actin binding proteins in a limiting pool of monomers. We first show that spontaneous nucleation of actin filaments naturally leads to heterogeneities in filament length distribution. We then investigate the effects of filament growth inhibition by capping proteins and growth promotion by formin proteins on filament length distribution. We find that filament length heterogeneity can be increased by growth inhibition, whereas growth promoters do not significantly affect length heterogeneities. Interestingly, a competition between filament growth inhibitors and growth promoters can give rise to bimodal filament length distribution as well as a highly heterogeneous length distribution with large statistical dispersion. We quantitatively predict how heterogeneity in actin filament length can be modulated by tuning F-actin nucleation and growth rates in order to create distinct filament subpopulations with different lengths.

SIGNIFICANCEActin filaments organize into different functional network architectures within eukaryotic cells. To maintain different actin network architectures, actin filaments of different lengths are often required. How cells regulate the length heterogeneity in actin filaments is not well understood. Here we show that the modulation of filament growth rates and nucleation rate by binding partners like formin and capping proteins can regulate the emergent length heterogeneity in the filament population and can create distinct sub-populations with different lengths. Our study sheds light into how actin binding proteins can control various aspects of filament size distribution by tuning their concentration or expression levels in the cell.

sentences: Direct reprogramming of human fibroblasts into insulin-producing cells by transcription factors Direct lineage reprogramming of one somatic cell into another bypassing an intermediate pluripotent state has emerged as an alternative to embryonic or induced pluripotent stem cell differentiation to generate clinically relevant cell types. One cell type of clinical interest is the pancreatic {beta} cell that secretes insulin and whose loss and/or dysfunction leads to diabetes. Generation of functional {beta}-like cells from developmentally related somatic cell types (pancreas, liver, gut) has been achieved via enforced expression of defined sets of transcription factors. However, clinical applicability of these findings is challenging because the starting cell types are not easily obtainable. Skin fibroblasts are accessible and easily manipulated cells that could be a better option, but available studies indicate that their competence to give rise to {beta} cells through similar direct reprogramming approaches is limited. Here, using human skin fibroblasts and a protocol that ensures high and consistent expression of adenovirus-encoded reprogramming factors, we show that the transcription factor cocktail consisting of Pdx1, Ngn3, MafA, Pax4 and Nkx2-2 activates key {beta} cell genes and down-regulates the fibroblast transcriptional program. The converted cells produce insulin and exhibit intracellular calcium responses to glucose and/or membrane depolarization. Furthermore, they secrete insulin in response to glucose in vitro and after transplantation in vivo. These findings demonstrate that transcription factor-mediated direct reprogramming of human fibroblasts is a feasible strategy to generate insulin-producing cells.

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Prostaglandin PGE2 receptor EP4 regulates microglial phagocytosis and increases susceptibility to diet-induced obesity BackgroundIn rodents, susceptibility to diet-induced obesity requires microglial activation, but the molecular components of this pathway remain incompletely defined. Prostaglandin E2 (PGE2) levels increase in the mediobasal hypothalamus during high fat diet (HFD) feeding, and the PGE2 receptor EP4 regulates microglial activation state and phagocytic activity, suggesting a potential role for microglial EP4 signaling in obesity pathogenesis.

MethodMetabolic phenotyping, as assessed by body weight, energy expenditure, glucose, and insulin tolerance, was performed in microglia-specific EP4 knockout (MG-EP4 KO) mice and littermate controls on HFD. Morphological and gene expression analysis of microglia, and a histological survey of microglia-neuron interactions in the arcuate nucleus was performed. Phagocytosis was assessed using in vivo and in vitro pharmacological techniques.

ResultsMicroglial EP4 deletion markedly reduced weight gain and food intake in response to HFD feeding. In correspondence with this lean phenotype, insulin sensitivity was also improved in the HFD-fed MG-EP4 KO mice though glucose tolerance remained surprisingly unaffected. Mechanistically, EP4-deficient microglia showed an attenuated phagocytic state marked by reduced CD68 expression and fewer contacts with POMC neuron soma and processes. These cellular changes observed in the microglial EP4 knockout mice corresponded with an increased density of POMC neurites extending into the paraventricular nucleus.

ConclusionThese findings reveal that microglial EP4 signaling promotes body weight gain and insulin resistance during HFD feeding. Furthermore, the data suggest that curbing microglial phagocytic function may preserve POMC cytoarchitecture and PVN input to limit overconsumption during diet-induced obesity.

sentences: Hormonal contraceptives alter amphetamine place preference and responsivity in the intact female rat. Hormonal contraceptives (HCs) are commonly used among reproductive aged women and alter the physiological state of the user by interfering with endogenous hormone concentrations and their actions on the reproductive tract. As hormones such as estradiol and progesterone modulate the incidence of substance abuse disorders in women, it is important to consider the influence HCs have on the female brain and behavior. This experiment explores how female sex steroid hormonal states associated with the rat estrous cycle, and modulating those states with HCs, influences measures of drug preference and responsivity. First, rats underwent food-light Pavlovian conditioning to measure conditioned orienting, a known predictor of amphetamine (AMP) place preference. Then, rats were conditioned and tested for AMP place preference with either an HC-implant or during estrous cycle stages associated with different ovarian hormone levels (i.e., proestrus (P) or metestrus/diestrus (M/D) while recording ultrasonic vocalizations (USVs) as an index of hedonic responsivity. Because of dopamines (DA) role in modulation of AMP actions, DA cell activity and availability were examined using tyrosine hydroxylase and FOS immunohistochemistry after final AMP challenge. Conditioned orienting did not differ between cycling and HC-implanted. P rats emitted more USVs during conditioning, showed higher AMP place preference throughout testing, and had higher DA cell activity in the substantia nigra compared to M/D and HC-implanted rats. Sex steroid hormone serum concentration and uterine horn thickness predicted some but not all of these measures. This experiment suggests ovarian hormones affect drug preference and responsivity, while providing novel insight into how hormone-altering contraceptives may reduce these measures.

sentences: A high content microscopy screening identifies genes involved in cell width control in Bacillus subtilis How cells control their shape and size is a fundamental question of biology. In most bacteria, cell shape is imposed by the peptidoglycan (PG) polymeric meshwork that surrounds the cell. Thus, bacterial cell morphogenesis results from the coordinated action of the proteins assembling and degrading the PG shell. Remarkably, during steady-state growth, most bacteria maintain a defined shape along generations, suggesting that error-proof mechanisms tightly control the process. In the rod-shaped model for Gram-positive bacteria Bacillus subtilis, the average cell length varies as a function of the growth rate but the cell diameter remains constant throughout the cell cycle and across growth conditions. Here, in an attempt to shed light on the cellular circuits controlling bacterial cell width, we developed a screen to identify genetic determinants of cell width in B. subtilis. Using high-content screening (HCS) fluorescence microscopy and semi-automated measurement of single-cell dimensions, we screened a library of ~ 4000 single knockout mutants. We identified 13 mutations significantly altering cell diameter, in genes that belong to several functional groups. In particular, our results indicate that metabolism plays a major role in cell width control in B. subtilis.

sentences: Intracellular Oxidative Stress Levels are Significantly Associated with the Green Autofluorescence Intensity of Buthionine Sulfoximine-Treated B16-F10 Cells Since oxidative stress is a critical common pathological factor of numerous diseases, it is critical to find biomarkers for non-invasive evaluations of the levels of oxidative stress in the body. Our previous studies have indicated that epidermal green autofluorescence (AF) is a novel biomarker of this type: The oxidative stress inducer buthionine sulfoximine (BSO) can dose-dependently increase the epidermal green AF of mice, with BSO doses being significantly associated with the AF intensity. However, it is necessary to use skin cell cultures to investigate the mechanisms underlying the relationships between BSO and the green AF intensity. In our current study we found that BSO concentration-dependently increased the green AF intensity of B16-F10 cells a skin cell line, with BSO concentrations being significantly associated with the AF intensity. BSO also concentration-dependently increased the intracellular DCF signals an index of ROS levels. The green AF intensity of the cells was also significantly associated with the intracellular ROS levels. Moreover, we found that the green AF intensity was significantly associated with the cell death induced by BSO. Collectively, our study has provided first evidence indicating that the green AF intensity of skin cells is significantly associated with both intracellular ROS levels and cell death of the skin cells exposed to oxidative stress, which has indicated that green AF is a novel biomarker for both oxidative stress and cell death.

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Imprecise recombinant viruses evolve via a fitness-driven, iterative process of polymerase template-switching events. Recombination is a common feature of many positive-strand RNA viruses, playing an important role in virus evolution. However, to date, there is limited understanding of the mechanisms behind the process. Utilising in vitro assays, we have previously shown that the template-switching event of recombination is a random and ubiquitous process that often leads to recombinant viruses with imprecise genomes containing sequence duplications. Subsequently, a process termed resolution, that has yet to be mechanistically studied, removes these duplicated sequences resulting in a virus population of wild type length genomes. Using defined imprecise recombinant viruses together with Oxford Nanopore and Illumina high throughput next generation sequencing technologies we have investigated the process of resolution. We show that genome resolution involves subsequent rounds of template-switching recombination with viral fitness resulting in the survival of a small subset of recombinant genomes. This alters our previously held understanding that recombination and resolution are independent steps of the process, and instead demonstrates that viruses undergo frequent and continuous recombination events over a prolonged period until the fittest viruses, predominantly those with wild type length genomes, dominate the population.

Author SummaryViruses with positive-sense RNA genomes, such as poliovirus, have several mechanisms by which they evolve. One of these is the process of recombination involving the large-scale exchange of genetic information. Recombination occurs during replication when the viral polymerase switches from copying one genome to another. However, the polymerase does not always accurately switch between the two, resulting in sequence duplications or deletions, and genomes that are referred to as imprecise. Over multiple rounds of replication sequence duplications are lost and genomes are resolved to wild type length, but it is unclear how this occurs. Here we used synthetic polioviruses containing defined sequence duplications to determine that the genome population undergoes repeated rounds of recombination until sequence duplications are lost and viruses with precise, wild type length genomes are selected for. This selection is based on the overall fitness of the virus population, with less fit imprecise viruses evolving more quickly. Our study suggests that recombination is a continual process where virus fitness drives the selection of a small subset of recombinant variants. These data are important for understanding how novel viruses evolve via recombination and how this process can be blocked to prevent novel and dangerous pathogens from arising.

sentences: Otolith-based discrimination of cod ecotypes and the effect of growth rate. Otolith shape has previously been used to identify ecotypes within the Icelandic cod (Gadus morhua) stock, using DST profiles to validate the results. Fish otolith shape variation has repeatedly been found to be largely determined by growth rate. To examine the effect of growth rate on the relationship between otolith shape and cod ecotypes (using the Pan I genotype as a proxy for ecotype), 826 archived sagittal otoliths collected over a 52 year sampling period were retrieved, the individual growth rate calculated, and otolith shape described using both Normalized Elliptic Fourier transform and Discrete Wavelet transform. Discriminant functions of otolith shape yielded high ecotype classification success, whether using Fourier or Wavelet descriptors, but only when excluding a heterozygous genotype from the analysis. The otolith shape variability of this genotype lowered the classification success, while otolith shape, in turn, was significantly affected by growth rate and cohort. Growth rate differences previously reported for the ecotypes were present, but were less marked than expected and indeed, growth rate variance attributable to ecotype identity was dwarfed by cohort- and location-related variance in growth. Such a strong effect of growth rate suggests that cod ecotype discrimination based on otolith shape is sensitive to both temporal and spatial variations in growth, which can mask the effect of ecotype-related growth rate differences on otolith shape.

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Evaluation of the effects of library preparation procedure and sample characteristics on the accuracy of metagenomic profiles Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities, or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera(R) Flex kit, perform when applied to metagenomic DNA. We measured each methods ability to produce metagenomic data that accurately represents the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral-associated, mouse-gut-associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each methods performance, indicating that careful consideration may be needed when selecting between methods, especially for low complexity communities. However, cost-effective preparation methods we assessed are generally comparable to the current gold standard Nextera(R) DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies.

IMPORTANCEMetagenomic library preparation methods and sequencing technologies continue to advance rapidly, allowing researchers to characterize microbial communities in previously underexplored environmental samples and systems. However, widely-accepted standardized library preparation methods can be cost-prohibitive. Newly available approaches may be less expensive, but their efficacy in comparison to standardized methods remains unknown. In this study, we compared five different metagenomic library preparation methods. We evaluated each method across a range of microbial communities varying in complexity and quantity of input DNA. Our findings demonstrate the importance of considering sample properties, including community type, composition, and DNA amount, when choosing the most appropriate metagenomic library preparation method.

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Functional coupling between target selection and acquisition in the superior colliculus To survive in unpredictable environments, animals must continuously evaluate their surroundings for behavioral targets, such as food and shelter, and direct their movements to acquire those targets. Although the ability to accurately select and acquire spatial targets depends on a shared network of brain regions, how these processes are linked by neural circuits remains unknown. The superior colliculus (SC) mediates the selection of spatial targets and remains active during orienting movements to acquire targets, which suggests the underexamined possibility that common SC circuits underlie both selection and acquisition processes. Here, we test the hypothesis that SC functional circuitry couples target selection and acquisition using a default motor plan generated by selection-related neuronal activity. Single-unit recordings from intermediate and deep layer SC neurons in male mice performing a spatial choice task demonstrated that choice-predictive neurons, including optogenetically identified GABAergic SC neurons whose activity was causally related to target selection, exhibit increased activity during movement to the target. By strategically recording from both rostral and caudal SC neurons, we also revealed an overall caudal-to-rostral shift in activity as targets were acquired. Finally, we used an attractor model to examine how target selection activity in the SC could generate a rostral shift in activity during target acquisition using only intrinsic SC circuitry. Overall, our results suggest a functional coupling between SC circuits that underlie target selection and acquisition, elucidating a key mechanism for goal-directed behavior.

Significance StatementThe ability to quickly select and acquire spatial targets is essential to animal survival. Neural circuits underlying these processes are shared in an interconnected network of brain regions, however it is unclear how circuits link decision-making processes with motor commands to execute choices. Here, we examine single-unit activity in the superior colliculus (SC) as mice select and acquire spatial targets to test the hypothesis that choice-related activity promotes target acquisition by generating a default motor plan for orienting movements. By demonstrating that choice-predictive neurons increase their firing rates during movement and capturing the dynamics of SC activity with an attractor model of intrinsic SC circuitry, our results support a role for SC circuits in coupling target selection and acquisition.

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The role of petal transpiration in floral humidity generation Floral humidity, an area of elevated humidity in the headspace of flowers, has been detected across angiosperms and may function as a pollinator cue for insect pollinators. It is believed floral humidity is produced predominantly through a combination of evaporation of both liquid nectar and transpirational water loss from the flower. However, the role of transpiration in floral humidity generation has not been tested and is largely inferred by continued humidity production when nectar is removed from flowers. Understanding the extent that transpiration contributes to floral humidity has important implications for understanding the function of floral humidity. We test whether transpiration contributes to the floral humidity generation of two species previously identified to produce elevated floral humidity, Calystegia silvatica and Eschscholzia californica. Floral humidity production of flowers that underwent an antitranspirant treatment, petrolatum gel which blocks transpiration from treated tissues, is compared to flowers that did not receive such treatments. Gel treatments reduced floral humidity production to approximately a third of that produced by untreated flowers in C. silvatica, and half of that in E. californica. This confirms, the previously untested, inferences that transpiration has a large contribution to floral humidity generation and that this contribution may vary between species.

HIGHLIGHTWe confirm, the previously untested, inferences that transpiration has a large contribution to floral humidity generation and show that this contribution may vary between species.

sentences: Fluency shaping increases integration of the command-to-execution and the auditory-to-motor pathways in persistent developmental stuttering Fluency-shaping enhances the speech fluency of persons who stutter, yet underlying conditions and neuroplasticity-related mechanisms are largely unknown. While speech production-related brain activity in stuttering is well studied, it is unclear whether therapy repairs networks of altered sensorimotor integration, imprecise neural timing and sequencing, faulty error monitoring, or insufficient speech planning. Here, we tested the impact of one-year fluency-shaping therapy on resting-state fMRI connectivity within sets of brain regions subserving these speech functions. We analyzed resting-state data of 22 patients who participated in a fluency-shaping program, 18 patients not participating in therapy, and 28 fluent control participants, measured one year apart. Improved fluency was accompanied by an increased synchronization within the sensorimotor integration network. Specifically, two connections were strengthened; the left inferior frontal gyrus showed increased connectivity with the precentral gyrus at the representation of the left laryngeal motor cortex, and the left inferior frontal gyrus showed increased connectivity with the right superior temporal gyrus. Thus, therapy-associated neural remediation was based on a strengthened integration of the command-to-execution pathway together with an increased auditory-to-motor coupling. Since we investigated task-free brain activity, we assume that our findings are not biased to network activity involved in compensation but represent long-term focal neuroplasticity effects.

sentences: Using dual EEG to analyse event-locked changes in child-adult neural connectivity Current approaches typically measure the connectivity between interacting physiological systems as a time-invariant property. This approach obscures crucial information about how connectivity between interacting systems is established and maintained. Here, we describe methods, and present computational algorithms, that will allow researchers to address this deficit. We focus on how two different approaches to measuring connectivity, namely concurrent (e.g., power correlations, phase locking) and sequential (e.g., Granger causality), can be applied to three aspects of the brain signal, namely amplitude, power, and phase. We guide the reader through worked examples using mainly simulated data on how to leverage these methods to measure changes in interbrain connectivity between adults and children/infants relative to events identified within continuous EEG data during a free-flowing naturalistic interaction. For each, we aim to provide a detailed explanation of the interpretation of the analysis and how they can be usefully used when studying early social interactions.

sentences: Lessons on fruiting body morphogenesis from genomes and transcriptomes of Agaricomycetes Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are among the most complex structures produced by fungi. Unlike vegetative hyphae, fruiting bodies grow determinately and follow a genetically encoded developmental program that orchestrates tissue differentiation, growth and sexual sporulation. In spite of more than a century of research, our understanding of the molecular details of fruiting body morphogenesis is limited and a general synthesis on the genetics of this complex process is lacking. In this paper, we aim to comprehensively identify conserved genes related to fruiting body morphogenesis and distill novel functional hypotheses for functionally poorly characterized genes. As a result of this analysis, we report 921 conserved developmentally expressed gene families, only a few dozens of which have previously been reported in fruiting body development. Based on literature data, conserved expression patterns and functional annotations, we provide informed hypotheses on the potential role of these gene families in fruiting body development, yielding the most complete description of molecular processes in fruiting body morphogenesis to date. We discuss genes related to the initiation of fruiting, differentiation, growth, cell surface and cell wall, defense, transcriptional regulation as well as signal transduction. Based on these data we derive a general model of fruiting body development, which includes an early, proliferative phase that is mostly concerned with laying out the mushroom body plan (via cell division and differentiation), and a second phase of growth via cell expansion as well as meiotic events and sporulation. Altogether, our discussions cover 1480 genes of Coprinopsis cinerea, and their orthologs in Agaricus bisporus, Cyclocybe aegerita, Armillaria ostoyae, Auriculariopsis ampla, Laccaria bicolor, Lentinula edodes, Lentinus tigrinus, Mycena kentingensis, Phanerochaete chrysosporium, Pleurotus ostreatus, and Schizophyllum commune, providing functional hypotheses for [~]10% of genes in the genomes of these species. Although experimental evidence for the role of these genes will need to be established in the future, our data provide a roadmap for guiding functional analyses of fruiting related genes in the Agaricomycetes. We anticipate that the gene compendium presented here, combined with developments in functional genomics approaches will contribute to uncovering the genetic bases of one of the most spectacular multicellular developmental processes in fungi.

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Dynamic in situ confinement triggers ligand-free neuropeptide receptor signaling Membrane receptors are central to cell-cell communication. Receptor clustering at the plasma membrane modulates physiological responses, and microscale receptor organization is critical for downstream signaling. Spatially restricted cluster formation of the neuropeptide Y2 hormone receptor (Y2R) was observed in vivo; however, the relevance of this confinement is not fully understood. Here, we controlled Y2R clustering in situ by a multivalent chelator nanotool in prestructured matrices. Fast Y2R enrichment in microscale arrays triggered a ligand-independent downstream signaling determined by an increase in cytosolic calcium, cell spreading and migration. We reveal that ligand-independent signaling by confinement differs from ligand-induced activation in the recruitment of arrestin-3 as downstream effector, which was recruited to confined regions only in presence of the ligand. The employed multivalent nanotool facilitated a dynamic receptor enrichment with high exchange in the confined regions, comparable to microscale condensates. This concept enables in situ organization of membrane receptors and the exploration of ligand-independent receptor signaling.

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sentences: Urban fox squirrels exhibit tolerance to humans but respond to stimuli from natural predators Animals in urban areas that experience frequent exposure to humans often behave differently than those in less urban areas, such as exhibiting less vigilance or anti-predator behavior. These behavioral shifts may be an adaptive response to urbanization, but it may be costly if animals in urban areas also exhibit reduced anti-predator behavior in the presence of natural predators. In trials with only a human observer as the stimulus, urban squirrels exhibited reduced vigilance and anti-predator behavior compared to those in less urban areas. Next, we exposed squirrels in multiple urban and less urban sites to acoustic playbacks of a control stimulus (non-predatory bird calls), a natural predator (hawk), and dogs and recorded their vigilance and three different anti-predator behaviors when a human approached them while either broadcasting one of these three playbacks or no playbacks at all. Squirrels at urban sites also did not differ in their behavioral responses to the playbacks from possible predators (hawks or dogs) when they were compared to those at less urban sites exposed to these playbacks. Urban squirrels also exhibited increased vigilance and anti-predator behavior when exposed to a human paired with hawk playbacks compared to the control playbacks. Together, our results indicate that urban squirrels did perceive and assess risk to the natural predator appropriately despite exhibiting increased tolerance to humans. These results provide little support for the hypothesis that increased tolerance to humans causes animals to lose their fear of natural predators.

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Cilia stimulatory and antibacterial activities of bitter receptor agonist diphenhydramine: insights into potential complimentary strategies for CF nasal infections BACKGROUNDBitter compounds increase ciliary beating and nitric oxide (NO) production in nasal epithelial cells through T2Rs in motile cilia. We examined expression of cilia T2Rs and both host and bacterial responses to T2R14 agonist diphenhydramine.

METHODUsing cultured human nasal epithelial cells grown at air liquid interface, we measured expression of T2Rs via qPCR. We measured effects of diphenhydramine on ciliary beat frequency via high-speed imaging and nitric oxide production via fluorescent dye DAF-FM. We measured effects of diphenhydramine on growth of lab and clinical strains of Pseudomonas aeruginosa. We measured biofilm formation of P. aeruginosa using crystal violet staining and surface attachment of P. aeruginosa to cystic fibrosis bronchial epithelial (CBFE41o-) cells using CFU counting.

RESULTST2R expression increased with mucocilliary differentiation and did not vary between CF and non-CF ALIs. Treatment with P. aeruginosa flagellin decreased expression of diphenhydramine-responsive T2R14 and 40, among other isoforms. Diphenhydramine increased both NO and CBF. Increases in CBF were disrupted after flagellin treatment. Diphenhydramine impaired growth, biofilm production, and surface attachment of P. aeruginosa.

CONCLUSIONST2R expression is similar between normal and CF cells but decreases with flagellin treatment. Utilizing T2R agonists as therapeutics within the context of CF, P. aeruginosa infections may require co-treatment with anti-inflammatories to prevent the reduction of T2R expression with TLR activation. T2R agonist diphenhydramine increases NO production and CBF while also decreasing bacterial growth and biofilm production, and thus diphenhydramine or derivate compounds may have potential clinical usefulness in CF infections as a topical therapy.

HIGHLIGHTSO_LIT2R14 agonist diphenhydramine increases nitric oxide production and cilia beating
C_LIO_LIFlagellin decreases T2R14 expression in primary airway epithelial cells
C_LIO_LIT2R14 agonist Diphenhydramine inhibits Pseudomonas growth and biofilm formation
C_LI

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Graphene oxide/silver nanoparticle ink formulations rapidly inhibit influenza A virus and OC43 coronavirus infection in vitro Respiratory tract infections present a significant risk to the human population, both through seasonal circulation and novel introductions with pandemic potential. There is a strong need for antiviral compounds with broad antimicrobial activity that can be coated onto filtration systems and personal protective equipment to augment their ability to remove infectious particles from the environment. Graphene oxide and silver nanoparticles are both materials with documented antimicrobial properties. Here, we tested the in vitro antiviral properties of several graphene oxide-silver nanoparticle composite materials, which were prepared through three different methods: reduction with silver salt, direct addition of silver nanospheres, and direct addition of silver nanospheres to thiolized graphene. These materials were tested over short time scales for their antiviral activity against two enveloped RNA viruses, influenza A virus and OC43 coronavirus, by performing viral plaque assays after exposure of the viruses to each material. It was found that the graphene oxide - silver nanoparticle materials generated by direct addition of the silver nanospheres were able to completely inhibit plaque formation by both viruses within one minute of exposure. Materials generated by the other two methods had varying levels of efficacy against influenza A virus. These studies indicate that graphene oxide-silver nanoparticle composite materials can rapidly neutralize RNA viruses and demonstrate their potential for use in a wide range of applications.

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sentences: In Poetry, if Meter has to Help Memory, it Takes its Time To test the idea that poetic meter emerged as a cognitive schema to aid verbal memory, we have focused on classical Italian poetry and on its three basic components of meter: rhyme, accent and verse length. Meaningless poems were generated by introducing prosody-invariant non-words into passages from Dantes Divina Commedia and Ariostos Orlando Furioso, which were then further manipulated by selectively ablating rhymes, modifying accent patterns or altering the number of syllables. The resulting four versions of each non-poem were presented in a fully balanced design to cohorts of high school educated Italian native speakers, who were then asked to retrieve 3 target non-words. Surprisingly, we found that the integrity of Dantes meter has no significant effect on memory performance. With passages derived from Ariosto, instead, removing each component downgrades memory by an amount proportional to its contribution to perceived metric plausibility, with rhymes having the strongest effects, followed by accents and then by verse length. Counterintuitively, the fully metric versions required longer reaction times, implying that activating metric schemata involves a cognitive cost. Within schema theories, this finding provides evidence for high-level interactions between procedural and episodic memory.

sentences: A Mean Shift Algorithm for Drift Correction in Localization Microscopy Single molecule localization microscopy (SMLM) techniques transcend the diffraction limit of visible light by localizing isolated emitters sampled stochastically. This time-lapse imaging necessitates long acquisition times, over which sample drift can become large relative to the localization precision. Here we present a novel, efficient, and robust method for estimating drift using a simple peak-finding algorithm based on mean shifts that is effective for SMLM in 2 or 3 dimensions.

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Reconstructed signaling and regulatory networks identify potential drugs for SARS-CoV-2 infection Several molecular datasets have been recently compiled to characterize the activity of SARS-CoV-2 within human cells. Here we extend computational methods to integrate several different types of sequence, functional and interaction data to reconstruct networks and pathways activated by the virus in host cells. We identify key proteins in these networks and further intersect them with genes differentially expressed at conditions that are known to impact viral activity. Several of the top ranked genes do not directly interact with virus proteins. We experimentally tested treatments for a number of the predicted targets. We show that blocking one of the predicted indirect targets significantly reduces viral loads in stem cell-derived alveolar epithelial type II cells (iAT2s).

Software and interactive visualizationhttps://github.com/phoenixding/sdremsc

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Molecular Architecture of the Chikungunya Virus Replication Complex All positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) for viral RNA synthesis in virus-infected cells. However, how these multi-component RCs assemble and function in synthesizing, processing, and transporting viral RNAs to the cytosol remains poorly defined. Here, we determined both the structure of the core RNA replicase of chikungunya virus (family Togaviridae) at a near-atomic level and the native RC architecture in its cellular context at the subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase (nsP1+2+4), the viral RNA-dependent RNA polymerase nsP4, in complex with nsP2 helicase-protease, was found to co-fold with the membrane-anchored nsP1 RNA-capping dodecameric ring and is located asymmetrically within nsP1 central pore. This complex forms the minimal core RNA replicase, while the addition of a large cytoplasmic ring next to the C-terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses. At last, our study will direct the needed development of antiviral therapies targeting RCs of pathogenic viruses.

SummaryCryoEM structure of the chikungunya virus replication complex reveals a multicomponent RNA synthesis nanomachine embedded in the plasma membrane of the host cell.

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Dietary Exposure to Antibiotic Residues Facilitates Metabolic Disorder by Altering the Gut Microbiota and Bile Acid Composition Antibiotics used as growth promoters in livestock and animal husbandry can be detected in animal-derived food. Epidemiological studies have implicated that exposure to these antibiotic residues in food may be associated to childhood obesity. Herein, the effect of exposure to residual dose of tylosin--an antibiotic growth promoter--on host metabolism and gut microbiota was explored in vivo. Theoretical maximal daily intake (TMDI) doses of tylosin were found to facilitate high-fat diet-induced obesity, induce insulin resistance, and perturb the composition of gut microbiota in mice. The obesity-related phenotypes were transferrable to germ-free recipient mice, indicating that the effects of TMDI dose of tylosin on obesity and insulin resistance occurred mainly via alteration of the gut microbiota. Tylosin TMDI exposure restricted to early life, which is the critical period of gut microbiota development, altered the abundance of specific bacteria related to host metabolic homeostasis later in life. Moreover, early-life exposure to tylosin TMDI was sufficient to modify the ratio of primary to secondary bile acids, thereby inducing lasting metabolic consequences via the downstream FGF15 signaling pathway. Altogether, these findings demonstrate that exposure to very low dose of antibiotic residues, whether continuously or in early life, can exert long-lasting effects on host metabolism by altering gut microbiota and its metabolites.

ImportanceEvidence has indicated that chronic exposure to antibiotic residues in food could contribute to obesity. However, few studies have investigated the effect of chronic exposure to very low-dose antibiotic residue in food (~1000-fold lower than the therapeutic dose) on gut microbiota and host metabolism. Our study demonstrates that even with limited exposure in early life, a residual dose of tylosin causes lasting metabolic disturbances through altering gut microbiota and its metabolites. Our findings reveal that the gut microbiota is susceptible to previously ignored environmental factors.

sentences: Micromechanical Characterisation of 3D Bioprinted neural cell models using Brillouin Microscopy Biofabrication of artificial 3D in vitro neural cell models that closely mimic the central nervous system (CNS) is an emerging field of research with applications from fundamental biology to regenerative medicine, and far reaching benefits for the economy, healthcare and the ethical use of animals. The micromechanical properties of such models are an important factor dictating the success of modelling outcomes in relation to accurate reproduction of the processes in native tissues. Characterising the micromechanical properties of such models non-destructively and over a prolonged span of time, however, are key challenges. Brillouin microscopy (BM) could provide a solution to this problem since this technology is non-invasive, label-free and is capable of microscale 3D imaging. In this work, the viscoelasticity of 3D bioprinted neural cell models consisting of NG 108-15 neuronal cells and GelMA hydrogels of various concentrations were investigated using BM. We demonstrate changes in the micro- and macro-scale mechanical properties of these models over a 7 day period, in which the hydrogel component of the model are found to soften as the cells grow, multiply and form stiffer spheroid-type structures. These findings signify the necessity to resolve in microscopic detail the mechanics of in vitro 3D tissue models and suggest Brillouin microscopy to be a suitable technology to bridge this gap.

sentences: Transcriptome signature of cell viability predicts drug response and drug interaction for Tuberculosis The treatment of tuberculosis (TB), which kills 1.8 million each year, remains difficult, especially with the emergence of multidrug resistant strains of Mycobacterium tuberculosis (Mtb). While there is an urgent need for new drug regimens to treat TB, the process of drug evaluation is slow and inefficient owing to the slow growth rate of the pathogen, the complexity of performing bacteriologic assays in a high-containment facility, and the context-dependent variability in drug sensitivity of the pathogen. Here, we report the development of "DRonA" and "MLSynergy", algorithms to perform rapid drug response assays and predict response of Mtb to novel drug combinations. Using a novel transcriptome signature for cell viability, DRonA accurately detects bacterial killing by diverse mechanisms in broth culture, macrophage infection and patient sputum, providing an efficient, and more sensitive alternative to time- and resource-intensive bacteriologic assays. Further, MLSynergy builds on DRonA to predict novel synergistic and antagonistic multi-drug combinations using transcriptomes of Mtb treated with single drugs. Together DRonA and MLSynergy represent a generalizable framework for rapid monitoring of drug effects in host-relevant contexts and accelerate the discovery of efficacious high-order drug combinations.

sentences: Stable Potassium Isotopes (41K/39K) Track Transcellular and Paracellular Potassium Transport in Biological Systems Here we present measurements of the stable isotope ratios of potassium (41K/39K) in three biological systems. We show that the ratio of 41K to 39K varies systematically: between the single-celled green alga Chlamydomonas reinhardtii and growth medium; between muscles of both euryhaline and stenohaline marine teleosts and seawater; and between blood plasma and red blood cells, muscles, cerebrospinal fluid, brain tissues, and urine in the terrestrial mammal Rattus norvegicus. Considered in the context of our current understanding of K+ transport in these biological systems, our results provide evidence that the fractionation of K isotopes depends on transport pathway and transmembrane transport machinery: K+ channels and paracellular transport through tight-junctions favor 39K whereas K+ pumps and co-transporters exhibit less isotopic fractionation. These results indicate that stable K isotopes can provide unique quantitative insights into the machinery and dynamics of K+ homeostasis in biological systems.

sentences: R7 photoreceptor axon targeting depends on the relative levels of lost and found expression in R7 and its synaptic partners As neural circuits form, growing processes select the correct synaptic partners through interactions between cell surface proteins. The presence of such proteins on two neuronal processes may lead to either adhesion or repulsion; however, the consequences of mismatched expression have rarely been explored. Here we show that the Drosophila CUB-LDL protein Lost and found (Loaf) is required in the UV-sensitive R7 photoreceptor for normal axon targeting only when Loaf is also present in its synaptic partners. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or expressing it in their postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more cell surface proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections.

sentences: Structural and functional characteristics of SARS-CoV-2 Omicron subvariant BA.2 spike The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. We have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and animal model with previously prevalent variants. BA.2 S can fuse membranes more efficiently than Omicron BA.1, mainly due to lack of a BA.1-specific mutation that may retard the receptor engagement, but still less efficiently than other variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility for the Omicron subvariants.

sentences: Genomic databanks and targeted assays help characterise domestic mosquito incursions Biosecurity strategies aiming to restrict the spread of invasive pests can benefit from knowing where new incursions have come from. This knowledge can be acquired using genomic databanks, where genetic variation in incursion samples is compared against variation in reference samples. Here, we use genomic databanks to investigate domestic incursions of two mosquito species in Australia. We used a deep learning method to trace a 2021 invasion of Aedes aegypti in Tennant Creek, Northern Territory, to Townsville, Queensland, and to trace Ae. albopictus incursions between several invaded and uninvaded islands in the Torres Strait. We observed high precision of tracing despite 30-70 generations separating incursions and source samples. Targeted assays also improved precision, in this case by comparing Wolbachia infection data to mitochondrial DNA variation in Ae. aegypti. For Ae. albopictus, incursions detected in 2021 were traced to different islands than incursions detected in 2019. Patterns of relatedness and inbreeding indicated that Tennant Creek was likely invaded by Ae. aegypti from one family, whereas Torres Strait incursions involved distinct kinship groups. Our results highlight the value of repeated tracing with genomic databanks that remain informative over years, and demonstrate how additional targeted assays (e.g. Wolbachia) can improve inferences.

sentences: State Dependent Coupling of Hippocampal Oscillations Oscillations occurring simultaneously in a given area represent a physiological unit of brain states. They allow for temporal segmentation of spikes and support distinct behaviors. To establish how multiple oscillatory components co-varies simultaneously and influence neuronal firing during sleep and wakefulness, we describe a multi-variate analytical framework for constructing the state space of hippocampal oscillations. Examining the co-occurrence patterns of oscillations on the state space, across species, uncovered the presence of network constraints and distinct set of cross-frequency interactions during wakefulness as compared to sleep. We demonstrated how the state space can be used as a canvas to map the neural firing and found that distinct neurons during navigation were tuned to different set of simultaneously occurring oscillations during sleep. This multivariate analytical framework provides a window to move beyond classical bivariate pipelines, for investigating oscillations and neuronal firing, thereby allowing to factor-in the complexity of oscillation-population interactions.

sentences: The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology Nab2 encodes a conserved polyadenosine RNA-binding protein (RBP) with broad roles in post-transcriptional regulation, including in poly(A) RNA export, poly(A) tail length control, transcription termination, and mRNA splicing. Mutation of the Nab2 human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability, but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part because no comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships likely remain unidentified. Here we present evidence that Drosophila melanogaster Nab2 interacts with the RBP Ataxin-2 (Atx2), a neuronal translational regulator, and implicate these proteins in coordinate regulation of neuronal morphology and adult viability. We then present the first high-throughput identifications of Nab2- and Atx2-associated RNAs in Drosophila brain neurons using an RNA immunoprecipitation-sequencing (RIP-Seq) approach. Critically, the RNA interactomes of each RBP overlap, and Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with a small fraction of all polyadenylated RNAs. The identities of shared associated transcripts (e.g. drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g. Arpc2, tea, respectively) promise insight into neuronal functions of and interactions between each RBP. Significantly, Nab2-associated RNAs are overrepresented for internal A-rich motifs, suggesting these sequences may partially mediate Nab2 target selection. Taken together, these data demonstrate that Nab2 opposingly regulates neuronal morphology and shares associated neuronal RNAs with Atx2, and that Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest.

sentences: Attentional Modulations of Alpha Power Are Sensitive to the Task-relevance of Auditory Spatial Information The topographical distribution of oscillatory power in the alpha band is known to vary depending on the current focus of spatial attention. Here, we investigated to what extend univariate and multivariate measures of post-stimulus alpha power are sensitive to the required spatial specificity of a task. To this end, we varied the perceptual load and the spatial demand in an auditory search paradigm. A centrally presented sound at the beginning of each trial indicated the to-be-localized target sound. This spatially unspecific pre-cue was followed by a sound array, containing either two (low perceptual load) or four (high perceptual load) simultaneously presented lateralized sound stimuli. In separate task blocks, participants were instructed either to report whether the target was located on the left or the right side of the sound array (low spatial demand) or to indicate the exact target location (high spatial demand). Univariate alpha lateralization magnitude was neither affected by perceptual load nor by spatial demand. However, an analysis of onset latencies revealed that alpha lateralization emerged earlier in low (vs. high) perceptual load trials as well as in low (vs. high) spatial demand trials. Finally, we trained a classifier to decode the specific target location based on the multivariate alpha power scalp topography. A comparison of decoding accuracy in the low and high spatial demand conditions suggests that the amount of spatial information present in the scalp distribution of alpha-band power increases as the task demands a higher degree of spatial specificity. Altogether, the results offer new insights into how the dynamic adaption of alpha-band oscillations in response to changing task demands is associated with post-stimulus attentional processing.

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In silico analysis of predicted differential MHC binding and CD8+ T-cell immune escape of SARS-CoV-2 B.1.1.529 variant mutant epitopes IntroductionThe B.1.1.529 (Omicron) SARS-CoV-2 variant has raised global concerns due to its high number of mutations and its rapid spread. It is of major importance to understand the impact of this variant on the acquired and induced immunity. Several preliminary studies have reported the impact of antibody binding and to this date, there are few studies on Omicrons CD8+ T-cell immune escape.

MethodsWe first assessed the impact of Omicron and B.1.617.2 (Delta) variant mutations on the SARS-CoV-2 spike epitopes submitted to the Immune Epitope Database (IEDB) with positive out-come on MHC ligand or T-cell assays (n=411). From those epitopes modified by a mutation, we found the corresponding homologous epitopes in Omicron and Delta. We then ran the netMHCpan computational MHC binding prediction on the pairs of IEDB epitopes and matching homologous epitopes over top 5 MHC I alleles on some selected populations. Lastly, we applied a Fisher test to find mutations enriched for homologous epitopes with decreased predicted binding affinity.

ResultsWe found 31 and 78 IEDB epitopes modified by Delta and Omicron mutations, respectively. The IEDB spike protein epitopes redundantly cover the protein sequence. The WT pMHC with a strong predicted binding tend to have homologous mutated pMHC with decreased binding. A similar trend is observed in Delta over all HLA genes, while in Omicron only for HLA-B and HLA-C. Finally, we obtained one and seven mutations enriched for homologous mutated pMHC with decreased MHC binding affinity in Delta and Omicron, respectively. Three of the Omicron mutations, VYY143-145del, K417N and Y505H, are replacing an aromatic or large amino acid, which are reported to be enriched in immunogenic epitopes. K417N is common with Beta variants, while Y505H and VYY143-145del are novel Omicron mutations.

ConclusionIn summary, pMHC with Delta and Omicron mutations show decreased MHC binding affinity, which results in a trend specific to SARS-CoV-2 variants. Such epitopes may decrease overall presentation on different HLA alleles suggesting evasion from CD8+ T-cell responses in specific HLA alleles. However, our results show B.1.1.529 (Omicron) will not totally evade the immune system through a CD8+ immune escape mechanism. Yet, we identified mutations in B.1.1.529 (Omicron) introducing amino acids associated with increased immunogenicity.

AvailabilityAll the code and results from this study are available at https://github.com/TRON-bioinformatics/omicron-analysis.

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Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation, to promote viral infection and symptom development in N. benthamiana Phytohormone gibberellin (GA) is an important plant signaling molecule that regulates plant growth and defense against abiotic and biotic stresses. To date, the molecular mechanism of the plant responses to viral infection mediated by GA is still undetermined. DELLA is a repressor of GA signaling and is recognized by the F-box protein, a component of the SCFSLY1/GID2 complex. The recognized DELLA is degraded by the ubiquitin-26S proteasome, leading to the activation of the GA signaling. Here, we report that ageratum leaf curl Sichuan virus (ALCScV)-infected N. benthamiana plants showed dwarfing symptom and abnormal flower development. The infection of ALCScV alters the expressions of GA pathway-related genes and decreases the content of endogenous GA significantly in N. benthamiana. Further, ALCScV-encoded C4 protein interacts with the DELLA protein NbGAI, and interferes with the interaction between NbGAI and NbGID2 to prevent the degradation of NbGAI, leading to the inhibition of the GA signaling pathway. Silencing of NbGAI or exogenous GA3 treatment significantly reduces viral accumulation and disease symptoms in N. benthamiana plants. The same results were proved by the experiments with C4 protein encoded by tobacco curly shoot virus (TbCSV). Therefore, we propose a novel mechanism of geminivirus C4 proteins controling virus infection and disease symptom development through interfering GA signaling pathway.

Author SummaryGibberellins (GAs) are plant hormones that are essential for many developmental processes in plants. It has indicated that plant virus infection can induce abnormal flower development and influence GA pathway resulting the plant dwarfing symptom, but the underlying mechanisms is still not well described. Here, we demonstrate that geminivirus - encoded C4 protein regulates the GA signaling pathway to promote viral accumulation and disease symptom development. Through directly interacting with NbGAI, the C4 protein interferes with the interaction between NbGAI and NbGID2, which inhibits the degradation of NbGAI. As a result, the GA signaling pathway is blocked, and the infected plants display symptoms of typical dwarfing and delayed flowering. Our results reveal a novel mechanism by which geminivirus C4 proteins influence viral pathogenicity via interfering the GA signaling pathway, and provide new insights into the interaction between virus and host.

sentences: Musculoskeletal modelling of sprawling and parasagittal forelimbs provides insight into synapsid postural transition The sprawling-parasagittal postural shift was a major transition during synapsid evolution and is considered key to mammalian ecological diversity. Despite a good fossil record, debate remains over when the shift to parasagittal posture occurred, primarily due to limited comparative biomechanical data on extant species. Here, we built forelimb musculoskeletal models of three extant taxa that bracket the sprawling-parasagittal transition: a tegu lizard, an echidna, and an opossum. We measured shoulder joint range of motion (ROM) about all three degrees of rotational freedom and characterized shoulder muscle moment arms (MMAs) across the entire pose space. Our results show that both the opossum and the tegu had high shoulder joint ROM, and both were substantially higher than the echidna. However, the opossum occupied a distinct region of pose space characterized by high humeral retraction angles. There are clear interspecific differences in MMAs related to posture, with the sprawling tegu and echidna emphasizing humeral depression, and the parasagittal opossum emphasizing humeral elevation. There are also notable differences between our sprawling taxa, with the echidna possessing much greater moment arms for humeral pronation than the tegu. We demonstrate clear functional variation between locomotor grades and use these data to hypothesize major shifts in forelimb function and posture along the mammalian stem.

sentences: Divining Deamidation and Isomerization in Therapeutic Proteins: Effect of Neighboring Residue Deamidation of asparagine (ASN) and isomerization of aspartic acid (ASP) residues are among the most commonly observed spontaneous post-translational modifications (PTMs) in proteins. Understanding and predicting a protein sequences propensity for such PTMs can help expedite protein therapeutic discovery and development. In this study, we utilized proton-affinity calculations with semi-empirical quantum mechanics (QM) and {micro}s long equilibrium molecular dynamics (MD) simulations to investigate mechanistic roles of structure and chemical environment in dictating spontaneous degradation of asparagine and aspartic acid residues in 131 clinical-stage therapeutic antibodies. Backbone secondary structure, side-chain rotamer conformation and solvent accessibility were found as three key molecular indicators of ASP isomerization and ASN deamidation. Comparative analysis of backbone dihedral angles along with N-H proton affinity calculations provides a mechanistic explanation for the strong influence of the identity of the n+1 residue on the rate of ASP/ASN degradation. With these findings, we propose a minimalistic physics-based classification model that can be leveraged to predict deamidation and isomerization propensity of therapeutic proteins.

sentences: The rapid, massive growth of COVID-19 authors in the scientific literature We examined the extent to which the scientific workforce in different fields was engaged in publishing COVID-19-related papers. According to Scopus (data cut, August 1, 2021), 210,183 COVID-19-related publications included 720,801 unique authors, of which 360,005 authors had published at least 5 full papers in their career and 23,520 authors were at the top 2% of their scientific subfield based on a career-long composite citation indicator. The growth of COVID-19 authors was far more rapid and massive compared with cohorts of authors historically publishing on H1N1, Zika, Ebola, HIV/AIDS and tuberculosis. All 174 scientific subfields had some specialists who had published on COVID-19. In 109 of the 174 subfields of science, at least one in ten active, influential (top-2% composite citation indicator) authors in the subfield had authored something on COVID-19. 52 hyper-prolific authors had already at least 60 (and up to 227) COVID-19 publications each. Among the 300 authors with the highest composite citation indicator for their COVID-19 publications, most common countries were USA (n=67), China (n=52), UK (n=32), and Italy (n=18). The rapid and massive involvement of the scientific workforce in COVID-19-related work is unprecedented and creates opportunities and challenges. There is evidence for hyper-prolific productivity.

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Characterization of the adult zebrafish electrocardiogram BackgroundThe use of zebrafish to explore cardiac physiology has been widely adopted within the scientific community. Whether this animal model can be used to determine drug cardiac toxicity via electrocardiogram (ECG) analysis, is still an ongoing question. Several reports indicate that the recording configuration severely affects the ECG waveforms and its derived-parameters, emphasizing the need for improved characterization.

MethodsECGs were recorded from adult zebrafish hearts in 3 different configurations (unexposed heart, exposed heart and extracted heart) to identify the most reliable method to explore ECG recordings at baseline and in response to commonly used clinical therapies.

ResultsWe found that the exposed heart configuration provided the most reliable and reproducible ECG recordings of waveforms and intervals. We were unable to determine T-wave morphology in unexposed hearts. In extracted hearts, ECG intervals were lengthened and P-waves were unstable. However, in the exposed heart configuration, we were able to reliably record ECGs and subsequently establish the QT-RR relationship (Holzgrefe correction) in response to changes in heart rate.

ConclusionsThe exposed heart configuration appears to be the most reliable technique to record ECGs in adult zebrafish. In this configuration, the QT-RR relationship, an important parameter in cardiac toxicity evaluation, can be determined using the Holzgrefe correction.

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Selective whole genome amplification as a tool to enrich specimens with low Treponema pallidum genomic DNA copies for whole genome sequencing Downstream next generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA using the NEBNext(R) Microbiome DNA Enrichment Kit followed by MDA with the REPLI-g Single Cell Kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/l of sample for SWGA and >129 genomic copies/l for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now.

ImportanceSyphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions, and if left untreated, can progress through multiple stages with varied clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past two decades, WGS data is needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of a SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

sentences: Rampant tooth loss across 200 million years of frog evolution Teeth have been broadly maintained across most clades of vertebrates but have been lost completely at least once in actinopterygian fishes and several times in amniotes. Using phenotypic data collected from over 500 genera via micro-computed tomography, we provide the first rigorous assessment of the evolutionary history of dentition across all major lineages of amphibians. We demonstrate that dentition is invariably present in caecilians and salamanders, but teeth have been lost completely more than 20 times in frogs, a much higher occurrence of edentulism than in any other vertebrate group. The repeated loss of teeth in anurans is associated with a specialized diet of small invertebrate prey as well as shortening of the lower jaw, but it is not correlated with a reduction in body size. Frogs provide an unparalleled opportunity for investigating the molecular and developmental mechanisms of convergent tooth loss on a large phylogenetic scale.

sentences: Demography, education, and research trends in the interdisciplinary field of disease ecology Micro- and macroparasites are a leading cause of mortality for humans, animals, and plants, and there is great need to understand their origins, transmission dynamics, and impacts. Disease ecology formed as an interdisciplinary field in the 1970s to fill this need and has recently rapidly grown in size and influence. Because interdisciplinary fields integrate diverse scientific expertise and training experiences, understanding their composition and research priorities is often difficult. Here, for the first time, we quantify the composition and educational experiences of a subset of disease ecology practitioners and identify topical trends in published research. We combined a large survey of self-declared disease ecologists with a literature synthesis involving machine-learning topic detection of over 18,500 disease ecology research articles. The number of graduate degrees earned by disease ecology practitioners has grown dramatically since the early 2000s. Similar to other science fields, we show that practitioners in disease ecology have diversified in the last decade in terms of gender identity and institution, with weaker diversification in race and ethnicity. Topic detection analysis revealed how the frequency of publications on certain topics have declined (e.g., HIV, serology), increased (e.g., the dilution effect, infectious disease in bats), remained relatively common (e.g., malaria ecology, influenza, vaccine research and development), or have consistently remained relatively infrequent (e.g., theoretical models, field experiments). Other topics, such as climate change, superspreading, emerging infectious diseases, and network analyses, have recently come to prominence. This study helps identify the major themes of disease ecology and demonstrates how publication frequency corresponds to emergent health and environmental threats. More broadly, our approach provides a framework to examine the composition and publication trends of other major research fields that cross traditional disciplinary boundaries.

sentences: The Point Mutations in SARS-CoV-2 Variants Induce Long-Range Dynamical Perturbations in Neutralizing Antibodies Monoclonal antibodies are emerging as a viable treatment for the coronavirus disease 19 (COVID-19). However, newly evolved variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can reduce the efficacy of currently available antibodies and can diminish vaccine-induced immunity. Here, we demonstrate that the microscopic dynamics of neutralizing monoclonal antibodies can be profoundly modified by the mutations present in the spike proteins of the SARS-COV-2 variants currently circulating in the world population. The dynamical perturbations within the antibody structure, which alter the thermodynamics of antigen recognition, are diverse and can depend both on the nature of the antibody and on the spatial location of the spike mutation. The correlation between the motion of the antibody and that of the spike receptor binding domain (RBD) can also be changed, modulating binding affinity. Using protein-graph-connectivity networks, we delineated the mutant-induced modifications in the information-flow along allosteric pathway throughout the antibody. Changes in the collective dynamics were spatially distributed both locally and across long-range distances within the antibody. On the receptor side, we identified an anchor-like structural element that prevents the detachment of the antibodies; individual mutations there can significantly affect the antibody binding propensity. Our study provides insight into how virus neutralization by monoclonal antibodies can be impacted by local mutations in the epitope via a change in dynamics. This realization adds a new layer of sophistication to the efforts for rational design of monoclonal antibodies against new variants of SARS-CoV2, taking the allostery in the antibody into consideration.

sentences: RNA-Seq is not required to determine stable reference genes for qPCR normalization Assessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes. Although numerous statistical approaches have been proposed to determine the best reference genes, they can give rise to conflicting results depending on experimental conditions. Hence, recent studies propose the use of RNA-Seq to identify stable genes followed by the application of different statistical approaches to determine the best set of reference genes for qPCR data normalization. In this study, we demonstrate that the statistical approach to determine the best reference genes from randomly selected candidates is more important than the preselection of  stable candidates from RNA-Seq data. Using a qPCR data normalization workflow that we have previously established; we show that qPCR data normalization using randomly chosen conventional reference genes renders the same results as stable reference genes selected from RNA-Seq data. We validated these observations in two distinct cross-sectional experimental conditions involving human iPSC derived microglial cells and mouse sciatic nerves. These results taken together show that given a robust statistical approach for reference gene selection, stable genes selected from RNA-Seq data do not offer any significant advantage over commonly used reference genes for normalizing qPCR assays.

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Pili Expression in Geobacter sulfurreducens Lacking the Putative Gene for the PilB Pilus Assembly Motor Multiple lines of evidence suggest that electrically conductive pili (e-pili) are an important conduit for long-range electron transport in Geobacter sulfurreducens, a common model microbe for the study of extracellular electron transport mechanisms. One strategy to study the function of e-pili has been to delete the gene for PilB, the pilus assembly motor protein, in order to prevent e-pili expression. However, we found that e-pili are still expressed after the gene for PilB is deleted. Conducting probe atomic force microscopy revealed filaments with the same diameter and similar current-voltage response as e-pili harvested from wild-type G. sulfurreducens or when e-pili are heterologously expressed from the G. sulfurreducens pilin gene in E. coli. Immunogold labeling demonstrated that a G. sulfurreducens strain expressing e-pili with a His-tag continued to express His-tag labelled e-pili when the PilB gene was deleted. Strains with the PilB gene deleted produced maximum current densities comparable to wild-type controls. These results demonstrate that deleting the gene for PilB is not an appropriate strategy for constructing strains of G. sulfurreducens without e-pili, necessitating a reinterpretation of the results of previous studies that have employed this approach.

ImportanceGeobacter sulfurreducens is a model microbe for the study of biogeochemically and technologically significant processes such as the reduction of Fe(III) oxides in soils and sediments; bioelectrochemical applications that produce electric current from waste organic matter or drive useful processes with the consumption of renewable electricity; direct interspecies electron transfer in anaerobic digestors and methanogenic soils and sediments; and metal corrosion. The phenotypes associated with gene deletions are an important strategy for determining the mechanisms for extracellular electron transfer in G. sulfurreducens. The results reported here demonstrate that a gene deletion previously thought to prevent the expression of electrically conductive pili in G. sulfurreducens does not have the intended result. Conductive pili continue to be expressed. This finding is important for interpreting the results of several previous studies on conductive pili function.

sentences: Pupil dilation and the slow wave ERP reflect surprise about choice outcome resulting from intrinsic variability in decision confidence Central to human and animal cognition is the ability to learn from feedback in order to optimize future rewards. Such a learning signal might be encoded and broadcasted by the brains arousal systems, including the noradrenergic locus coeruleus. Pupil responses and the positive slow wave component of event-related potentials reflect rapid changes in the arousal level of the brain. Here we ask whether and how these variables may reflect surprise: the mismatch between ones expectation about being correct and the outcome of a decision, when expectations fluctuate due to internal factors (e.g., engagement). We show that during an elementary decision-task in the face of uncertainty both physiological markers of phasic arousal reflect surprise. We further show that pupil responses and slow wave ERP are unrelated to each other, and that prediction error computations depend on feedback awareness. These results further advance our understanding of the role of central arousal systems in decision-making under uncertainty.

sentences: A large chromosomal inversion shapes gene expression in seaweed flies (Coelopa frigida) Inversions often underlie complex adaptive traits, but the genic targets inside them are largely unknown. Gene expression profiling provides a powerful way to link inversions with their phenotypic consequences. We examined the effects of the Cf-Inv(1) inversion in the seaweed fly Coelopa frigida on gene expression variation across sexes and life stages. Our analyses revealed that Cf-Inv(1) shapes global expression patterns but the extent of this effect is variable with much stronger effects in adults than larvae. Furthermore, within adults, both common as well as sex specific patterns were found. The vast majority of these differentially expressed genes mapped to Cf-Inv(1). However, genes that were differentially expressed in a single context (i.e. in males, females or larvae) were more likely to be located outside of Cf-Inv(1). By combining our findings with genomic scans for environmentally associated SNPs, we were able to pinpoint candidate variants in the inversion that may underlie mechanistic pathways that determine phenotypes. Together the results in this study, combined with previous findings, support the notion that the polymorphic Cf-Inv(1) inversion in this species is a major factor shaping both coding and regulatory variation resulting in highly complex adaptive effects.

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Testing the accuracy of 3D automatic landmarking via genome-wide association studies BackgroundVarious advances in 3D automatic phenotyping and particularly in landmark-based geometric morphometric methods have been made, but only a few studies have tested the reliability of such automatic procedures in morphometric analyses. It is generally accepted that automatic landmarking compromises the capture of the actual biological variation, and this not only affects its performance to effectively detect differences among sample means but also the structure of covariance matrices. However, no studies have directly tested the actual impact of such landmarking approaches in analyses requiring a large number of specimens and for which the precision of phenotyping is crucial to capture an actual biological signal adequately.

ResultsHere, we use a recently developed 3D atlas-based automatic landmarking method to test its accuracy in detecting QTLs associated with craniofacial development of the house mouse skull and lower jaws for a large number of specimens (circa 700) that were previously phenotyped via a semiautomatic landmarking method complemented with manual adjustment. We compare both landmarking methods with univariate and multivariate mapping of the skull and the lower jaws. In the univariate mapping, the automatic approach failed to recover the same SNPs and found only 1 out of 17 previously identified QTLs for the skull, but found one new QTL. Similarly, for the lower jaws, the automatic approach failed to recover the same SNPs but found 2 neighbouring SNPs for 1 out of 8 previously identified QTLs. For centroid size, the same general results were recovered by the automatic method for both the skull and lower jaws, with the same peak SNP being found for the lower jaws. In the multivariate mapping, the automatic approach did not detect the same markers nor QTLs having their regions overlapping with the ones identified with the semi-automatic procedure for the skull, while the same marker, which is also the peak SNP and sole QTL, was recovered by the automatic pipeline for lower jaws.

ConclusionOur results confirm the notion that information is lost in the automated landmarking procedure but somewhat dependent on the analyzed structure. The automatic method seems to capture certain types of structures slightly better, such as lower jaws whose shape is almost entirely summarized by its outline and could be assimilated as a 2D flat object. By contrast, the more apparent 3D features exhibited by a structure such as the skull are not adequately captured by the automatic method. We conclude that using 3D atlas-based automatic landmarking methods requires careful consideration of the experimental question and the cautious interpretation of their results.

sentences: Tracking SARS-CoV-2 Omicron diverse spike gene mutations identifies multiple inter-variant recombination events The current pandemic of COVID-19 is fueled by more infectious emergent Omicron variants. Ongoing concerns of emergent variants include possible recombinants, as genome recombination is an important evolutionary mechanism for the emergence and re-emergence of human viral pathogens. Although recombination events among SARS-CoV-1 and MERS-CoV were well-documented, it has been difficult to detect the recombination signatures in SARS-CoV-2 variants due to their high degree of sequence similarity. In this study, we identified diverse recombination events between two Omicron major subvariants (BA.1 and BA.2) and other variants of concern (VOCs) and variants of interest (VOIs), suggesting that co-infection and subsequent genome recombination play important roles in the ongoing evolution of SARS-CoV-2. Through scanning high-quality completed Omicron spike gene sequences, eighteen core mutations of BA.1 variants (frequency >99%) were identified (eight in NTD, five near the S1/S2 cleavage site, and five in S2). BA.2 variants share three additional amino acid deletions with the Alpha variants. BA.1 subvariants share nine common amino acid mutations (three more than BA.2) in the spike protein with most VOCs, suggesting a possible recombination origin of Omicron from these VOCs. There are three more Alpha-related mutations (del69-70, del144) in BA.1 than BA.2, and therefore BA.1 may be phylogenetically closer to the Alpha variant. Revertant mutations are found in some dominant mutations (frequency >95%) in the BA.1 subvariant. Most notably, multiple additional amino acid mutations in the Delta spike protein were also identified in the recently emerged Omicron isolates, which implied possible recombination events occurred between the Omicron and Delta variants during the on-going pandemic. Monitoring the evolving SARS-CoV-2 genomes especially for recombination is critically important for recognition of abrupt changes to viral attributes including its epitopes which may call for vaccine modifications.

sentences: Distinct Neural Representations of Decision Uncertainty in Metacognition and Mentalizing Metacognition and mentalizing are both associated with meta-level mental state representations. Specifically, metacognition refers to monitoring ones own cognitive processes, while mentalizing refers to monitoring others cognitive processes. However, this self-other dichotomy is insufficient to delineate the two high-level mental processes. We here used functional magnetic resonance imaging (fMRI) to systematically investigate the neural representations of different levels of decision uncertainty in monitoring different targets (the current self, the past self, and others) performing a perceptual decision-making task. Our results reveal diverse formats of intrinsic mental state representations of decision uncertainty in mentalizing, separate from the associations with external information. External information was commonly represented in the right inferior parietal lobe (IPL) across the mentalizing tasks. However, the meta-level mental states of decision uncertainty attributed to others were uniquely represented in the dorsomedial prefrontal cortex (dmPFC), rather than the temporoparietal junction (TPJ) that also equivalently represented the object-level mental states of decision inaccuracy attributed to others. Further, the object-level and meta-level mental states of decision uncertainty, when attributed to the past self, were represented in the precuneus and the lateral frontopolar cortex (lFPC), respectively. In contrast, the dorsal anterior cingulate cortex (dACC) consistently represented both decision uncertainty in metacognition and estimate uncertainty during monitoring the different mentalizing processes, but not the inferred decision uncertainty in mentalizing. Hence, our findings identify neural signatures to clearly delineate metacognition and mentalizing and further imply distinct neural computations on the mental states of decision uncertainty during metacognition and mentalizing.

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Conserved FimK truncation coincides with increased expression of type 3 fimbriae and cultured bladder epithelial cell association in Klebsiella quasipneumoniae Klebsiella spp. commonly cause both uncomplicated urinary tract infection (UTI) and recurrent UTI (rUTI). Klebsiella quasipneumoniae, a relatively newly defined species of Klebsiella, has been shown to be metabolically distinct from Klebsiella pneumoniae, but its urovirulence mechanisms have not been defined. K. pneumoniae uses both type 1 and type 3 fimbriae to attach to host epithelial cells. The type 1 fimbrial operon is well-conserved between Escherichia coli and K. pneumoniae with the exception of fimK, which is unique to Klebsiella spp. FimK contains an N-terminal DNA binding domain and a C-terminal phosphodiesterase (PDE) domain that has been hypothesized to cross-regulate type 3 fimbriae expression via modulation of cellular levels of cyclic di-GMP. Here, we find that a conserved premature stop codon in K. quasipneumoniae fimK results in truncation of the C-terminal PDE domain and that the bladder epithelial cell association and invasion of uropathogenic K. quasipneumoniae strain KqPF9 is dependent on type 3 but not type 1 fimbriae. Further, we show that basal expression of both type 1 and type 3 fimbrial operons as well as bladder epithelial cell association is elevated in KqPF9 relative to uropathogenic K. pneumoniae TOP52 and that complementation of KqPF9{Delta}fimK with the TOP52 fimK allele reduced type 3 fimbrial expression and bladder epithelial cell attachment. Taken together these data suggest that the C-terminal PDE of FimK can modulate type 3 fimbrial expression in K. pneumoniae and its absence in K. quasipneumoniae may lead to a loss of type 3 fimbrial cross-regulation.

ImportanceK. quasipneumoniae is often indicated as the cause of many opportunistic infections including urinary tract infection (UTI), which affects >50% of women worldwide. However, virulence mechanisms of K. quasipneumoniae remain uninvestigated. Prior to this work, K. quasipneumoniae and K. pneumoniae had only been distinguished phenotypically by metabolic differences. This work contributes to the understanding of K. quasipneumoniae virulence phenotypes by evaluating the contribution of type 1 and type 3 fimbriae, which are critical colonization factors encoded by all Klebsiella spp., to K. quasipneumoniae bladder epithelial cell attachment. We identify clear phenotypic differences in both bladder epithelial cell attachment between uropathogenic K. pneumoniae and K. quasipneumoniae. Importantly, we find that a structural difference in the fimbrial regulatory gene fimK may contribute to differential co-regulation of type 1 and type 3 fimbriae between the two Klebsiella species.

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Radial-axial transport coordination enhances sugar translocation in the phloem vasculature of plants Mass transport of photosynthates in the phloem of plants is necessary for describing plant carbon allocation, productivity, and responses to water and thermal stress. Several hypotheses about optimization of phloem structure and function and limitations of phloem transport under drought have been proposed, and tested with models and anatomical data. However, the true impact of radial water exchange of phloem conduits with their surroundings on mass transport of photosynthates has not been addressed. Here, the physics of the Munch mechanism of sugar transport is re-evaluated to include local variations in viscosity resulting from the radial water exchange in two dimensions (axial and radial). Model results show that radial water exchange pushes sucrose away from conduit walls thereby reducing wall frictional stress due to a decrease in sap viscosity and an increase in sugar concentration in the central region of the conduit. These two co-occurring effects lead to increased sugar front speed and axial mass transport across a wide range of phloem conduit lengths. Thus, sugar transport operates more efficiently than predicted by previous models that ignore these two effects. A faster front speed leads to higher phloem resiliency under drought because more sugar can be transported with a smaller pressure gradient.

SummaryThe overall speed of sap increased by including a concentrationdependent viscosity in axial and radial directions.

sentences: Incorporating Target-Specific Pharmacophoric Information Into Deep Generative Models For Fragment Elaboration Despite recent interest in deep generative models for scaffold elaboration, their applicability to fragment-to-lead campaigns has so far been limited. This is primarily due to their inability to account for local protein structure or a users design hypothesis. We propose a novel method for fragment elaboration, STRIFE that overcomes these issues. STRIFE takes as input Fragment Hotspot Maps (FHMs) extracted from a protein target, and processes them to provide meaningful and interpretable structural information to its generative model, which in turn is able to rapidly generate elaborations with complementary pharmacophores to the protein. In a large-scale evaluation, STRIFE outperforms existing, structure-unaware, fragment elaboration methods in proposing highly ligand efficient elaborations. In addition to automatically extracting pharmacophoric information from a protein targets FHM, STRIFE optionally allows the user to specify their own design hypotheses.

sentences: Targeting the translational machinery in gastrointestinal stromal tumors (GIST) - a new therapeutic vulnerability Although KIT-mutant GISTs can be effectively treated with tyrosine kinase inhibitors (TKIs), many patients develop resistance to imatinib mesylate (IM) as well as the FDA-approved later-line agents sunitinib, regorafenib and ripretinib. Resistance mechanisms mainly involve secondary mutations in the KIT receptor tyrosine kinase gene indicating continued dependency on the KIT signaling pathway. The fact that the type of secondary mutation confers either sensitivity or resistance towards TKIs and the notion that secondary mutations exhibit intra- and intertumoral heterogeneity complicates the optimal choice of treatment in the imatinib-resistant setting. Therefore, new strategies that target KIT independently of its underlying mutations are urgently needed. Homoharringtonine (HHT) is a first-in-class inhibitor of protein biosynthesis and is FDA-approved for chronic myeloid leukemia (CML) that is resistant to at least two TKIs. HHT has also shown activity in KIT-mutant mastocytosis models, which are intrinsically resistant to imatinib and most other TKIs. We hypothesized that HHT could be effective in GIST through downregulation of KIT expression and subsequent decrease of KIT activation and downstream signaling. Testing several GIST cell line models, HHT led to a significant reduction in nascent protein synthesis and was highly effective in the nanomolar range in IM-sensitive and IM-resistant GIST cell lines. HHT treatment resulted in a rapid and complete abolishment of KIT expression and activation, while KIT mRNA levels were minimally affected. The response to HHT involved induction of apoptosis as well as cell cycle arrest. The antitumor activity of HHT was confirmed in a GIST xenograft model. Taken together, inhibition of protein biosynthesis is a promising strategy to overcome TKI resistance in GIST.

sentences: Low antigen abundance limits efficient T-cell recognition of highly conserved regions of SARS-CoV-2 Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8+ T-cell epitopes across the nucleocapsid (N), spike (S) and ORF3a proteins of SARS-CoV-2 and five CD8+ T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8+ T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad pre-existing immunity in the community.

sentences: Substrate stiffness modulates integrin α5 expression and ECM-associated gene expression in fibroblasts Biomaterials, like polydimethylsiloxane (PDMS), are soft, biocompatible, and tuneable, which makes them useful to delineate specific substrate factors that regulate the complex landscape of cell-substrate interactions. We used a commercial formulation of PDMS to fabricate substrates with moduli 40 kPa, 300 kPa, and 1.5 MPa, and cultured HMF3S fibroblasts on them. Gene expression analysis was performed by RT-PCR and Western blotting. Cellular and nuclear morphologies were analyzed using confocal imaging, and MMP-2 and MMP-9 activities were determined with gelatin zymography. Results, comparing mechanotransduction on PDMS substrates with control petridishes, show that substrate stiffness modulates cell morphologies and proliferations. Cell nuclei were rounded on compliant substrates and correlated with increased tubulin expression. Proliferations were higher on stiffer substrates with cell cycle arrest on softer substrates. Integrin 5 expression decreased on lower stiffness substrates, and correlated with inefficient TGF-{beta} activation. Changes to the activated state of the fibroblast on higher stiffness substrates were linked to altered TGF-{beta} secretion. Collagen I, collagen III, and MMP-2 expression levels were lower on compliant PDMS substrates as compared to stiffer ones; there was little MMP-9 activity on substrates. These results demonstrate critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts which is highly relevant in diseases like tissue fibrosis.

sentences: Endocytic Trafficking Promotes Vacuolar Enlargements for Fast Cell Expansion Rates in Plants The vacuole has a space-filling function, allowing a particularly rapid plant cell expansion with very little increase in cytosolic content (Lofke et al., 2015; Scheuring et al., 2016; Dunser et al., 2019). Despite its importance for cell size determination in plants, very little is known about the mechanisms that define vacuolar size. Here we show that the cellular and vacuolar size expansions are coordinated. By developing a pharmacological tool, we enabled the investigation of membrane delivery to the vacuole during cellular expansion. Counterintuitively, our data reveal that endocytic trafficking from the plasma membrane to the vacuole is enhanced in the course of rapid root cell expansion. While this "compromise" mechanism may theoretically at first decelerate cell surface enlargements, it fuels vacuolar expansion and, thereby, ensures the coordinated augmentation of vacuolar occupancy in dynamically expanding plant cells.

sentences: Chromatin interaction aware gene regulatory modeling with graph attention networks Linking distal enhancers to genes and modeling their impact on target gene expression are longstanding unresolved problems in regulatory genomics and critical for interpreting non-coding genetic variation. Here we present a new deep learning approach called GraphReg that exploits 3D interactions from chromosome conformation capture assays in order to predict gene expression from 1D epigenomic data or genomic DNA sequence. By using graph attention networks to exploit the connectivity of distal elements up to 2Mb away in the genome, GraphReg more faithfully models gene regulation and more accurately predicts gene expression levels than state-of-the-art deep learning methods for this task. Feature attribution used with GraphReg accurately identifies functional enhancers of genes, as validated by CRISPRi-FlowFISH and TAP-seq assays, outperforming both CNNs and the recently proposed Activity-by-Contact model. Sequence-based GraphReg also accurately predicts direct transcription factor (TF) targets as validated by CRISPRi TF knockout experiments via in silico ablation of TF binding motifs. GraphReg therefore represents an important advance in modeling the regulatory impact of epigenomic and sequence elements.

sentences: Molecular approaches reveal speciation between red and blue flowered plants in the Mediterranean Lysimachia arvensis and Lysimachia monelli (Primulaceae) Flower colour constitutes a pivotal evolutionary force in speciation. The Mediterranean Lysimachia arvensis and L. monelli are morphologically variable species having both blue or red flowered plants. Previous studies suggested that L. arvensis plants differing in colour are diverging lineages, but this variation has not been considered in a phylogenetic context. We reconstruct the phylogenetic signal and the ancestral states of flower colour of Mediterranean Lysimachia species by using nuclear (ITS) and three plastid markers. Blue and red specimens are nested in two independent clades in the ITS tree, thus supporting that L. arvensis and L. monelli are polyphyletic, whereas low phylogenetic resolution was found in plastid markers. Blue-flowered L. arvensis is reconstructed sister to L. talaverae in a monophyletic clade sister to the remaining Lysimachia. Red-flowered L. arvensis is reconstructed sister to red-flowered L. monelli in a monophyletic clade sister to blue-flowered L. monelli and L. foemina. Our results suggest that colour lineages in L. arvensis and L. monelli constitute different species, but flower colour did not promote the separation of these lineages. We propose a new name for blue-flowered L. arvensis (L. loeflingii) and a new combination for red-flowered L. monelli (L. collina).

sentences: Multi-Omics Analysis of Fibroblasts from the Invasive Tumor Edge Reveals that Tumor-Stroma Crosstalk Induces O-glycosylation of the CDK4-pRB Axis The invasive leading edge represents a potential gateway for tumor invasion. We hypothesize that crosstalk between tumor and stromal cells within the tumor microenvironment (TME) results in the activation of key biological pathways depending on their location in the tumor (edge vs core). Here, we highlight phenotypic differences between Tumor-Adjacent-Fibroblasts (TAFs) from the invasive edge and Cancer-Associated Fibroblasts (CAFs) from the tumor core, established from human lung adenocarcinomas. We use an innovative multi-omics approach that includes genomics, proteomics and, O-glycoproteomics to characterize crosstalk between TAFs and cancer cells. Our analysis shows that O-glycosylation, an essential post-translational modification resulting from sugar metabolism, alters key biological pathways including the CDK4-pRB axis in the stroma, and indirectly modulates pro-invasive features of cancer cells. In summary, aside from improving the efficacy of CDK4 inhibitors anti-cancer agents, the O-glycoproteome poses a new consideration for important biological processes involved in tumor-stroma crosstalk.

sentences: Sablefish (Anoplopoma fimbria) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions.

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Selection of software and database for metagenomics sequence analysis impacts the outcome of microbial profiling and pathogen detection AimShotgun metagenomic sequencing analysis is widely used for microbial profiling of biological specimens and pathogen detection. However, very little is known about the technical biases caused by the choice of analysis software and databases. In this study, we evaluated shotgun metagenomics taxonomical profiling software to characterize the microbial compositions of biological samples collected from wild rodents.

Method and ResultsUsing nine of the most widely used metagenomics software and four different databases, we analyzed shotgun metagenomic sequence data from three sets of wild rodent tissue samples. We demonstrated the discrepancies in results when different databases and software were used, which cause significant variation in microbial community characterizations. Our analysis also showed that these software differed in their ability to detect the presence of Leptospira, a major zoonotic pathogen of one health importance.

ConclusionsSignificant differences in compositional profiles for the same dataset while using different databases and software combinations can result in confounding biological conclusions in microbial profiling.

Significance and Impact of StudyThis study cautions that the selection of software and databases to analyze metagenomics data can influence the outcome and biological interpretation.

sentences: Time-resolved FRET screening identifies small molecular modifiers of mutant Huntingtin conformational inflexibility in patient-derived cells Huntingtons disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1 encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTTs conformational flexibility in human patient fibroblasts. We applied an antibody-based time-resolved Forster resonance energy transfer (TR-FRET) immunoassay, measuring endogenous HTT flexibility using two validated HTT-specific antibodies. The ratio of TR-FRET signal at 4{degrees}C and 20{degrees}C differs between wtHTT and mHTT and allowed to perform a high-throughput screening using HTT flexibility as a read-out. We identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. This novel screening approach has the potential to identify previously unknown HD drugs and drug targets.

sentences: Precise identification of cancer cells from allelic imbalances in single cell transcriptomes A fundamental step of tumour single cell mRNA analysis is separating cancer and non-cancer cells. We show that the common approach to separation, using shifts in average expression, can lead to erroneous biological conclusions. By contrast, allelic imbalances representing copy number changes directly detect the cancer genotype and accurately separate cancer from non-cancer cells. Our findings provide a definitive approach to identifying cancer cells from single cell mRNA sequencing data.

sentences: Extended-Ensemble Docking to Probe Evolution of Ligand Binding Sites in Proteins Undergoing Large-Scale Structural Changes for Their Function Proteins can sample a broad landscape as they undergo conformational transition between different functional states. As key players in almost all cellular processes, proteins are important drug targets. Considering the different conformational states of a protein is therefore central for a successful drug-design strategy. Here we introduce a novel docking protocol, termed as extended-ensemble docking, pertaining to proteins that undergo large-scale (global) conformational changes during their function. In its application to multidrug ABC-transporter P-glycoprotein (Pgp), extensive non-equilibrium molecular dynamics simulations employing system-specific collective variables capturing the alternate access mechanism of Pgp, are first used to construct the transition cycle of the transporter. An extended set of conformational states representing the full transition between the inward- and the outward-facing states of Pgp, is then used to seed high-throughput docking calculations of a set of known substrates, non-substrates, and modulators of the transporter. Large differences are observed in the predicted binding affinities to the conformational ensemble, with compounds showing stronger binding affinities to intermediate conformations compared to the starting crystal structure. Hierarchical clustering of the individual binding modes of the different compounds shows all ligands preferably bind to the large central cavity of the protein, formed at the apex of the transmembrane domain (TMD), whereas only small binding populations are observed in the previously described R and H sites present within the individual TMD leaflets. Based on the results, the central cavity is further divided into two major subsites: first subsite preferably binds smaller substrates and high-affinity inhibitors, whereas the second one shows preference for larger substrates and low-affinity modulators. These central sites along with the low-affinity interaction sites present within the individual TMD leaflets may respectively correspond to the proposed high- and low-affinity binding sites in Pgp. We propose further optimization strategy for developing more potent inhibitor of Pgp, based on increasing its specificity to the extended ensemble of the protein instead of using a single protein structure, as well as its selectivity for the high-affinity binding site. In contrast to earlier in-silico studies using single static structures of Pgp, our results show better agreement with experimental studies, pointing to the importance of incorporating the global conformational flexibility of proteins in future drug-discovery endeavors.

sentences: Goal-specific brain MRI harmonization There is significant interest in pooling magnetic resonance image (MRI) data from multiple datasets to enable mega-analysis. Harmonization is typically performed to reduce heterogeneity when pooling MRI data across datasets. Most MRI harmonization algorithms do not explicitly consider downstream application performance during harmonization. However, the choice of downstream application might influence what might be considered as study-specific confounds. Therefore, ignoring downstream applications during harmonization might potentially limit downstream performance. Here we propose a goal-specific harmonization framework that utilizes downstream application performance to regularize the harmonization procedure. Our framework can be integrated with a wide variety of harmonization models based on deep neural networks, such as the recently proposed conditional variational autoencoder (cVAE) harmonization model. Three datasets from three different continents with a total of 2787 participants and 10085 anatomical T1 scans were used for evaluation. We found that cVAE removed more dataset differences than the widely used ComBat model, but at the expense of removing desirable biological information as measured by downstream prediction of mini mental state examination (MMSE) scores and clinical diagnoses. On the other hand, our goal-specific cVAE (gcVAE) was able to remove as much dataset differences as cVAE, while improving downstream cross-sectional prediction of MMSE scores and clinical diagnoses.

sentences: Guidance by followers ensures long-range coordination of cell migration through α-Catenin mechanoperception Morphogenesis, wound healing and some cancer metastases depend upon migration of cell collectives that need to be guided to their destination as well as coordinated with other cell movements. During zebrafish gastrulation, extension of the embryonic axis is led by the mesendodermal polster that migrates towards the animal pole, followed by axial mesoderm that undergoes convergence and extension. We here investigate how polster cells are guided towards the animal pole. Using a combination of precise laser ablations, advanced transplants and functional as well as in silico approaches, we establish that each polster cell is oriented by its immediate follower cells. Each cell perceives the migration of followers, through E-Cadherin/-Catenin mechanotransduction, and aligns with them. Directional information therefore propagates from cell to cell over the whole tissue. Such guidance of migrating cells by followers ensures long-range coordination of movements and developmental robustness.

sentences: Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats (Rattus norvegicus); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.

sentences: Reduced Hippocampal Inhibition and Enhanced Autism-Epilepsy Comorbidity in Mice Lacking Neuropilin 2 The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2 deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2-/- mice have reduced number of parvalbumin, somatostatin and Neuropeptide Y cells, mainly in CA1. Whole cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/- and Nrp2-/- mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2 deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration and higher severity in Nrp2-/- compared to Nrp2+/+ animals. Finally, Nrp2+/- and Nrp2-/-, but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2-/- mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.

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CovRadar: Continuously tracking and filtering SARS-CoV-2 mutations for molecular surveillance The SARS-CoV-2 pandemic underlined the importance of molecular surveillance to track the evolution of the virus and inform public health interventions. Fast analysis, easy visualization and convenient filtering of the latest virus sequences are essential for this purpose. However, access to computational resources, the lack of bioinformatics expertise, and the sheer volume of sequences in public databases complicate surveillance efforts. CovRadar combines an analytical pipeline and a web application designed for the molecular surveillance of the spike gene of SARS-CoV-2, an important vaccine target. The intuitive web front-end focuses on mutations rather than viral lineages and provides easy access to frequencies and spatio-temporal distributions from global sample collections. The data is regularly updated based on a scalable and reproducible analytical back-end. With this platform, we aim to give users, those with or without bioinformatics skills or sufficient computational resources, the possibility to track and explore mutational changes in the SARS-CoV-2 spike gene and to filter, download, and further analyze data that meet their questions and needs. Advanced computational users have the ability to apply the analytical pipeline and data visualization methods locally on their own data. CovRadar is freely accessible at https://covradar.net, source code is available at https://gitlab.com/dacs-hpi/covradar.

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sentences: Genetic diversity of local and introduced cassava germplasm in Burundi using DArTseq molecular analyses In Burundi, most of small-scale farmers still grow traditional cassava landraces that are adapted to local conditions and have been selected for consumer preferred attributes. They tend to be susceptible, in varying degrees, to devastating cassava viral diseases such as Cassava Brown Steak Disease (CBSD) and Cassava Mosaic Disease (CMD) with production annual losses of US$1 billion annually. For long term resistance to the disease, several breeding strategies have been proposed. A sound basis for a breeding program is to understand the genetic diversity of both landraces and elite introduced breeding cultivars. This will also assist in efforts to conserve landraces ahead of the broad distribution of improved varieties which have the possibility of replacing landraces. Our study aimed at determining the genetic diversity and relationships of local landraces and introduced elite germplasm using morphological and simple nucleotide polymorphism (SNP) markers as well as identifying a core set of germplasm from the local varieties to be used in the cassava breeding program. A total of 118 cultivars were characterized for morphological trait variation based on leaf, stem and root traits, and genetic variation using SNP markers. Results of morphological characterization based on Wards Method revealed three main clusters and five accessions sharing similar characteristics. Molecular characterization identified over 18,000 SNPs and six main clusters and three pairs of duplicates which should be pooled together as one cultivar to avoid redundancy. Results of population genetic analysis showed low genetic distance between populations and between local landraces and elite germplasm. Accessions that shared similar morphological traits were divergent at the molecular level indicating that clustering using morphological traits was inconsistent. Despite the variabilities found within the collection, it was observed that cassava germplasm in Burundi have a narrow genetic base.

sentences: Sex Differences in Brain Tumor Glutamine Metabolism Reveal Sex-Specific Vulnerabilities to Treatment Sex differences in normal metabolism are well described, but whether they persist in cancerous tissue is unknown. We assessed metabolite abundance in glioblastoma surgical specimens and found that male glioblastomas are enriched for amino acids, including glutamine. Using PET imaging, we found that gliomas in male patients exhibit significantly higher glutamine uptake. These sex differences were well-modeled in murine transformed astrocytes, in which male cells imported and metabolized more glutamine and were more sensitive to glutaminase 1 (GLS1) inhibition. The sensitivity to GLS1 inhibition in males was driven by their dependence on glutamine-derived glutamate for -ketoglutarate synthesis and TCA cycle replenishment. Females were resistant to GLS1 inhibition through greater pyruvate carboxylase-mediated TCA cycle replenishment. Thus, clinically important sex differences exist in targetable elements of metabolism. Recognition of sex-biased metabolism is an opportunity to improve treatments for all patients through further laboratory and clinical research.

sentences: Functional, transcriptional, and microbial shifts associated with healthy pulmonary aging: insights from rhesus macaques Older individuals are at increased risk of developing severe respiratory infections due to age-related changes in the immunological, microbial, and functional landscape of the lung. However, our understanding of the impact of age on the respiratory tract remains limited as samples from healthy humans are challenging to obtain and confounding variables such as smoking and environmental pollutant exposure make it difficult to assess the true impact of aging. On the other hand, studies in rodent models are biased by their specific pathogen free status. In this study, we utilize a rhesus macaque model of healthy aging to examine the functional, immunological, and microbial consequences of aging in the lung. Pulmonary function testing in this large (n=34 adult, n=49 aged) cross-sectional study established age and sex differences similar to humans supporting the translational accuracy of this model. Additionally, an increased abundance of myeloid cells (alveolar and infiltrating macrophages) and a concomitant decrease in T-cells were also observed in aged animals. Single cell RNA sequencing indicated a transcriptional shift in the pulmonary CD8+ T-cell population from GRZMB expressing cells to IFN expressing cells, while frequency of IL-1B expressing alveolar macrophages was significantly reduced. Interestingly, the lung microbiome of many animals was dominated by a single microbe, Tropheryma spp., the prevalence of which decreased with age. These data provide a comprehensive picture of the functional, microbial and immunological changes of the lung in healthy macaque aging and provide insight into the increased prevalence and severity of respiratory disease in the elderly.

sentences: Yeast proteins reversibly aggregate like amphiphilic molecules More than a hundred proteins in yeast reversibly aggregate and phase-separate in response to various stressors, such as nutrient depletion and heat shock. We know little about the sequence and structural features behind this ability, which has not been characterized on a proteome-wide level. To identify the distinctive features of aggregation-prone regions, we apply machine learning algorithms to genome-scale limited proteolysis-mass spectrometry data from 96 yeast proteins that phase-separate upon heat shock. We find that the aggregation-prone regions (APRs) of our study proteins are significantly enriched in aliphatic residues and depleted in positively charged amino acids. Aggregator proteins with longer APRs show a greater propensity to aggregate, a relationship that can be explained by equilibrium statistical thermodynamics. Altogether, our observations suggest that proteome-wide reversible protein aggregation is mediated by sequence-encoded properties. Aggregating proteins resemble supra-molecular amphiphiles, where APRs are the hydrophobic parts, and non-APRs are the hydrophilic parts.

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Bacterial community response to species overrepresentation or omission is strongly influenced by life in spatially structured habitats Variations in type and strength of interspecific interactions in natural bacterial communities (e.g., synergistic to inhibitory) affect species composition and community functioning. The extent of interspecific interactions is often modulated by environmental factors that constrain diffusion pathways and cell mobility and limit community spatial arrangement. We studied how spatially structured habitats affect interspecific interactions and influence the resulting bacterial community composition. We used a bacterial community made of 11 well-characterized species that grew in porous habitats (comprised of glass beads) under controlled hydration conditions or in liquid habitats. We manipulated the initial community composition by overrepresenting or removing selected members, and observed community composition over time. Life in porous media reduced the number and strength of interspecific interactions compared to mixed liquid culture, likely due to spatial niche partitioning in porous habitats. The community converged to similar species composition irrespective of the initial species mix, however, the dominant bacterial species was markedly different between liquid culture and structured porous habitats. Moreover, differences in water saturation levels of the porous medium affected community assembly highlighting the need to account for habitat structure and physical conditions to better understand and interpret assembly of bacterial communities. We point at the modulation of bacterial interactions due to spatial structuring as a potential mechanism for promoting community stability and species coexistence, as observed in various natural environments such as soil or human gut.

ImportanceBacteria live as complex multispecies communities essential for healthy and functioning ecosystems ranging from soil to the human gut. The bacterial species that form these communities can have positive or negative impact on each other, promoting or inhibiting each others growth. Yet, the factors controlling the balance of such interactions in nature, and how these influence the community, are not fully understood. Here, we show that bacterial interactions are modified by life in spatially structured bacterial habitats. These conditions exert important control over the resulting bacterial community regardless of initial species composition. The study demonstrates limitations of inferences from bacterial communities grown in liquid culture relative to behaviour in structured natural habitats such as soil.

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Trophic status and local conditions affect microbial potential for denitrification versus internal nitrogen cycling in lake sediments The nitrogen (N) cycle is of global importance as N is an essential element and a limiting nutrient in terrestrial and aquatic ecosystems. Excessive anthropogenic N fertilizer usage threatens sensitive downstream aquatic ecosystems. Although freshwater lake sediments remove N through various microbial transformation processes, few studies have investigated the microbial communities involved. In an integrated biogeochemical and microbiological study on a eutrophic and oligotrophic lake, we estimated N removal rates in the sediments from porewater concentration gradients. Simultaneously, the abundance of different microbial N transformation genes was investigated using metagenomics on a seasonal and spatial scale. We observed that contrasting nutrient concentrations in the sediments were reflected in distinct microbial community compositions and significant differences in the abundance of various N transformation genes. Within each lake, we observed a more pronounced spatial than seasonal variability. The eutrophic Lake Baldegg showed a higher denitrification potential with higher nosZ gene (N2O reductase) abundance and higher nirS:nirK (nitrite reductase) ratio, indicating a greater capacity for complete denitrification. Correspondingly, this lake had a higher N removal efficiency. The oligotrophic Lake Sarnen, in contrast, had a higher potential for DNRA and nitrification, and specifically a high abundance of Nitrospirae, including some capable of comammox. In general, the oligotrophic lake ecosystems had a higher microbial diversity, thus acting as an important habitat for oligotrophic microbes. Our results demonstrate that knowledge of the genomic N transformation potential is important for interpreting N process rates and understanding the limitations of the N cycle response to environmental drivers.

Importance{paragraph}Anthropogenic nitrogen (N) inputs can lead to eutrophication in aquatic systems, specifically in N limited coastal ecosystems. Lakes act as N sinks by transforming reactive N to N2 through denitrification or anammox. The N cycle in lake sediments is mediated by microbial processes and affected by environmental drivers such as the amount and quality of settling organic material or nitrate concentration. However, the microbial communities mediating the different N transformation processes and their impact on N removal in freshwater lake sediments remain largely unknown. We provide the first seasonally and spatially resolved metagenomic analysis of the N cycle in the sediments of two lakes with different trophic states. We show that the trophic state of lakes provokes other microbial communities with characteristic key players and functional potential for N transformation.

sentences: Multiscale three-dimensional imaging of intact human organs down to the cellular scale using hierarchical phase-contrast tomography Human organs are complex, three-dimensional and multiscale systems. Spatially mapping the human body down through its hierarchy, from entire organs to their individual functional units and specialised cells, is a major obstacle to fully understanding health and disease. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique utilising the European Synchrotron Radiation Facilitys Extremely Brilliant Source: the worlds first high-energy 4th generation X-ray source. HiP-CT enabled three-dimensional and non-destructive imaging at near-micron resolution in soft tissues at one hundred thousand times the voxel size whilst maintaining the organs structure. We applied HiP-CT to image five intact human parenchymal organs: brain, lung, heart, kidney and spleen. These were hierarchically assessed with HiP-CT, providing a structural overview of the whole organ alongside detail of the organs individual functional units and cells. The potential applications of HiP-CT were demonstrated through quantification and morphometry of glomeruli in an intact human kidney, and identification of regional changes to the architecture of the air-tissue interface and alveolar morphology in the lung of a deceased COVID-19 patient. Overall, we show that HiP-CT is a powerful tool which can provide a comprehensive picture of structural information for whole intact human organs, encompassing precise details on functional units and their constituent cells to better understand human health and disease.

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Induced fit with replica exchange improves protein complex structure prediction Despite the progress in prediction of protein complexes over the last decade, recent blind protein complex structure prediction challenges revealed limited success rates (less than 20% models with DockQ score > 0.4) on targets that exhibit significant conformational change upon binding. To overcome limitations in capturing backbone motions, we developed a new, aggressive sampling method that incorporates temperature replica exchange Monte Carlo (T-REMC) and conformational sampling techniques within docking protocols in Rosetta. Our method, ReplicaDock 2.0, mimics induced-fit mechanism of protein binding to sample backbone motions across putative interface residues on-the-fly, thereby recapitulating binding-partner induced conformational changes. Furthermore, ReplicaDock 2.0 clocks in at 150-500 CPU hours per target (protein-size dependent); a runtime that is significantly faster than Molecular Dynamics based approaches. For a benchmark set of 88 proteins with moderate to high flexibility (unbound-to-bound iRMSD over 1.2 [A]), ReplicaDock 2.0 successfully docks 61% of moderately flexible complexes and 35% of highly flexible complexes. Additionally, we demonstrate that by biasing backbone sampling particularly towards residues comprising flexible loops or hinge domains, highly flexible targets can be predicted to under 2 [A] accuracy. This indicates that additional gains are possible when mobile protein segments are known.

Significance StatementProteins bind each other in a highly specific and regulated manner, and these associated dynamics of binding are intimately linked to their function. Conventional techniques of structure determination such as cryo-EM, X-ray crystallography and NMR are time-consuming and arduous. Using a temperature-replica exchange Monte Carlo approach that mimics the kinetic mechanism of "induced fit" binding, we improved prediction of protein complex structures, particularly for targets that exhibit considerable conformational changes upon binding (Interface root mean square deviation (unbound-bound) > 1.2 [A]. Capturing these binding-induced conformational changes in proteins can aid us in better understanding biological mechanisms and suggest intervention strategies for disease mechanisms.

sentences: Role of collagen-like protein BclA3 in the assembly of the exosporium layer of Clostridioides difficile spores. Newly formed spores are essential for persistence of C. difficile in the host, transmission to a new susceptible host (Deakin et al., 2012b) and recurrence of CDI. BclA3 and BclA2 Spore surface proteins are expressed during sporulation under the control of mother-cell specific sigma factors of the RNA polymerase, SigE and SigK. Deletion of bclA3 leads to spores with an electron-dense exosporium layer that lacks bump-like structures in the electron-dense layer and hair-like projections, both structures typically found in the wild type spore. Therefore, in this work, we have addressed the role of the exosporium collagen-like BclA3 glycoprotein in the assembly of the exosporium layer. Immunogold labelling of BclA2CTD and BclA3CTD indicates that both proteins are located in the hairs, with BclA2 located outermost of BclA3. Absence of BclA3 leads to spores with no hair-like projections, and absence of bumps in thick exosporium spores, a phenotype also expressed in by the deletion of the collagen-like region of BclA3. Overall, these results provide insights into the role of BclA3 in the assembly of the exosporium layer of C. difficile spores.

sentences: Supervised Phenotype Discovery from Multimodal Brain Imaging Data-driven discovery of image-derived phenotypes (IDPs) from large-scale multimodal brain imaging data has enormous potential for neuroscientific and clinical research by linking IDPs to subjects demographic, behavioural, clinical and cognitive measures (i.e., non-imaging derived phenotypes or nIDPs). However, current approaches are primarily based on unsupervised approaches, without use of information in nIDPs. In this paper, we proposed Supervised BigFLICA (SuperBigFLICA), a semi-supervised, multimodal, and multi-task fusion approach for IDP discovery, which simultaneously integrates information from multiple imaging modalities as well as multiple nIDPs. SuperBigFLICA is computationally efficient and largely bypasses the need for parameter tuning. Using the UK Biobank brain imaging dataset with around 40,000 subjects and 47 modalities, along with more than 17,000 nIDPs, we showed that SuperBigFLICA enhances the prediction power of nIDPs, bench-marked against IDPs derived by conventional expert-knowledge and unsupervised-learning approaches (with average nIDP prediction accuracy improvements of up to 46%). It also enables learning of generic imaging features that can predict new nIDPs. Further empirical analysis of the SuperBigFLICA algorithm demonstrates its robustness in different prediction tasks and the ability to derive biologically meaningful IDPs in predicting health outcomes and cognitive nIDPs, such as fluid intelligence and hypertension scores.

sentences: Exceptional ancient DNA preservation and fibre remains of a Sasanian saltmine sheep mummy in Chehrabad, Iran Mummified remains have long attracted interest as a potential source of ancient DNA. However, mummification is a rare process that requires an anhydrous environment to rapidly dehydrate and preserve tissue before complete decomposition occurs. We present the whole genome sequences of a [~]1600 year old naturally mummified sheep recovered from Chehr[a]b[a]d, a salt mine in northwestern Iran. Comparative analyses of published ancient sequences revealed remarkable DNA integrity of this mummy. Hallmarks of postmortem damage, fragmentation and hydrolytic deamination, are substantially reduced, likely due to the high-salinity of this taphonomic environment. Metagenomic analyses reflect the profound influence of high salt content on decomposition; its microbial profile is predominated by halophilic archaea and bacteria, possibly contributing to the preservation of this sample. Applying population genomic analyses we find consistent clustering of this sheep with Southwest Asian modern breeds, suggesting ancestry continuity. Genotyping of a locus influencing the woolly phenotype showed the existence of an ancestral "hairy" allele in this sheep, consistent with hair fibre imaging, further elucidating Sasanian-period animal husbandry.

sentences: Genetic tools for the stable overexpression of circular RNAs Circular RNAs (circRNAs) are a class of non-coding RNAs that feature a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient overexpression of circRNAs in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs. We further showed that circRNA transposons can be supplied to mouse livers via hydrodynamic tail vein injection, resulting in ectopic circRNA expression in a hepatocellular carcinoma mouse model. Furthermore, we generated genetically engineered mice harboring circRNA expression constructs. We demonstrate that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. Overall, these tools expand the genetic toolkit available for the functional characterization of circRNAs of interest.

sentences: Attention Decorrelates Sensory and Motor Signals in the Mouse Visual Cortex Visually-guided behaviors depend on the activity of cortical networks receiving visual inputs and transforming these signals to guide appropriate actions. However, non-retinal inputs, carrying motor signals as well as cognitive and attentional modulatory signals, also activate these cortical regions. How these networks avoid interference between coincident signals ensuring reliable visual behaviors is poorly understood. Here, we observed neural responses in the dorsal-parietal cortex of mice during a visual discrimination task driven by visual stimuli and movements. We found that visual and motor signals interacted according to two canonical mechanisms: divisive normalization and response demixing. Interactions were contextually modulated by the animals state of attention, with attention amplifying visual and motor signals and decorrelating them in a low-dimensional space of neural activations. These findings reveal canonical computational principles operating in dorsal-parietal networks that enable separation of incoming signals for reliable visually-guided behaviors during interactions with the environment.

sentences: Force-dependent interactions between talin and full-length vinculin Talin and vinculin are part of a multi-component system involved in mechanosensing in cell-matrix adhesions. Both exist in auto-inhibited forms, and activation of vinculin requires binding to mechanically activated talin, yet how forces affect talins interaction with vinculin has not been investigated. Here by quantifying the force-dependent talin-vinculin interactions and kinetics using single-molecule analysis, we show that mechanical exposure of a single vinculin binding site (VBS) in talin is sufficient to relieve the autoinhibition of vinculin resulting in high-affinity binding. We provide evidence that the vinculin undergoes dynamic fluctuations between an auto-inhibited closed conformation and an open conformation that is stabilized upon binding to the VBS. Furthermore, we discover an additional level of regulation in which the mechanically exposed VBS binds vinculin significantly more tightly than the isolated VBS alone. Molecular dynamics simulations reveal the basis of this new regulatory mechanism, identifying a sensitive force-dependent change in the conformation of an exposed VBS that modulates binding. Together, these results provide a comprehensive understanding of how the interplay between force and autoinhibition provides exquisite complexity within this major mechanosensing axis.

sentences: MrHAMER yields highly accurate single molecule viral sequences enabling analysis of intra-host evolution Technical challenges remain in the sequencing of RNA viruses due to their high intra-host diversity. This bottleneck is particularly pronounced when interrogating long-range co-evolution given the read-length limitations of next-generation sequencing platforms. This has hampered the direct observation of long-range genetic interactions that code for protein-protein interfaces with relevance in both drug and vaccine development. Here we overcome these technical limitations by developing a nanopore-based long-range viral sequencing pipeline that yields accurate single molecule sequences of circulating virions from clinical samples. We demonstrate its utility in observing the evolution of individual HIV Gag-Pol genomes in response to antiviral pressure. Our pipeline, called Multi-read Hairpin Mediated Error-correction Reaction (MrHAMER), yields >1000s viral genomes per sample at 99.9% accuracy, maintains the original proportion of sequenced virions present in a complex mixture, and allows the detection of rare viral genomes with their associated mutations present at <1% frequency. This method facilitates scalable investigation of genetic correlates of resistance to both antiviral therapy and immune pressure, and enable the identification of novel host-viral and viral-viral interfaces that can be modulated for therapeutic benefit.

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Rhythmicity of Intestinal IgA Responses Confers Oscillatory Commensal Microbiota Mutualism Mutualistic interactions with the commensal microbiota are enforced through a range of immune responses that confer metabolic benefits for the host and ensure tissue health and homeostasis. Immunoglobulin (Ig)A responses directly determine the composition of commensal species that colonize the intestinal tract but require significant metabolic resources to fuel antibody production by tissue-resident plasma cells. Here we demonstrate IgA responses are subject to diurnal regulation by dietary-derived metabolic cues and a cell-intrinsic circadian clock. Rhythmicity in IgA secretion conferred oscillatory patterns on the commensal microbial community and its associated metabolic activity, resulting in changes to metabolite availability over the course of the circadian day. Our findings suggest circadian networks comprising intestinal IgA, the diet and the microbiota align to ensure metabolic health.

One-Sentence SummaryWe demonstrate diurnal rhythms in intestinal IgA act to cross-regulate oscillations in the abundance of commensal microbes to foster mutualism.

sentences: Actin fence therapy with exogenous V12Rac1 protects against Acute Lung Injury High mortality in Acute Lung Injury (ALI) results from sustained proinflammatory signaling by alveolar receptors, such as TNF receptor type 1 (TNFR1). Factors that determine the sustained signaling are not known. Unexpectedly, optical imaging of live alveoli revealed a major TNF-induced surge of alveolar TNFR1 due to a Ca2+-dependent mechanism that decreased the cortical actin fence. Mouse mortality due to inhaled LPS was associated with cofilin activation, actin loss and the TNFR1 surge. The constitutively active form of the GTPase, Rac1 (V12Rac1), given intranasally as a non-covalent construct with a cell-permeable peptide, enhanced alveolar F-actin and blocked the TNFR1 surge. V12Rac1 also protected against ALI-induced mortality resulting from intranasal (i.n.) instillation of LPS, or of Pseudomonas aeruginosa. We propose a new therapeutic paradigm in which actin enhancement by exogenous Rac1 strengthens the alveolar actin fence, protecting against proinflammatory receptor hyperexpression, hence blocking ALI.

sentences: In macrophages fatty acid oxidation spares glutamate for use in diverse metabolic pathways required for alternative activation Fatty acid oxidation (FAO) is upregulated in IL-4-stimulated (alternatively activated) macrophages (M(IL-4)). We examined the effect of loss of function of the enzyme Cpt1a, which facilitates the entry of long chain fatty acids (FA) into mitochondria for FAO, on alternative activation. Expression of M(IL-4) markers ARG1, CD301 and RELM, was impaired in tamoxifen-treated ERT2Cre x Cpt1afl/fl macrophages and in macrophages expressing shRNA targeting Cpt1a (Cpt1a-shRNA). In contrast, VaviCre x Cpt1afl/fl and LysmCre x Cpt1afl/fl M(IL-4) responded normally to IL-4. Reduced alternative activation due to Cpt1a loss of function was linked to decreased cellular pools of -ketoglutarate, glutamate, and glutathione, diminished commitment of glucose carbon to serine/glycine synthesis, and decreased expression of genes in the Nrf2-oxidative stress response pathway. Consistent with this, reactive oxygen species were increased. Restoration of glutathione pools with N-acetyl cysteine normalized oxidative stress and allowed alternative activation in the face of Cpt1a-deficiency, pointing to a role for FAO in the control of ROS and as being important for alternative activation. In VaviCre x Cpt1afl/fl M(IL-4), glutamine uptake was increased, compensating for the loss of FAO to meet necessary metabolic demands, to allow alternative activation. The data indicate that macrophages are able to regulate glutamine metabolism to compensate for chronic disruption of FAO to meet metabolic needs.

sentences: A multimodal neuroprosthetic interface to record, modulate and classify electrophysiological correlates of cognitive function Most mental disorders are characterised by impaired cognitive function and behaviour control. Their often chronic reoccurring nature and the lack of efficient therapies necessitate the development of new treatment strategies. Brain-computer interfaces, equipped with multiple sensing and stimulation abilities, offer a new toolbox, whose suitability for diagnosis and therapy of mental disorders has not yet been explored. Here, we developed a soft and multimodal neuroprosthesis to measure and modulate prefrontal neurophysiological features of neuropsychiatric symptoms. We implanted the device epidurally above the medial prefrontal cortex of rats and obtained auditory event-related brain potentials reflecting intact neural stimulus processing and alcohol-induced neural impairments. Moreover, implant-driven electrical and pharmacological stimulation enabled successful modulation of neural activity. Finally, we developed machine learning algorithms which can deal with sparsity in the data and distinguish effects with high accuracy. Our work underlines the potential of multimodal bioelectronic systems to enable a personalised and optimised therapy.

sentences: Measuring brain beats: cardiac-aligned fast fMRI signals Blood and cerebrospinal fluid (CSF) pulse and flow throughout the brain, driven by the cardiac cycle. These fluid dynamics, which are essential to healthy brain function, are characterized by several noninvasive magnetic resonance imaging (MRI) methods. Recent developments in fast MRI, specifically simultaneous multislice (SMS) acquisition methods, provide a new opportunity to rapidly and broadly assess cardiac-driven flow, including CSF spaces, surface vessels and parenchymal vessels. We use these techniques to assess blood and CSF flow dynamics in brief (3.5 minute) scans on a conventional 3T MRI scanner. Cardiac pulses are measured with a photoplethysmograph (PPG) on the index finger, along with fMRI signals in the brain. We retrospectively analyze the fMRI signals gated to the heart beat. Highly reliable cardiac-gated fMRI temporal signals are observed in CSF and blood on the timescale of one heartbeat (test-retest reliability within subjects R2>0.50). Cardiac pulsations with a local minimum following systole are observed in blood vessels, with earlier extrema in the carotid and basilar arteries and in branches of the anterior, posterior and middle cerebral arteries and extrema [~]200 ms later in the superior sagittal, transverse and straight sinuses. CSF spaces in the ventricles and subarachnoid space showed cardiac pulsations with a local maximum following systole instead. Similar responses are observed, with less temporal detail, in slower resting state scans with slice timing retrospectively aligned to the cardiac pulse in the same manner. The SMS measurements rapidly, noninvasively and reliably sample brain-wide fMRI signal pulsations aligned to the heartbeat. The measurements estimate the amplitude and phase of cardiac driven fMRI pulsations in the CSF relative to those in the arteries, which is thought to be an estimate of the local intracranial impedance. Cardiac aligned fMRI signals can provide new insights about fluid dynamics or diagnostics for diseases where these dynamics are important.

sentences: Dissecting conformational rearrangements and allosteric modulation in metabotropic glutamate receptor activation Selective allosteric modulators bear great potential to fine-tune neurotransmitter-induced brain receptor responses. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated to different brain diseases. These multidomain class C GPCRs experience concerted structural rearrangements and rely on allosteric modulation of agonist action to be fully activated. Here we establish live cell compatible fluorescence labeling of mGlu2 by click chemistry through genetic code expansion. Using lanthanide resonance energy transfer, we establish multiple FRET sensors to monitor ligand effects on conformational changes in mGlu2 extracellular domain and subsequently dissect the underlying conformational states by smFRET. Using three distinct FRET sensors, we demonstrate that mGlu activation relies on a ligand-induced sampling of three conformational states. Orthosteric agonists act by promoting the closure of the mGlu2 ligand binding domains, leading to an equilibrium between an inactive intermediate and the active state. Allosteric modulator further push this equilibrium toward the active state, promoting and stabilizing the relative reorientation of the mGlu protomers. These results underline the complex and dynamic nature of such type of neuroreceptors, pointing out that ligands fine-tune activation by differentially acting on the equilibria between multiple states.

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The role of competition versus cooperation in microbial community coalescence New microbial communities often arise through the mixing of two or more separately assembled parent communities, a phenomenon that has been termed "community coalescence". Understanding how the interaction structures of complex parent communities determine the outcomes of coalescence events is an important challenge. While recent work has begun to elucidate the role of competition in coalescence, that of cooperation, a key interaction type commonly seen in microbial communities, is still largely unknown. Here, using a general consumer-resource model, we study the combined effects of competitive and cooperative interactions on the outcomes of coalescence events. In order to do so, we simulate coalescence events between pairs of communities with different degrees of competition for shared carbon resources and cooperation through cross-feeding on leaked metabolic by-products (facilitation). We also study how structural and functional properties of post-coalescence communities evolve when they are subjected to repeated coalescence events. We find that in coalescence events, the less competitive and more cooperative parent communities contribute a higher proportion of species to the new community, because this endows superior ability to deplete resources and resist invasions. Consequently, when a community is subjected to repeated coalescence events, it gradually evolves towards being less competitive and more cooperative, as well as more species rich, robust and efficient in resource use. Encounters between microbial communities are becoming increasingly frequent as a result of anthropogenic environmental change, and there is great interest in how the coalescence of microbial communities affects environmental and human health. Our study provides new insights into the mechanisms behind microbial community coalescence, and a framework to predict outcomes based on the interaction structures of parent communities.

Author summaryIn nature, new microbial communities often arise from the fusion of whole, previously separate communities (community coalescence). Despite the crucial role that interactions among microbes play in the dynamics of complex communities, our ability to predict how these affect the outcomes of coalescence events remains limited. Here, using a general mathematical model, we study how the structure of species interactions confers an advantage upon a microbial community when it encounters another, and how communities evolve after undergoing repeated coalescence events. We find that competitive interactions between species preclude their survival upon a coalescence event, while cooperative interactions are advantageous for post-coalescence survival. Furthermore, after a community is exposed to many coalescence events, the remaining species become less competitive and more cooperative. Ultimately, this drives the community evolution, yielding post-coalescence communities that are more species-rich, productive, and resistant to invasions. There are many potential environmental and health implications of microbial community coalescence, which will benefit from the theoretical insights that we offer here about the fundamental mechanisms underlying this phenomenon.

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pOPIN-GG: A resource for modular assembly in protein expression vectors The ability to recombinantly produce target proteins is essential to many biochemical, structural, and biophysical assays that allow for interrogation of molecular mechanisms behind protein function. Purification and solubility tags are routinely used to maximise the yield and ease of protein expression and purification from E. coli. A major hurdle in high-throughput protein expression trials is the cloning required to produce multiple constructs with different solubility tags. Here we report a modification of the well-established pOPIN expression vector suite to be compatible with modular cloning via Type IIS restriction enzymes. This allows users to rapidly generate multiple constructs with any desired tag, introducing modularity in the system and delivering compatibility with other modular cloning vector systems, for example streamlining the process of moving between expression hosts. We demonstrate these constructs maintain the expression capability of the original pOPIN vector suite and can also be used to efficiently express and purify protein complexes, making these vectors an excellent resource for high-throughput protein expression trials.

HighlightsO_LIpOPIN-GG expression vectors allow for modular cloning enabling rapid screening of purification and solubility tags at no loss of expression compared to previous vectors.
C_LIO_LICloning into the pOPIN-GG vectors can be performed from PCR products or from level 0 vectors containing the required parts.
C_LIO_LISeveral vectors with different resistances and origins of replication have been generated allowing the effective co-expression and purification of protein complexes.
C_LIO_LIAll pOPIN-GG vectors generated here are available on Addgene, as well as level 0 acceptors and tags.
C_LI

sentences: Fine mapping spatiotemporal mechanisms of genetic variants underlying cardiac traits and disease The causal variants and genes underlying thousands of cardiac GWAS signals have yet to be identified. Here, we leveraged spatiotemporal information on 966 RNA-seq cardiac samples and performed an expression quantitative trait locus (eQTL) analysis detecting eQTLs considering both eGenes and eIsoforms. We identified 2,578 eQTLs associated with a specific developmental stage-, tissue- and/or cell type. Colocalization between eQTL and GWAS signals of five cardiac traits identified variants with high posterior probabilities for being causal in 210 GWAS loci. Pulse pressure GWAS loci were enriched for colocalization with fetal- and smooth muscle- eQTLs; pulse rate with adult- and cardiac muscle- eQTLs; and atrial fibrillation with cardiac muscle- eQTLs. Fine mapping identified 79 credible sets with five or fewer SNPs, of which 15 were associated with spatiotemporal eQTLs. Our study shows that many cardiac GWAS variants impact traits and disease in a developmental stage-, tissue- and/or cell type-specific fashion.

sentences: Prenatal immune stress induces a prolonged blunting of microglia activation that impacts striatal connectivity Recent studies suggested that microglia, the primary brain immune cells, can affect circuit connectivity and neuronal function1-3. Microglia infiltrate the neuroepithelium early in embryonic development and are maintained in the brain throughout adulthood4,5. Several maternal environmental factors, such as aberrant microbiome, immune activation, and poor nutrition, can influence prenatal brain development6-8. Nevertheless, it is unknown how changes in the prenatal environment instruct the developmental trajectory of infiltrating microglia, which in turn affect brain development and function. Here we show that after maternal immune activation (MIA) microglia from the offspring have a long-lived decrease in immune reactivity (blunting) across the developmental trajectory. The blunted immune response was concomitant with changes in the chromatin accessibility and reduced transcription factor occupancy of the open chromatin. Single cell RNA sequencing revealed that MIA does not induce a distinct subpopulation but rather decreases the contribution to inflammatory microglia states. Prenatal replacement of MIA microglia with physiological infiltration of naive microglia ameliorated the immune blunting and restored a decrease in presynaptic vesicle release probability onto dopamine receptor type-two medium spiny neurons, indicating that aberrantly formed microglia due to an adverse prenatal environment impacts the long-term microglia reactivity and proper striatal circuit development.

sentences: eIF4E3 Forms an Active eIF4F Complex during Stresses (eIF4FS) Targeting mTOR and Re-Programs the Translatome. The eIF4E are a family of initiation factors that bind the mRNA 5 cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active only during stress and that it selects specific mRNA populations based on 5 TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have "moon-lighting" functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote "stress resistance" and adaptation.

sentences: Translation Inhibitory Elements from Hox a3 and a11 mRNAs use uORFs for translation inhibition During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5UTR. First, an Internal Ribosome Entry Site (IRES) enables cap-independent translation. The second regulon is a Translation Inhibitory Element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of Hox a3 and a11 TIE elements. Both TIEs possess an upstream Open Reading Frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In TIE a3, we identify a uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of TIE a11 is different, it also contains a uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a  start-stop. The a11  start-stop sequence is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hox a11 main ORF.

sentences: Vibrio cholerae biofilm dispersal regulator causes cell release from matrix through type IV pilus retraction The extracellular matrix is a defining feature of bacterial biofilms and provides structural stability to the community by binding cells to the surface and to each other. Transitions between bacterial biofilm initiation, growth, and dispersion require different regulatory programs, all of which result in modifications to the extracellular matrix composition, abundance, or functionality. However, the mechanisms by which individual cells in biofilms disengage from the matrix to enable their departure during biofilm dispersal are unclear. Here, we investigated active biofilm dispersal of Vibrio cholerae during nutrient starvation, resulting in the discovery of the conserved Vibrio biofilm dispersal regulator VbdR. We show that VbdR triggers biofilm dispersal by controlling cellular release from the biofilm matrix, which is achieved by inducing the retraction of the mannose-sensitive hemagglutinin (MSHA) type IV pili and the expression of a matrix protease IvaP. We further show that MSHA pili have numerous binding partners in the matrix and that the joint effect of MSHA pilus retraction and IvaP activity is necessary and sufficient for causing biofilm dispersal. These results highlight the crucial role of type IV pilus dynamics during biofilm dispersal and provide a new target for controlling V. cholerae biofilm abundance through the induction and manipulation of biofilm dispersal.

sentences: Correlated variability in primate superior colliculus depends on functional class Correlated variability (spike count correlations, rSC) in a population of neurons can constrain how information is read out, depending on behavioral task and neuronal tuning. Here we tested whether rSC also depends on neuronal functional class. We recorded from populations of neurons in macaque superior colliculus (SC), a structure that contains well-defined functional classes. We found that during a guided saccade task, different classes of neurons exhibited differing degrees of rSC. "Delay class" neurons displayed the highest rSC, especially during the delay epoch of saccade tasks that relied on working memory. This was only present among Delay class neurons within the same hemisphere. The dependence of rSC on functional class indicates that subpopulations of SC neurons occupy distinct circuit niches with distinct inputs. Such subpopulations should be accounted for differentially when attempting to model or infer population coding principles in the SC, or elsewhere in the primate brain.

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Novel regulatory and transcriptional networks associated with resistance to platinum-based chemotherapy in ovarian cancer BackgroundHigh-grade serous ovarian cancer (HGSOC) is a highly lethal gynecologic cancer, in part due to resistance to platinum-based chemotherapy reported among 20% of patients. This study aims to elucidate the biological mechanisms underlying chemotherapy resistance, which remain poorly understood.

MethodsSequencing data (mRNA and microRNA) from HGSOC patients were analyzed to identify differentially expressed genes and co-expressed transcript networks associated with chemotherapy response. Initial analyses used datasets from The Cancer Genome Atlas and then replicated in two independent cancer cohorts. Moreover, transcript expression datasets and genomics data (i.e. single nucleotide polymorphisms) were integrated to determine potential regulation of the associated mRNA networks by microRNAs and expression quantitative trait loci (eQTLs).

ResultsIn total, 196 differentially expressed mRNAs were enriched for adaptive immunity and translation, and 21 differentially expressed microRNAs were associated with angiogenesis. Moreover, co-expression network analysis identified two mRNA networks associated with chemotherapy response, which were enriched for ubiquitination and lipid metabolism, as well as three associated microRNA networks enriched for lipoprotein transport and oncogenic pathways. In addition, integrative analyses revealed potential regulation of the mRNA networks by the associated microRNAs and eQTLs.

ConclusionWe report novel transcriptional networks and pathways associated with resistance to platinum-based chemotherapy among HGSOC patients. These results aid our understanding of the effector networks and regulators of chemotherapy response, which will improve drug efficacy and identify novel therapeutic targets for ovarian cancer.

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Glucose-dependent miR-125b is a negative regulator of β-cell function Impaired pancreatic {beta}-cell function and insulin secretion are hallmarks of type 2 diabetes. MicroRNAs are short non-coding RNAs that silence gene expression, vital for the development and function of {beta}-cells. We have previously shown that {beta}-cell specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in {beta}-cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycaemia in {beta}-cells. Here we show that islet miR-125b-5p expression is up-regulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human {beta}-cell line EndoC{beta}-H1 shortened mitochondria and enhanced GSIS, whilst mice overexpressing miR-125b-5p selectively in {beta}-cells (MIR125B-Tg) were hyperglycaemic and glucose intolerant. MIR125B-Tg {beta}-cells contained enlarged lysosomal structures and showed reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Biocide's susceptibility and frequency of biocide resistance genes and the effect of exposure to sub-inhibitory concentrations of sodium hypochlorite on antibiotic susceptibility of Stenotrophomonas maltophilia The overuse of biocides in healthcare-facilities poses risk for emergence and spread of antibiotic resistance among nosocomial pathogens. Hospital-acquired infections due to S. maltophiliahave been increased. The objective of this study was to evaluate the susceptibility of S. maltophilia clinical isolates to commonly used biocides in hospitals, as well asthe frequency of biocides resistance genes among them. This study also intended to assess the effect of exposureof S. maltophilia isolates to sub-inhibitory concentrations of sodium hypochlorite upon the antimicrobial susceptibility patterns.This study included 97 S.maltophiliaisolates.Biofilm formation was determined by microtiter plate assay. The susceptibility tests of five biocides were studied against all S. maltophilia isolates by microbroth dilution method. Susceptibility of isolates to antibiotics by disk diffusion method were compared before and after exposure to sub-inhibitory concentrations of sodium hypochlorite. Presence of qacE, qacE{Delta}1, SugE genes was screened by PCR. Based on minimum inhibitory and bactericidal concentrations of biocidessodium hypochlorite 5% and ethyl alcohol 70% were the strongest and weakest against S. maltophilia isolates, respectively. The frequency of sugE gene resistance genes was found 90.7% in our clinical S. maltophilia isolates. None of the isolates carried qacE and qacE{Delta}1 gene. Exposure to sub-inhibitory concentration of sodium hypochlorite showed significantly change the susceptibility of isolates towards ceftazidime (P = .019), ticarcillin/clavulanate (P = .009), and chloramphenicol (P = .028).This study demonstrated that exposure to sub-inhibitory concentrations of sodium hypochlorite leads to reduced antibiotic susceptibility and development of multidrug-resistant S.maltophilia strains.

sentences: Social and asocial learning in zebrafish are encoded by a shared brain network that is differentially modulated by local activation Group living animals can use social and asocial cues to predict the presence of a reward or a punishment in the environment through associative learning. The degree to which social and asocial learning share the same mechanisms is still a matter of debate, and, so far, studies investigating the neuronal basis of these two types of learning are scarce and have been restricted to primates, including humans, and rodents. Here we have used a Pavlovian fear conditioning paradigm in which a social (fish image) or an asocial (circle image) conditioned stimulus (CS) have been paired with an unconditioned stimulus (US=food), and we have used the expression of the immediate early gene c-fos to map the neural circuits associated with social and asocial learning. Our results show that the learning performance is similar with social (fish image) and asocial (circle image) CSs. However, the brain regions involved in each learning type are distinct. Social learning is associated with an increased expression of c-fos in olfactory bulbs, ventral zone of ventral telencephalic area, ventral habenula and ventromedial thalamus, whereas asocial learning is associated with a decreased expression of c-fos in dorsal habenula and anterior tubercular nucleus. Using egonetworks, we further show that each learning type has an associated pattern of functional connectivity across brain regions. Moreover, a community analysis of the network data reveals four segregated functional submodules, which seem to be associated with different cognitive functions involved in the learning tasks: a generalized attention module, a visual response module, a social stimulus integration module and a learning module. Together, these results suggest that, although there are localized differences in brain activity between social and asocial learning, the two learning types share a common learning module and social learning also recruits a specific social stimulus integration module. Therefore, our results support the occurrence of a common general-purpose learning module, that is differentially modulated by localized activation in social and asocial learning.

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Glutathione regulates subcellular iron homeostasis via transcriptional activation of iron responsive genes in Arabidopsis While glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants, its involvement in modulating subcellular Fe homeostasis remained elusive. In this study, we report that the GSH depleted mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with significant down-regulation of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4 and the chloroplast Fe importer, AtPIC1. Moreover, the pad2-1 mutant accumulated higher Fe content in vacuoles and lower in chloroplasts compared with Col-0 under Fe limited condition. Exogenous GSH treatment could enhance the chloroplast Fe content in Col-0 plants but failed to do so in the nramp3nramp4 double mutant demonstrating the role of GSH in modulating subcellular Fe homeostasis. Pharmacological experiments, mutant analysis and promoter assay revealed that this regulation involved transcriptional activation of the transporter genes by a GSH-GSNO module. The Fe responsive bHLH transcription factors (TFs), AtbHLH29, AtbHLH38 and AtbHLH101 were identified to interact with the promoters of these genes which were in turn activated via S-nitrosylation. Together, the present study delineates the role of GSH-GSNO module in regulating subcellular Fe homeostasis by transcriptional activation of the Fe transporters AtNRAMP3, AtNRAMP4 and AtPIC1 via S-nitrosylation of the bHLH TFs during Fe deficiency.

Summary statementGlutathione regulates subcellular iron homeostasis under iron deficiency via GSNO dependent transcriptional activation of AtNRAMP3, AtNRAMP4 and AtPIC1 genes by S-nitrosylation of the iron responsive bHLH transcription factors, AtbHLH29, AtbHLH38 and AtbHLH101.

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Subthreshold repetitive transcranial magnetic stimulation drives structural synaptic plasticity in the young and aged motor cortex. BackgroundRepetitive transcranial magnetic stimulation (rTMS) is a non-invasive tool commonly used to drive neural plasticity in the young adult and aged brain. Recent data from mouse models have shown that even at subthreshold intensities (0.12 Tesla), rTMS can drive neuronal and glial plasticity in the motor cortex. However, the physiological mechanisms underlying subthreshold rTMS induced plasticity and whether these are altered with normal ageing are unclear.

ObjectiveTo assess the effect of subthreshold rTMS, using the intermittent theta burst stimulation (iTBS) protocol on structural synaptic plasticity in the mouse motor cortex of young and aged mice.

MethodsLongitudinal in vivo 2-photon microscopy was used to measure changes to the structural plasticity of pyramidal neuron dendritic spines in the motor cortex following a single train of subthreshold rTMS (in young adult and aged animals) or the same rTMS train administered on 4 consecutive days (in young adult animals only). Data were analysed with Bayesian hierarchical generalized linear regression models and interpreted with the aid of Bayes Factors (BF).

ResultsWe found strong evidence (BF>10) that subthreshold rTMS altered the rate of dendritic spine losses and gains, dependent on the number of stimulation sessions and that a single session of subthreshold rTMS was effective in driving structural synaptic plasticity in both young adult and aged mice.

ConclusionThese findings provide further evidence that rTMS drives synaptic plasticity in the brain and uncovers structural synaptic plasticity as a key mechanism of subthreshold rTMS induced plasticity.

sentences: Impact of cell culture on the transcriptomic programs of primary and iPSC-derived human alveolar type 2 cells The alveolar epithelial type 2 cell (AEC2) is the facultative progenitor of lung alveoli tasked to maintain distal lung homeostasis. AEC2 dysfunction has been implicated in the pathogenesis of a number of pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), highlighting the importance of human in vitro models of the alveolar epithelium. However, AEC2-like cells captured in cell culture have yet to be directly compared to their in vivo counterparts at single cell resolution. Here, we apply single cell RNA sequencing to perform head-to-head comparisons between the global transcriptomes of freshly isolated primary (1{degrees}) adult human AEC2s, their isogenic cultured progeny, and human iPSC-derived AEC2s (iAEC2s) cultured in identical conditions. We find each population occupies a distinct transcriptomic space with both types of cultured AEC2s (1{degrees} and iAEC2s) exhibiting similarities to and differences from freshly purified 1{degrees} cells. Across each cell type, we find an inverse relationship between proliferative states and AEC2 maturation states, with uncultured 1{degrees} AEC2s being most quiescent and mature, their cultured progeny being more proliferative/less mature, and cultured iAEC2s being most proliferative/least mature. iAEC2s also express significantly lower levels of major histocompatibility complex (MHC) genes compared to 1{degrees} cells, suggesting immunological immaturity. Cultures of either type of human AEC2 (1{degrees} or iAEC2) do not generate detectable type 1 alveolar cells in these defined conditions; however, iAEC2s after co-culture with fibroblasts can give rise to a subset of cells expressing "transitional cell markers" recently described in fibrotic lung tissue of patients with pulmonary fibrosis or in mouse models of pulmonary fibrosis. Hence, we provide direct comparisons of the transcriptomic programs of 1{degrees} and engineered AEC2s, two in vitro model systems that can be harnessed for studies of human lung health and disease.

sentences: Relative precision of the sibship and LD methods for estimating effective population size with genomics-scale datasets Computer simulations were used to compare relative precision of two widely-used single-sample methods for estimating effective population size (Ne)--the sibship method and the linkage-disequilibrium (LD) method. Emphasis is on performance when thousands of gene loci are used, which now can easily be achieved even for non-model species. Results show that unless Ne is very small, if at least 500-2000 diallelic loci are used, precision of the LD method is higher than the maximum possible precision for the sibship method, which occurs when all sibling relationships have been correctly identified. Results also show that when precision is high for both methods, their estimates of effective population size are high and positively correlated, which limits additional gains in precision that might be obtained by combining information from the two estimators.

sentences: High-resolution structure and dynamics of mitochondrial complex I - insights into the proton pumping mechanism Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1 MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I from Yarrowia lipolytica at 2.1 [A] resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which [~]100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4 [A] resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamics simulations, we define details of the proton translocation pathways, and offer new insights into the redox-coupled proton pumping mechanism of complex I.

sentences: Correction of multiple-blinking artefacts in photoactivated localisation microscopy Photoactivated localisation microscopy (PALM) produces an array of localisation coordinates by means of photoactivatable fluorescent proteins. However, observations are subject to fluorophore multiple-blinking and each protein is included in the dataset an unknown number of times at different positions, due to localisation error. This causes artificial clustering to be observed in the data. We present a workflow using calibration-free estimation of blinking dynamics and model-based clustering, to produce a corrected set of localisation coordinates now representing the true underlying fluorophore locations with enhanced localisation precision. These can be reliably tested for spatial randomness or analysed by other clustering approaches, and previously inestimable descriptors such as the absolute number of fluorophores per cluster are now quantifiable, which we validate with simulated data. Using experimental data, we confirm that the adaptor protein, LAT, is clustered at the T cell immunological synapse, with its nanoscale clustering properties depending on location and intracellular phosphorylatable tyrosine residues.

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Stable Interaction Of The UK B.1.1.7 lineage SARS-CoV-2 S1 Spike N501Y Mutant With ACE2 Revealed By Molecular Dynamics Simulation Corona Virus Disease of 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has caused a massive health crisis across the globe, with some genetic variants gaining enhanced infectivity and competitive fitness, and thus significantly aggravating the global health concern. In this regard, the recent SARS-CoV-2 alpha variant, B.1.1.7 lineage, reported from the United Kingdom (UK), is of great significance in that it contains several mutations that increase its infection and transmission rates as evident from clinical reports. Specifically, the N501Y mutation in the SARS-CoV-2 spike S1 receptor binding domain (S1-RBD) has been shown to possess an increased affinity for ACE2, although the basis for this is not entirely clear yet. Here, we dissect the mechanism underlying the increased affinity using molecular dynamics (MD) simulations of the available ACE2-S1-RBD complex structure (6M0J) and show a prolonged and stable interfacial interaction of the N501Y mutant S1-RBD with ACE2 compared to the wild type S1-RBD. Additionally, we find that the N501Y mutant S1-RBD displays altered dynamics that likely aids in its enhanced interaction with ACE2. By elucidating a mechanistic basis for the increased affinity of the N501Y mutant S1-RBD for ACE2, we believe that the results presented here will aid in developing therapeutic strategies against SARS-CoV-2 including designing drugs targeting the ACE2-S1-RBD interaction.

SignificanceThe emergence of the new SARS-CoV-2 lineage in the UK in December 2020 has further aggravated the COVID-19 pandemic due to an increased ability of the variant to infect human hosts, likely due to mutations in the viral S1 spike protein including the N501Y S1-RBD mutation that is located at the interface of S1-RBD and ACE2, the host cell receptor for SARS-CoV-2. Given its location at the interface, N501Y S1-RBD mutation can therefore potentially alter the interfacial interaction. Multiple, all-atom, explicit solvent MD simulations of the ACE2-S1-RBD complex carried here indicated a more stable interaction between the N501Y mutant S1-RBD and ACE2 through stabilizing interfacial interactions of residues at one end of the interface that are either sequentially or physically near the mutation site. These mechanistic details will aid in better understanding the mechanism by which the alpha variant has increased infectivity as well as in designing better therapeutics including ACE2-S1 spike protein inhibitors that will, in turn, help thwarting the current and future pandemic.

HighlightsO_LIN501 in the wild type SARS-CoV-2 S1-RBD forms unsustained hydrogen bonds with residues in the ACE2, namely Y41 and K353
C_LIO_LIY501 in the N501Y mutant SARS-CoV-2 S1-RBD is not capable of forming substantial hydrogen bonds with ACE2 within the time span of the current simulation
C_LIO_LIEvidence from analyzing the simulation results suggests that Y501 of S1-RBD could form other types of non-covalent interactions with ACE2, such as van der Waals interactions
C_LIO_LIN501Y S1-RBD mutation stabilizes the position of interfacial residues neighboring to the mutation site, as well as other non-interfacial residues that are distant from the mutation site
C_LIO_LIThese altered dynamics results in more stable interaction of S1-RBD with ACE2 which could be the main reason underlying the reported enhanced affinity of S1-RBD in the SARS-CoV-2 alpha variant (UK B.1.1.7 lineage) to ACE2
C_LI

sentences: Microglia become hypofunctional and release metalloproteases and tau seeds after phagocytosing live neurons with P301S tau aggregates The microtubule-associated protein tau aggregates in multiple neurodegenerative diseases, causing inflammation and changing the inflammatory signature of microglia by unknown mechanisms. We have shown that microglia phagocytose live neurons containing tau aggregates cultured from P301S tau transgenic mice due to neuronal tau aggregate-induced exposure of the  eat me signal phosphatidylserine. Here we show that after phagocytosis, microglia become hypophagocytic while releasing seed-competent insoluble tau aggregates. These microglia activate acidic {beta}-galactosidase, and release senescence-associated cytokines and matrix remodeling enzymes alongside tau, indicating a senescent phenotype. In particular, the marked NF{kappa}B-induced activation of matrix metalloprotease 3 (MMP3/stromelysinl) was replicated in the brains of P301S mutant tau transgenic mice, and in human brains from tauopathy patients. These data show that microglia that have been activated to ingest live neurons with tau aggregates behave hormetically, becoming hypofunctional while acting as vectors of tau aggregate spreading.

sentences: Pathogenic tau disrupts the cellular program that maintains neuronal identity Neurons in human Alzheimers disease acquire phenotypes that are also present in various cancers, including over-stabilization of the cytoskeleton, nuclear pleomorphism, decondensation of constitutive heterochromatin, and aberrant activation of the cell cycle. Unlike in cancer, in which cell cycle activation drives tumor formation, activation of the cell cycle in post-mitotic neurons is sufficient to induce neuronal death. Multiple lines of evidence suggest that abortive cell cycle activation is a consequence of pathogenic forms of tau, a protein that drives neurodegeneration in Alzheimers disease and related "tauopathies." We have combined network analysis of human Alzheimers disease and mouse tauopathy with mechanistic studies in Drosophila to discover that pathogenic forms of tau drive abortive cell cycle activation by disrupting the cellular program that maintains neuronal identity. Mechanistically, we identify Moesin, a prognostic biomarker for cancer and mediator of the epithelial-mesenchymal transition (EMT), as a major effector of tau-induced neurotoxicity. We find that aberrant activation of Moesin in neurons acts through the actin cytoskeleton to dysregulate the cellular program that maintains neuronal identity. Our study identifies mechanistic parallels between tauopathy and cancer and sets the stage for novel therapeutic approaches.

sentences: Safety and Immunogenicity Evaluation of Inactivated whole-virus-SARS-COV-2 In Mice As Emerging Vaccine Development In Egypt The current worldwide pandemic COVID-19 is causing severe human health problems, with high numbers of mortality rates and huge economic burdens that require an urgent demand for safe, and effective and vaccine development. Our study was the first trail to development and evaluation of safety and immune response to inactivated whole SARS-COV-2 virus vaccine adjuvanted with aluminium hydroxide. We used characterized SARS-COV-2 strain, severe acute respiratory syndrome coronavirus 2 isolates (SARS-CoV-2/human/EGY/Egy-SERVAC/2020) with accession numbers; MT981440; MT981439; MT981441; MT974071; MT974069 and MW250352 at GenBank that isolated from Egyptian patients SARS-CoV-2-positive. Development of the vaccine was carried out in a BSL - 3 facilities and the immunogenicity was determined in mice at two doses (55{micro}g and 100{micro}g per dose). All vaccinated mice were received a booster dose 14 days post first immunization. Our results demonstrated distinct cytopathic effect on the vero cell monolayers induced through SARS-COV-2 propagation and the viral particles were identified as Coronaviridae by transmission electron microscopy. SARS-CoV-2 was identified by RT-PCR performed on the cell culture. Immunogenicity of the developed vaccine indicated the high antigen-binding and neutralizing antibody titers, regardless the dose concentration, with excellent safety profiles.However, no deaths or clinical symptoms in mice groups. The efficacy of the inactivated vaccine formulation was tested by wild virus challenge the vaccinated mice and detection of viral replication in lung tissues. Vaccinated mice recorded complete protection from challenge infection three weeks post second dose. SARS-COV-2 replication was not observed in the lungs of mice following SARS-CoV-2 challenge, regardless of the level of serum neutralizing antibodies. This finding will support the future trials for evaluation an applicable SARS-CoV-2 vaccine candidate.

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Bacterial cell shape control by nutrient-dependent synthesis of cell division inhibitors By analysing cell shape and size of the bacterium Bacillus subtilis under nutrient perturbations, protein depletion, and antibiotic treatments we find that cell geometry is extremely robust, reflected in a well-conserved scaling relation between surface area (S) and volume (V), S ~ V{gamma}, with{gamma} = 0.85. We develop a molecular model supported by single-cell simulations to predict that the surface-to-volume scaling exponent{gamma} is regulated by nutrient-dependent production of metabolic enzymes that act as cell division inhibitors in bacteria. Using theory that is supported by experimental data, we predict the modes of cell shape transformations in different bacterial species and propose a mechanism of cell shape adaptation to different nutrient perturbations. For organisms with high surface-to-volume scaling exponent{gamma} , such as B. subtilis, cell width is not sensitive to growth rate changes, whereas organisms with low{gamma} , such as A. baumannii, cell shape adapts readily to growth rate changes.

SIGNIFICANCEHow bacteria regulate their size and shapes to optimise their growth fitness in different nutrient environments remains largely unknown. By analysing the surface area and volume of rod-shaped B. subtilis exposed to different nutrient conditions and antibiotics we find that cells preserve a power law scaling between surface area and volume. We show that the surface-to-volume scaling is extremely robust and is regulated by nutrient-dependent synthesis of cell division inhibitors. By analysing different bacterial types, we find that cells conserve the surface-to-volume scaling exponent that is typical for each species, implying distinct mechanisms for morphological adaptation in each organism.

sentences: APOBEC mutagenesis is low in most types of non-B DNA structures, unlike other types of cancer mutagenesis While somatic mutations are known to be enriched in genome regions with non-canonical DNA secondary structure, the impact of particular mutagens still needs to be elucidated. Here, we demonstrate that in human cancers, the APOBEC mutagenesis is not enriched in direct repeats, mirror repeats, short tandem repeats, and G-quadruplexes, and even decreased below its level in B-DNA for cancer samples with very high APOBEC activity. In contrast, we observe that the APOBEC-induced mutational density is positively associated with APOBEC activity in inverted repeats (cruciform structures), where the impact of cytosine at the 3-end of the hairpin loop is substantial. Surprisingly, the APOBEC-signature mutation density per TC motif in the single-stranded DNA of a G-quadruplex (G4) is lower than in the four-stranded part of G4 and in B-DNA. The APOBEC mutagenesis, as well as the UV-mutagenesis in melanoma samples are absent in Z-DNA regions, due to depletion of their mutational signature motifs.

sentences: A novel paradigm for optimal mass feature peak picking in large scale LC-MS datasets using the isopair: isoLock, autoCredential and anovAlign Untargeted metabolomics enables direct quantification of metabolites without apriori knowledge of their identity. Liquid chromatography mass spectrometry (LC-MS), a popular method to implement untargeted metabolomics, identifies metabolites via combined mass/charge (m/z) and retention time as mass features. Improvements in the sensitivity of mass spectrometers has increased the complexity of data produced, leading to computational obstacles. One outstanding challenge is calling metabolite mass feature peaks rapidly and accurately in large LC-MS datasets (dozens to thousands of samples) in the presence of measurement and other noise. While existing algorithms are useful, they have limitations that become pronounced at scale and lead to false positive metabolite predictions as well as signal dropouts. To overcome some of these shortcomings, biochemists have developed hybrid computational and carbon labeling techniques, such as credentialing. Credentialing can validate metabolite signals, but is laborious and its applicability is limited. We have developed a suite of three computational tools to overcome the challenges of unreliable algorithms and inefficient validation protocols: isolock, autoCredential and anovAlign. Isolock uses isopairs, or metabolite-istopologue pairs, to calculate and correct for mass drift noise across LC-MS runs. autoCredential leverages statistical features of LC-MS data to amplify naturally present 13C isotopologues and validate metabolites through isopairs. This obviates the need to artificially introduce carbon labeling. anovAlign, an anova-derived algorithm, is used to align retention time windows across samples to accurately delineate retention time windows for mass features. Using a large published clinical dataset as well as a plant dataset with biological replicates across time, genotype and treatment, we demonstrate that this suite of tools is more sensitive and reproducible than both an open source metabolomics pipelines, XCMS, and the commercial software progenesis QI. This software suite opens a new era for enhanced accuracy and increased throughput for untargeted metabolomics.

sentences: Adsorption of semiflexible polymers in crowded environments Macromolecular crowding is a feature of cellular and cell-free systems that, through depletion effects, can impact the interactions of semiflexible biopolymers with surfaces. In this work, we use computer simulations to study crowding-induced adsorption of semiflexible polymers on otherwise repulsive surfaces. Crowding particles are modeled explicitly, and we investigate the interplay between the bending stiffness of the polymer and the volume fraction and size of crowding particles. Adsorption is promoted by stiffer polymers, smaller crowding particles, and larger volume fractions of crowders. We characterize transitions from non-adsorbed to partially and strongly adsorbed states as a function of the bending stiffness. The crowding-induced transitions occur at smaller values of the bending stiffness as the volume fraction of crowders increases. Concomitant effects on the size and shape of the polymer are reflected by crowding- and stiffness-dependent changes to the radius of gyration. We also demonstrate that curvature of the confining surface can induce desorption when the bending stiffness is sufficiently large. The results of our simulations shed light on the interplay of crowding and bending stiffness on the spatial organization of biopolymers in encapsulated cellular and cell-free systems.

sentences: Predictive regulatory and metabolic network models for systems analysis of Clostridioides difficile Though Clostridioides difficile is among the most studied anaerobes, the interplay of metabolism and regulation that underlies its ability to colonize the human gut is unknown. We have compiled public resources into three models and a portal to support comprehensive systems analysis of C. difficile. First, by leveraging 151 transcriptomes from 11 studies we generated a regulatory model (EGRIN) that organizes 90% of C. difficile genes into 297 high quality conditional co-regulation modules. EGRIN predictions, validated with independent datasets, recapitulated and extended regulons of key transcription factors, implicating new genes for sporulation, carbohydrate transport and metabolism. Second, by advancing a metabolic model, we discovered that 15 amino acids, diverse carbohydrates, and 10 metabolic genes are essential for C. difficile growth within an intestinal environment. Finally, by integrating EGRIN with the metabolic model, we developed a PRIME model that revealed unprecedented insights into combinatorial control of essential processes for in vivo colonization of C. difficile and its interactions with commensals. We have developed an interactive web portal (http://networks.systemsbiology.net/cdiff-portal/) to disseminate all data, algorithms, and models to support collaborative systems analyses of C. difficile.

sentences: Development and Characterization of Recombinant Vesicular Stomatitis Virus (rVSV)-based Bivalent Vaccine Against COVID-19 Delta Variant and Influenza Virus COVID-19 and influenza are both highly contagious respiratory diseases with a wide range of severe symptoms and cause great disease burdens globally. It has become very urgent and important to develop a bivalent vaccine that is able to target these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant VSV (rVSV) vaccine candidates. These rVSV-based vaccines co-express SARS-CoV-2 Delta variant spike protein (SP) or the receptor binding domain (RBD) and four copies of the highly conserved M2 ectodomain (M2e) of influenza A fused with the Ebola glycoprotein DC-targeting/activation domain. Animal studies have shown that immunization with these bivalent rVSV vaccines induced efficient but variable levels of humoral and cell-mediated immune responses against both SARS-CoV-2 and influenza M2e protein. Significantly, our vaccine candidates induced production of high levels of neutralizing antibodies that protected cells against SARS-CoV-2 Delta and other SP-pseudovirus infections in culture. Furthermore, vaccination with the bivalent VSV vaccine via either intramuscular or intranasal route efficiently protected mice from the lethal challenge of H1N1 and H3N2 influenza viruses and significantly reduced viral load in the lungs. These studies provide convincing evidence for the high efficacy of this bivalent vaccine to prevent influenza replication and initiate robust immune responses against SARS-CoV-2 Delta variants. Further investigation of its efficacy to protect against SARS-CoV-2 Delta variants will provide substantial evidence for new avenues to control two contagious respiratory infections, COVID-19 and influenza.

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A survey of the mouse hindbrain in the fed and fasted state using single-nucleus RNA sequencing ObjectiveThe area postrema (AP) and the nucleus tractus solitaris (NTS), located in the hindbrain, are key nuclei that sense and integrate peripheral nutritional signals and, consequently, regulate feeding behaviour. While single cell transcriptomics have been used in mice to reveal the gene expression profile and heterogeneity of key hypothalamic populations, similar in-depth studies have not yet been performed in the hindbrain.

MethodsUsing single-nucleus RNA sequencing, we provide a detailed survey of 16,034 cells within the AP and NTS of the mouse, in the fed and fasted state.

ResultsOf these, 8910 are neurons that group into 30 clusters, with 4289 coming from mice fed ad libitum and 4621 from overnight fasted mice. 7124 nuclei are from non-neuronal cells, including oligodendrocytes, astrocytes and microglia. Interestingly, we identified that the oligodendrocyte population was particularly transcriptionally sensitive to an overnight fast. The receptors GLP1R, GIPR, GFRAL and CALCR, which bind GLP1, GIP, GDF15 and amylin respectively, are all expressed in the hindbrain and are major targets for anti-obesity therapeutics. We characterise the transcriptomes of these four populations and show that their gene expression profiles are not dramatically altered by an overnight fast. Notably, we find that roughly half of cells that express GIPR are oligodendrocytes. Additionally, we profile POMC expressing neurons within the hindbrain and demonstrate that 84% of POMC neurons express either PCSK1, PSCK2 or both, implying that melanocortin peptides are likely produced by these neurons.

ConclusionWe provide a detailed single-cell level characterisation of AP and NTS cells expressing receptors for key anti-obesity drugs that are either already approved for human use or are in clinical trials. This resource will help delineate the mechanisms underlying the effectiveness of these compounds, and also prove useful in the continued search for other novel therapeutic targets.

sentences: Comammox Nitrospira bacteria outnumber canonical nitrifiers irrespective of electron donor mode and availability Complete ammonia oxidizing bacteria coexist with canonical ammonia and nitrite oxidizing bacteria in a wide range of environments. Whether this is due to competitive or cooperative interactions, or a result of niche separation is not yet clear. Understanding the factors driving coexistence of nitrifiers is critical to manage nitrification processes occurring in engineered and natural ecosystems. In this study, microcosm-based experiments were used to investigate the impact of nitrogen source and loading on the population dynamics of nitrifiers in drinking water biofilter media. Shotgun sequencing of DNA followed by co-assembly and reconstruction of metagenome assembled genomes revealed clade A2 comammox bacteria were likely the primary nitrifiers within microcosms and increased in abundance over Nitrsomonas-like ammonia and Nitrospira-like nitrite oxidizing bacteria irrespective of nitrogen source type or loading. Changes in comammox bacterial abundance did not correlate with either ammonia or nitrite oxidizing bacterial abundance in urea amended systems where metabolic reconstruction indicated potential for cross feeding between ammonia and nitrite oxidizing bacteria. In contrast, comammox bacterial abundance demonstrated a negative correlation with nitrite oxidizers in ammonia amended systems. This suggests potentially weaker synergistic relationships between ammonia and nitrite oxidizers might enable comammox bacteria to displace nitrite oxidizers from complex nitrifying communities.

sentences: Two different cell-cycle processes determine the timing of cell division in Escherichia coli Cells must control the cell cycle to ensure that key processes are brought to completion. In Escherichia coli, it is controversial whether cell division is tied to chromosome replication or to a replication-independent inter-division process. A recent model suggests instead that both processes may limit cell division with comparable odds in single cells. Here, we tested this possibility experimentally by monitoring single-cell division and replication over multiple generations at slow growth. We then perturbed cell width, causing an increase of the time between replication termination and division. As a consequence, replication became decreasingly limiting for cell division, while correlations between birth and division and between subsequent replication-initiation events were maintained. Our experiments support the hypothesis that both chromosome replication and a replication-independent inter-division process can limit cell division: the two processes have balanced contributions in non-perturbed cells, while our width perturbations increase the odds of the replication-independent process being limiting.

sentences: The VersaLive platform enables pipette-compatible microfluidic mammalian cell culture for versatile applications Microfluidic-based cell culture allows for precise spatio-temporal regulation of microenvironment, live cell imaging and better recapitulation of physiological conditions, while minimizing reagents consumption. Despite their usefulness, most microfluidic systems are designed with one specific application in mind and require specialized equipment and expertise for their operation. All these requirements prevent microfluidic-based cell culture to be widely adopted. Here, we designed and implemented a versatile and easy-to-use perfusion cell culture microfluidic platform for multiple application (VersaLive) requiring only standard pipettes. Here, we showcase the multiple uses of VersaLive (e.g., time-lapse live cell imaging, immunostaining, cell recovery, cell lysis) on mammalian cell lines and primary cells. VersaLive can replace standard cell culture formats in several applications, thus decreasing costs and increasing reproducibility across laboratories. The layout, documentation and protocols are open-source and available online at https://versalive.tigem.it/.

sentences: Face processing in the infant brain after pandemic lockdown The role of visual experience in the development of face processing has long been debated. We present a new angle on this question through a serendipitous study that cannot easily be repeated. Infants viewed short blocks of faces during fMRI in a repetition suppression task. The same identity was presented multiple times in half of the blocks (Repeat condition) and different identities were presented once each in the other half (Novel condition). In adults, the fusiform face area (FFA) tends to show greater neural activity for Novel vs. Repeat blocks in such designs, suggesting that it can distinguish same vs. different face identities. As part of an ongoing study, we collected data before the COVID-19 pandemic and after an initial State lockdown was lifted. The resulting sample of 12 infants (9-24 months) divided equally into pre- and post-lockdown groups with matching ages and data quantity/quality. The groups had strikingly different FFA responses: pre-lockdown infants showed repetition suppression (Novel>Repeat), whereas post-lockdown infants showed the opposite (Repeat>Novel), often referred to as repetition enhancement. These findings provide speculative evidence that altered visual experience during the lockdown, or other correlated environmental changes, may have affected face processing in the infant brain.

sentences: Limiting factors for queen conch (Lobatus gigas) reproduction: A simulation-based evaluation Queen conch are among the most economically, socially, and culturally important fishery resources in the Caribbean. Despite a multitude of fisheries management measures enacted across the region, populations are depleted and failing to recover. It is believed that queen conch are highly susceptible to depensatory processes, impacting reproductive success and contributing to the lack of recovery. We developed a model of reproductive dynamics to evaluate how variations in biological factors such as population density, movement speeds, movement restrictions, rest periods between mating events, sexual facilitation, and perception of conspecifics affect reproductive success and overall reproductive output. We compared simulation results to empirical observations of mating and spawning frequencies from conch populations in the central Bahamas and Florida Keys. Our results confirm that low probability of mate finding associated with decreased population density is the primary driver behind observed breeding behavior in the field, although additional factors also play important roles. In particular, sexual facilitation and perception of conspecifics may explain observed lack of mating at low densities and differences between mating frequencies in the central Bahamas and Florida Keys, respectively. Our simulations suggest densities greater than 200 adults/ha are needed for high levels of spawning output, supporting the suggestion that effective management strategies for queen conch should aim to protect high-density reproductive aggregations and critical breeding habitats.

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Challenges of γ9δ2TCR affinity maturation when using phage display BackgroundOver the past years we showed that the efficacy of {beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) depends on the functional avidity of the {gamma}9{delta}2TCR. We hypothesized that functional avidity mediated through {gamma}9{delta}2TCR in the TEG format could be further enhanced by increasing affinity of the {gamma}9{delta}2TCR.

MethodsWe attempted to overcome limited affinity of natural occurring {gamma}9{delta}2TCRs through affinity maturation by phage display using a library containing mutations in CDR1 and CDR2 of both TCR chains.

ConclusionAffinity maturation of {gamma}9{delta}2TCR by using phage display was not successful. The largest hurdle was the periplasmic expression of {gamma}9{delta}2TCR constructs in E.coli which is a prerequisite for successful phage display. The underlying reason for this lack of expression was the instability of the single chain (sc)TCR format. Expression of scTCR formats in HEK293F cells yielded only 15-20% correctly folded scTCR.

sentences: The sperm protein Spaca6 is essential for fertilization in zebrafish Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, yet its function has not been investigated outside of mammals. Here we show that zebrafish spaca6 encodes for a sperm membrane protein which is essential for fertilization. Zebrafish spaca6 knockout males are sterile. Furthermore, Spaca6-deficient sperm have normal morphology, are motile, and can approach the egg, but fail to bind to the egg and therefore cannot complete fertilization. Interestingly, sperm lacking Spaca6 have decreased levels of another essential and conserved sperm fertility factor, Dcst2, revealing a previously unknown dependence of Dcst2 expression on Spaca6. Together, our results show that zebrafish Spaca6 regulates Dcst2 levels and is required for binding between the sperm membrane and the oolemma. This is in contrast to murine SPACA6, which was reported not to be required for sperm-egg membrane binding but necessary for fusion. These findings demonstrate that Spaca6 is essential for zebrafish fertilization and is a conserved sperm factor in vertebrate reproduction.

sentences: Limitations of bacterial culture, viral PCR, and tulathromycin susceptibility test methods from upper respiratory tract samples in predicting the outcome of tulathromycin control or treatment of bovine respiratory disease in high risk feeder heifers A cross-sectional prospective cohort study to correlate BRD clinical outcomes for tulathromycin metaphylaxis/treatment for bovine respiratory disease (BRD) with the results of bacterial culture and tulathromycin susceptibility from isolates of deep nasopharyngeal swabs (DNS) as well as viral polymerase chain reaction (PCR) results from nasal swabs revealed poor correlation of bacterial culture and tulathromycin susceptibility with response to tulathromycin metaphylaxis or treatment. 1031 heifers, assumed to be at high-risk (>40% expected BRD morbidity rates), were procured and transported to a research feedlot in Texas. Isolation rates from DNS collected on arrival and at first treatment respectively were: Mannheimia haemolytica (10.9% & 34.1%); Pasteurella multocida (10.4% & 7.4%); Mycoplasma bovis (1.0% & 36.6%); and Histophilus somni (0.7% & 6.3%). Prevalence of BRD viral nucleic acid on nasal swabs collected at first treatment were: PI-3V (34.1%); BVDV (26.3%); BoHV-1 (10.8%); and BRSV (54.1%). Increased relative risk of treatment failure was associated with positive viral PCR results, PI-3V (1.2644), BVDV (1.3917), BHV-1 (1.5156), and BRSV (1.3474) from nasal swabs collected at first pull and culture of M. haemolytica (1.2284) from DNS collected at arrival; however, no other statistically predictable risk of treatment outcomes were measured from DNS for bacterial isolation or tulathromycin susceptibility for M. haemolytica or P. multocida at arrival or first treatment. Predictive values of bacterial culture and tulathromycin susceptibility were substantially lower than the 85% level expected with susceptibility testing. These results indicate tulathromycin susceptibility testing of isolates of M. haemolytica or P. multocida from DNS collected on arrival or at first pull unreliably predict clinical efficacy of tulathromycin for BRD control or treatment most likely due to impacts of unpredictable risk factors and other viral and/or bacterial BRD comorbidities.

sentences: A consensus view on the folding mechanism of protein G, L and their mutants Much of our understanding of folding mechanisms comes from interpretations of experimental {phi} and {psi} value analysis - relating the differences in stability of the transition state ensemble (TSE) and folded state. We introduce a unified approach combining simulations and Bayesian inference to provide atomistic detail for the folding mechanism of protein G, L and their mutants. Protein G and L fold to similar topologies despite low sequence similarity, but differ in their folding pathways. A fast folding redesign of protein G, NuG2, switches folding pathways and folds through a similar pathway with protein L. A redesign of protein L also leads to faster folding, respecting the original folding pathway. Our Bayesian inference approach starts from the same prior on all systems and correctly identifies the folding mechanism for each of the four proteins - a success of the force field and sampling strategy. The approach is computationally efficient and correctly identifies the TSE and intermediate structures along the folding pathway in good agreement with experiments. We complement our findings by using two orthogonal approaches that differ in computational cost and interpretability. Adaptive sampling MD combined with Markov State Model provide a kinetic model that confirms the more complex folding mechanism of protein G and its mutant. Finally, a novel fragment decomposition approach using AlphaFold identifies preferences for secondary structure element combinations that follows the order of events observed in the folding pathways.

sentences: Unsupervised Clustering characterizes the CPG dinucleotides distribution of the Andes hantavirus Hantaviruses belong to the family of Bunyaviridae, and small mammals host them. Humans are infected either by inhaling virus-containing aerosols or through contact with animal droppings. Even if rodents host the pathogenic species and humans are dead-end hosts, they get accidentally infected. Andes Orthohantavirusus (ANDV) seems to be the only species with documented person-to-person transmission. Hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) are both serious syndromes associated with hantavirus infections. For both syndromes, the mortality rate is near 40%. Decades of studies already highlighted the CpG repression in RNA viruses, and both the estimation of the CpG odds ratio and the correlation with their genome polarity were dominant factors to figure out the CpG bias. We conducted the differential analysis of the CpG odds ratio for all the orthohantaviruses on the full segmented genomes (L, M, S). The results suggested the statistical significance of the three groups. The "Small" genomes resulted in the more informative from the CpG odd ratio point of view. We calculated the CpG odds ratio for all the Orthohantaviruses within these segments, and besides, we estimated the correlation coefficient with the relative coding sequences (CDS). Preliminary results firstly confirmed the CpG odds ratio as the lowest among all the nucleotides. Second, highlighted the Andes virus as that whose CpG odds ratio within CDS is highest. The use of these two measures as features for unsupervised clustering algorithms has brought to the identification of four different sub-groups inside of the Orthohantaviridae family. The evidence is that the Andes Hantavirus exhibits a peculiar CpG odds ratio distribution, probably linked to its unique prerogative to pass from human-to-human.

sentences: An open, analysis-ready, and quality controlled resource for pediatric brain white-matter research We created resources to facilitate research on the role of human brain microstructure in the development of mental health disorders, based on openly-available diffusion MRI (dMRI) data from the Healthy Brain Network (HBN) study. First, we curated the HBN dMRI data (N=2747) into the Brain Imaging Data Structure and preprocessed it according to best-practices, including denoising and correcting for motion effects, susceptibility-related distortions, and eddy currents. Preprocessed, analysis-ready data was made openly available. Data quality plays a key role in the analysis of dMRI, and we provide automated quality control (QC) scores for every scan, as part of the data release. To scale QC to this large dataset, we trained a neural network through the combination of a small data subset scored by experts and a larger set scored by community scientists. The network performs QC highly concordant with that of experts on a held out set (ROC-AUC = 0.947). A further analysis of the neural network demonstrates that it relies on image features with relevance to QC. Altogether, this work both delivers a resource for transdiagnostic research in brain connectivity and pediatric mental health and serves as a novel tool for automated QC of large datasets.

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A complete reference genome improves analysis of human genetic variation Compared to its predecessors, the Telomere-to-Telomere CHM13 genome adds nearly 200 Mbp of sequence, corrects thousands of structural errors, and unlocks the most complex regions of the human genome to clinical and functional study. Here we demonstrate how the new reference universally improves read mapping and variant calling for 3,202 and 17 globally diverse samples sequenced with short and long reads, respectively. We identify hundreds of thousands of novel variants per sample--a new frontier for evolutionary and biomedical discovery. Simultaneously, the new reference eliminates tens of thousands of spurious variants per sample, including up to 12-fold reduction of false positives in 269 medically relevant genes. The vast improvement in variant discovery coupled with population and functional genomic resources position T2T-CHM13 to replace GRCh38 as the prevailing reference for human genetics.

One Sentence SummaryThe T2T-CHM13 reference genome universally improves the analysis of human genetic variation.

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Bacterial community dynamics during embryonic development of the little skate (Leucoraja erinacea) BackgroundMicrobial transmission from parent to offspring is hypothesized to be widespread in vertebrates. However, evidence for this is limited as many evolutionarily important clades remain unexamined. There is currently no data on the microbiota associated with any Chondrichthyan species during embryonic development, despite the global distribution, ecological importance, and phylogenetic position of this clade. In this study, we take the first steps towards filling this gap by investigating the microbiota associated with embryonic development in the little skate, Leucoraja erinacea, a common North Atlantic species and popular system for chondrichthyan biology.

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

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

ConclusionsOur study is the first exploration of the chondrichthyan microbiota throughout ontogeny and provides the first evidence of vertical transmission in this group.

sentences: Disrupted microglial iron homeostasis in progressive multiple sclerosis Multiple Sclerosis (MS) is a chronic autoimmune disease affecting the central nervous system (CNS). Despite therapies that reduce relapses, many patients eventually develop secondary progressive MS (SPMS), characterized by ongoing and irreversible neurodegeneration and worsening clinical symptoms. Microglia are the resident innate immune cells of the CNS. While the cellular and molecular determinants of disability progression in MS remain incompletely understood, they are thought to include non-resolving microglial activation and chronic oxidative injury. In this study, our aim was to better characterize microglia in SPMS tissues to identify disease-related changes at the single cell level. We performed single nucleus RNA-seq (snRNA-seq) on cryopreserved post-mortem brain cortex and identified disease associated changes in multiple cell types and in particular distinct SPMS-enriched microglia subsets. When compared to the cluster most enriched in healthy controls (i.e. homeostatic microglia), we found a number of SPMS-enriched clusters with transcriptional profiles reflecting increased oxidative stress and perturbed iron homeostasis. Using histology and RNA-scope, we confirmed the presence of iron accumulating, ferritin-light chain (FTL)-expressing microglia in situ. Among disease-enriched clusters, we found evidence for divergent responses to iron accumulation and identified the antioxidant enzyme GPX4 as a key fate determinant. These data help elucidate processes that occur in progressive MS brains, and highlight novel nodes for therapeutic intervention.

sentences: Crumbs complex-directed apical membrane dynamics in epithelial cell ingression Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cell lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. Here, we show that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. Our findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer.

sentences: Synthetic cell-based materials extract positional information from morphogen gradients Dynamic biomaterials composed of synthetic cellular structures have the potential to adapt and functionally differentiate guided by physical and chemical cues from their environment. Inspired by developing biological systems, which efficiently extract positional information from chemical morphogen gradients in the presence of environmental uncertainties, we here investigate the analogous question: how well can a synthetic cell determine its position within a synthetic multicellular structure? In order to calculate positional information in such systems, we created and analyzed a large number of replicas of synthetic cellular assemblies, which were composed of emulsion droplets connected via lipid bilayer membranes. The droplets contained cell-free two-node feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. We found that in our system, simple anterior-posterior differentiation is possible, but positional information is limited by gene expression noise, and is also critically affected by the temporal evolution of the morphogen gradient and the life-time of the cell-free expression system contained in the synthetic cells. Using a 3D printing approach, we demonstrate morphogen-based differentiation also in larger tissue-like assemblies.

sentences: Processing body dynamics drive non-genetic MEK inhibitors tolerance by fine-tuning KRAS and NRAS translation Overactivation of the Mitogen-activated protein kinase (MAPK) pathway is a critical driver of many human cancers. However, therapies targeting this pathway have proven effective in only a few cancers, as cancers inevitably develop resistance. Puzzling observations suggest that MAPK targeting fails in tumors due to early compensatory RAS overexpression, albeit by unexplained mechanisms. We identified a novel mechanism of drug tolerance to MEK inhibitors (MEKi) that involves Processing Bodies (PBs), a membraneless organelle (MLO). MEKi promoted translation of the oncogenes KRAS and NRAS, which in turn triggered BRAF phosphorylation. This overexpression, which occurred in the absence of neotranscription, depended on PB dissolution as the source of the RAS mRNA. Moreover, in response to MEKi removal, the process was dynamic as PBs rapidly reformed and reduced MAPK signaling. These results highlight a dynamic spatiotemporal negative feedback loop of MAPK signaling via RAS mRNA sequestration. Furthermore, we observed a phenotype with a low number of PBs along with strong KRAS and NRAS induction capacities. Overall, we describe a new intricate mechanism involving PBs in the translational regulation of essential cellular signaling pathways like MAPKs, paving the way for future therapies altering MLO and thereby improving targeted cancer therapies.

sentences: Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis Despite the existence of well-characterized, canonical mutations that confer high-level drug resistance to Mycobacterium tuberculosis (Mtb), there is evidence that drug resistance mechanisms are more complex than simple acquisition of such mutations. Recent studies have shown that Mtb can acquire non-canonical resistance-associated mutations that confer survival advantages in the presence of certain drugs, likely acting as stepping-stones for acquisition of high-level resistance. Rv2752c/rnj, encoding RNase J, is disproportionately mutated in drug-resistant clinical Mtb isolates. Here we show that deletion of rnj confers increased tolerance to lethal concentrations of several drugs. RNAseq revealed that RNase J affects expression of a subset of genes enriched for PE/PPE genes and stable RNAs and is key for proper 23S rRNA maturation. Gene expression differences implicated two sRNAs and ppe50-ppe51 as important contributors to the drug tolerance phenotype. In addition, we found that in the absence of RNase J, many short RNA fragments accumulate because they are degraded at slower rates. We show that the accumulated transcript fragments are targets of RNase J and are characterized by strong secondary structure and high G+C content, indicating that RNase J has a rate-limiting role in degradation of highly structured RNAs. Taken together, our results demonstrate that RNase J indirectly affects drug tolerance, as well as reveal the endogenous roles of RNase J in mycobacterial RNA metabolism.

sentences: 2-kupl: mapping-free variant detection from DNA-seq data of matched samples The detection of genome variants, including point mutations, indels and structural variants, is a fundamental and challenging computational problem. We address here the problem of variant detection between two deep-sequencing (DNA-seq) samples, such as two human samples from an individual patient, or two samples from distinct bacterial strains. The preferred strategy in such a case is to align each sample to a common reference genome, collect all variants and compare these variants between samples. Such mapping-based protocols have several limitations. DNA sequences with large indels, aggregated mutations and structural variants are hard to map to the reference. Furthermore, DNA sequences cannot be mapped reliably to genomic low complexity regions and repeats. Herein, we introduce 2-kupl, a k-mer based, mapping-free protocol to detect variants between two DNA-seq samples. On simulated and actual data, 2-kupl achieves a higher precision than other mapping-free protocols. Applying 2-kupl to prostate cancer whole exome data, we identify a number of candidate variants in hard-to-map regions and propose potential novel recurrent variants in this disease.

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Effect of knee joint loading on chondrocyte mechano-vulnerability and severity of post-traumatic osteoarthritis induced by ACL-injury in mice ObjectiveThe objective of this study is to understand the role of altered in vivo mechanical environments in knee joints post anterior cruciate ligament (ACL)-injury in chondrocyte vulnerability against mechanical stimuli and in the progression of post-traumatic osteoarthritis (PT-OA).

MethodsDifferential in vivo mechanical environments were induced by unilateral ACL-injury (uni-ACL-I) and bilateral ACL-injury (bi-ACL-I) in 8-week-old female C57BL/6 mice. The gait parameters, the mechano-vulnerability of in situ chondrocytes, Youngs moduli of cartilage extracellular matrix (ECM), and the histological assessment of OA severity (OARSI score) were compared between control and experimental groups at 0[~]8-weeks post-ACL-injury.

ResultsWe found that bi-ACL-I mice experience higher joint-loading on their both injured limbs, but uni-ACL-I mice balance their joint-loading between injured and uninjured hind limbs resulting in a reduced joint-loading during gait. We also found that at 4- and 8-week post-injury the higher weight-bearing hind limbs (i.e., bi-ACL-I) had the increased area of chondrocyte death induced by impact loading and higher OARSI score than the lower weight-bearing limbs (uni-ACL-I). Additionally, we found that at 8-weeks post-injury the ECM became stiffer in bi-ACL-I joints and softer in uni-ACL-I joints.

ConclusionsOur results show that ACL-injured limbs with lower in vivo joint-loading develops PT-OA significantly slower than injured limbs with higher joint-loading during gait. Our data also indicate that articular chondrocytes in severe PT-OA are more fragile from mechanical impacts than chondrocytes in healthy or mild PT-OA. Thus, preserving physiologic joint-loads on injured joints will reduce chondrocyte death post-injury and may delay PT-OA progression.

sentences: A bivariate zero-inflated negative binomial model and its applications to biomedical settings SO_SCPLOWUMMARYC_SCPLOWThe zero-inflated negative binomial (ZINB) distribution has been widely used for count data analyses in various biomedical settings due to its capacity of modeling excess zeros and overdispersion. When there are correlated count variables, a bivariate model is essential for understanding their full distributional features. For this purpose, we develop a Bivariate Zero-Inflated Negative Binomial (BZINB) model that has a simple latent variable framework and parameters with intuitive interpretations. Using this model, we examine two biomedical data examples where the counts are zero-inflated--single cell RNA sequencing (scRNA-seq) data and dental caries count indices. In scRNA-seq data example, a correlation between a pair of genes is estimated after adjusting for the effects of dropout events represented by excess zeros. In the dental caries data, we analyze how the treatment with Xylitol mints affects the marginal mean and other patterns of response manifested in the two dental caries traits. An R package  bzinb is available on CRAN.

sentences: Motor cortex activity across movement speeds is predicted by network-level strategies for generating muscle activity Learned movements can be skillfully performed at different paces. What neural strategies produce this flexibility? Can they be predicted and understood by network modeling? We trained monkeys to perform a cycling task at different speeds, and trained artificial recurrent networks to generate the empirical muscle-activity patterns. Network solutions reflected the principle that smooth well-behaved dynamics require low trajectory tangling. Network solutions had a consistent form, which yielded quantitative and qualitative predictions. To evaluate predictions, we analyzed motor cortex activity recorded during the same task. Responses supported the hypothesis that the dominant neural signals reflect not muscle activity, but network-level strategies for generating muscle activity. Single-neuron responses were better accounted for by network activity than by muscle activity. Similarly, neural population trajectories shared their organization not with muscle trajectories, but with network solutions. Thus, cortical activity could be understood based on the need to generate muscle activity via dynamics that allow smooth, robust control over movement speed.

sentences: Paradoxical relationship between speed and accuracy in olfactory figure-background segregation In natural settings, many stimuli impinge on our sensory organs simultaneously. Parsing these sensory stimuli into perceptual objects is a fundamental task faced by all sensory systems. Similar to other sensory modalities, increased odor backgrounds decrease the detectability of target odors by the olfactory system. The mechanisms by which background odors interfere with the detection and identification of target odors are unknown. Here we utilized the framework of the Drift Diffusion Model (DDM) to consider possible interference mechanisms in an odor detection task. We consider effects of background odors on both signal and noise in the decision-making dynamics, and show that these produce different predictions about decision accuracy and speed. To test these predictions, we trained mice to detect target odors that are embedded in random background mixtures in a two-alternative choice task. Trial duration was independent of behavioral reaction times to avoid motivating rapid responses. We found that the behavioral data is most consistent with background odors acting by adding noise to the decision-making dynamics. The added noise decreases the correct rate, but also decreases decision times, thereby creating a paradoxical relationship between speed and accuracy of target detection, where mice make faster and less accurate decisions in the presence of background odors.

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A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism in Bacteroides thetaiotaomicron Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes. Here we report the identification of RBPs that are broadly distributed across the Bacteroidetes, with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron {Delta}rbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The {Delta}rbpB mutant had reduced expression of BT1871, encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes.

ImportanceThe human colon houses hundreds of bacterial species, including many belonging to the genus Bacteroides, that aid in breaking down our food to keep us healthy. Bacteroides have many genes responsible for breaking down different dietary carbohydrates and complex regulatory mechanisms ensure that specific genes are only expressed when the right carbohydrates are available. In this study, we discovered that Bacteroides use a family of RNA-binding proteins as global regulators to coordinate expression of carbohydrate utilization genes. The ability to turn different carbohydrate utilization genes on and off in response to changing nutrient conditions is critical for Bacteroides to live successfully in the gut, and thus the new regulators we have identified may be important for life in the host.

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Diversity and Divergence: Evolution of defense chemistry in the tropical tree genus Inga O_LIPlants are widely recognized as chemical factories, with each species producing dozens to hundreds of unique secondary metabolites. These compounds shape the interactions between plants and their natural enemies. Here we explore how plants generate chemical diversity, and what evolutionary processes have led to novel compounds and unique chemical profiles.
C_LIO_LIWe comprehensively characterized the chemical profile of one-third of the species of tropical rainforest trees in the genus Inga ([~] 100, Fabaceae) and applied phylogenetic comparative methods to understand the mode of chemical defense evolution.
C_LIO_LIWe show that: 1) Each Inga species produces exceptionally high levels of phytochemical diversity, despite costs, tradeoffs and biosynthetic constraints. 2) Closely related species have highly divergent defense profiles, with individual compounds, major compound classes and complete profiles showing little to no phylogenetic signal. 3) We show that the evolution of a species chemical profile shows a signature of divergent adaptation, implying that it is advantageous for a species to have distinct chemistry from close relatives to avoid shared natural enemies. 4) Finally, we hypothesize a model where deep homology of biosynthetic pathways and rapid changes in regulatory mechanisms may better explain the observed large shifts in defense chemicals between closely related taxa.
C_LI

sentences: Optimisation-based modelling for drug discovery in malaria The discovery of new antimalarial medicines with novel mechanisms of action is important, given the ability of parasites to develop resistance to current treatments. Through the Open Source Malaria project that aims to discover new medications for malaria, several series of compounds have been obtained and tested. Analysis of the effective fragments in these compounds is important in order to derive means of optimal drug design and improve the relevant pharmaceutical application. We have previously reported a novel optimisation-based method for quantitative structure-activity relationship modelling, modSAR, that provides explainable modelling of ligand activity through a mathematical programming formulation. Briefly, modSAR clusters small molecules according to chemical similarity, determines the optimal split of each cluster into appropriate regions, and derives piecewise linear regression equations to predict the inhibitory effect of small molecules. Here, we report application of modSAR in the analysis of OSM anti-malarial compounds and illustrate how rules generated by the model can provide interpretable results for the contribution of individual ECFP fingerprints in predicting ligand activity, and contribute to the search for effective drug treatments.

sentences: Centrosomal Enrichment and Proteasomal Degradation of SYS-1/-β-catenin Requires the Microtubule Motor Dynein The C. elegans Wnt/{beta}-catenin Asymmetry (W{beta}A) pathway utilizes asymmetric regulation of SYS- 1/{beta}-catenin and POP-1/TCF coactivators. W{beta}A differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased towards their appropriate cell fate. Despite the induction of asymmetry, {beta}-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Due to the mitosis-specific localization of SYS-1 to centrosomes and enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNAi-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe depletion of microtubules by nocodazole treatment or RNAi of dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose a model whereby retrograde microtubule-mediated trafficking enables SYS-1 enrichment at centrosomes, enhancing its eventual proteasomal degradation. These studies support the link between centrosomal localization and enhancement of proteasomal degradation, particularly for proteins not generally considered  centrosomal.

sentences: A common SEC23B missense mutation in congenital dyserythropoietic anemia type II leads to growth restriction and chronic pancreatitis in mice Human loss-of-function mutations in SEC23B result in congenital dyserythropoietic anemia type II (CDAII). Complete deficiency of SEC23B in mice leads to perinatal death caused by massive degeneration of professional secretory tissues. Functions of SEC23B in postnatal mice are unclear. In this study, we generated knockout (KO) mice with deletion of exons 5 and 6 of Sec23b and knockin (KI) mice with the E109K mutation, the most common CDAII missense mutation. Despite decreased SEC23B protein level, Sec23bki/ki mice showed no obvious abnormalities. Hemizygous (Sec23bki/ko) mice exhibit a partial lethal phenotype, with only half of expected mice survive past weaning. Sec23bki/ko pancreas had exocrine insufficiency and chronic pancreatitis histology, as well as increased ER stress and apoptosis. Moreover, Sec23bki/ko mice exhibited severe growth restriction accompanied by growth hormone (GH) insensitivity, reminiscent of the Laron syndrome. Growth restriction is not associated with hepatocyte-specific Sec23b deletion, suggesting non-liver origin of the phenotype. Inflammation associated with chronic pancreatic deficiency may explain GH insensitivity in Sec23bki/ko mice. Our results indicate that phenotype severities are linked to the residual functions of SEC23B, demonstrating the importance of SEC23B in pancreatic acinar function in adult mice. The Sec23bki/ko mice provide a novel model of chronic pancreatitis and growth retardation.

sentences: Conserved secreted effectors determine endophytic growth and multi-host plant compatibility in a vascular wilt fungus Fungal interactions with plant roots, either beneficial or detrimental, have a major impact on agriculture and ecosystems. The soil inhabiting ascomycete Fusarium oxysporum (Fo) constitutes a species complex of worldwide distribution causing vascular wilt in more than a hundred different crops. Individual isolates of the fungus exhibit host-specific pathogenicity, determined by proteinaceous effectors termed secreted in xylem (SIX). However, such isolates can also colonize roots of non-host plants asymptomatically as endophytes, or even protect them against pathogenic isolates. The molecular determinants of multi-host plant colonization are currently unknown. Here, we identified a set of fungal effectors termed ERCs (Early Root Compatibility effectors), which are secreted during early biotrophic growth of Fo on both host and non-host plants. In contrast to the strain-specific SIX effectors, which are encoded on accessory genomic regions, ERCs are encoded on core regions and are found across the entire Fo species complex as well as in other phytopathogens, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence on the host plant and rapid activation of plant immune responses, while in a non-pathogenic isolate it led to impaired root colonization and loss of biocontrol ability. Strikingly, some ERCs also contribute to Fo infection on the non-vascular land plant Marchantia polymorpha. Our results reveal an evolutionarily conserved mechanism for multi-host colonization by root infecting fungi.

sentences: On the Etiology of Aesthetic Chills: a Behavioral Genetic Study Aesthetic chills, broadly defined as a somatic marker of peak emotional-hedonic responses, are experienced by individuals across a variety of human cultures. Yet individuals vary widely in the propensity of feeling them. These individual differences have been studied in relation to demographics, personality, and neurobiological and physiological factors, but no study to date has explored the genetic etiological sources of variation. To partition genetic and environmental sources of variation in the propensity of feeling aesthetic chills, we fitted a biometrical genetic model to data from 14127 twins (from 8995 pairs), collected by the Netherlands Twin Register. Both genetic and unique environmental factors accounted for variance in aesthetic chills, with heritability estimated at .36 ([.33, .39] 95% CI). We found females more prone than males to report feeling aesthetic chills. However, a test for genotype x sex interaction did not show evidence that heritability differs between sexes. We thus show that the propensity of feeling aesthetic chills is not shaped by nurture alone, but it also reflects underlying genetic propensities.

sentences: Differential serotonergic modulation of principal neurons and interneurons in the anterior piriform cortex Originating from the brainstem raphe nuclei, serotonin is an important neuromodulator involved in a variety of physiological and pathological functions. Specific optogenetic stimulation of serotonergic neurons results in the divisive suppression of spontaneous, but not sensory evoked activity in the majority of neurons in the primary olfactory cortex and an increase in firing in a minority of neurons. To reveal the mechanisms involved in this dual serotonergic control of cortical activity we used a combination of in vitro electrophysiological recordings from identified neurons in the primary olfactory cortex, optogenetics and pharmacology and found that serotonin suppressed the activity of principal neurons, but excited local interneurons. The results have important implications in sensory information processing and other functions of the olfactory cortex and related brain areas.

sentences: Downregulation of NCL attenuates tumor formation and growth in cervical cancer by targeting the PI3K/AKT pathway Nucleolin (NCL, C23) is a multifunctional phosphoprotein that plays a vital role in modulating the survival, proliferation and apoptosis of cancer cells. However, the effects of NCL on cervical cancer and the underlying mechanisms behind this are poorly understood. In the study presented here, Hela cells were transfected with shRNAs targeting the endogenous NCL gene (sh-NCL-Hela). NCL knockdown inhibited cell proliferation and promoted apoptosis both in vivo and in vitro. Mechanistic studies revealed that NCL knockdown inhibited the PI3K/AKT pathway by upregulating FGF, ITGA, TNXB, VEGF, Caspase 3, and Bax, as well as by downregulating AKT, GNB4, CDK6, IL6R, LAMA, PDGFD, PPP2RSA and BCL-2. In addition, the expression levels of apoptosis-related genes after using a PI3K inhibitor LY294002 were consistent with shRNA studies, while treatment with a 740Y-P agonist showed the opposite effect. Altogether, this study uncovered that downregulation of NCL may be a novel treatment strategy for cervical cancer.

sentences: The therapeutic potential of D-Serine in reducing expression of the cytopathic genotoxin colibactin Some Escherichia coli strains belonging mainly to the B2 phylogroup harbour the pks island, a 54 kb genomic island encoding the biosynthesis genes for a genotoxic compound named colibactin. In eukaryotic cells, colibactin can induce DNA damage, cell cycle arrest and chromosomal instability. Moreover, production of colibactin has been implicated in the development of colorectal cancer. In this study, we demonstrate the inhibitory effect of D-Serine on the expression of the pks island in two colibactin-producing strains, CFT073 and Nissle 1917, and determine the implications for cytopathic effects on host cells. To investigate the specificity of the inhibitory effect of D-Serine, we also tested a comprehensive panel of proteinogenic L-amino acids and corresponding D-enantiomers for their ability to modulate clbB transcription using RT-qPCR. Several D-amino acids exhibited the ability to inhibit expression of clbB, with D-Serine exerting the strongest repressing activity (3.81-fold in CFT073; 3.80-fold in Nissle 1917) and thus, we focussed additional experiments on D-Serine. To investigate the cellular effect, we investigated if repression of colibactin by D-Serine could reduce the cytopathic responses normally observed during infection of HeLa cells with pks+ strains. Levels of {gamma}-H2AX (a marker of DNA double strand breaks) were reduced 2.75-fold in cells infected with D-Serine treatment. Moreover, exposure of pks+ E. coli to D-Serine during infection caused a reduction in cellular senescence that was observable at 72 h post infection. The recent finding of an association between pks-carrying commensal E. coli and CRC, highlights the necessity for the development of colibactin targeting therapeutics. Here we show that D-Serine can reduce expression of colibactin, and inhibit downstream cellular cytopathy, illuminating its therapeutic potential to prevent colibactin-associated disease.

sentences: T cell response following anti COVID-19 BNT162b2 vaccination is maintained against the SARS-CoV-2 Omicron B.1.1.529 variant of concern The progression of the COVID-19 pandemic leads to the emergence of variants of concern (VOC), which may compromise the efficacy of the currently administered vaccines. Antigenic drift can potentially bring about a reduced protective T cell immunity and consequently to more severe disease manifestations. To assess this possibility, the T cell responses to the wild-type, Wuhan-1 SARS-CoV-2 ancestral spike protein and Omicron B.1.1.529 spike protein were compared. Accordingly, peripheral blood mononuclear cells (PBMC) were collected from 8 healthy volunteers 4-5 months following a third vaccination with BNT162b2, and stimulated with overlapping peptide libraries representing the spike of either the ancestral or Omicron SARS-CoV- 2 virus variants. Quantification of the specific T cells was carried out by a fluorescent ELISPOT assay, monitoring interferon-gamma (IFNg), interleukin-10 (IL-10) and interleukin-4 (IL-4) secreting cells. For all the examined individuals, comparable level of reactivity to both forms of spike protein were determined. In addition, a dominant Th1 response was observed, manifested mainly by IFNg secreting cells and only limited numbers of IL-10 and IL-4 secreting cells. The data demonstrates a stable T cell activity to the emerging Omicron variant in the tested individuals, therefore the protective immunity to the variant following BNT162b2 vaccination is not significantly affected.

sentences: Quantitative analysis of transcription start site selection in Saccharomyces cerevisiae determines contributions of DNA sequence and RNA Polymerase II activity DNA sequence at Transcription Start Sites (TSSs) is a key determinant of initiation by RNA Polymerase II (Pol II). To function as a TSS, an initiation compatible sequence must be specified by a promoter in an appropriate chromatin context. We report the development of a method for quantitative analysis of transcription initiation by Pol II that involves construction of DNA libraries of barcoded promoter variants, production of RNA transcripts, and analysis of transcript 5 ends and transcript yields (Pol II MAssively Systematic Transcript End Readout, "Pol II MASTER"). Using Pol II MASTER, we measure the efficiency of transcription initiation during "promoter scanning" by Saccharomyces cerevisiae Pol II for ~1 million unique TSS sequences. Furthermore, we employ Pol II MASTER to determine how Pol II activity, known to widely alter TSS selection in vivo, alters TSS efficiencies across our promoter variants. Pol II MASTER recapitulates known critical qualities of Saccharomyces cerevisiae TSS -8, -1, and +1 positions while demonstrating that surrounding sequences modulate initiation efficiency over a wide range. We discover functional interactions between neighboring sequence positions, indicating that adjacent positions likely function together. We demonstrate that initiation efficiencies are altered for +1 A TSSs relative to +1 G TSSs when Pol II activity is perturbed through genetic means. Pol II MASTER provides data for predictive models of TSS initiation efficiency at genomic promoters.

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Reference transcriptome data in silkworm, Bombyx mori BackgroundThe silkworm Bombyx mori is a lepidopteran model insect with biological and industrial importance. Its high-quality genome sequence has recently become available and the utilization of this information in combination with extensive transcriptomic analyses is expected to provide an elaborate gene model. It will also be possible to clarify gene expression in detail using this approach.

ResultsWe herein performed RNA-seq analysis of ten major tissues/subparts of silkworm larvae. Sequences were mapped onto the reference genome assembly and reference transcriptome data was successfully constructed. The reference data provided a nearly complete sequence for sericin-1, a major silk gene with a complex structure. We also markedly improved the gene model for other genes. Transcriptomic expression was investigated in each tissue and a number of transcripts were identified that were exclusively expressed in tissues such as the testis. Transcripts strongly expressed in the midgut formed tight genomic clusters, suggesting that they originated from tandem gene duplication. Transcriptional factor genes expressed in specific tissues or the silk gland subparts were also identified.

ConclusionsWe successfully constructed reference transcriptome data in the silkworm and found that a number of transcripts showed unique expression profiles. These results will facilitate basic studies on the silkworm and accelerate its applications, which will contribute to further advances in lepidopteran and entomological research and the practical use of these insects.

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Epithelial and neutrophil interactions and coordinated response to Shigella in a human intestinal enteroid-neutrophil co-culture model Polymorphonuclear neutrophils (PMN) are recruited to the gastrointestinal mucosa in response to inflammation, injury, and infection. Herein, we report the development and the characterization of an ex vivo tissue co-culture model consisting of human primary intestinal enteroid monolayers and PMN, and a mechanistic interrogation of PMN-epithelial cell interaction and response to Shigella, a primary cause of childhood dysentery. Cellular adaptation and tissue integration, barrier function, PMN phenotypic and functional attributes, and innate immune responses were examined. PMN within the enteroid monolayers acquired a distinct activated/migratory phenotype that was influenced by direct epithelial cell contact as well as by molecular signals. Seeded on the basal side of the intestinal monolayer, PMN intercalated within the epithelial cells and moved paracellularly toward the apical side. Co-cultured PMN also increased basal secretion of IL-8. Shigella added to the apical surface of the monolayers evoked additional PMN phenotypic adaptations, including increased expression of cell surface markers associated with chemotaxis and cell degranulation (CD47, CD66b, and CD88). Apical Shigella infection triggered rapid transmigration of PMN to the luminal side, NET formation as well as bacterial phagocytosis and killing. Shigella infection modulated cytokine production in the co-culture; apical MCP-1, TNF-, and basolateral IL-8 production were downregulated, while basolateral IL-6 secretion was increased. We demonstrated, for the first time, PMN phenotypic adaptation, mobilization, and coordinated epithelial cell-PMN innate response upon Shigella infection in the human intestinal environment. The enteroid monolayer-PMN co-culture represents a technical innovation for mechanistic interrogation of gastrointestinal physiology, host-microbe interaction, innate immunity, and evaluation of preventive/therapeutic tools.

ImportanceStudies of mucosal immunity and microbial host cell interaction have traditionally relied on animal models and in vitro tissue culture using immortalized cancer cell lines, which render non-physiological and often unreliable results. Herein we report the development and characterization of an ex vivo enteroid-PMN co-culture consisting of normal human intestinal epithelium and a mechanistic interrogation of PMN and epithelial cell interaction and function in the context of Shigella infection. We demonstrated tissue-driven phenotypic and functional adaptation of PMN and a coordinated epithelial cell and PMN response to Shigella, a primary cause of dysentery in young children in the developing world.

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Zebrafish her3 knockout impacts developmental and cancer-related gene signatures HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle. However, the mechanisms of HES3 cooperation in fusion-positive rhabdomyosarcoma are unclear and are likely related to her3/HES3s role in neurogenesis. To investigate HES3s function during development, we generated a zebrafish CRISPR/Cas9 knockout of her3, the zebrafish ortholog of HES3. Loss of her3 is not embryonic lethal and adults exhibit expected Mendelian ratios. Embryonic her3 zebrafish mutants are significantly smaller than wildtype and a subset present with lens defects as adults. Transcriptomic analysis of her3 mutant embryos indicates that genes involved in organ development, such as pctp and grinab, are significantly downregulated. Further, differentially expressed genes in her3 knockout embryos are enriched for HOX and SOX10 motifs. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases. Altogether, this new model is a powerful system to study her3/HES3-mediated neural development and its misappropriation in cancer contexts.

Summary StatementHere, we generate and characterize a zebrafish her3/HES3 knockout to elucidate the functional role of her3/HES3, a transcriptional repressor, in neural development and tumorigenic processes.

sentences: Sensorimotor feedback loops are selectively sensitive to reward While it is well established that motivational factors such as earning more money for performing well improve motor performance, how the motor system implements this improvement remains unclear. For instance, feedback-based control, which uses sensory feedback from the body to correct for errors in movement, improves with greater reward. But feedback control encompasses many feedback loops with diverse characteristics such as the brain regions involved and their response time. Which specific loops drive these performance improvements with reward is unknown, even though their diversity makes it unlikely that they are contributing uniformly. This lack of mechanistic insight leads to practical limitations in applications using reward, such as clinical rehabilitation, athletic coaching, and brain-inspired robotics. We systematically tested the effect of reward on the latency (how long for a corrective response to arise?) and gain (how large is the corrective response?) of eight distinct sensorimotor feedback loops in humans. Only the feedback responses known to rely on prefrontal associative cortices showed sensitivity to reward, while feedback responses that relied mainly on premotor and sensorimotor cortex did not show sensitivity to reward. Our results may have implications regarding feedback control performance in pathologies showing a cognitive decline, or on athletic coaching. For instance, coaching methodologies that rely on reinforcement or "reward shaping" may need to specifically target aspects of movement that rely on reward-sensitive feedback responses.

sentences: Low-coverage reduced representation sequencing reveals subtle within-island genetic structure in Aldabra giant tortoises Aldabrachelys gigantea (Aldabra giant tortoise) is one of only two giant tortoise species left in the world and survives as a single wild population of over 100,000 individuals on Aldabra Atoll, Seychelles. Despite this large current population size, the species faces an uncertain future because of its extremely restricted distribution range and high vulnerability to the projected consequences of climate change. Captive-bred A. gigantea are increasingly used in rewilding programs across the region, where they are introduced to replace extinct giant tortoises in an attempt to functionally resurrect degraded island ecosystems. However, there has been little consideration of the current levels of genetic variation and differentiation within and among the islands on Aldabra. As previous microsatellite studies were inconclusive, we combined low-coverage and double digest restriction associated DNA (ddRAD) sequencing to analyze samples from 33 tortoises (11 from each main island). Using 5,426 variant sites within the tortoise genome, we detected patterns of population structure within two of the three studied islands, but no differentiation between the islands. These unexpected results highlight the importance of using genome-wide genetic markers to capture higher-resolution genetic structure to inform future management plans, even in a seemingly panmictic population. We show that low-coverage ddRAD sequencing provides an affordable alternative approach to conservation genomic projects of non-model species with large genomes.

sentences: Cardiac and gastric interoception have distinct neural substrates The functional roles of interoception or senses that arise from within the body have been thought to guide agents in adaptive behavior by informing various bodily states like heart rate or energy status. However, direct evidence that different types of interoception are processed or represented differently in the human brain is still lacking. Hence, we directly compared the neural activation for cardiac (awareness related to heartbeats) and gastric (awareness related to the stomach) interoception in the same population (N = 31). Participants were asked to focus on their heart and stomach sensations in a magnetic resonance imaging scanner. The results showed that neural activation underlying gastric interoception encompassed the sensorimotor and reward-related regions when compared with cardiac interoception, including the occipitotemporal visual cortices, bilateral primary motor cortex, primary somatosensory cortex, left orbitofrontal cortex, and bilateral hippocampal regions. Conversely, cardiac interoception selectively activated the right frontal operculum/insula, in contrast to gastric interoception. Moreover, we found that the left dorsal middle insula encoded cardiac and gastric interoception differently, but the posterior insula was unaffected by the multivoxel pattern analysis. Our results present novel data demonstrating that cardiac and gastric interoception have distinct neural substrates particularly relevant to the functions for each interoception.

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

sentences: The RNA helicases DDX5 and DDX17 facilitate neural differentiation of human pluripotent stem cells NTERA2 Understanding human neurogenesis is critical toward regenerative medicine for neurodegeneration. However, little is known how neural differentiation is regulated by RNA helicases, which comprise a diverse class of RNA remodeling enzymes. We show here that expression of the DEAD boxcontaining RNA helicases DDX5 and DDX17 is abundant throughout retinoic acid-induced neural differentiation of the human pluripotent stem cell (hPSC) line NTERA2, and is mostly localized within the nucleus. Using ChIP-seq, we identify that the two RNA helicases occupy chromatin genome-wide at regions associated with neurogenesis- and differentiation-related genes in both hPSCs and their neural derivatives. Further, RNA-seq analyses indicate both DDX5 and DDX17 are mutually required for controlling transcriptional expression of these genes. We show that the two RNA helicases are not important for maintenance of stem cell state of hPSCs. In contrast, they facilitate early neural differentiation of hPSCs, generation of neurospheres from the stem cells, and expression of key neurogenic transcription factors during neural differentiation. Importantly, DDX5 and DDX17 are important for differentiation of hPSCs toward NESTIN- and TUBB3-positive cells, which represent neural progenitors and mature neurons. Collectively, our findings suggest the role of DDX5 and DDX17 in transcriptional regulation of genes involved in neurogenesis, and hence in neural differentiation of hPSCs.

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Evidence for efficient non-evaporative leaf-to-air heat dissipation in a pine forest under drought conditions O_LIDrier climates predicted for many regions can result in reduced evaporative cooling leading to leaf heat stress and enhanced mortality. To what extent non-evaporative cooling can contribute to plant resilience to the increasingly stressful conditions is poorly known at present.
C_LIO_LIUsing a novel, high accuracy infrared system for continuous measurements of leaf temperature in mature trees under field conditions, we assessed leaf-to-air temperature differences {Delta}Tleaf-air of pine needles during drought.
C_LIO_LIOn mid-summer days, {Delta}Tleaf-air remained <1.5 {degrees}C, both in trees exposed to summer drought, and in those provided with a supplement irrigation having a 10x higher transpiration rate. The non-evaporative cooling in the drought-exposed trees must be facilitated by low resistance to heat transfer generating large H. {Delta}Tleaf-air was weakly related to variations in the radiation load and mean wind speed in the lower part of the canopy, but highly dependent on canopy structure and within-canopy turbulence that enhanced the sensible heat flux H.
C_LIO_LINon-evaporative cooling is demonstrated as an effective cooling mechanism in needle-leaf trees, which can be a critical factor in forest resistance to drying climates. The generation of a large H at the leaf scale provides a basis for the development of the previously identified canopy-scale  convector effect.
C_LI

sentences: Anti-Bat Ultrasound Production in Moths is Globally and Phylogenetically Widespread Warning signals are well known in the visual system, but rare in other modalities. Some moths produce ultrasonic sounds to warn bats of noxious taste or to mimic unpalatable models. Here we report results from a long-term study across the globe, assaying moth response to playback of bat echolocation. We tested 252 genera, spanning most families of large-bodied moths, and outline anti-bat ultrasound production in 52 genera, with eight new subfamily origins described. Based on acoustic analysis of ultrasonic emissions and palatability experiments with bats, it seems that acoustic warning and mimicry are the raison detre for sound production in most moths. However, some moths use high-density ultrasound capable of jamming bat sonar. In fact, we find preliminary evidence of independent origins of sonar jamming in at least six subfamilies. Palatability data indicates that jamming and warning are not mutually exclusive strategies. To explore the possible organization of anti-bat warning sounds into acoustic mimicry rings, we intensively studied a community of moths in Ecuador and found five distinct acoustic clusters using machine learning algorithms. While these data represent an early understanding of acoustic aposematism and mimicry across this megadiverse insect order, it is likely that ultrasonically-signaling moths comprise one of the largest mimicry complexes on earth.

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Characterization of cocaine addiction-like behavior in heterogeneous stock rats Twin studies suggest that approximately 50% of the vulnerability to cocaine use disorder is determined by genetic factors, but genome-wide association studies (GWAS) in humans have only begun to identify specific genes that confer this risk. The identification of a set of single nucleotide polymorphisms (SNPs) associated with increased vulnerability to develop compulsive cocaine use represents a major goal for understanding of the genetic risk factors to cocaine use disorder and facilitating the identification of novel druggable targets.

Here we characterized addiction-like behaviors in heterogeneous stock (HS) rats, a unique outbred strain of rats characterized by high genetic variability that has been developed to mimic genetic variability in humans. HS rats were allowed to self-administer cocaine 6h/daily for 14 days. Animals were also screened for compulsive cocaine use, using progressive-ratio (PR) and responding despite adverse consequences (contingent foot shocks). To minimize cohort-specific effects, we used large cohorts (n = 46-60) and normalized the level of responding within cohorts using a Z-score. To take advantage of the three behaviors related to compulsive intake and further identify subjects that are consistently vulnerable vs. resilient to compulsive cocaine use we computed an Addiction index by averaging normalized responding (Z-scores) for the three behavioral tests. Results showed high individual variability between vulnerable and resilient rats that is likely to facilitate detection of gene variants associated with vulnerable vs. resilient individuals. Such data will have considerable translational value for designing pharmacogenetic studies in humans.

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ACTIVA: realistic single-cell RNA-seq generation with automatic cell-type identification using introspective variational autoencoders MotivationSingle-cell RNA sequencing (scRNAseq) technologies allow for measurements of gene expression at a single-cell resolution. This provides researchers with a tremendous advantage for detecting heterogeneity, delineating cellular maps, or identifying rare subpopulations. However, a critical complication remains the low number of single-cell observations due to limitations by the rarity of a subpopulation, tissue degradation, or cost. This absence of sufficient data may cause inaccuracy or irreproducibility of downstream analysis. In this work, we present ACTIVA (Automated Cell-Type-informed Introspective Variational Autoencoder): a novel framework for generating realistic synthetic data using a single-stream adversarial variational autoencoder conditioned with cell-type information. Within a single framework, ACTIVA can generate data representative of the entire population, or specific subpopulations on demand, as opposed to two separate models (such as scGAN and cscGAN). Data generation and augmentation with ACTIVA can enhance scRNAseq pipelines and analysis, such as benchmarking new algorithms, studying the accuracy of classifiers, and detecting marker genes. ACTIVA will facilitate analysis of smaller datasets, potentially reducing the number of patients and animals necessary in initial studies.

ResultsWe train and evaluate models on multiple public scRNAseq datasets. In comparison to GAN-based models (scGAN and cscGAN), we demonstrate that ACTIVA generates cells that are more realistic and harder for classifiers to identify as synthetic, which also have better pair-wise correlations between genes. We show that data augmentation with ACTIVA significantly improves the classification of rare subtypes (more than 45% improvement compared to not augmenting and 4% better than cscGAN) all while reducing training time by an order of magnitude in comparison to both models.

Availability of data and codeLinks to raw, pre- and post-processed data, source code and tutorials are available at https://github.com/SindiLab.

Supplementary informationSupplementary material can be found as a separate file with the same pre-print submission.

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The uptake of avermectins in Caenorhabditis elegans is dependent on Intra-Flagellar Transport and other protein trafficking pathways Parasitic nematodes are globally important and place a heavy disease burden on infected humans, crops and livestock, while commonly administered anthelmintics used for treatment are being rendered ineffective by increasing levels of resistance. Although the modes of action and resistance mechanisms caused by detoxification and target site insensitivity for these compounds is well documented, the mechanisms for uptake, which can also cause resistance, are still poorly defined. It has recently been shown in the model nematode Caenorhabditis elegans that the avermectins or macrocyclic lactones such as ivermectin and moxidectin gain entry though the sensory cilia of the amphid neurons. This study interrogated the molecular mechanisms involved in the uptake of avermectins using a combination of forward genetics and targeted resistance screening approaches along with visualising a BODIPY labelled ivermectin analog and confirmed the importance of intraflagellar transport in this process. This approach also identified the protein trafficking pathways used by the downstream effectors and the components of the ciliary basal body that are required for effector entry into these non-motile structures. Mutations in many of the genes under investigation also resulted in resistance to the unrelated anthelmintic drugs albendazole and levamisole, giving insights into the potential mechanisms of multidrug resistance observed in field isolates of the parasitic nematodes that are a scourge of ruminant livestock. In total 50 novel C. elegans anthelmintic survival associated genes were identified in this study, three of which (daf-6, rab-35 and inx-19) are associated with broad spectrum cross resistance. When combined with previously known resistance genes, there are now 53 resistance associated genes which are directly involved in amphid, cilia and IFT function.

Author SummaryNematodes represent significant pathogens of man and domestic animals and control relies heavily on limited classes of Anthelminitic drugs. Single and multi-drug resistance is a growing problem however mechanisms of anthelmintic drug resistance and drug uptake by nematodes remain to be clearly elucidated. In Caenorhabditis elegans there has been an association between amphid and dye filling defects with resistance to avermectins however the effector and causal mechanisms remain elusive. This study uses a combination of fluorescently labelled anthelmintics and anthelmintic resistance screens to probe the uptake mechanisms for these drugs. The role of the sensory amphids in the uptake of avermectins was confirmed. The avermectins enter the distal segment of the cilia using an effector which is delivered by the UNC-119 and UNC-33/UNC-44 transport systems to the base of the cilia, followed by distal appendage dependent entry and transport along the cilia by the intraflagellar transport pathway. Of the genes investigated, three (daf-6, rab-35 and inx-19) were linked to cross resistance against all the anthelmintics tested (Ivermectin, Moxidectin, Albendazole and Levamisole). This study gives further insight into how important classes of anthelmintics enter nematodes and highlights the potential for this process to give rise to anthelmintic resistance.

sentences: Single molecule analysis reveals positive allosteric modulators are required to stabilize the active state of a metabotropic glutamate receptor Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity, and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the Gi protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.

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Dual regulation of the actin cytoskeleton by CARMIL-GAP CARMIL (Capping protein Arp2/3 Myosin I Linker) proteins are multi-domain scaffold proteins that regulate actin dynamics by regulating the activity of Capping Protein (CP). Here we characterize CARMIL-GAP, a Dictyostelium CARMIL isoform that contains a ~130 residue insert that, by homology, is a GTPase activating (GAP) domain for Rho-related GTPases. Consistently, this GAP domain binds Dictyostelium Rac1a and accelerates its rate of GTP hydrolysis. CARMIL-GAP concentrates with F-actin in phagocytic cups and at the leading edge of chemotaxing cells, and CARMIL-GAP null cells exhibit pronounced defects in phagocytosis and chemotactic streaming. Importantly, these defects are fully rescued by expressing GFP-tagged CARMIL-GAP in CARMIL-GAP null cells. Finally, rescue with versions of CARMIL-GAP that lack either GAP activity or the ability to regulate CP show that while both activities contribute significantly to CARMIL-GAP function, the GAP activity plays the bigger role. Together, our results add to the growing evidence that CARMIL proteins influence actin dynamics by regulating signaling molecules as well as CP, and that the continuous cycling of the Rho GTPases nucleotide state is often required to drive Rho-dependent biological processes.

SUMMARY STATEMENTThe assembly of actin filaments supports a wide array of fundamental cellular functions, including cell migration and phagocytosis. Actin assembly is controlled by a host of regulatory proteins, with Capping Protein being one of the most important. Capping Protein is in turn regulated by the CARMIL family of proteins. Actin assembly is also controlled by signaling pathways that often converge on Rho-related GTPases like Rac1. These GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. Guanine nucleotide exchange factors (GEFs) and guanine nucleotide activating proteins (GAPs) drive Rho-related GTPases to their GTP-bound and GDP-bound states, respectively. Here we characterized a version of CARMIL that contains within it a GAP domain for Rac1. We show that CARMIL-GAP supports the actin-based processes of cell migration and phagocytosis. We also show that while CARMIL-GAPs ability to regulate Capping Protein and the nucleotide state of Rac1 are both important for its cellular functions, its ability to regulate Rac1 via its GAP domain plays the bigger role. Finally, our data support the emerging concept that the continuous cycling of Rho GTPases between their GTP- bound and GDP-bound states is often required to drive Rho-dependent biological processes.

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Transfer learning improves outcome predictions for ASD from gene expression in blood BackgroundPredicting outcomes on human genetic studies is difficult because the number of variables (genes) is often much larger than the number of observations (human subject tissue samples). We investigated means for improving model performance on the types of under-constrained problems that are typical in human genetics, where the number of strongly correlated genes (features) may exceed 10,000, and the number of study participants (observations) may be limited to under 1,000.

MethodsWe created  train,  validate and  test datasets from 240 microarray observations from 127 subjects diagnosed with autism spectrum disorder (ASD) and 113  typically developing (TD) subjects. We trained a neural network model (a.k.a., the  naive model) on 10,422 genes using the  train dataset, composed of 70 ASD and 65 TD subjects, and we restricted the model to one, fully-connected hidden layer to minimize the number of trainable parameters, including a dropout layer to help prevent overfitting. We experimented with alternative network architectures and tuned the hyperparameters using the  validate dataset, and performed a single, final evaluation using the holdout  test dataset. Next, we trained a neural network model using the identical architecture and identical genes to predict tissue type in GTEx data. We transferred that learning by replacing the top layer of the GTEx model with a layer to predict ASD outcome and we retrained the new layer on the ASD dataset, again using the identical 10,422 genes.

FindingsThe  naive neural network model had AUROC=0.58 for the task of predicting ASD outcomes, which saw a statistically significant 7.8% improvement from transfer learning.

InterpretationWe demonstrated that neural network learning could be transferred from models trained on large RNA-Seq gene expression to a model trained on a small, microarray gene expression dataset with clinical utility for mitigating over-training on small sample sizes. Incidentally, we built a highly accurate classifier of tissue type with which to perform the transfer learning.

FundingThis work was supported in part by NIMH R01-MH110558 (E.C., N.E.L.)

Author SummaryImage recognition and natural language processing have enjoyed great success in reusing the computational efforts and data sources to overcome the problem of over-training a neural network on a limited dataset. Other domains using deep learning, including genomics and clinical applications, have been slower to benefit from transfer learning. Here we demonstrate data preparation and modeling techniques that allow genomics researchers to take advantage of transfer learning in order to increase the utility of limited clinical datasets. We show that a non-pre-trained,  naive model performance can be improved by 7.8% by transferring learning from a highly performant model trained on GTEx data to solve a similar problem.

sentences: Recurrent Independent Pseudogenization Events of the Sperm Fertilization Gene ZP3r in Apes and Monkeys In mice, ZP3r/sp56 is a binding partner to the egg coat protein ZP3 and may mediate induction of the acrosome reaction. ZP3r, as a member of the RCA cluster, is surrounded by paralogs, some of which have been shown to be evolving under positive selection. Sequence divergence paired with paralogous relationships with neighboring genes, has complicated the accurate identification of the human ZP3r ortholog. Here, we phylogenetically and syntenically resolve that the human ortholog of ZP3r is the pseudogene C4BPAP1. We investigate the evolution of this gene within primates. We observe independent pseudogenization events of ZP3r in all Apes with the exception of Orangutans, and many monkey species. ZP3r in both primates that retain ZP3r and rodents contains positively selected sites. We hypothesize that redundant mechanisms mediate ZP3 recognition in mammals and ZP3rs relative importance to ZP recognition varies across species.

sentences: Ferroptosis assassinates tumor (FAST) In 2020, nearly 20 million peoples got cancer and nearly 10 million peoples died of cancer, indicating the current therapies do not meet the cancer treatment and cancer remains a great threat to human health and life. New therapies are still in urgent demand. In a recent study, we developed a new effective cancer therapy, gene-interfered ferroptosis therapy (GIFT), by combining cancer cell-specific knockdown of two iron efflux genes (FPN and LCN2) with iron nanoparticles (FeNPs). GIFT shows wide antitumor activity, high cancer specificity, certain cancer eradication potential, and biosafety. To further improve the therapy, we here develop an updated GIFT named as Ferroptosis ASsassinates Tumor (FAST) by knocking down five additional ferroptosis-resistance genes (FSP1, FTH1, GPX4, SLC7A11, NRF2). As a result, we found that FAST showed more significant antitumor activity than GIFT. Especially, FAST eradicated three different types of tumors (leukemia, colon cancer and lung metastatic melanoma) from over 50 percent of cancer mice, making the mice to survive up to 250 days without tumor relapse. FAST also significantly inhibited and prevented growth of spontaneous breast cancer and improved survival in mice. Additionally, FAST showed high pan-antitumor efficacy, high cancer specificity, and in vivo safety.

sentences: Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche Tissue homeostasis often requires a properly placed niche to support stem cells. Morphogenetic processes that position a niche are just being described. For the Drosophila testis we recently showed that pro niche cells, specified at disparate positions during early gonadogenesis, must assemble into one collective at the anterior of the gonad. We now find that Slit and FGF signals emanating from adjacent visceral mesoderm regulate assembly. In response to signaling, niche cells express islet, which we find is also required for niche assembly. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. Function of such niches is severely disrupted, with niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. Our work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.

sentences: Allosteric activation of CwlD amidase activity by the GerS lipoprotein during Clostridioides difficile spore formation Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. Spore germination depends on the degradation of the protective spore peptidoglycan layer known as the spore cortex. Cortex degradation is mediated by enzymes that recognize the spore-specific peptidoglycan modification, muramic-{partial}-lactam (MAL). In C. difficile, MAL synthesis depends on the activity of the CwlD amidase and the GerS lipoprotein, which directly binds CwlD. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind zinc stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to zinc, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of zinc co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

sentences: Evaluating the reliability, validity, and utility of overlapping networks: Implications for cognitive control Brain regions can be consistently clustered together into a small number of large-scale networks based on the correlation of their activity across time. However, the methods typically used to assign regions to these networks typically force non- or minimal overlap between the networks, losing information about how particular regions may connect to multiple networks. However, new methods are emerging that allow networks to overlap. Here, we examined the reliability and validity of one such assignment method, the mixed membership algorithm, and the utility of overlapping assignment for identifying potential gaps in existing network models of behavior. Analyses were conducted on resting state data from a large set of participants. First, we split the data into two halves and generated group-level overlapping networks for each subgroup. We then used a bootstrapped Dice similarity analysis to demonstrate that the binarized network topographies showed good agreement between subgroups. Next, we generated nonoverlapping networks in the combined data using a typical k-means clustering method. Using the same Dice Similarity analysis, we compared these nonoverlapping networks both with previously published nonoverlapping networks (Yeo et al, 2011) and with overlapping networks likewise generated for the combined data. The nonoverlapping networks generally recapitulated known networks in the literature, and the overlapping networks captured large portions of one to three nonoverlapping network topographies. Following this, we used a relative entropy analysis to assess the uniformity of each regions distribution of connections across the overlapping networks. While many regions were indeed assigned to one network, more were assigned to multiple networks. Furthermore, regions assigned to multiple networks did not necessarily show stronger connection to one network over the others. Finally, we assessed the network membership of regions of the fronto-parietal and cingulo-opercular networks of the Dual-Networks model of cognitive control. In combination, the regions were members of all of the overlapping networks, and the majority were assigned to multiple networks. Together, these results indicate that overlapping assignment produces consistent and biologically plausible network topographies, and that a complete network model of cognitive control, or indeed any network model of behavior needs to be updated to account for multiple-network membership and the complex interactions that occur between the relevant networks as a function of their overlapping nature.

sentences: Oncogenic and Tumor Suppressor Functions for Lymphoid Enhancer Factor 1 in a Murine Model of T Acute Lymphoblastic Leukemia T lymphocyte acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease affecting T cells at multiple stages of their development and is characterized by frequent genomic alterations. The transcription factor LEF1 is inactivated through mutation in a subset of T-ALL cases but elevated LEF1 expression and activating mutations have also been identified in this disease. Here we show, in a murine model of T-ALL, that the developmental timing of Lef1 mutation impacts its ability to function as a cooperative tumor suppressor or oncogene. T cell transformation in the presence of LEF1 allows leukemic cells to become addicted to its presence. In contrast, deletion prior to transformation both accelerates leukemogenesis and results in leukemic cells with altered expression of genes controlling receptor signaling pathways. Our data demonstrate that the developmental timing of Lef1 mutations impact its apparent oncogenic or tumor suppressive characteristics and demonstrate the utility of mouse models for understanding the cooperation and consequence of mutational order in leukemogenesis.

sentences: Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation, and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe small RNAs, pseudogenes, and transposable elements, and highlight tissue specific expression and methylation patterns. Resequencing of forty wild accessions provides insights into genome-wide genetic variation as well as QTL regions for flowering time and a seedling color phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

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Engineered endosymbionts capable of directing mammalian cell gene expression Modular methods for directing mammalian gene expression would enable advances in tissue regeneration, enhance cell-based therapeutics and improve modulation of immune responses. To address this challenge, engineered endosymbionts (EES) that escape endosomal destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to control host cell gene expression were developed. Microscopy confirmed that EES escape phagosomes, replicate within the cytoplasm, and can secrete reporter proteins into the cytoplasm that were then transported to the nucleus. Synthetic operons encoding the mammalian transcription factors, Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the EES genome. Using controlled induction, these EES were shown to direct gene expression in J774A.1 macrophage/monocyte cells and modulate the host cell fates. Expressing mammalian transcription factors from engineered intracellular bacteria as endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes.

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sentences: Plankton energy flows using a global size-structured and trait-based model Plankton community models are critical tools for understanding the processes that shape marine plankton communities, how plankton communities impact biogeochemical cycles, and the feedbacks between community structure and function. Here, using the flexible Marine Biogeochemistry Library (MARBL), we present the Size-based Plankton Ecological TRAits (MARBL-SPECTRA) model, which is designed to represent a diverse plankton community while remaining computationally tractable. MARBL-SPECTRA is composed of nine phytoplankton and six zooplankton size classes represented using allometric scaling relationships for physiological traits and interactions within multiple functional types. MARBL-SPECTRA is embedded within the global ocean component of the Community Earth System Model (CESM) and simulates large-scale, emergent patterns in phytoplankton growth limitation, plankton phenology, plankton generation time, and trophic transfer efficiency. The model qualitatively reproduces observed global patterns of surface nutrients, chlorophyll biomass, net primary production, and the biogeographies of a range of plankton size classes. In addition, the model simulates how predator:prey dynamics and trophic efficiency vary across gradients in total ecosystem productivity. Shorter food chains that export proportionally more carbon from the surface to the ocean interior occur in productive regions, whereas in oligotrophic regions, the food chains are relatively long and export less organic matter from the surface. The union of functional type modelling with size-resolved, trait-based modelling approaches allows MARBL-SPECTRA to capture both large-scale elemental cycles and the structure of planktonic food webs affecting trophic transfer efficiency.

sentences: Examining the Neural Correlates of Error Awareness in a Large fMRI Study Goal-directed behaviour is dependent upon the ability to detect errors and implement appropriate post-error adjustments. Accordingly, several studies have explored the neural activity underlying error-monitoring processes, identifying the insula cortex as crucial for error awareness and reporting mixed findings with respect to the anterior cingulate cortex. Variable patterns of activation have previously been attributed to insufficient statistical power. We therefore sought to clarify the neural correlates of error awareness in a large event-related functional magnetic resonance imaging (MRI) study. Four hundred and two healthy participants undertook the error awareness task, a motor Go/No-Go response inhibition paradigm in which participants were required to indicate their awareness of commission errors. Compared to unaware errors, aware errors were accompanied by significantly greater activity in a network of regions including the insula cortex, supramarginal gyrus, and midline structures such as the anterior cingulate cortex and supplementary motor area. Error awareness activity was related to indices of task performance and dimensional measures of psychopathology in selected regions including the insula, supramarginal gyrus and supplementary motor area. Taken together, we identified a robust and reliable neural network associated with error awareness.

sentences: The influence of leaf anatomical traits on photosynthetic in coffee leaves in Yunnan of China Leaf photosynthesis is largely determined by anatomical features. This work aimed to reveal the quantitative effects of Coffea arabica leaf anatomical structure on photosynthesis. Pearsons correlation and path analysis was used to explore the correlation between leaf structure and photosynthesis. The comprehensive evaluation value of the correlation between leaf anatomical traits and photosynthetic parameters is calculated by using the Criteria Importance Though Intercrieria Correlation (CRITIC) method to obtain the objective weight of each photosynthetic parameter. Most of the leafs anatomical traits significantly (P<0.05) to highly significantly (P<0.01) correlated with photosynthetic parameters, suggesting that anatomical traits greatly affected photosynthetic capacity in Coffea arabica leaf. Likewise, path coefficient analysis revealed that all the leaf anatomical traits influenced photosynthetic capacity directly and indirectly. The results of the comprehensive evaluation showed that leaf thickness (LT) and stomatal density (d) are the anatomical characteristics most closely related to photosynthesis. These results may help pinpoint anatomical features required to gain higher photosynthesis in coffee leaves.

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Transmembrane Kinases are essential for plant development Transmembrane kinase family proteins (TMKs) have been implicated in regulating both auxin signaling and plant development. To obtain insights of the potential TMKs function in plant development, we regenerated new full sets of tmk mutants, and discovered many new phenotypes, such as defective organogenesis, smaller rosette leaves, fertility defects and selfing population defects in different combination of tmk mutants. Taken together, our results demonstrated that TMKs participated in multiple aspects of plant development, which provided a great reference for any future research.

One-sentence summaryMultiple tmk mutants analysis illustrated the key roles of TMKs in plant development.

sentences: The phosphate starvation response recruits the TOR pathway to regulate growth in Arabidopsis cell cultures AO_SCPLOWBSTRACTC_SCPLOWIn eukaryotes, TOR (Target Of Rapamycin) is a conserved regulator of growth that integrates both endogenous and exogenous signals. These signals include the internal nutritional status, and in plants, TOR has been shown to be regulated by carbon, nitrogen and sulfur availability. In this study, we show that in Arabidopsis the TOR pathway also integrates phosphorus availability to actively modulate the cell cycle, which in turn regulates the intracellular content of amino acids and organic acids. We observed a substantial overlap between the phenotypic, metabolic and transcriptomic responses of TOR inactivation and phosphorus starvation in Arabidopsis cell culture. Although phosphorus availability modulates TOR activity, changes in the levels of TOR activity do not alter the expression of marker genes for phosphorus status. These data prompted us to place the sensing of phosphorus availability upstream of the modulation of TOR activity which, in turn, regulates the cell cycle and primary metabolism to adjust plant growth in plants.

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Polyacrylamide co-N,N'-methylene bisacrylamide monoliths for high peak capacity HILIC-HRMS of intact proteins at low trifluoroacetic acid content In this study, we optimized a polymerization mixture to synthesize polyacrylamide-co-N,N-methylene bisacrylamide monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of crosslinker and the porogen composition on the separation performance of the resulting columns were studied.

The homogeneity of the structure and the different porosities were examined through scanning electron microscopy. Further characterization of the monolithic structure revealed a permeable (Kf between 2.5 x 10-15 and 1.40 x 10-13 m2) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min.

Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 m core-shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA) the peak capacities of the two columns were similar (31 and 36 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained its separation performance and selectivity (peak capacity 26), whereas the packed column showed greatly reduced performance (peak capacity 7), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.

sentences: Mechanodetection of neighbor plants elicits adaptive leaf movements through calcium dynamics Plants detect their neighbors via various cues, including reflected light and touching of leaf tips, which elicit in upward leaf movement (hyponasty). It is currently unknown how touch is sensed and how the signal is transferred from the leaf tip to the petiole base that drives hyponasty. Here, we show that touch-induced hyponasty involves a signal transduction pathway that is distinct from light-mediated hyponasty. We found that mechanostimulation of the leaf tip upon touching causes cytosolic calcium ([Ca2+]cyt induction in leaf tip trichomes that spreads towards the petiole. Both perturbation of the calcium response and the absence of trichomes inhibit touch-induced hyponasty. Finally, using plant competition assays, we show that touch-induced hyponasty is adaptive in dense stands of Arabidopsis. We thus establish a novel, adaptive mechanism regulating hyponastic leaf movement in response to mechanostimulation by neighbors in dense vegetation.

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Systemic IgG repertoire as a biomarker for translocating gut microbiota members While the microbiota has been associated with diseases states, how specific alterations in composition, function, or localization contribute to pathologies remains unclear. The ability of defined microbes to translocate has been linked to diseases including inflammatory bowel disease (IBD) and was shown to promote responses to immune checkpoint therapy. However, scalable and unbiased tools to uncover microbes with enhanced ability to translocate are limited. Herein, we developed an approach to utilize systemic IgG in an unbiased, culture-independent, and high-throughput fashion as a biomarker to identify gut microbiota members that are capable of translocation across the gastrointestinal barrier. We validate these findings in a cohort of human subjects, and highlight a number of microbial taxa against which elevated IgG responses are unique to subjects with IBD including Collinsella, Bifidobacterium, Faecalibacterium, and Blautia spp. Collinsella and Bifidobacterium taxa identified as translocators and targets of immunity in IBD also exhibited heightened bacterial activity and growth rates in Crohns disease subjects. Our approach may represent a complementary tool to illuminate privileged interactions between host and its microbiota, and may provide an additional lens by which to uncover microbes linked to disease processes.

One Sentence SummaryCirculating microbiota-specific IgG can identify gut microbiota constituents capable of crossing the gut barrier.

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A two-level, dynamic fitness landscape of hepatitis C virus revealed by self-organized haplotype maps Fitness landscapes reflect the adaptive potential of viruses. There is no information on how fitness peaks evolve when a virus replicates extensively in a controlled cell culture environment. Here we report the construction of Self-Organized Maps (SOMs), based on deep sequencing reads of three amplicons of the NS5A-NS5B-coding region of hepatitis C virus (HCV). A two-dimensional neural network was constructed and organized according to sequence relatedness. The third dimension of the fitness profile was given by the haplotype frequencies at each neuron. Fitness maps were derived for 44 HCV populations that share a common ancestor that was passaged up to 210 times in human hepatoma Huh-7.5 cells. As the virus increased its adaptation to the cells, the number of fitness peaks expanded, and their distribution shifted in sequence space. The landscape consisted of an extended basal platform, and a lower number of protruding higher fitness peaks. The function that relates fitness level and peak abundance corresponds a power law, a relationship observed with other complex natural phenomena. The dense basal platform may serve as spring-board to attain high fitness peaks. The study documents a highly dynamic, double-layer fitness landscape of HCV when evolving in a monotonous cell culture environment. This information may help interpreting HCV fitness landscapes in complex in vivo environments.

IMPORTANCEThe study provides for the first time the fitness landscape of a virus in the course of its adaptation to a cell culture environment, in absence of external selective constraints. The deep sequencing-based self-organized maps document a two-layer fitness distribution with an ample basal platform, and a lower number of protruding, high fitness peaks. This landscape structure offers potential benefits for virus resilience to mutational inputs.

sentences: Integrated epigenome and transcriptome analysis of normal and arrested meiotic initiation during mouse spermatogenesis The transition from mitotic to meiotic cell cycles is a transcriptional event that entails the activation of genes important for meiosis and requires germline-specific retinoic acid (RA) signaling target, Stra8. To identify novel transcription factors underlying mammalian meiotic initiation, we conducted integrative snATAC-seq and scRNA-seq analysis using wild-type and Stra8-deficient mouse testicular cells to map the chromatin accessibility and gene expression landscapes of normal and genetically arrested meiotic initiation. Our results identified a cluster of putative inhibitory transcription factors for meiotic initiation, which we consider "meiotic inhibitors". STRA8 binds to the regulatory sequences of these meiotic inhibitors and represses their expression upon meiotic initiation. In Stra8-deficient cells that suffer meiotic initiation arrest, the chromatin accessibility of these meiotic inhibitors is increased, concurrent with their uncontrolled and sustained expression. Among these meiotic inhibitors include KLF4, MAX, and MAZ. Importantly, by analyzing the single cell transcriptomes of human testes, our data show that these putative meiotic inhibitor genes are upregulated in early germ cells from patients with spermatogenic failure. Our study suggests that proper repression of meiotic inhibitors is essential for both mouse and human spermatogenesis.

sentences: Dual effects of presynaptic membrane mimetics on α-synuclein amyloid aggregation Aggregation of intrinsically disordered -synuclein (SN) under various conditions is closely related to synucleinopathies. Although various biological membranes have shown to alter the structure and aggregation propensity of SN, a thorough understanding of the molecular and mechanical mechanism of amyloidogenesis in membranes remains unanswered. Herein, we examined the structural changes, binding properties, and amyloidogenicity of three variations of SN mutants under two types of liposomes, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and presynaptic vesicle mimetic (Mimic) membranes. While neutrally charged DOPC membranes elicited marginal changes in the structure and amyloid fibrillation of SNs, negatively charged Mimic membranes induced dramatic helical folding and biphasic amyloid generation. At low concentration of Mimic membranes, the amyloid fibrillation of SNs was promoted in a dose-dependent manner. However, further increases in the concentration constrained the fibrillation process. These results suggest the dual effect of Mimic membranes on regulating the amyloidogenesis of SN, which is rationalized by the amyloidogenic structure of SN and condensation-dilution of local SN concentration. Finally, we propose physicochemical properties of SN and membrane surfaces, and their propensity to drive electrostatic interactions as decisive factors of amyloidogenesis.

sentences: Neurotensin neurons in the central extended amygdala control energy balance Overeating and a sedentary life style are major causes of obesity and related metabolic disorders. Identification of the neurobiological processes that regulate energy balance will facilitate development of interventions for these disorders. Here we show that the Neurotensin-expressing neurons in the mouse IPAC (IPACNts), a nucleus of the central extended amygdala, bidirectionally coordinate hedonic feeding and physical activity, thereby regulating energy balance, metabolic processes and bodyweight. IPACNts are preferentially activated by consumption of highly palatable food or exposure to its taste and smell. Activating IPACNts promotes food intake in a palatability-dependent manner and decreases locomotion. Conversely, inhibiting IPACNts selectively reduces palatable food intake and dramatically enhances physical activity and energy expenditure, and in parallel stimulates physiological responses that oppose diet-induced obesity and metabolic dysfunctions. Thus, a single neuronal population, Neurotensin-expressing neurons in the IPAC, acts to control obesogenic and leptogenic processes by synergistically coordinating energy intake and expenditure with metabolism.

sentences: The Psychometrics of Automatic Speech Recognition Deep neural networks have had considerable success in neuroscience as models of the visual system, and recent work has suggested this may also extend to the auditory system. We tested the behaviour of a range of state of the art deep learning-based automatic speech recognition systems on a wide collection of manipulated sounds used in standard human psychometric experiments. While some systems showed qualitative agreement with humans in certain tests, in others all tested systems diverged markedly from humans. In particular, all systems used spectral invariance, temporal fine structure and speech periodicity differently from humans. We conclude that despite some promising results, none of the tested automatic speech recognition systems can yet act as a strong proxy for human speech recognition. However, we note that the more recent systems with better performance also tend to better match human results, suggesting that continued cross-fertilisation of ideas between human and automatic speech recognition may be fruitful. Our open source toolbox allows researchers to assess future automatic speech recognition systems or add additional psychoacoustic measures.

sentences: PRMT5 promotes symmetric dimethylation of RNA processing proteins and modulates activated T cell alternative splicing and Ca2+/NFAT signaling Protein Arginine Methyltransferase (PRMT) 5 is the major type 2 methyltransferase catalyzing symmetric dimethylation (SDM) of arginine. PRMT5 inhibition or deletion in CD4 Th cells reduces TcR engagement-induced IL-2 production and Th cell expansion and confers protection against experimental autoimmune encephalomyelitis (EAE), the animal model of Multiple Sclerosis. However, the mechanisms by which PRMT5 modulates T helper (Th) cell proliferation are still not completely understood and neither are the methylation targets in T cells. In this manuscript, we uncover the role of PRMT5 on alternative splicing (AS) in activated T cells and identify several targets of PRMT5 SDM involved in splicing. In addition, we find a possible link between PRMT5 mediated AS of Trpm4 (Transient Receptor Potential Cation Channel Subfamily M Member 4) and TcR/NFAT signaling/IL-2 production. This understanding may guide development of drugs targeting these processes to benefit patients with T cell-mediated diseases.

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Chemophoresis Engine: Universal Principle of ATPase-driven Cargo Transport Cell polarity regulates the orientation of the cytoskeleton members that directs intracellular transport for cargo-like organelles, using chemical gradients sustained by ATP or GTP hydrolysis. However, how cargo transports are directly mediated by chemical gradients remains unknown. We previously proposed a physical mechanism that enables directed movement of cargos, referred to as chemophoresis. According to the mechanism, a cargo with reaction sites is subjected to a chemophoresis force in the direction of the increased concentration. Based on this, we introduce an extended model, the chemophoresis engine, as a general mechanism of cargo motion, which transforms chemical free energy into directed motion through the catalytic ATP hydrolysis. We applied the engine to plasmid motion in a parABS system to demonstrate the the self-organization system for directed plasmid movement and pattern dynamics of ParA-ATP concentration, thereby explaining plasmid equi-positioning and pole-to-pole oscillation observed in bacterial cells and in vitro experiments. We mathematically show the existence and stability of the plasmid-surfing pattern, which allows the cargo-directed motion through the symmetry-breaking transition of the ParA-ATP spatiotemporal pattern. Finally, based on its generality, we discuss the chemophoresis engine as a universal principle of hydrolysis-driven intracellular transport.

SIGNIFICANCEThe formation of organelle/macromolecule patterns depending on chemical concentration under non-equilibrium conditions, first observed during macroscopic morphogenesis (1), has recently been observed at the intracellular level as well, and its relevance as intracellular morphogen has been demonstrated in the case of bacterial cell division. These studies have discussed how cargos maintain positional information provided by chemical concentration gradients/localization. However, how cargo transports are directly mediated by chemical gradients remains unknown. Based on the previously proposed mechanism of chemotaxis-like behavior of cargos (referred to as chemophoresis, (2, 3)), we introduce a chemophoresis engine as a physicochemical mechanism of cargo motion, which transforms chemical free energy to directed motion. The engine is based on the chemophoresis force to make cargoes move in the direction of the increasing ATPase(-ATP) concentration and an enhanced catalytic ATPase hydrolysis at the positions of the cargoes. Applying the engine to ATPase-driven movement of plasmid-DNAs in bacterial cells, we constructed a mathematical model to demonstrate the self-organization for directed plasmid motion and pattern dynamics of ATPase concentration, as is consistent with in vitro and in vivo experiments. We propose that this chemophoresis engine works as a universal principle of hydrolysis-driven intracellular transport.

sentences: Novel insights into the cold resistance of Hevea brasiliensis through coexpression networks Hevea brasiliensis, a tropical tree species from the Amazon rainforest, is the main source of natural rubber worldwide. Due to the high pressure of fungal diseases in hot, humid regions, rubber plantations have been moved to "escape areas", which are dryer and have lower temperatures during the winter. Here, we combined gene expression data of a primary (GT1) and a secondary (RRIM600) young rubber tree clones, which present different cold tolerance strategies, to analyze rubber tree gene expression regulation during 24 h of cold exposure (10{degrees}C). Together with traditional differential expression approaches, a RNA sequencing (RNA-seq) gene coexpression network (GCN) comprising 27,220 genes was established in which the genes were grouped into 832 clusters. In the GCN, most of the rubber tree molecular responses to cold stress were grouped in 26 clusters, which were divided into three GCN modules: a downregulated group comprising 12 clusters and two upregulated groups comprising eleven and three clusters. Considering the three modules identified, the general Hevea response to short-term cold exposure involved downregulation of gibberellin (GA) signaling, complex regulation of jasmonic acid (JA) stress responses and programmed cell death (PCD) and upregulation of ethylene responsive genes. The hub genes of the cold-responsive modules were subsequently identified and analyzed. As a result of the GCN strategy applied in this study, we could not only access individual DEGs related to the Hevea cold response, but also provide insights into a deeper cascade of associated mechanisms involved in the response to cold stress in young rubber trees. Our results may represent the genetic stress responses of the species, developed during its evolution, since the varieties chosen for this work are genotypes that were selected during the early years of rubber tree domestication. The understanding of H. brasiliensis cold response mechanisms can greatly improve the breeding strategies for this crop, which has a narrow genetic base, is impacted by climate change and is the only source for large-scale rubber production.

sentences: Single-Molecule Tracking of Chromatin-Associated Proteins in the C. elegans Gonad 1.Biomolecules are distributed within cells by molecular-scale diffusion and binding events that are invisible in standard fluorescence microscopy. These molecular search kinetics are key to understanding nuclear signaling and chromosome organization, and can be directly observed by single-molecule tracking microscopy. Here, we report a method to track individual proteins within intact C. elegans gonads and apply it to study the molecular dynamics of the axis, a proteinaceous backbone that organizes meiotic chromosomes. Using either fluorescent proteins or enzymatically ligated dyes, we obtain multi-second trajectories with a localization precision of 15-25 nm in nuclei actively undergoing meiosis. Correlation with a reference channel allows for accurate measurement of protein dynamics, compensating for movements of the nuclei and chromatin within the gonad. We find that axis proteins exhibit either static binding to chromatin or free diffusion in the nucleoplasm, and we separately quantify the motion parameters of these distinct populations. Freely diffusing axis proteins selectively explore chromatin-rich regions, suggesting they are circumventing the central phase-separated region of the nucleus. This work demonstrates that single-molecule microscopy can infer nanoscale-resolution dynamics within living tissue, expanding the possible applications of this technique.

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Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: only one of the two isoforms is kinetically active Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a Neglected Tropical Disease endemic to 36 African countries, with approximately 70 million people currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. Thus, both effective and affordable treatments are urgently needed. The causative agent of HAT is the protozoan Trypanosoma brucei ssp. Annotation of the T. brucei genome confirms previous observations that T. brucei is a purine auxotroph. Incapable of de novo purine synthesis, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purines from their hosts for the synthesis of purine monophosphates. Complete and accurate genome annotations in combination with the identification and characterization of the catalytic activity of purine salvage enzymes enables the development of target-specific therapies in addition to providing a deeper understanding of purine metabolism in T. brucei. In trypanosomes, purine phosphoribosyltransferases represent promising drug targets due to their essential and central role in purine salvage. Enzymes involved in adenine and adenosine salvage, such as adenine phosphoribosyltransferases (APRTs, EC 2.4.2.7), are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs. Analysis of the T. brucei genome shows two putative aprt genes: APRT1 (Tb927.7.1780) and APRT2 (Tb927.7.1790). Here we report studies of the catalytic activity of each putative APRT, revealing that of the two T. brucei putative APRTs, only APRT1 is kinetically active, thereby signifying a genomic misannotation of Tb927.7.1790 (putative APRT2). Reliable genome annotation is necessary to establish potential drug targets and identify enzymes involved in adenine and adenosine-based prodrug activation.

Author SummaryNeglected Tropical Diseases, are defined by the World Health Organization as a diverse group of 20 different diseases that disproportionally affect the worlds poorest populations, including Human African Trypanosomiasis (HAT). HAT is endemic to 36 African countries and approximately 70 million people worldwide are currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. The causative agent of HAT is Trypanosoma brucei ssp. Unlike humans, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purine bases from their hosts for the synthesis of DNA and RNA. Here we report on the characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei. Our studies reveal that of the two putative APRTs, only APRT1 is kinetically active under physiological conditions. Accurate genome annotations in combination with the characterization of purine salvage enzymes allows the development of target-specific therapies. Enzymes involved in adenine and adenosine salvage, such as APRTs, are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs.

sentences: High-Resolution, Large Imaging Volume, and Multi-View Single Objective Light-Sheet Microscopy Recent developments in Oblique Plane Microscopy (OPM) have shown that it can achieve high spatio-temporal resolution. Here we describe a single objective light-sheet microscope based on oblique plane illumination that achieves: (i) large field of view and high-resolution imaging via a custom remote focusing objective; (ii) fast volumetric imaging by means of light-sheet stabilised stage scanning - a novel scanning modality that extends the imaging volume without compromising imaging speed nor quality; (iii) multi-view imaging by alternating the orientation of light-sheet illumination and detection to improve the image quality on large samples; (iv) simpler design and ergonomics by remote placement of coverslips to allow inverted imaging, enabling imaging across scales in a high-throughput format. Overall, we achieved a resolution of 450 nm laterally and 2 m axially and a field of view of 3000 m x 800 m x 300 m. We demonstrate the speed, field of view, resolution and versatility of our novel instrument by imaging various systems, including zebrafish whole brain activity, Drosophila egg chamber development, and zebrafish development - up to nine embryos simultaneously.

sentences: Detection and Interspecies Comparison of SARS-CoV-2 Delta Variant (AY.3) in Feces from a Domestic Cat and Human Samples Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have spilled over from humans to companion and wild animals since the inception of the global COVID-19 pandemic. However, whole genome sequencing data of the viral genomes that infect non-human animal species has been scant. Here, we detected and sequenced a SARS-CoV-2 delta variant (AY.3) in fecal samples from an 11-year-old domestic house cat previously exposed to an owner who tested positive for SARS-CoV-2. Molecular testing of two fecal samples collected 7 days apart yielded relatively high levels of viral RNA. Sequencing of the feline-derived viral genomes showed the two to be identical, and differing by between 4 and 14 single nucleotide polymorphisms in pairwise comparisons to human-derived lineage AY.3 sequences collected in the same geographic area and time period. However, several mutations unique to the feline samples reveal their divergence from this cohort on phylogenetic analysis. These results demonstrate continued spillover infections of emerging SARS-CoV-2 variants that threaten human and animal health, as well as highlight the importance of collecting fecal samples when testing for SARS-CoV-2 in animals. To the authors knowledge, this is the first published case of a SARS-CoV-2 delta variant in a domestic cat in the United States.

sentences: Automated assignment of cell identity from single-cell multiplexed imaging and proteomic data The creation of scalable single-cell and highly-multiplexed imaging technologies that profile the protein expression and phosphorylation status of heterogeneous cellular populations has led to multiple insights into disease processes including cancer initiation and progression. A major analytical challenge in interpreting the resulting data is the assignment of cells to a priori known cell types in a robust and interpretable manner. Existing approaches typically solve this by clustering cells followed by manual annotation of individual clusters or by strategies that gate protein expression at predefined thresholds. However, these often require several subjective analysis choices such as selecting the number of clusters and do not automatically assign cell types in line with prior biological knowledge. They further lack the ability to explicitly assign cells to an unknown or uncharacterized type, which exist in most highly multiplexed imaging experiments due to the limited number of markers quantified. To address these issues we present Astir, a probabilistic model to assign cells to cell types by integrating prior knowledge of marker proteins. Astir uses deep recognition neural networks for fast Bayesian inference, allowing for cell type annotations at the million-cell scale and in the absence of previously annotated reference data across multiple experimental modalities and antibody panels. We demonstrate that Astir outperforms existing approaches in terms of accuracy and robustness by applying it to over 2.1 million single cells from several suspension and imaging mass cytometry and microscopy datasets in multiple tissue contexts. We further showcase that Astir can be used for the fast analysis of the spatial architecture of the tumour microenvironment, automatically quantifying the immune influx and spatial heterogeneity of patient samples. Astir is freely available as an open source Python package at https://www.github.com/camlab-bioml/astir.

sentences: Perturbation of ACE2 structural ensembles by SARS-CoV-2 spike protein binding The human ACE2 enzyme serves as a critical first recognition point of coronaviruses, including SARS-CoV-2. In particular, the extracellular domain of ACE2 interacts directly with the S1 tailspike protein of the SARS-CoV-2 virion through a broad protein-protein interface. Although this interaction has been characterized by X-ray crystallography and Cryo-EM, these structures do not reveal significant differences in ACE2 structure upon S1 protein binding. In this work, using several all-atom molecular dynamics simulations, we show persistent differences in ACE2 structure upon binding. These differences are determined with the Linear Discriminant Analysis (LDA) machine learning method and validated using independent training and testing datasets, including long trajectories generated by D. E. Shaw Research on the Anton 2 supercomputer. In addition, long trajectories for 78 potent ACE2-binding compounds, also generated by D. E. Shaw Research, were projected onto the LDA classification vector in order to determine whether the ligand-bound ACE2 structures were compatible with S1 protein binding. This allows us to predict which compounds are "apo-like" vs "complex-like", as well as to pinpoint long-range ligand-induced allosteric changes of ACE2 structure.

sentences: Long-term consequences of alcohol use in early adolescent mice: focus on neuroadaptations in GR, CRF and BDNF Our aim was to assess the cognitive and emotional state, as well as related-changes in the glucocorticoid receptor (GR), the corticotropin-releasing factor (CRF) and the brain-derived neurotrophic factor (BDNF) expression of adolescent C57BL/6J male mice after a five-week two-bottle choice protocol (postnatal day (pd) 21 to pd52). Additionally, we wanted to analyse whether the behavioural and neurobiological effects observed in late adolescence (pd62) lasted until adulthood (pd84). Behavioural testing revealed that alcohol during early adolescence increased anxiety-like and compulsive-related behaviours, which was maintained in adulthood. Concerning cognition, working memory was only altered in late adolescent mice, whereas object location test performance was impaired in both ages. In contrast, novel object recognition remained unaltered. Immunohistochemical analysis showed that alcohol during adolescence diminished BDNF+ cells in the cingulate cortex, the hippocampal CA1 layer and the central amygdala. Regarding hypothalamic-pituitary-adrenal axis (HPA) functioning, alcohol abuse increased the GR and CRF expression in the hypothalamic paraventricular nucleus and the central amygdala. Besides this, GR density was also higher in the prelimbic cortex and the basolateral amygdala, regardless of the animals age. Our findings suggest that adolescent alcohol exposure led to long-term behavioural alterations, along with changes in BDNF, GR and CRF expression in limbic brain areas involved in stress response, emotional regulation, and cognition.

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Vibrio cholerae El Tor strains linked to global cholera are homogeneous by pulsed-field gel electrophoresis Vibrio cholerae O1 El Tor, causative agent of the ongoing seventh cholera pandemic, is native to the aquatic environment of the Ganges Delta, Bay of Bengal (GDBB). Recent studies traced pandemic strains to the GDBB and proposed global spread of cholera had occurred via intercontinental transmission. In the research presented here, NotI-digested genomic DNA extracted from V. cholerae O1 clinical and environmental strains isolated in Bangladesh during 2004 - 2014 was analyzed by pulsed-field gel electrophoresis (PFGE). Results of cluster analysis showed 94.67% of the V. cholerae isolates belonged to clade A and included the majority of clinical isolates of spatio-temporal origin and representing different cholera endemic foci. The rest of the strains were estuarine, all environmental isolates from Mathbaria, Bangladesh, and occurred as singletons, clustered in clades B and C, or in the small clades D and E. Cluster analysis of the Bangladeshi strains and including 157 El Tor strains from thirteen countries in Asia, Africa, and the Americas revealed 85% of the total set of isolates belonged to clade A, indicating all were related, yet did not form an homogeneous cluster. Overall, 15% of the global strains comprised multiple small clades or segregated as singletons. Three sub-clades could be discerned within the major clade A, reflecting distinct lineages of V. cholerae El Tor associated with cholera in Asia, Africa, and the Americas. The presence in Asia and the Americas of non-pandemic V. cholerae El Tor populations differing by PFGE and from strains associated with cholera globally suggests different ecotypes are resident in distant geographies.

Author SummaryCholera is a major health threat, especially in the Ganges Delta, Bay of Bengal (GDBB). Vibrio cholerae, causative agent of cholera, is native to the GDBB aquatic environment. Recent genomic studies suggest GDBB is the cholera hotspot where the disease spreads globally via human activity. Pulsed-field gel electrophoresis (PFGE) of NotI-digested genomic DNA from V. cholerae El Tor endemic cholera strains was done, including Bangladesh aquatic environment and clinical strains from distant geographical regions representing three cholera-prone continents. Results showed the majority of pandemic strains belonged to a major cluster, suggesting clonal relatedness. Ecotypes were detected, indicating geographically specific lineages. It is concluded that epidemic strains in Bangladesh and thirteen countries of Asia, Africa, and the Americas are geographically adapted, with independent evolution of the bacterium in respective geographical regions.

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BOLD fMRI signals of visual white matter encode visuotopic information and predict effective connectivity between visual areas The functional significance of BOLD signals in white matter (WM) remains unclear. The current study investigated whether 7T BOLD fMRI signal from visual WM tracts contains high fidelity retinotopic information and whether it correlates with the effective connectivity between visual areas. Population receptive field (pRF) analysis of the 7T retinotopy dataset from the Human Connectome Project revealed clear contralateral retinotopic representations from two visual WM bundles: optic radiation (OR) and vertical occipital fasciculus (VOF). The retinotopic organization of OR is consistent with post-mortem studies. The pRF size of WM voxels also increases with eccentricity. Based on the retinotopic maps of OR, we investigated whether BOLD signals in OR during visual stimulation are related to the resting-state effective connectivity between the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Results show that visually-evoked BOLD responses in OR correlate with the feedforward and feedback connectivity between the LGN and V1 during resting state. These findings demonstrate that WM BOLD signals contain high fidelity information such as visual field maps, and also predict the functional connectivity of brain areas.

Significance statementWhite matter (WM) tracks conduct spiking activity between distant neurons. Weak fluctuations of BOLD signals in the WM can be detected with fMRI, but their functional relevance remains largely unknown. Here we characterized the visual field map properties of two major visual WM bundles: the optic radiation (OR) and vertical occipital fasciculus (VOF). Population receptive field analysis of the WM BOLD signals revealed clear visual field maps in both WM tracts. Effective connectivity analysis further showed that visually evoked BOLD responses in OR can predict the resting thalamo-cortical functional connectivity. These findings demonstrate that WM BOLD signals contain highly specific functional information and could directly index the functional connectivity between brain areas.

sentences: Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression level with cell volume While most genes expression levels are proportional to cell volumes, some genes exhibit nonlinear scaling between their expression levels and cell volume. Therefore, their mRNA and protein concentrations change as the cell volume increases, which often have crucial biological functions such as cell-cycle regulation. However, the biophysical mechanism underlying the nonlinear scaling between gene expression and cell volume is still unclear. In this work, we show that the nonlinear scaling is a direct consequence of the heterogeneous recruitment abilities of promoters to RNA polymerases based on a gene expression model at the whole-cell level. Those genes with weaker (stronger) recruitment abilities than the average ability spontaneously exhibit superlinear (sublinear) scaling with cell volume. Analysis of the promoter sequences and the nonlinear scaling of Saccharomyces cerevisiaes mRNA levels shows that motifs associated with transcription regulation are indeed enriched in genes exhibiting nonlinear scaling, in concert with our model.

sentences: Simulation of foraging behavior using a decision-making agent with Bayesian and inverse Bayesian inference: Levy and Brownian walk-like search patterns derived from differences in prey distributions It has been stated that in human migratory behavior, the step length series may have temporal correlation and that there is some relationship between this time dependency and the fact that the frequency distribution of step length follows a power-law distribution. Furthermore, the frequency of occurrence of the step length in some large marine organisms has been found to switch between power-law and exponential distributions, depending on the difficulty of prey acquisition. However, to date it has not been clarified how the aforementioned three phenomena arise: the positive correlation created in the step length series, the relation between the positive correlation of the step length series and the form of an individuals step length distribution, and the switching between power-law and exponential distributions depending on the abundance of prey. This study simulated foraging behavior using the Bayesian decision-making agent simultaneously performing both knowledge learning and knowledge-based inference to analyze how the aforementioned three phenomena arise. In the agent with learning and inference, past experiences were stored as hypotheses (knowledge) and they were used in current foraging behavior; at the same time, the hypothesis continued to be updated based on new experiences. The simulation results show that the agent with both learning and inference has a mechanism that simultaneously causes all the phenomena.

sentences: Assessment of the suitability of marker fragments DNA sequences of the internal transcribed spacer region (ITS), beta-tubulin gene (TUB) and elongation factor (EF1) genes for diagnostics of Fusarium tricinctum species complex in Russian in grain products By results of laboratory researches from plant samples of grain crops species Fusarium avenaceum, Fusarium tricinctum and Fusarium acuminatum related to Fusarium tricinctum species complex were isolated and identified, based on cultural and morphological characteristics, molecular-genetic analysis of 3 DNA fragments was performed: internal transcribed spacer (ITS) site, beta-tubulin gene site (TUB), elongation factor gene site (EF1) showed insignificant intraspecific and interspecific variability at ITS and EF1 sites, which may negatively affect the reliability of target species identification, TUB site was more effective for distinguishing and identifying the studied species, due to significant intraspecific and interspecific variability

sentences: Stress-dependent dynamic and reversible formation of cytoskeleton-like filaments and gel-transition by tardigrade tolerance proteins. Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through comprehensive analysis, we identified 336 such proteins, collectively dubbed "Desolvataion-induced ReversiblY condensing Proteins (DRYPs)". Unexpectedly, we rediscovered CAHS proteins as highly enriched in DRYPs, 3 of which were major components of DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. CAHS filamentation increases cell stiffness to resist deformation and improves resistance to dehydration-like stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeletal proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and also contribute to the exceptional physical stability in a dehydrated state.

sentences: Extracellular cues accelerate neurogenesis of induced pluripotent stem cell derived neurons Neurogenesis is a complex process encompassing neuronal progenitor cell expansion/proliferation and differentiation, followed by neuron maturation. In vivo models are most commonly used to study neurogenesis; however, human induced pluripotent stem cell-derived (iPSC) neurons are increasingly used to establish cellular models of human neurological processes. Unfortunately, the differentiation and maturation of iPSC-derived neurons varies in methodology, is asynchronous, and has restricted experimental utility because of extended differentiation/maturation times. To accelerate and standardize iPS neuronal maturation, we differentiated and matured feeder layer-free iPSC-derived neuronal cultures under physiological oxygen levels (5%), and modified the underlying extracellular matrix and medium composition. Our results demonstrate that calretinin gene expression occurred earlier under our optimized iPS conditions and the corresponding "neurogenesis burst" associated with proliferative expansion occurred more synchronously, reliably emerging two and three weeks after differentiation. As expected, the expression of mature neuronal markers (i.e., NeuN+/Calbindin+) started at 4-weeks post-differentiation. qPCR microarray, western blot and single cell analyses using high content imaging indicated that 4-week iPS neuronal cultures were non-cycling with decreased expression of cyclin D1 and Ki67. Our data demonstrate that extracellular cues influence the kinetics of neurogenesis models and that feeder layer-free iPSC-derived neurogenesis can be reproducibly miniaturized.

sentences: Extreme sampling for genetic rare variant association analysis of dichotomous traits with focus on infectious disease susceptibility As genomic sequencing becomes more accurate and less costly, large cohorts and consortiums of cohorts are providing high power for rare variant association studies for many conditions. When large sample sizes are not attainable and the phenotype under study is continuous, an extreme phenotypes design can provide high statistical power with a small to moderate sample size. We extend the extreme phenotypes design to the dichotomous infectious disease outcome by sampling on extremes of the pathogenic exposure instead of sampling on extremes of phenotype. We use a likelihood ratio test (LRT) to test the significance of association between infection status and presence of susceptibility rare variants. More than 10 billion simulations are studied to assess the method. The method results in high sample enrichment for rare variants affecting susceptibility. Greater than 90% power to detect rare variant associations is attained in reasonable scenarios. The ordinary case-control design requires orders of magnitude more samples to achieve the same power. The Type I error rate of the LRT is accurate even for p-values < 10-7. We find that erroroneous exposure assessment can lead to power loss more severe than excluding the observations with errors. Nevertheless, careful sampling on exposure extremes can make a study feasible by providing adequate statistical power. Limitations of this method are not unique to this design, and the power is never less than that of the ordinary case-control design. The method applies without modification to other dichotomous outcomes that have strong association with a continuous covariate.

sentences: Evaluation of a Recessive Male Floret Color in Cultivated Northern Wild Rice (Zizania palustris L.) for Pollen-Mediated Gene Flow Studies Northern wild rice (NWR; Zizania palustris L.) is a wind-pollinated, annual, aquatic grass that grows naturally in the Great Lakes Region (GLR) of the United States and Canada, and is also cultivated in flooded paddies, predominantly in California and Minnesota. A better understanding of pollen-mediated gene flow is needed within the species for both conservation and breeding efforts as cultivation occurs within the species natural range and spatially-isolated, paddy structures are limited within breeding programs. Widely cited pollen travel research in NWR demonstrated that pollen could travel at least 3200m. However, a population segregating for male sterility was used as the pollen recipient in the study and was determined to not be adequate for NWR pollen travel studies. Here, we present the characterization of a recessive white male floret (WMF) population in contrast to the dominant, purple male floret (PMF) color of cultivated NWR along with estimates of pollen-mediated gene flow in a cultivated paddy setting. Studies conducted in 2018 and 2019 revealed that the primary amount of pollen-mediated gene flow occurred within the first 7m from the PMF donor source with no gene flow detected past 63m. These results suggest that the likelihood of pollen-mediated gene flow between cultivated NWR and natural stands remains low. We also identified a strong linkage between male floret, auricle, and culm color. This study demonstrates that the WMF trait is an excellent candidate for use in pollen-mediated gene flow studies in NWR.

sentences: Morph-dependent effect of nematode infection on host movement in the land snail Cepaea nemoralis (Mollusca, Gastropoda) Host behaviour can be influenced by parasitic risk and infection through a variety of direct and indirect mechanisms. We can expect individuals expressing different phenotypes to also differ in the ways their behaviour is altered by parasites. We used the land snail Cepaea nemoralis, a species with various shell colour morphs differing in behaviour and physiology, as a model to study the link between parasite response and individual behaviour variation. We analysed metazoan parasite abundance and its relation to behaviour (movement and food intake) in snails from three shell morphs (from light unbanded to darker five-banded) and from two neighbouring populations from contrasted environments. Snails were parasitized by mites, trematodes and nematodes, from rarest to most frequent. We confirm that terrestrial gastropods can defend against infection by trapping parasitic nematodes in their shell. We show that nematode encapsulation can uncover past infections even when a population is currently nematode-free, possibly due to parasite seasonality. We present new observations suggesting that not only nematodes but also mites might be encapsulated in shells. Vulnerability to parasites was morph-dependent, with darker snails harbouring fewer nematodes. Behaviour (movement and food intake) was dependent on nematode, but not trematode infection. Individuals with higher nematode load ate less, irrespective of morph and population. The morph that was most vulnerable to parasitism (unbanded snails) showed reduced activity in the nematode-infected population compared to the one that was nematode-free at sampling time. As a result, parasite risk may cancel previously described behavioural differences between morphs. We discuss the possible mechanisms explaining morph-dependent responses to parasites, and how parasite risk may be an unseen force shaping Cepaea nemoralis morph variation in space and time. We conclude on the possible ecological consequences of the link, mediated by shell colour, between thermal and immune responses.

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5-aminolevulinic acid-photodynamic therapy ameliorates cutaneous granuloma by killing drug-resistant Mycobacterium marinum BackgroundAlthough 5-aminolevulinic acid photodynamic therapy (ALA-PDT) has been extensively used to treat to various skin disease, the application of ALA-PDT on cutaneous infection caused by Mycobacterium marinum (M. marinum), especially by drug-resistant M. marinum is still not clear.

ObjectivesWe evaluated the therapeutic efficacy of ALA-PDT on M. marinum in a mouse infection model and tested its killing effect on M. marinum in vitro. Finally, we investigated the clinical effect of ALA-PDT in treating cutaneous granuloma caused by drug-resistant M. marinum.

Materials and methodsWe isolated total 9 strains of M. marinum from patients and confirmed by morphological and molecular approaches. The strains were identified by anti-mycobacterial susceptibility. Then we evaluated the killing effect of ALA-PDT on M. marinum in vitro and in a mouse model to observe the antimycobacterial effect of ALA-PDT. Therapeutic efficacy was further assessed in patients with cutaneous granuloma caused by drug-resistant M. marinum.

ResultsWe demonstrated that the ALA-PDT directly killed M. marinum in vitro. The paws cutaneous lesions of mice caused by M. marinum were fully recovered 2 weeks after ALA-PDT treatment. However, there was no significant difference for immune cells in peripheral blood before and after ALA-PDT therapy. Finally, ALA-PDT proved to be effective in treat two patients with cutaneous infection caused by drug-resistant M. marinum.

ConclusionsThe results suggest that ALA-PDT is effective in treating M. marinum cutaneous infections by killing M. marinum directly, independent of systemic immune responses. The data highlight the ALA-PDT as a promising therapeutic choice for M. marinum infection, especially for drug-resistant strains.

sentences: The Impact of Control Interface on Features of Heart Rate Variability AO_SCPLOWBSTRACTC_SCPLOWShared human-robot control for assistive machines can improve the independence of individuals with motor impairments. Monitoring elevated levels of workload can enable the assistive autonomy to adjust the control-sharing in an assist-as-needed way, to achieve a balance between user fatigue, stress and independent control. In this work, we aim to investigate how heart-rate variability features can be utilized to monitor elevated levels of mental workload while operating a powered wheelchair, and how that utilization might vary under different control interfaces. To that end, we conducted a 22 person study with three commercial interfaces. Our results show that the validity and reliability of using the ultra-short-term heart-rate variability features as predictors for workload indeed are affected by the type of interface in use.

sentences: The perceived position of a moving object is reset by temporal, not spatial limits When the internal texture of a Gabor patch drifts orthogonally to its physical path, its perceived motion deviates dramatically from its physical path. The local position shifts accumulate to such an extent that a 45{degrees} oblique physical path appears to be vertical. However, at some point, a limit is reached and the path resets back to its veridical location, whereupon a new accumulation starts, making the new perceived path segment appear parallel to the pre-reset segment, but offset horizontally from it. Here, we tested whether spontaneous resets of this motion-induced position shift depend on the time or the distance over which position errors accrue, or both. We introduced a temporal gap in the middle of the path that forced the illusory path to reset back to its veridical physical position. This gap-triggered reset allowed us to measure the magnitude of the illusory offset up to that point. We found that perceived offset was less than expected for the angle of illusory drift, indicating that spontaneous resets had occurred prior to the gap-induced reset. The position offset decreased when the pre-gap duration increased but approximately doubled when the path length doubled. This pattern of perceived offsets is best accounted for by spontaneous resets that occur randomly over time at a constant rate, independently of the distance traveled. Our results suggest a temporal, not spatial, limit for the accumulation of position errors that underlies this illusion.

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Single-neuron bursts encode pathological oscillations in Parkinson's disease and essential tremor Deep brain stimulation procedures offer an invaluable opportunity to study disease through intracranial recordings from awake patients. Herein, we address the relationship between singleneuron and aggregate-level (local field potential; LFP) activities in the subthalamic nucleus (STN) and thalamic ventral intermediate nucleus (Vim) of patients with Parkinsons disease (n=19) and essential tremor (n=16), respectively. Both disorders have been characterized by pathologically elevated LFP oscillations, as well as an increased tendency for neuronal bursting. Our findings suggest that periodic single-neuron bursts encode both pathophysiological beta (13-33Hz; STN) and tremor (4-10Hz; Vim) LFP oscillations, evidenced by strong time-frequency and phase-coupling relationships between the bursting and LFP signals. Spiking activity occurring outside of bursts had no relationship to the LFP. In STN, bursting activity most commonly preceded the LFP oscillation, suggesting that neuronal bursting generated within STN may give rise to an aggregate-level LFP oscillation. In Vim, LFP oscillations most commonly preceded bursting activity, suggesting that neuronal firing may be entrained by periodic afferent inputs. In both STN and Vim, the phasecoupling relationship between LFP and high-frequency oscillation (HFO) signals closely resembled the relationships between the LFP and single-neuron bursting. This suggests that periodic singleneuron bursting is likely representative of a higher spatial and temporal resolution readout of periodic increases in the amplitude of HFOs, which themselves may be a higher resolution readout of aggregate-level LFP oscillations. Overall, our results may reconcile "rate" and "oscillation" models of Parkinsons disease and shed light onto the single-neuron basis and origin of pathophysiological oscillations in movement disorders.

SignificanceIn surgical patients with Parkinsons disease and essential tremor, we leverage intracranial recordings to establish a link between pathophysiological phenomena across various scales of observation (spatio-temporal resolutions). We provide insights and reconcile theories about aberrant neurocircuit phenomena which underly theses debilitating, medically refractory movement disorders. Furthermore, our connectivity analyses between single-neuron and local field potential activities may shed light on the origin of the deleterious neural oscillations underlying these disorders. Ultimately, our findings may aid in the development or investigation of targeted therapies to address or correct underlying neurocircuit dysfunction, which can include neuropharmaceuticals, but also novel neuromodulatory strategies like closed-loop deep brain stimulation targeting pathophysiological oscillations and phase-dependent stimulation methods seeking to stimulate "at the right time/phase."

sentences: A Novel Chronic in vivo Oral Cadmium Exposure-Washout Mouse Model for Studying Cadmium Toxicity and Complex Diabetogenic Effects Type II diabetes mellitus (T2DM) is characterized by insulin resistance, {beta}-cell dysfunction and hyperglycemia. In addition to well known risk factors such as lifestyle and genetic risk score, accumulation of environmental toxicants in organs relevant to glucose metabolism is increasingly recognized as additional risk factors for T2DM. Here, we describe the development of an in vivo oral cadmium (Cd) exposure model. It was shown that oral Cd exposure in drinking water followed by washout and high fat diet (HFD) in C57BL/6N mice results in islet Cd bioaccumulation comparable to that found in native human islets while mitigating the anorexic effects of Cd to achieve the same weight gain required to induce insulin resistance as in Cd naive control mice. Inter individual variation in plasma glucose and insulin levels as well as islet Cd bioaccumulation was observed in both female and male mice. Regression analysis showed an inverse correlation between islet Cd level and plasma insulin following a glucose challenge in males but not in females. This finding highlights the need to account for inter individual target tissue Cd concentrations when interpreting results from in vivo Cd exposure models. No effect of Cd on insulin secretion was observed in islets ex vivo, highlighting differences between in vivo and ex vivo cadmium exposure models. In summary, our oral in vivo Cd exposure-washout with HFD model resulted in islet Cd bioaccumulation that is relevant in the context of environmental cadmium exposure in humans. Here, we showed that islet Cd bioaccumulation is associated with complex cadmium-mediated changes in glucose clearance and {beta}-cell function. The model described here will serve as a useful tool to further examine the relationship between Cd exposure, islet Cd bioaccumulation, dysglycemia and their underlying mechanisms.

sentences: Development and validation of Arc nanobodies: new tools for probing Arc dynamics and function Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N-and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc and analysis of capsid domain specific functions.

sentences: Influence of claustrum on cortex varies by area, layer, and cell type The claustrum is a small subcortical structure with widespread connections with disparate regions of the cortex. These far-reaching projections have led to many hypotheses concerning its function. However, we know little about how claustrum input affects neural activity in cortex, particularly beyond frontal areas. Here, using optogenetics and multi-regional Neuropixels recordings from over 15,000 neurons in awake mice, we demonstrate that the effect of claustrum input differs depending on brain area, layer, and cell type. Brief claustrum stimulation produces approximately 1 spike per claustrum neuron, which affects many fast-spiking (FS; putative inhibitory) but very few regular-spiking (RS; putative excitatory) cortical neurons. Prolonged claustrum stimulation affects many more cortical FS and RS neurons. More inhibition occurs in frontal regions and deeper layers, while more excitation occurs in posterior regions and superficial layers. These differences imply that the function of claustrum input to cortex depends on the area, supporting the idea that claustro-cortical circuits are organized into functional modules.

sentences: SPIN - Species by Proteome INvestigation Genetic species determination has become an indispensable tool in forensics, archaeology, ecology, and food authentication. The available methods are either suited for detecting a single taxon across many samples or for screening a wide range of species across a few samples. Here, we introduce "Species by Proteome INvestigation" (SPIN), a proteomics workflow capable of querying over 150 mammalian species in 7.2 minutes of mass spectrometry (MS) analysis. Streamlined and automated sample preparation by protein aggregation capture, high-speed chromatography and data-independent acquisition, and a confident species inference algorithm facilitate processing hundreds of samples per day. We demonstrate the correct classification of known references, reproducible species identification in degraded Iron-Age material from Scandinavia, and test the limits of our methods with Middle and Upper Palaeolithic bones from Southern European sites with late Neanderthal occupation. While this initial study is focused on modern and archaeological mammalian bone, SPIN will be open and expandable with other biological tissues and taxa.

sentences: Antisense therapy in a new rat model of Alexander disease reverses GFAP pathology, white matter deficits, and motor impairment. Alexander disease (AxD) is a devastating leukodystrophy caused by gain of function mutations in GFAP, and the only available treatments are supportive. Recent advances in antisense oligonucleotide (ASO) therapy have demonstrated that transcript targeting can be a successful strategy for human neurodegenerative diseases amenable to this approach. We have previously used mouse models of AxD to show that Gfap-targeted ASO suppresses protein accumulation and reverses pathology; however, the mice have a mild phenotype with no apparent leukodystrophy or overt clinical features and are therefore limited for assessing functional outcomes. In this report we introduce a new rat model of AxD that exhibits hallmark pathology with GFAP aggregation in the form of Rosenthal fibers, widespread astrogliosis, and white matter deficits. These animals develop normally during the first postnatal weeks but fail to thrive after weaning and develop severe motor deficits as they mature, with approximately 15 % dying of unknown cause between 6 to 12 weeks of age. In this model, a single treatment with Gfap-targeted ASO provides long lasting suppression, reverses GFAP pathology, and depending on age of treatment, prevents or mitigates white matter deficits and motor impairment. This is the first report of an animal model of AxD with myelin pathology and motor impairment, recapitulating prominent features of the human disease. We use this model to show that ASO therapy has the potential to not only prevent but also reverse many aspects of disease.

sentences: H3K4me1-marked Enhancer Activation in Resistant Prostate Cancers Implicates SOX4 and MENIN Inhibition as Therapeutic Strategies Chromatin elements and regulators play important roles during progression of prostate cancer, however, their involvement in response to therapy is less well understood. Using comprehensive chromatin profiling of patient-derived tumors, we find that enhancer elements marked by H3K4me1 are highly enriched in aggressive therapy-resistant prostate cancers on important resistance-driving genes, such as those involved in FOXA1, NOTCH and TGF-{beta} signaling. Importantly, by targeting H3K4me1-elements through inhibition of the MLL complex, a H3K4 methyltransferase, we reduced the proliferative capacity and H3K4me1-associated loci in enzalutamide-resistant prostate cancer lines. We identify AR, FOXA1, HOXB13 and SOX4 as a subset of core TFs that are critical for establishing transcriptional networks via active enhancer reprogramming during acquisition of resistance to therapy. Knock-down of SOX4 reduced cell proliferation and disrupted the H3K4me1 enhancer landscape, further suggesting a role for this TF in therapy-resistance. Overall, our data implicate H3K4me1-marked enhancers as a key epigenetic feature of therapy-resistance, implicate SOX4 in enhancer reprogramming and suggest use of MLL/MENIN inhibitors as a potential therapeutic strategy in high-grade and locally advanced prostate cancers that do not respond to traditional therapies.

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The Interaction of Angiotensin II and Milk Fat Globule Epidermal Growth Factor VIII in Proinflammatory Arterial Remodeling BackgroundAngiotensin II (Ang II) and milk fat globule-epidermal growth factor VIII (MFG-E8) are involved in age-associated arterial remodeling; however, the inflammatory role of MFG-E8 in Ang II associated arterial remodeling with aging remains unknown.

Methods and ResultsIn this study, 30-week-old MFG-E8 knock out (KO) and age-matched wild-type (WT) mice were infused with Ang II or saline. After infusion the with Ang II, the aortic molecular, cellular, and structural remodeling were observed in mice and compared to those infused with saline, but these effects were dependent on the expression of MFG-E8: (1) In the WT mice, Ang II infusion substantially increased intimal-medial thickness, elastic lamina degradation, collagen deposition, and the proliferation of VSMCs; in contrast, in the KO mice, these effects were significantly reduced; (2) In the WT mice, Ang II treatment significantly increased the activation and expression of MMP2, TGF-{beta}1, and its downstream signaling molecule p-SMAD2, and collagen type I production, however, in the KO mice, these molecular effects were significantly reduced; (3) In the WT mice, Ang II treatment increased inflammatory p-NF-{kappa}B p65, MCP1, TNF-, ICAM1, and VCAM1 molecular expression, while conversely, in the KO mice, no significant inflammatory changes were found; (4) Importantly, compared to untreated control mice with a wide range of age from 4-96 weeks, Ang II infused "younger" mice produced an "older" arterial inflammatory phenotype, which was alleviated by MFG-E8 deficiency.

ConclusionsMFG-E8 mediates Ang II associated arterial inflammatory remodeling. Targeting MFG-E8 is a novel molecular approach to curb adverse arterial remodeling during aging and hypertension.

CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Both Ang II and MFG-E8 increases are involved in proinflammatory arterial remodeling mediating the molecular, cellular and tissue events in aging and hypertension.
* MFG-E8 is essential for Ang II induced and age-associated adverse arterial remodeling via the increase of proinflammation, intimal medial thickening, elastin fragmentation, collagen deposition, and VSMC proliferation.

What Are the Clinical Implications?Since MFG-E8 mediates Ang II induced proinflammation in arterial wall remodeling in aging and hypertension, targeting MFG-E8 is a potential molecular approach to curb inflammatory arterial remodeling, maintaining the health of the vascular system during aging and hypertension.

sentences: All2: A tool for selecting mosaic mutations from comprehensive multi-cell comparisons Accurate discovery of somatic mutations in a cell is a challenge that partially lays in immaturity of dedicated analytical approaches. Approaches comparing cells genome to a control bulk sample miss common mutations, while approaches to find such mutations from bulk suffer from low sensitivity. We developed a tool, All2, which enables accurate filtering of mutations in a cell from exhaustive comparison of cells genomes to each other without data for bulk(s). Based on all pair-wise comparisons, every variant call (point mutation, indel, and structural variant) is classified as either a germline variant, mosaic mutation, or false positive. As All2 allows for considering dropped-out regions, it is applicable to whole genome and exome analysis of cloned and amplified cells. By applying the approach to a variety of available data, we showed that its application reduces false positives, enables sensitive discovery of high frequency mutations, and is indispensable for conducting high resolution cell lineage tracing. All2 is freely available at https://github.com/abyzovlab/All2.

sentences: Multimodal sensorimotor integration of visual and kinaesthetic afferents modulates motor circuits in humans Optimal motor control requires the effective integration of multi-modal information. Visual information of movement performed by others even enhances potentials in the upper motor neurons, through the mirror-neuron system. On the other hand, it is known that motor control is intimately associated with afferent proprioceptive information. Kinaesthetic information is also generated by passive, external-driven movements. In the context of sensory integration, its an important question, how such passive kinaesthetic information and visually perceived movements are integrated. We studied the effects of visual and kinaesthetic information in combination, as well as isolated, on sensorimotor-integration - compared to a control condition. For this, we measured the change in the excitability of motor cortex (M1) using low-intensity TMS. We hypothesised that both visual motoneurons and kinaesthetic motoneurons could enhance the excitability of motor responses. We found that passive wrist movements increase the motor excitability, suggesting that kinaesthetic motoneurons do exist. The kinaesthetic influence on the motor threshold was even stronger than the visual information. Moreover, the simultaneous visual and passive kinaesthetic information increased the cortical excitability more than each of them independently. Thus, for the first time, we found evidence for the integration of passive kinaesthetic- and visual-sensory stimuli.

sentences: Validation of the first PGT-whole genome sequencing approach including mitochondrial variants Whole genome screening is currently not available in preimplantation genetic testing (PGT) due to poor performance of whole genome amplification methods. Here, we present the first validation study using our latest whole genome amplification (WGA) protocol, which yields amplified DNA with comparable quality to genomic DNA when perfoming whole genome sequencing assays. We started by validating PGT for aneuploidy (PGT-A) using our WGA protocol on cell lines and donated human embryos, where amplification success rates were >99.9% and 96.2% respectively. Accuracy on both was >99.9% on aneuploidy status. We next validated whole genome sequencing using amplified and genomic DNA from the Genome in the Bottle reference sample NA12878 to demonstrate accuracy, specificity, sensitivity, and precision of our WGA methods. Amplified DNA and genomic DNA are comparable in this case, with 99.99% accuracy, 99.99% specificity, 98.0% sensitivity and 98.1% precision with our WGA protocol. We additionally examined variant calls between biopsies and the remaining embryos from which they were derived. Again, 99.99% accuracy, 99.99% specificity, 98.1% sensitivity and 98.0% precision were observed. Mitochondrial heteroplasmy between biopsies and embryos was validated, and 99.9% accuracy, 100% specificity, 99.1% sensitivity and 100% precision were observed. Finally, we demonstrated the ability to improve the accuracy of certain types of mendelian variants in our data to close to 100% by leveraging parental and a born childs genome to perform linkage analysis.

sentences: On stability of Canonical Correlation Analysis and Partial Least Squares with application to brain-behavior associations Associations between datasets, each comprising many features, can be discovered through multivariate methods like Canonical Correlation Analysis (CCA) or Partial Least Squares (PLS). Application of CCA/PLS to high-dimensional datasets raises critical questions about reliability and interpretability. To study this, we developed a generative modeling framework to simulate synthetic datasets, parameterized by dimensionality, variance structure, and association strength. We found that CCA/PLS associations could be highly inaccurate when the number of samples per feature is relatively small. For PLS, profiles of feature weights exhibit detrimental bias toward leading principal component axes. We confirmed these trends in state-of-the-art neuroimaging datasets, Human Connectome Project (n{approx}1000) and UK Biobank (n=20000), finding that only the latter comprised sufficient samples for stable estimates. Analysis of the neuroimaging literature using CCA to map brain-behavior relationships revealed also that the commonly employed sample sizes yield unstable CCA solutions. Finally, we provide a calculator of dataset properties required for CCA/PLS stability. Collectively, we characterize how to limit detrimental effects of overfitting on CCA/PLS stability, and provide recommendations for future studies.

sentences: Mosquito-bacteria interactions during larval development trigger metabolic changes with carry-over effects on adult fitness In animals with distinct life stages such as holometabolous insects, adult phenotypic variation is often shaped by the environment of immature stages, including their interactions with microbes colonizing larval habitats. Such carry-over effects were previously observed for several adult traits of the mosquito Aedes aegypti after larval exposure to different bacteria, but the mechanistic underpinnings are unknown. Here, we investigated the molecular changes triggered by gnotobiotic larval exposure to different bacteria in Ae. aegypti. We initially screened a panel of 16 bacterial isolates from natural mosquito breeding sites to determine their ability to influence adult life-history traits. We subsequently focused on four bacterial isolates (belonging to Flavobacterium, Lysobacter, Paenibacillus, and Enterobacteriaceae) with significant carry-over effects on adult survival and found that they were associated with distinct transcriptomic profiles throughout mosquito development. Moreover, we detected carry-over effects at the level of gene expression for the Flavobacterium and Paenibacillus isolates. The most prominent transcriptomic changes in gnotobiotic larvae reflected a profound remodeling of lipid metabolism, which translated into phenotypic differences in lipid storage and starvation resistance at the adult stage. Together, our findings indicate that larval exposure to environmental bacteria trigger substantial physiological changes that impact adult fitness, uncovering a mechanism underlying carry-over effects of mosquito-bacteria interactions during larval development.

sentences: Huygens synchronization of medial septal pacemaker neurons generates hippocampal theta oscillation Episodic learning and memory retrieval are critically dependent on a hippocampal 4-12 Hz oscillatory  clock signal, the theta oscillation. This clock is largely externally paced, by a network of GABAergic neurons in the medial septum (MS). Theoretical studies suggested a range of hypotheses how this network may achieve theta synchrony; however, experimental evidence is still lacking. By recording multiple single MS neurons and hippocampal local field potential oscillations simultaneously, with both acute and chronically implanted silicon probes, we show that MS pacemaker units oscillate at individual frequencies within the theta range in rodents. Synchronization of MS neuron frequencies, accompanied by an elevation of firing rates, was found to parallel hippocampal theta formation in multiple rodent model systems. This suggests a general mechanism for theta synchronization, akin to the synchronization of weakly coupled pendulum clocks observed by Huygens in the 17th century. We optogenetically identified the MS pacemaker units as parvalbumin-expressing GABAergic neurons, while the previously enigmatic MS glutamatergic neurons were mostly theta-activated non-rhythmic cells. Our data were consistent with a network model of partially connected single-compartment inhibitory pacemaker neurons, in which synchronization and de-synchronization in the frequency domain upon waxing and waning tonic excitatory drive was sufficient to toggle MS network output between theta and non-theta states. These results provide experimental and theoretical support to a frequency-synchronization mechanism for pacing hippocampal theta, which may serve as an inspirational prototype for the countless examples of synchronization processes in the central nervous system from Nematoda to Anthropoda to Chordate and Vertebrate phyla.

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Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC, and SFH-1) in Enterobacterales isolated from surface waters in Japan ObjectivesCarbapenemase-producing Enterobacterales (CPE) pose serious threats to public health. Compared with clinical CPE, the genetic characteristics of environmental CPE are not well understood. This study aimed to characterize the genetic determinants of carbapenem resistance in CPE isolated from environmental waters in Japan.

MethodsEighty-five water samples were collected from rivers and a lake in Japan. CPE were identified using selective media, and genome sequencing was performed for the obtained isolates (n = 21).

ResultsVarious rare/novel carbapenemases were identified: GES-5 in Raoultella planticola (n = 1), FRI-8 and FRI-11 in Enterobacter spp. (n = 8), IMI-22 and IMI-23 in Serratia ureilytica (n = 3), and SFC-1, SFC-2 and SFH-1 in Serratia fonticola (n = 9). Genomes of 11 isolates could be closed, allowing the elucidation of the genetic contexts of the carbapenemase genes. The blaGES-5 gene was located within a class 1 integron, In2071 (cassette array, blaGES-5-aacA3-aadA16), on a 33 kb IncP6 plasmid. The blaFRI-8 genes were carried on IncFII(Yp) plasmids ranging in size from 191 kb to 244 kb, and the blaFRI-11 genes were carried on 70 kb and 74 kb IncFII(pECLA)/IncR plasmids. The blaIMI-22 and blaIMI-23 genes were colocated on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes.

ConclusionsThis study revealed the presence of rare/novel carbapenemases among CPE in aquatic environments, suggesting that the environment may act as a potential reservoir of these minor carbapenemases.

sentences: Male mate choice unresolved in the mangrove rivulus Mate choice has the potential to drive phenotypic evolution because it can determine traits that increase an individuals likelihood to reproduce (courtship behaviors, elaborate ornamentation). These traits, however, can also be detrimental for health or survival, often antagonizing the evolution of extreme phenotypes. Mangrove rivulus fish (Kryptolebias marmoratus) develop as self-fertilizing simultaneous hermaphrodites. Hermaphrodites overwhelmingly self-fertilize their eggs internally, but occasionally oviposit unfertilized eggs. Some individuals change sex to male after sexual maturity, essentially forgoing the reproductive assurance of selfing. In a continuing effort to understand how sex change to male is maintained this species, I designed an experiment to determine whether males act as choosers to increase their likelihood of finding unfertilized eggs for reproduction. I hypothesized that males would prefer to associate with younger hermaphrodites when given a dichotomous choice, as they lay a greater proportion of unfertilized eggs compared to older hermaphrodites. The males in this study did not show a preference for either the younger or older hermaphrodite but exhibited greater within individual variance across subtrials than among individual variation. I discuss alternative hypotheses concerning male mate choice in mangrove rivulus, which may illuminate hypotheses to be tested in this and other hermaphroditic species.

sentences: Genetic architecture of natural variations of cardiac performance in flies Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. We investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP)1. Genome Wide Associations Studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that we identified were used to map potential regulatory non-coding regions, which in turn were employed to predict Transcription Factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart specific knockdown. Additionally, we showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, we showed that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, we documented correlations between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 and EGR2 in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans.

sentences: Robustness of phylogenetic inference to model misspecification caused by pairwise epistasis Likelihood-based phylogenetic inference posits a probabilistic model of character state change along branches of a phylogenetic tree. These models typically assume statistical independence of sites in the sequence alignment. This is a restrictive assumption that facilitates computational tractability, but ignores how epistasis, the effect of genetic background on mutational effects, influences the evolution of functional sequences. We consider the effect of using a misspecified site-independent model on the accuracy of Bayesian phylogenetic inference in the setting of pairwise-site epistasis. Previous work has shown that as alignment length increases, tree reconstruction accuracy also increases. Here, we present a simulation study demonstrating that accuracy increases with alignment size even if the additional sites are epistatically coupled. We introduce an alignment-based test statistic that is a diagnostic for pair-wise epistasis and can be used in posterior predictive checks.

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GIP: An open-source computational pipeline for mapping genomic instability from protists to cancer cells Genome instability has been recognized as a key driver for microbial and cancer adaptation and thus plays a central role in many human pathologies. Even though genome instability encompasses different types of genomic alterations, most available genome analysis software are limited to just one kind mutation or analytical step. To overcome this limitation and better understand the role of genetic changes in enhancing pathogenicity we established GIP, a novel, powerful bioinformatic pipeline for comparative genome analysis. Here we show its application to whole genome sequencing datasets of Leishmania, Plasmodium, Candida, and cancer. Applying GIP on available data sets validated our pipeline and demonstrated the power of our analysis tool to drive biological discovery. Applied to Plasmodium vivax genomes, our pipeline allowed us to uncover the convergent amplification of erythrocyte binding proteins and to identify a nullisomic strain. Re-analyzing genomes of drug adapted Candida albicans strains revealed correlated copy number variations of functionally related genes, strongly supporting a mechanism of epistatic adaptation through interacting gene-dosage changes. Our results illustrate how GIP can be used for the identification of aneuploidy, gene copy number variations, changes in nucleic acid sequences, and chromosomal rearrangements. Altogether, GIP can shed light on the genetic bases of cell adaptation and drive disease biomarker discovery.

One Sentence SummaryGIP - a novel pipeline for detecting, comparing and visualizing genome instability.

sentences: Contextual extinction of drug-associated discriminative stimuli fails to attenuate drug-vs-food choice in rhesus monkeys Relapse within the context of a substance use disorder can be triggered by cues that function as discriminative stimuli to signal contingencies of drug availability and promote drug-taking behavior. Extinction procedures can weaken this association between drug-associated cues and drug-taking behavior and may reduce the probability of relapse. This study evaluated a regimen of extinction training on cocaine and heroin self-administration in rhesus monkeys under a drug-vs.-food choice procedure. Behavior was initially maintained under a concurrent schedule of food (1-g food pellets; fixed-ratio 100 schedule) and cocaine injections (0-0.1 mg/kg/injection; fixed-ratio 10) (n=4 males) or heroin injections (0-0.01 mg/kg/injection; fixed-ratio 10) (n=3 females and 1 male) during daily 2-h choice sessions. Subsequently, choice sessions were supplemented by daily 20-h extinction sessions for 14 consecutive days. During extinction sessions, drug-associated discriminative stimuli were presented, but responding produced saline injections. Drug continued to be available during choice sessions. Prior to extinction, both cocaine- and heroin-maintained dose-dependent increases in drug-vs.-food choice. Exposure to 14 extinction sessions failed to significantly decrease drug choice and increase food choice. These preclinical results do not support the effectiveness of extinguishing drug-associated discriminative stimuli as a non-pharmacological treatment strategy for reducing drug choice.

sentences: Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda) Biological aging is connected to life history variation across ecological scales, as well as informing a basic understanding of age-related declines to organismal function. Altered DNA methylation dynamics are a conserved aspect of biological aging and have recently been modeled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic aging, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe aging in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 8% of its lifespan. Although patterns of CHH methylation showed little association with age, both CpG and CHG methylation contexts were strongly associated with aging, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological aging in plants.

sentences: Ongoing habenular activity is driven by forebrain networks and modulated by olfactory stimuli Ongoing neural activity, which represents internal brain states, is constantly modulated by the sensory information that is generated by the environment. In this study, we show that the habenular circuits act as a major brain hub integrating the structured ongoing activity of the limbic forebrain circuitry and the olfactory information. We demonstrate that ancestral homologs of amygdala and hippocampus in zebrafish forebrain are the major drivers of ongoing habenular activity. We also reveal that odor stimuli can modulate the activity of specific habenular neurons that are driven by this forebrain circuitry. Our results highlight a major role for the olfactory system in regulating the ongoing activity of the habenula and the forebrain, thereby altering brains internal states.

sentences: The extracellular sialidase NEU3 induces neutrophil priming Some extracellular glycoconjugates have sialic acid as the terminal sugar, and sialidases are enzymes that remove this sugar. Mammals have four sialidases, but their biological functions are unclear. In this report, we show that incubation of human neutrophils with the human sialidase NEU3, but not NEU1, NEU2 or NEU4, inducess human male and female neutrophils to change from a round to a more amoeboid morphology, causes the primed neutrophil markers CD66, CD11B, and CD18 to localize to the cell cortex, and decreases the localization of the unprimed neutrophil markers CD43 and CD62L at the cell cortex. NEU3, but not the other 3 sialidases, also causes human male and female neutrophils to increase their F-actin content. The inhibition of NEU3 by the NEU3 inhibitor 2-acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these results indicate that NEU3 can prime human male and female neutrophils, and that NEU3 is a potential regulator of inflammation.

sentences: Effects of Ancestry and Hypopigmentation Alleles on Skin Color in a Caribbean Native American Population Interest in the genetic basis of variation in skin pigmentation in Native American populations led us to seek indigenous populations of the Western Hemisphere with African and minimal European admixture to study the effect of Native American ancestry on skin color. Admixture analysis from DNA collected from 458 individuals in the Kalinago territory of the Commonwealth of Dominica showed shared ancestry with East Asians at K=3 and 55% Native American, 32% African, and 11% European ancestry at K=6, the highest Native American ancestry of Caribbean populations. Skin pigmentation was 20 to 80 melanin units, averaging 46. Three albino individuals were homozygous for multi-nucleotide polymorphism OCA2NW273KV of African origin, whose population allele frequency was 0.03 and single allele effect size was -8 melanin units. Hypopigmenting allele frequencies for SLC24A5A111T and SLC45A2L374F were 0.14 and 0.05, whose single allele effect sizes were -6 and -3, respectively. Skin color plots of individuals lacking known hypopigmenting alleles suggests that Native American Ancestry reduced pigmentation by more than 20 melanin units (low and high estimates 21.8 and 28.5). Shared ancestry with East Asians at K=3 suggests potential sharing of one or more pigmentation alleles.

sentences: Myosins generate contractile force and maintain organization in the cytokinetic contractile ring During cytokinesis cells assemble an actomyosin contractile ring whose tension constricts and divides cells, but the ring tension was rarely measured. Actomyosin force generation is well understood for the regular sarcomeric architecture of striated muscle, but recent super-resolution studies of fission yeast contractile rings revealed organizational building blocks that are not sarcomeres but irregularly positioned plasma membrane-anchored protein complexes called nodes. Here we measured contractile ring tensions in fission yeast protoplast cells. The myosin II isoforms Myo2 and Myp2 generated the tension, with a ~2-fold greater contribution from Myo2. Simulations of a molecularly detailed ring model revealed a sliding node mechanism for tension, where nodes hosting tense actin filaments were pulled bidirectionally around the ring. Myo2 and Myp2 chaperoned self-assembling components into the ring organization, and anchored the ring against bridging instabilities. Thus, beyond force production, Myo2 and Myp2 are the principal organizers, bundlers and anchors of the contractile ring.

sentences: Suppression of aggregate and amyloid formation by a novel intrinsically disordered region in metazoan Hsp110 chaperones Molecular chaperones maintain protein homeostasis (proteostasis) by ensuring the proper folding of polypeptides. Loss of proteostasis has been linked to the onset of numerous neurodegenerative disorders including Alzheimers, Parkinsons, and Huntingtons disease. Hsp110 is related to the canonical Hsp70 class of protein folding molecular chaperones and interacts with Hsp70 as a nucleotide exchange factor (NEF), promoting rapid cycling of ADP for ATP. In addition to its NEF activity, Hsp110 possesses an Hsp70-like substrate binding domain (SBD) whose biological roles remain undefined. Previous work in Drosophila melanogaster has shown that loss of the sole Hsp110 gene (Hsc70cb) accelerates the aggregation of polyglutamine (polyQ)-expanded human Huntingtin, while its overexpression protects against polyQ-mediated neuronal cell death. We hypothesize that in addition to its role as an Hsp70 NEF, Drosophila Hsp110 may function in the fly as a protective protein "holdase", preventing the aggregation of unfolded polypeptides via the SBD-{beta} subdomain. Using an in vitro protein aggregation assay we demonstrate for the first time that Drosophila Hsp110 effectively prevents aggregation of the model substrate citrate synthase. We also report the discovery of a redundant and heretofore unknown potent holdase capacity in a 138 amino-acid region of Hsp110 carboxyl-terminal to both SBD-{beta} and SBD- (henceforth called the C-terminal extension). This sequence is highly conserved in metazoan Hsp110 genes, completely absent from fungal representatives, including Saccharomyces cerevisiae SSE1, and is computationally predicted to contain an intrinsically disordered region (IDR). We demonstrate that this IDR sequence within the human Hsp110s, Apg-1 and Hsp105, inhibits the formation of amyloid A{beta}-42 and -synuclein fibrils in vitro but cannot mediate fibril disassembly. Together these findings demonstrate the existence of a second independent, passive holdase property of metazoan Hsp110 chaperones capable of suppressing both general protein aggregation and amyloidogenesis and raise the possibility of exploitation of this IDR for therapeutic benefit in combating neurodegenerative disease.

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Functional and Highly Crosslinkable HIV-1 Envelope Glycoproteins Enriched in a Pretriggered Conformation Binding to the receptor, CD4, drives the pretriggered, "closed" (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer into more "open" conformations (States 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the State-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize States 2/3. HIV-1 Env metastability has created challenges for defining the State-1 structure and developing immunogens mimicking this labile conformation. The availability of functional State-1 Envs that can be efficiently crosslinked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4 and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered State-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable Tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly crosslinkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state.

IMPORTANCEThe development of an efficient vaccine is critical for combating HIV-1 infection worldwide. However, the instability of the pretriggered shape (State 1) of the viral envelope glycoprotein (Env) makes it difficult to raise neutralizing antibodies against HIV-1. Here, by introducing multiple changes in Env, we derived two HIV-1 Env variants that are enriched in State 1 and can be efficiently crosslinked to maintain this shape. These Env complexes are more stable in detergent, assisting their purification. Thus, our study provides a path to a better characterization of the native pretriggered Env, which should assist vaccine development.

sentences: Comparing divergence landscapes from reduced-representation and whole-genome re-sequencing in the yellow-rumped warbler (Setophaga coronata) species complex Researchers seeking to generate genomic data for non-model organisms are faced with a number of trade-offs when deciding which method to use. The selection of reduced representation approaches versus whole genome re-sequencing will ultimately affect the marker density, sequencing depth, and the number of individuals that can multiplexed. These factors can affect researchers ability to accurately characterize certain genomic features, such as landscapes of divergence--how FST varies across the genomes. To provide insight into the effect of sequencing method on the estimation of divergence landscapes, we applied an identical bioinformatic pipeline to three generations of sequencing data (GBS, ddRAD, and WGS) produced for the same system, the yellow-rumped warbler species complex. We compare divergence landscapes generated using each method for the myrtle warbler (Setophaga coronata coronata) and the Audubons warbler (S. c. auduboni), and for Audubons warblers with deeply divergent mtDNA resulting from mitochondrial introgression. We found that most high-FST peaks were not detected in the ddRAD dataset, and that while both GBS and WGS were able to identify the presence of large peaks, WGS was superior at a finer scale. Comparing Audubons warblers with divergent mitochondrial haplotypes, only WGS allowed us to identify small (10-20kb) regions of elevated differentiation, one of which contained the nuclear-encoded mitochondrial gene NDUFAF3. We calculated the cost per base pair for each method and found it was comparable between GBS and WGS, but significantly higher for ddRAD. These comparisons highlight the advantages of WGS over reduced representation methods when characterizing landscapes of divergence.

sentences: Time-lagged Flux in the Transition Path Ensemble: Flux Maximization and Relation to Transition Path Theory Transition path ensemble is of special interest in reaction coordinate identification as it consists of reactive trajectories that start from the reactant state and end in the product one. As a theoretical framework for describing the transition path ensemble, the transition path theory has been introduced more than ten years ago and so far its applications have been only illustrated in several low-dimensional systems. Given the transition path ensemble, expressions for calculating flux, current (a vector field), and principal curve are derived here in the space of collective variables from the transition path theory and they are applicable to time-series obtained from molecular dynamics simulations of high-dimensional systems, i.e., the position coordinates as a function of time in the transition path ensemble. The connection of the transition path theory is made to a density-weighted average flux, a quantity proposed in a previous work to appraise the relevance of a coordinate to the reaction coordinate [W. Li, J. Chem. Phys. 156, 054117 (2022)]. Most importantly, as an extension of the existing quantities, time-lagged quantities such as flux and current are also proposed. The main insights and objects provided by these time-lagged quantities are illustrated in the application to the alanine peptide in vacuum.

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Impact of acute visual experience on development of LGN receptive fields in the ferret Selectivity for direction of motion is a key feature of primary visual cortical neurons. Visual experience is required for direction selectivity in carnivore and primate visual cortex, but the circuit mechanisms of its formation remain incompletely understood. Here we examined how developing lateral geniculate nucleus (LGN) neurons may contribute to cortical direction selectivity. Using in vivo electrophysiology techniques, we examined LGN receptive field properties of visually naive female ferrets before and after exposure to 6 hours of motion stimuli in order to assess the effect of acute visual experience on LGN cell development. We found that acute experience with motion stimuli did not significantly affect the weak orientation or direction selectivity of LGN neurons. In addition, we found that neither latency nor sustainedness or transience of LGN neurons significantly changed with acute experience. These results suggest that the direction selectivity that emerges in cortex after acute experience is computed in cortex and cannot be explained by changes in LGN cells.

Significance StatementThe development of typical neural circuitry requires experience-independent and experience-dependent factors. In the visual cortex of carnivores and primates, selectivity for motion arises as a result of experience, but we do not understand whether the major brain area that sits between the retina and the visual cortex - the lateral geniculate nucleus of the thalamus - also participates. Here we found that lateral geniculate neurons do not exhibit changes as a result of several hours of visual experience with moving stimuli, at a time when visual cortical neurons undergo a rapid change. We conclude that lateral geniculate neurons do not participate in this plasticity, and that changes in cortex are likely responsible for the development of direction selectivity in carnivores and primates.

sentences: Repertoire of naturally acquired maternal antibodies transferred to infants for protection against shigellosis Shigella is the second leading cause of diarrheal diseases, accounting for >200,000 infections and >50,000 deaths in children under 5 years of age annually worldwide. The incidence of Shigella-induced diarrhea is relatively low during the first year of life and increases substantially, reaching its peak between 11 to 24 months of age. This epidemiological trend hints at an early protective immunity of maternal origin and an increase in disease incidence when maternally acquired immunity wanes. The magnitude, type, antigenic diversity, and antimicrobial activity of maternal antibodies transferred via placenta that can prevent shigellosis during early infancy are not known. To address this knowledge gap, Shigella-specific antibodies directed against the lipopolysaccharide (LPS) and virulence factors (IpaB, IpaC, IpaD, IpaH, and VirG), and antibody-mediated serum bactericidal (SBA) and opsonophagocytic killing antibody (OPKA) activity were measured in maternal and cord blood sera from a longitudinal cohort of mother-infant pairs living in rural Malawi. Protein-specific (very high levels) and Shigella LPS IgG were detected in maternal and cord blood sera; efficiency of placental transfer was 100% and 60%, respectively, and had preferential IgG subclass distribution (protein-specific IgG1 > LPS-specific IgG2). In contrast, SBA and OPKA activity in cord blood was substantially lower as compared to maternal serum and varied among Shigella serotypes. LPS was identified as the primary target of SBA and OPKA activity. Maternal sera had remarkably elevated Shigella flexneri 2a LPS IgM, indicative of recent exposure. Our study revealed a broad repertoire of maternally acquired antibodies in infants living in a Shigella-endemic region and highlights the abundance of protein-specific antibodies and their likely contribution to disease prevention during the first months of life. These results contribute new knowledge on maternal infant immunity and target antigens that can inform the development of vaccines or therapeutics that can extend protection after maternally transferred immunity wanes.

sentences: A pH-eQTL interaction at the RIT2-SYT4 Parkinson's disease risk locus in the substantia nigra Parkinsons disease (PD) causes severe motor and cognitive disabilities that result from the progressive loss of dopamine neurons in the substantia nigra. The rs12456492 variant in the RIT2 gene has been repeatedly associated with increased risk for Parkinsons disease. From a transcriptomic perspective, a meta-analysis found that RIT2 gene expression is correlated with pH in the human brain. To assess these pH associations in relation to PD risk, we examined the two datasets that assayed rs12456492, gene expression, and pH in the postmortem human brain. Using the BrainEAC dataset, we replicate the positive correlation between RIT2 gene expression and pH in the human brain (n=100). Furthermore, we found that the relationship between expression and pH is influenced by rs12456492. When tested across ten brain regions, this interaction is specifically found in the substantia nigra. A similar association was found for the co-localized SYT4 gene. In addition, SYT4 associations are stronger in a combined model with both genes, and the SYT4 interaction appears to be specific to males. In the GTEx dataset, the pH associations involving rs12456492 and expression of either SYT4 and RIT2 was not seen. This null finding may be due to the short postmortem intervals (PMI) of the GTEx tissue samples. In the BrainEAC data, we tested the effect of PMI and only observed the interactions in the longer PMI samples. These previously unknown associations suggest novel mechanistic roles for rs12456492, RIT2, and SYT4 in the regulation of pH in the substantia nigra.

sentences: An allosteric agonist activates BK channels by perturbing coupling between Ca2+ binding and pore opening BK type Ca2+-activated K+ channels activate in response to both the membrane voltage and intracellular Ca2+ with distinct mechanisms. Ca2+ binds to the cytosolic domain (CTD) to open the pore across the membrane, but the mechanism that couples Ca2+ binding to pore opening is not clear. Here we show that a compound, BC5, identified using in silico screening, interacts with BK channels at the interface between the CTD and the transmembrane voltage sensing domain (VSD) and enhances channel activity by specifically affecting the Ca2+ dependent mechanism. BC5 activates the channel in the absence of Ca2+ binding but Ca2+ binding inhibits BC5 effects. Thus, BC5 perturbs the pathway that couples Ca2+ binding to pore opening to allosterically affect both, which is supported by atomistic simulations and mutagensis. The results suggest that the CTD- VSD interaction makes a major contribution to the mechanism of Ca2+ dependent activation and is an important site for allosteric agonists to modulate BK channel activation.

sentences: An integrated strategy reveals complex glycosylation of erythropoietin using top-down and bottom-up mass spectrometry The characterization of glycoproteins, like erythropoietin, is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis of a first-generation erythropoietin (epoetin beta), including a developed top-down mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). Extending the coverage of our newly developed Python script to phosphorylated N-glycans enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin. The site-specificity of N-glycans was revealed at glycopeptide level by pGlyco software using different proteases. In total, 215 N-glycan compositions were identified from N-glycan and glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two different O-glycan compositions, based on the mass shifts between non-O-glycosylated and O-glycosylated species. This integrated strategy allows the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

sentences: Exploring the dynamic mechanisms of steady-state visual evoked potentials at the whole-brain level: evidence from a computational study Periodic visual stimulation can induce stable steady-state visual evoked potentials (SSVEPs) distributed in multiple brain regions and has potential applications in both neural engineering and cognitive neuroscience. However, the underlying dynamic mechanisms of SSVEPs at the whole-brain level are still not completely understood. Here, we addressed this issue by simulating the rich dynamics of SSVEPs with a large-scale brain model designed with constraints of neuroimaging data acquired from the human brain. By eliciting activity of the occipital areas using an external periodic stimulus, our model was capable of replicating both the spatial distributions and response features of SSVEPs that were observed in experiments. In particular, we confirmed that alpha-band (8-12 Hz) stimulation could evoke stronger SSVEP responses; this frequency sensitivity was due to nonlinear entrainment and resonance, and could be modulated by endogenous factors in the brain. Interestingly, the stimulus-evoked brain networks also exhibited significant superiority in topological properties near this frequency-sensitivity range, and stronger SSVEP responses were demonstrated to be supported by more efficient functional connectivity at the neural activity level. These findings not only provide insights into the mechanistic understanding of SSVEPs at the whole-brain level but also indicate a bright future for large-scale brain modeling in characterizing the complicated dynamics and functions of the brain.

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Crisscross multilayering of cell sheets Simple hydrostatic skeletons such as the Hydras consist of two stacked layers of cells perpendicularly oriented. Although this crisscross architecture can be recapitulated in vitro, little is known on the formation of such multilayers starting from a monolayer. In the present article, we show that bilayering of myoblasts results from the organization and activity of the cells originally in the monolayer which can be described as a contractile active nematic. As expected, most of the +1/2 topological defects that are associated with this nematic order self-propel. However, a subpopulation of these defects remains immobile. Perpendicular bilayering occurs exclusively at these motionless defects. Indeed, cells located at the head of these defects converge toward the (immobile) core and accumulate there until they start migrating on top of the tail of the first layer while the tail cells migrate in the opposite direction under the head cells. Since the cells keep their initial orientations, the two stacked layers end up perpendicularly oriented. This concerted process leading to a bilayer is dependent on the apical secretion of Extra Cellular Matrix (ECM) by the cells. Indeed, we evidence the presence of ECM between the cell layers and at the apical surface of the topmost layer. ECM molecules are oriented in the direction of the cells that produce them, which may guide the migration of the subsequent cell layers on their apical side.

Significance StatementHydrostatic skeletons such as that of the Hydra consist of two stacked layers of cells perpendicularly oriented whose coordinated contraction allows for complex movements. Such crisscross organization is also observed with myoblasts in culture. Confluent monolayers organize in well-aligned domains between which topological defects position themselves. Although these singularities are generally self-propelled, a fraction of them remains motionless. Perpendicular bilayering occurs exclusively at these particular pinned defects. Cells first accumulate at the head of the defects until they split in two perpendicular layers migrating in an antiparallel way on top of each other. Such a concerted process is highly dependent on the precise organization of the cell-secreted Extra Cellular Matrix (ECM).

sentences: Supergene formation is associated with a major shift in genome-wide patterns of diversity in a butterfly Selection shapes genetic diversity around target mutations, yet little is known about how selection on specific loci affects the genetic trajectories of populations, including their genome-wide patterns of diversity and demographic responses. Adaptive introgression provides a way to assess how adaptive evolution at one locus impacts whole-genome biology. Here we study the patterns of genetic variation and geographic structure in a neotropical butterfly, Heliconius numata, and its closely related allies in the so-called melpomene-silvaniform subclade. H. numata is known to have evolved a supergene via the introgression of an adaptive inversion about 2.2 million years ago, triggering a polymorphism maintained by balancing selection. This locus controls variation in wing patterns involved in mimicry associations with distinct groups of co-mimics, and butterflies show disassortative mate preferences and heterozygote advantage at this locus. We contrasted patterns of genetic diversity and structure 1) among extant polymorphic and monomorphic populations of H. numata, 2) between H. numata and its close relatives, and 3) between ancestral lineages in a phylogenetic framework. We show that H. numata populations which carry the introgressed inversions in a balanced polymorphism show markedly distinct patterns of diversity compared to all other taxa. They show the highest diversity and demographic estimates in the entire clade, as well as a remarkably low level of geographic structure and isolation by distance across the entire Amazon basin. By contrast, monomorphic populations of H. numata as well as its sister species and their ancestral lineages all show the lowest effective population sizes and genetic diversity in the clade, and higher levels of geographical structure across the continent. This suggests that the large effective population size of polymorphic populations could be a property associated with harbouring the supergene. Our results are consistent with the hypothesis that the adaptive introgression of the inversion triggered a shift from directional to balancing selection and a change in gene flow due to disassortative mating, causing a general increase in genetic diversity and the homogenisation of genomes at the continental scale.

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Host transcriptional response to SARS-CoV-2 infection in COVID-19 patients BackgroundOne of the most perplexing aspects of infection with the SARS-CoV-2 virus has been the variable response elicited in its human hosts. Investigating the transcriptional changes in individuals affected by COVID-19 can help understand and predict the degree of illness and guide clinical outcomes in diverse backgrounds.

MethodsAnalysis of host transcriptome variations via RNA sequencing from naso/oropharyngeal swabs of COVID-19 patients.

ResultsWe report strong upregulation of the innate immune response, especially type I interferon pathway, upon SARS-CoV-2 infection. Upregulated genes were subjected to a comparative meta-analysis using global datasets to identify a common network of interferon stimulated and viral response genes that mediate the host response and resolution of infection. A large proportion of mis-regulated genes showed a reduction in expression level, suggesting an overall decrease in host mRNA production. Significantly downregulated genes included those encoding olfactory, taste and neuro-sensory receptors. Many pro-inflammatory markers and cytokines were also downregulated or remained unchanged in the COVID-19 patients. Finally, a large number of non-coding RNAs were identified as down-regulated, with a few of the lncRNAs associated with functional roles in directing the response to viral infection.

ConclusionsSARS-CoV-2 infection results in the robust activation of the bodys innate immunity. Reduction of gene expression is well correlated with the clinical manifestations and symptoms of COVID-19 such as the loss of smell and taste, and myocardial and neurological complications. This study provides a critical dataset of genes that will enhance our understanding of the nature and prognosis of COVID-19.

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Spatial phylogenetics of Japanese ferns: Patterns, processes, and implications for conservation PremiseBiodiversity is often only measured with species richness. However, this ignores evolutionary history and is not sufficient for making conservation decisions. Here, we characterize multiple facets and drivers of biodiversity to understand how these relate to bioregions and conservation status in the ferns of Japan.

MethodsWe compiled a community dataset of 1,239 20 km x 20 km grid-cells including 672 taxa based on > 300,000 specimen records. We combined this with a phylogeny and functional traits to analyze taxonomic, phylogenetic, and functional diversity, and modeled biodiversity metrics in response to environmental factors and reproductive mode. Hierarchical clustering was used to delimit bioregions. Conservation status and threats were assessed by comparing the overlap of significantly diverse grid-cells with conservation zones and range maps of native Japanese deer.

ResultsTaxonomic richness was highest at mid-latitudes. Phylogenetic and functional diversity and phylogenetic endemism were highest in small southern islands. Relative phylogenetic and functional diversity were high at high and low latitudes, and low at mid-latitudes. Grid-cells were grouped into three (phylogenetic) or four (taxonomic) major bioregions. Temperature and apomixis were identified as drivers of biodiversity patterns. Conservation status was generally high for grid-cells with significantly high biodiversity, but the threat due to herbivory by deer was greater for taxonomic richness than other metrics.

ConclusionsOur integrative approach reveals previously undetected patterns and drivers of biodiversity in the ferns of Japan. Future conservation efforts should recognize that threats can vary by biodiversity metric and consider multiple multiple metrics when establishing conservation priorities.

sentences: A large panel of chicken cells are invaded in vivo by Salmonella Typhimurium even when depleted of all known invasion factors Poultry are the main source of human infection by Salmonella. As infected poultry are asymptomatic, the identification of infected poultry farms is difficult. Controlling animal infections is thus of primary importance. As cell tropism is known to govern disease, our aim was therefore to identify the infected host-cell types in chicks and the role of the three known invasion factors in this process (T3SS-1, Rck and PagN). Chicks were inoculated with wild-type or isogenic fluorescent Salmonella Typhimurium mutants via the intraperitoneal route. Our results show that liver, spleen, gall bladder and aortic vessels could be foci of infection and that phagocytic and non-phagocytic cells, including immune, epithelial and endothelial cells, are invaded in vivo in each organ. Moreover, a mutant defective for the T3SS-1, Rck and PagN remained able to colonize organs as the wild-type strain and invaded non-phagocytic cells in each organ studied. As the infection of gall bladder was not really described in chicks, invasion of gall bladder cells was confirmed by immunohistochemistry and infection was shown to last several weeks after inoculation of chicks. All together, these findings provide new insights into the dynamics of Salmonella spread in vivo in chicks at the organ and cellular levels.

sentences: Network-based pathogenicity prediction for variants of uncertain significance While the number of genome sequences continues to increase, the functions of many detected gene variants remain to be identified. These variants of uncertain significance constitute a major barrier to precision medicine 1-3. Although many computational methods have been developed to predict the function of these variants, they all rely on individual gene features and do not consider complex molecular relationships. Here we develop PathoGN, a molecular network-based approach for predicting variant pathogenicity. PathoGN significantly outperforms existing methods using benchmark datasets. Moreover, PathoGN successfully predicts the pathogenicity of 3,994 variants of uncertain significance in the real-world database ClinVar and designates potential pathogenicity. This is the first computational method for the clinical interpretation of variants using biomolecular networks, and we anticipate our method to be broadly useful for the clinical interpretation of variants and for assigning biological function to unknown variants at the genomic scale.

sentences: Temporal fine structure influences voicing confusions for consonant identification in multi-talker babble To understand the mechanisms of speech perception in everyday listening environments, it is important to elucidate the relative contributions of different acoustic cues in transmitting phonetic content. Previous studies suggest that the envelope of speech in different frequency bands conveys most speech content, while the temporal fine structure (TFS) can aid in segregating target speech from background noise. However, the role of TFS in conveying phonetic content beyond what envelopes convey for intact speech in complex acoustic scenes is poorly understood. The present study addressed this question using online psychophysical experiments to measure the identification of consonants in multi-talker babble for intelligibility-matched intact and 64-channel envelope-vocoded stimuli. Consonant confusion patterns revealed that listeners had a greater tendency in the vocoded (versus intact) condition to be biased toward reporting that they heard an unvoiced consonant, despite envelope and place cues being largely preserved. This result was replicated when babble instances were varied across independent experiments, suggesting that TFS conveys voicing information beyond what is conveyed by envelopes for intact speech in babble. Given that multi-talker babble is a masker that is ubiquitous in everyday environments, this finding has implications for the design of assistive listening devices such as cochlear implants.

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Brain Connectivity meets Reservoir Computing The connectivity of Artificial Neural Networks (ANNs) is different from the one observed in Biological Neural Networks (BNNs). Can the wiring of actual brains help improve ANNs architectures? Can we learn from ANNs about what network features support computation in the brain when solving a task?

ANNs architectures are carefully engineered and have crucial importance in many recent performance improvements. On the other hand, BNNs exhibit complex emergent connectivity patterns. At the individual level, BNNs connectivity results from brain development and plasticity processes, while at the species level, adaptive reconfigurations during evolution also play a major role shaping connectivity.

Ubiquitous features of brain connectivity have been identified in recent years, but their role in the brains ability to perform concrete computations remains poorly understood. Computational neuroscience studies reveal the influence of specific brain connectivity features only on abstract dynamical properties, although the implications of real brain networks topologies on machine learning or cognitive tasks have been barely explored.

Here we present a cross-species study with a hybrid approach integrating real brain connectomes and Bio-Echo State Networks, which we use to solve concrete memory tasks, allowing us to probe the potential computational implications of real brain connectivity patterns on task solving.

We find results consistent across species and tasks, showing that biologically inspired networks perform as well as classical echo state networks, provided a minimum level of randomness and diversity of connections is allowed. We also present a framework, bio2art, to map and scale up real connectomes that can be integrated into recurrent ANNs. This approach also allows us to show the crucial importance of the diversity of interareal connectivity patterns, stressing the importance of stochastic processes determining neural networks connectivity in general.

sentences: Computing Minimal Boolean Models of Gene Regulatory Networks Models of Gene Regulatory Networks (GRNs) capture the dynamics of the regulatory processes that occur within the cell as a means to understand the variability observed in gene expression between different conditions. Arguably the simplest mathematical construct used for modeling is the Boolean network, which dictates a set of logical rules for transition between states described as Boolean vectors. Due to the complexity of gene regulation and the limitations of experimental technologies, in most cases knowledge about regulatory interactions and Boolean states is partial. In addition, the logical rules themselves are not known a-priori. Our goal in this work is to present a methodology for inferring this information from the data, and to provide a measure for comparing network states under different biological conditions. We present a novel methodology for integrating experimental data and performing a search for the optimal consistent structure via optimization of a linear objective function under a set of linear constraints. We also present a statistical approach for testing the similarity of inferred network states under different conditions. Finally, we extend our methodology into a heuristic that can handle large datasets that are generated by single-cell RNA-Sequencing(scRNA-Seq). We demonstrate the effectiveness of these tools using a public scRNA-Seq dataset and the GRN that is associated with it. Our methodology will enable researchers to obtain a better understanding of the dynamics of gene regulatory networks and their biological role.

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The Legionella Lqs-LvbR regulatory network controls temperature-dependent growth onset and bacterial cell density Legionella species are facultative intracellular pathogens, which can cause a life-threatening pneumonia termed Legionnaires disease. Legionella pneumophila employs the Legionella quorum sensing (Lqs)-LvbR network to regulate virulence and motility, but its role for growth in media is ill-defined. Compared to the parental L. pneumophila strain JR32, a {Delta}lqsR mutant showed a reduced lag phase at 30{degrees}C and reached a higher cell density at 45{degrees}C, while the {Delta}lqsA, {Delta}lqsS and {Delta}lqsT mutants exhibited a longer lag phase and reached only a lower cell density. A {Delta}lvbR mutant resumed growth like the parental strain at 30{degrees}C, but exhibited a substantially reduced cell density at 45{degrees}C. Thus, LvbR is an important cell density regulator at elevated temperatures. A quantitative analysis of temperature-dependent growth characteristics of environmental and clinical strains revealed that L. pneumophila strains grew in AYE medium after distinct lag phases with similar rates at 30{degrees}C, reached different cell densities at the optimal growth temperature of 40{degrees}C, and no longer grew at 50{degrees}C. Legionella longbeachae reached a rather low cell density at 40{degrees}C and did not grow at and beyond 45{degrees}C. Genes encoding components of the Lqs-LvbR network were present in the genomes of the environmental and clinical L. pneumophila isolates, and the PlqsR, PlqsA, PlqsS and PlvbR promoters from strain JR32 were active in these strains. Taken together, our results indicate that the Lqs-LvbR network governs the temperature-dependent growth onset and cell density of the L. pneumophila reference strain JR32, and possibly also of environmental and clinical L. pneumophila isolates.

ImportanceEnvironmental bacteria of the genus Legionella are the causative agents of the severe pneumonia Legionnaires disease, the incidence of which is worldwide on the rise. Legionella pneumophila and Legionella longbeachae are the clinically most relevant species. The opportunistic pathogens are inhaled through contaminated aerosols and replicate in human lung macrophages with a similar mechanism as in their natural hosts, free-living amoebae. Given their prevalence in natural and technical water systems, an efficient control of Legionella spp. by physical, chemical or biological means will reduce the incidence of Legionnaires disease. Here we report that the Legionella quorum sensing (Lqs) system and the pleiotropic transcription factor LvbR govern the temperature-dependent growth onset and cell density of bacterial cultures. Hence, the growth of L. pneumophila in water systems is not only determined by the temperature and nutrient availability, but also by quorum sensing, i.e., density- and signaling molecule-dependent gene regulation.

sentences: Ca2+-mediated higher-order assembly of b0,+AT-rBAT is a key step for system b0,+ biogenesis and cystinuria Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, which causes recurrent kidney stones and occasionally severe kidney failure. Mutations of the two responsible proteins, rBAT and b0,+AT, which comprise system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Although recent structural insights into human b0,+AT-rBAT suggested a model for transport-inactivating mutations, the mechanisms by which type I mutations trigger trafficking deficiencies are not well understood. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+-mediated higher-order assembly of system b0,+ is the key to its trafficking on the cell surface. We show that Ca2+ stabilizes the interface between two rBAT molecules to mediate super-dimerization, and this in turn facilitates the N-glycan maturation of system b0,+. A common cystinuria mutant T216M and mutations that disrupt the Ca2+ site in rBAT cause the loss of higher-order assemblies, resulting in protein trafficking deficiency. Mutations at the super-dimer interface reproduce the mis-trafficking phenotype, demonstrating that super-dimerization is essential for cellular function. Cell-based transport assays confirmed the importance of the Ca2+ site and super-dimerization, and additionally suggested which residues are involved in cationic amino acid recognition. Taken together, our results provide the molecular basis of type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and trafficking diseases in general.

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Neurotransmitter regulation rather than cell-intrinsic properties shape the high-pass filtering properties of olfactory bulb glomeruli GABAergic periglomerular (PG) cells in the olfactory bulb are proposed to mediate an intraglomerular "high-pass" filter through inhibition targeted onto a glomerulus. With this mechanism, weak stimuli (e.g., an odor with a low affinity for an odorant receptor) mainly produce PG cell-driven inhibition but strong stimuli generate enough excitation to overcome inhibition. PG cells may be particularly susceptible to being activated by weak stimuli due to their intrinsically small size and high input resistance. Here, we used dual-cell patch-clamp recordings and imaging methods in bulb slices obtained from wild-type and transgenic rats with labeled GABAergic cells to test a number of predictions of the high-pass filtering model. We first directly compared the responsiveness of PG cells with that of external tufted cells (eTCs), which are a class of excitatory interneurons that reside in a parallel but opposing position in the glomerular circuitry. PG cells were in fact found to be no more responsive than eTCs at low levels of sensory neuron activity. While PG cells required smaller currents to be excited, this advantage was offset by the fact that a given level of sensory neuron activity produced much smaller synaptic currents. We did however identify other factors that shaped the excitation/inhibition balance in a manner that would support a high-pass filter, including glial glutamate transporters and presynaptic metabotropic glutamate receptors. We conclude that a single glomerulus may act as a high-pass filter to enhance the contrast between different olfactory stimuli through mechanisms that are largely independent cell intrinsic properties.

Key Points SummaryO_LIGABAergic periglomerular (PG) cells in the olfactory bulb are proposed to mediate a "high-pass" filter at a single glomerulus that selectively blocks weak stimulus signals. Their efficacy may reflect their intrinsically small size and high input resistance, which allows them to be easily excited.
C_LIO_LIWe found that PG cells were in fact no more likely to be activated by weak stimuli than excitatory neurons. PG cells spiked more readily in response to a fixed current input, but this advantage for excitability was offset by small synaptic currents.
C_LIO_LIGlomeruli nevertheless display an excitation/inhibition balance that can support a high pass filter, becoming much more favorable with increasing stimulus strength. Factors shaping the filter include glial glutamate transporters and presynaptic metabotropic glutamate receptors.
C_LIO_LIWe conclude that a single glomerulus may act as a high-pass filter to enhance stimulus contrast through mechanisms that are largely independent of cell-intrinsic properties.
C_LI

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Characterization of a Novel Type Homoserine Dehydrogenase Only with High Oxidation Activity from Arthrobacter nicotinovorans Homoserine dehydrogenase (HSD) is a key enzyme in the synthesis pathway of the aspartate family of amino acids. HSD can catalyze the reversible reaction of L-aspartate-{beta}-semialdehyde (L-ASA) to L-homoserine (L-Hse). In direct contrast, growth characteristic studies of some bacterial such as Arthrobacter nicotinovorans showed that the bacterium could grow well in medium with L-homoserine as sole carbon, nitrogen and energy source, but the genes responsible for the degradation of L-Hse remain unknown. Based on the function and sequence analysis of HSD, one putative homoserine dehydrogenase from A.nicotinovorans was named AnHSD, which was different from those HSDs that from the aspartic acid metabolic pathway, might be responsible for the degradation of L-Hse. Surprisingly, the analysis showed that the purified AnHSD exhibited specific L-Hse oxidation activity without reducing activity. At pH 10.0 and 40 {degrees}C, The Km and Kcat of AnHSD was 6.30 {+/-} 1.03 mM and 462.71 s-1, respectively. AnHSD was partiality for NAD+ cofactor, as well as insensitive to feedback inhibition of downstream amino acids of aspartic acid family. The physiological role of AnHSD in A.nicotinovorans is discussed. These findings provide a novel insight for a better understanding of an alternative genetic pathway for L-Hse catabolism which was dominated by the novel HSD.

ImportanceL-homoserine is an important building block for the synthesis of L-threonine, L-methionine, L-lysine which from aspartic acid family amino acids. However, some bacteria can make use of L-homoserine as a sole carbon and nitrogen source. Although the microbial degradation of L-homoserine has been studied several times, the genes involved and the molecular mechanisms remain unclear. In this study, we show that AnHSD responsible for the catabolism of L-homoserine in strain Arthrobacter nicotinovorans, as a special homoserine dehydrogenase with high diversity exists in Arthrobacter, Microbacterium, Rhizobium. We report for the first time that this novel homoserine dehydrogenase is now proposed to play a crucial role in that L-homoserine can use as a sole carbon and nitrogen source. This study is aimed at elucidating the enzymatic properties and function features of homoserine dehydrogenase from Arthrobacter nicotinovorans. These findings provide new insight into the catabolism of L-homoserine in bacteria.

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Protease-activatable biosensors of SARS-CoV-2 infection for cell-based drug, neutralisation and virological assays Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Author summaryTechniques for measuring infection with SARS-CoV-2 in the laboratory are laborious and time-consuming, and different laboratories use different approaches. There is therefore no generally agreed way to quantitate neutralising antibodies against SARS-CoV-2, which block infection with the virus and protect people from COVID-19. In this study, we describe a new way to measure SARS-CoV-2 infection, which is much simpler and faster than existing methods. It relies on the production of a specific protease enzyme by the virus, which is able to cleave and activate an engineered protein biosensor in infected cells. This biosensor emits light in the presence of viral infection, and the amount of light released is used as a readout for the amount of infectious SARS-CoV-2 present. The signal is very sensitive, so the number of infected cells required is very small, and the method can be scaled-up to test many samples at once. In particular, we demonstrate how it can be used to detect different variants of SARS-CoV-2, and quantitate neutralising antibodies against these viruses.

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Sero-Prevalence and Risk Factor of  Peste Des Petits Ruminants in Sheep and Goats of Ethiopia: Systematic Review and Meta- Analysis BackgroundPeste des petits ruminant (PPR) is the most common prevalent viral disease of sheep and goats that impacts productivity and international animal trade in the world and also in Ethiopia. Despite the huge economic consequences related to PPR, little is known about the sero-prevalence of this disease at the country levels. The objective of this systematic review and meta-analysis was to estimate a single-group summary for sero-prevalence of PPR disease in small ruminants of Ethiopia and assess the potential risk factor to contribute the sero-prevalence estimate.

MethodologyArticle on PPR in sheep and goats were searched in PubMed, Web of Science, Google Scholar, reference lists and African online source of articles that had been conducted between 1994 to 2020 and using inclusion and exclusion criteria with restricted to those studies published in English language.

ResultsA total of 13 published papers containing 46 district level studies were included for analyses. The single-group summary of PPR disease sero-prevalence in small ruminant was estimated to be 27.71% (95 % CI: 21.46 - 33.96). Overall, the estimated pooled sero-prevalence at country level in sheep was 33.56% (95% CI: 18.72-48.41) and in goats 25.14% (95% CI: 15.68-34.59). Significant heterogeneity (I2 > 80%) was noted in all pooled estimates. The visual inspection of the funnel plot demonstrated the presence of possible publication bias which could be associated with the small number of studies and longtime interval.

ConclusionsThis quantitative review showed that the pooled sero-prevalence to be high and regional prevalence estimates of PPR presented here will be useful in raising awareness and advocating the Governments to engage in initiatives PPR control and prevention.

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Coding Recognition of the Dose-Effect Interdependence of Small Biomolecules Encrypted on Paired Chromatographic-Based Microassay Arrays The discovery of small biomolecules suffers from the lack of a comprehensive framework to express the intrinsic correlation between bioactivity and contributing small molecules in complex samples with molecular and bioactivity diversity. Here, by mapping a samples 2D-HPTLC fingerprint to microplates, the paired chromatographic-based microassay arrays are created, which can be used as quasi-chip to characterize multiple attributes of chromatographic components; and as the array differential expression of the bioactivity and molecular attributes of irregular chromatographic spots for dose-effect interdependent encoding; and also as the automatic-collimated array mosaics of the multi-attributes of each component itself encrypted by its chromatographic fingerprint. Based on this homologous framework, we propose a correlating recognition strategy for small-biomolecules through their self-consistent chromatographic behavior characteristics. In the approach, the small-biomolecule recognition in diverse compounds is transformed into a constraint satisfaction problem, which is addressed through examining the dose-effect interdependence of the homologous 2D code pairs by array matching algorithm, instead of preparing diverse compound monomers of complex test sample for identifying item-by-item. Furtherly, considering the dose-effect interdependent 2D code pairs as links and the digital-specific quasimolecular ions as nodes, an extendable self-consistent framework that correlates mammalian cell phenotypic and target-based bioassays with small biomolecules is established. Therefore, the small molecule contributions and the correlations of bioactivities, as well as their pathway can be comprehensively revealed, so as to improve the reliability and efficiency of screening. This strategy was successfully applied to galangal, and practiced the high-throughput digital preliminary screening of small-biomolecules in a natural product.

HighlightRapid self-recognition of small biomolecules in diverse samples without pre-isolation Recognized by dose-effect interdependency developed from homologous TLC fingerprint Matching of HPTLC-based molecular imprinting and bioautography on microassay arrays Microarray-based differential expression of substance attributes instead of spot scan An array framework for combining phenotype-based and target-based assays with TLC-MS

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sentences: Multiple mechanisms of photoreceptor spectral tuning following loss of UV color vision in Heliconius butterflies Color vision modifications occur in animals via a process known as spectral tuning. In Heliconius butterflies, a genus-specific UVRh opsin duplication led to the evolution of UV color discrimination in Heliconius erato females, a rare trait among butterflies. In the H. melpomene and H. ismenius lineages, the UV2 receptor has been lost. Here we compare how loss of the UV2 photoreceptor has altered the visual system of these butterflies. We compare visual system evolution in three Heliconius butterfly species using a combination of intracellular recordings, ATAC-seq, and antibody staining. We identify several spectral tuning mechanisms including adaptive evolution of opsins, deployment of two types of filtering pigments, and co-expression of two distinct opsins in the same cell. Our data show that opsin gain and loss is driving rapid divergence in Heliconius visual systems via tuning of multiple spectral classes of photoreceptor in distinct lineages, potentially contributing to ongoing speciation in this genus.

sentences: Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe The number, distribution and composition of nuclear pore complexes (NPCs) in the nuclear envelope (NE) varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization is controlled, we analyzed NPC number and distribution in the fission yeast Schizosaccharomyces pombe using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions. Our data revealed that NPC density is maintained across a wide range of nuclear sizes. Regions of reduced NPC density are observed over the nucleolus and surrounding the spindle pole body (SPB). Lem2-mediated tethering of the centromeres to the SPB is required to maintain NPC exclusion, which is important for timely mitotic progression. These findings provide a quantitative understanding of NPC number and distribution in S. pombe and show that interactions between the centromere and the NE influences local NPC distribution.

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A base-resolution panorama of the in vivo impact of cytosine methylation on transcription factor binding While methylation of CpG dinucleotides is traditionally considered antagonistic to the DNA-binding activity of most transcription factors (TFs), recent in vitro studies have revealed a more complex picture, suggesting that over a third of TFs may preferentially bind to methylated sequences. Expanding these in vitro observations to in vivo TF binding preferences, however, is challenging, as the effect of methylation of individual CpG sites cannot be easily isolated from the confounding effects of DNA accessibility and regional DNA methylation. As a result, the in vivo methylation preferences of most TFs remain uncharacterized.

Here, we introduce joint accessibility-methylation-sequence (JAMS) models, which connect the strength of the binding signal observed in ChIP-seq to the DNA accessibility of the binding site, regional methylation level, DNA sequence, and base-resolution cytosine methylation. We show that JAMS models quantitatively explain the TF binding strength, recapitulate cell type-specific TF binding, and have high precision for inferring intra-motif methylation effects. Analysis of 2209 ChIP-seq experiments resulted in high-confidence JAMS models for 260 TFs, revealing that 45% of TFs are inhibited by intra-motif methylation in vivo. In contrast, only 16 TFs (6%) preferentially bind to methylated sites, including 11 novel methyl-binding TFs that are mostly from the multi-zinc finger family of TFs.

Our study substantially expands the repertoire of in vivo methyl-binding TFs, but also suggests that most TFs that prefer methylated CpGs in vitro present themselves as methylation agnostic in vivo, potentially due to the balancing effect of competition with other methyl-binding proteins.

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Spectral clustering of single-cell multi-omics data on multilayer graphs Single-cell sequencing technologies that simultaneously generate multimodal cellular profiles present opportunities for improved understanding of cell heterogeneity in tissues. How the multimodal information can be integrated to obtain a common cell type identification, however, poses a computational challenge. Multilayer graphs provide a natural representation of multi-omic single-cell sequencing datasets, and finding cell clusters may be understood as a multilayer graph partition problem.

We introduce two spectral algorithms on multilayer graphs, spectral clustering on multilayer graphs (SCML) and the weighted locally linear (WLL) method, to cluster cells in multi-omic single-cell sequencing datasets. We connect these algorithms through a unifying mathematical framework that represents each layer using a Hamiltonian operator and a mixture of its eigenstates to integrate the multiple graph layers, demonstrating in the process that the WLL method is a rigorous multilayer spectral graph theoretic reformulation of the popular Seurat weighted nearest neighbor (WNN) algorithm. Implementing our algorithms and applying them to a CITE-seq dataset of cord blood mononuclear cells yields results similar to the Seurat WNN analysis. Our work thus extends spectral methods to multimodal single-cell data analysis.

The code used in this study can be found at https://github.com/jssong-lab/sc-spectrum

sentences: DetecDiv, a generalist deep-learning platform for automated cell division tracking and survival analysis Automating the extraction of meaningful temporal information from sequences of microscopy images represents a major challenge to characterize dynamical biological processes. So far, strong limitations in the ability to quantitatively analyze single-cell trajectories have prevented large-scale investigations to assess the dynamics of entry into replicative senescence in yeast. Here, we have developed DetecDiv, a microfluidic-based image acquisition platform combined with deep learning-based software for high-throughput single-cell division tracking. We show that DetecDiv can automatically reconstruct cellular replicative lifespans with high accuracy and performs similarly with various imaging platforms and geometries of microfluidic traps. In addition, this methodology provides comprehensive temporal cellular metrics using time-series classification and image semantic segmentation. Last, we show that this method can be further applied to automatically quantify the dynamics of cellular adaptation and the real-time cell survival upon exposure to environmental stress. Hence, this methodology provides an all-in-one toolbox for high-throughput phenotyping for cell cycle, stress response, and replicative lifespan assays.

sentences: Predicting Brain Regions Related to Alzheimer's Disease Based on Global Feature Alzheimers disease (AD) is a common neurodegenerative disease in the elderly, early diagnosis and timely treatment are very important to delay the course of the disease. In the past, most of the brain regions related to AD were identified based on the imaging method, which can only identify some atrophic brain regions. In this work, we used mathematical models to find out the potential brain regions related to AD. First, diffusion tensor imaging (DTI) was used to construct the brain structural network. Next, we set a new local feature index 2hop-connectivity to measure the correlation among different areas. The 2hop-connectivity utilizes the higher-order information of the graph structure relative to the traditional graph theory metrics. And for this, we proposed a novel algorithm named 2hopRWR to measure 2hop-connectivity. At last, we proposed a new index GFS (Global Feature Score) based on a global feature by combing 5 local features: degree centrality, betweenness centrality, closeness centrality, the number of maximal cliques, and 2hop-connectivity, to judge which brain regions are likely related to Alzheimers Disease. As a result, all the top ten brain regions in the GFS scoring difference between the AD group and the non-AD group were related to AD by literature verification. Literature validation results comparing GFS with local features showed that GFS outperforms individual local features. Finally, the results of the canonical correlation analysis showed that the GFS was significantly correlated with the scores of the mini-mental state examination (MMSE) scale and the Montreal cognitive assessment (MoCA) scale. So, we believe the GFS can also be used as a new index to assist in diagnosis and objective monitoring of disease progression. Besides, the method proposed in this paper can be used as a differential network analysis method in other areas of network analysis.

sentences: REPINs are facultative genomic symbionts of bacterial genomes Mobile genetic elements (MGEs), such as transposons and insertion sequences, propagate within bacterial genomes, but persistence times in individual lineages are short. For long-term survival, MGEs must continuously invade new hosts by horizontal transfer. Theoretically, MGEs that persist for millions of years in single lineages, and are thus subject to vertical inheritance, should not exist. Here we make the case for an exception -- a class of MGE termed REPIN. REPINs are non-autonomous MGEs whose duplication depends on non-jumping RAYT transposases. Comparisons of REPINs and typical MGEs show that replication rates of REPINs are orders of magnitude lower, REPIN population size fluctuations correlate with changes in available genome space, REPIN conservation depends on RAYT function, and REPIN diversity accumulates within host lineages. From these data it follows that REPINs persist for millions of years within single host lineages. Such long-term persistence is expected to generate conflicts arising from the diverging effects of selection acting simultaneously on REPINs and host genomes. Evidence of conflict comes from analyses of REPIN abundance and diversity in two distantly related bacterial species. Taken together, our data lead to the conclusion that REPINs are ancient Darwinian replicators that have evolved enduring, beneficial relationships, with eubacterial genomes.

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CD47-SIRPα controls ADCC killing of primary T cells by PMN through a combination of trogocytosis and NADPH oxidase activation. Immunotherapies targeting the "dont eat me" myeloid checkpoint constituted by CD47 SIRP interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti-CD47-SIRP therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies, and the strong ADCC effector function of Poly Morpho Nuclear Cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb-dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRP checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRP blockade and ROS induction. Although previous studies had concluded that disruption of SIRP signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRP also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRP immune checkpoint and to design agents able to enhance myeloid cells function while limiting adverse effects to healthy cells able to participate in the anti-tumor immune response.

SynopsisDose-limiting toxicities demonstrate the need to investigate anti-CD47-SIRP therapy effects on non-tumor CD47-bearing cells. We demonstrate that anti-CD47 mAbs stimulate potent killing of T cells by PMN through a novel combination of trogocytosis and ROS regulated by SIRP.

Graphical Abstract

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sentences: Uncovering the Organization of Neural Circuits with Generalized Phase Locking Analysis Despite the considerable progress of in vivo neural recording techniques, inferring the biophysical mechanisms underlying large scale coordination of brain activity from neural data remains challenging. One obstacle is the difficulty to link functional connectivity measures to mechanistic models of network activity. We address this question for the case of spike-field coupling, which quantifies the synchronization between neurophysiological activities observed at two different scales: on the one hand, the action potential produced by neurons, on the other hand a mesoscopic "field" signal, reflecting subthreshold activities. We develop Generalized Phase Locking Analysis (GPLA) as a multichannel extension of univariate spike-field coupling that can be interpreted based on neural field models of mesoscopic network activity. GPLA estimates the dominant spatio-temporal distributions of field activity and neural ensembles, and the strength of the coupling between them. We demonstrate the statistical benefits and interpretability of this approach in various biophysical neuronal network models and Utah array recordings. In particular, GPLA, uncovers the contribution of recurrent microcircuits to the spatio-temporal dynamics observed in multi-channel recordings.

sentences: Inflammation modulates regeneration in the acute or chronically damaged zebrafish retina Unlike mammals, zebrafish regenerate in response to retinal damage. Because microglia are activated by retinal damage, we investigated their role during regeneration following acute or chronic damage. At three weeks-post-fertilization (wpf), fish exhibiting NMDA-induced acute damage or cone photoreceptor-specific chronic degeneration, the gold rush (gosh) mutant, displayed reactive microglia and Muller glia proliferation. Retinas treated to inhibit the immune response lacked reactive microglia and possessed fewer PCNA-positive cells, while LPS treatment increased microglia and PCNA-labeled cells. NMDA-injured retinas upregulated the expression of il-1{beta} and tnf- pro-inflammatory cytokine genes, followed by increased expression of il-10 and arg1 anti-inflammatory/remodeling cytokine genes. An early and transiently TNF- pro-inflammatory microglia population was identified in the NMDA-damaged retina. In contrast, gosh mutant retinas exhibited a mild increase of pro-inflammatory cytokine gene expression concurrently with a greater increased in anti-inflammatory/remodeling cytokine gene expression. Few TNF- pro-inflammatory microglia were observed in the gosh retina. How inflammation regulates regeneration in zebrafish would provide important clues towards improving the therapeutic strategies for repairing injured mammalian tissues.

sentences: Structural variant selection for high-altitude adaptation using single-molecule long-read sequencing Structural variants (SVs) can be important drivers of human adaptation with strong effects, but previous studies have focused primarily on common variants with weak effects. Here, we used large-scale single-molecule long-read sequencing of 320 Tibetan and Han samples, to show that SVs are key drivers of selection under high-altitude adaptation. We expand the landscape of global SVs, apply robust models of selection and population differentiation combining SVs, SNPs and InDels, and use epigenomic analyses to predict driver enhancers, target genes, upstream regulators, and biological functions, which we validate using enhancer reporter and DNA pull-down assays. We reveal diverse Tibetan-specific SVs affecting the cis- and trans-regulatory circuitry of diverse biological functions, including hypoxia response, energy metabolism, lung function, etc. Our study greatly expands the global SV landscape, reveals the central role of gene-regulatory circuitry rewiring in human adaptation, and illustrates the diverse functional roles that SVs can play in human biology.

sentences: Pathology and immunity after SARS-CoV-2 infection in male ferrets is affected by age and inoculation route Improving COVID-19 intervention strategies partly relies on animal models to study SARS-CoV-2 disease and immunity. In our pursuit to establish a model for severe COVID-19, we inoculated young and adult male ferrets intranasally or intratracheally with SARS-CoV-2. Intranasal inoculation established an infection in all ferrets, with viral dissemination into the brain and gut. Upon intratracheal inoculation only adult ferrets became infected. However, neither inoculation route induced observable COVID-19 symptoms. Despite this, a persistent inflammation in the nose was prominent in especially young ferrets and follicular hyperplasia in the bronchi developed 21 days post infection. These effects -if sustained- might resemble long-COVID. Respiratory and systemic cellular responses and antibody responses were induced only in animals with an established infection. We conclude that intranasally-infected ferrets resemble asymptomatic COVID-19 and possibly aspects of long-COVID. Combined with the increasing portfolio to measure adaptive immunity, ferrets are a relevant model for SARS-CoV-2 vaccine research.

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Chronic caffeine treatment disrupts the circadian rhythm in Drosophila The circadian clock governs the timing of sleep-wake cycles as well as of other behavioural, physiological and metabolic processes. While the endogenous circadian clock mediates the timing of sleep, homeostatic mechanisms modulate the amount and depth of sleep. Evidence from previous studies showed that caffeine intake promotes wakefulness, whereas adult-stage specific caffeine treatment not only suppresses sleep but also delays the phase of circadian rhythm in Drosophila. In humans, caffeine is consumed on a daily basis and hence it is important to understand the effect of prolonged caffeine intake on circadian and homeostatic regulation of sleep. In the present study we examined the differential effect of acute and chronic caffeine treatment on sleep ontogeny as well as on circadian and homeostatic regulation of sleep in Drosophila. The results of our study showed that acute caffeine treatment reduces day and night sleep in mature flies through the homeostatic pathway whereas it reduced only the day sleep in young flies. Chronic caffeine treatment did not exert any significant effect on sleep in young flies. On the other hand, it delayed the timing of sleep in mature flies and in addition flies under higher caffeine concentration reduced the morning and evening anticipatory activity under 12 hour: 12 hour light: dark cycles. These flies also exhibited either a longer free running period or arrhythmicity under constant darkness. The results of our study showed that acute caffeine treatment suppresses sleep through the homeostatic pathway whereas prolonged caffeine treatment disrupts the circadian rhythm in mature flies.

Statement of significanceIn the present study, we assessed the effect of caffeine on sleep ontogeny and circadian rhythm by using Drosophila as a model organism. We found that acute treatment of caffeine reduced sleep in both young and mature flies. Young flies were tolerant to chronic caffeine treatment whereas it affected the timing of sleep in mature flies. As in mammals, caffeine slows down the circadian clock in flies. Most importantly, prolonged caffeine treatment disrupted the circadian rhythm in Drosophila. Our studies provide new insights into the effect of prolonged daily caffeine intake on circadian clock and sleep.

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Transcriptional mapping of the macaque retina and RPE-choroid reveals conserved inter-tissue transcription drivers and signaling pathways PurposeThe macula and fovea comprise a highly sensitive visual detection tissue that is susceptible to common disease processes like age-related macular degeneration (AMD). Our understanding of the molecular determinants of high acuity vision remains unclear, as few model organisms possess a human-like fovea. We explore transcription factor networks and receptor-ligand interactions to elucidate tissue interactions in the macula and peripheral retina and concomitant changes in the underlying retinal pigment epithelium (RPE)/choroid.

MethodsPoly-A selected, 100 bp paired-end RNA-sequencing (RNA-seq) was performed across the macular/foveal, perimacular, and temporal peripheral regions of the neural retina and RPE/choroid tissues of four adult Rhesus macaque eyes to characterize region- and tissue-specific gene expression. RNA-seq reads were mapped to both the macaque and human genomes for maximum alignment and analyzed for differential expression and Gene Ontology (GO) enrichment.

ResultsComparison of the neural retina and RPE/choroid tissues indicated distinct, contiguously changing gene expression profiles from fovea through perimacula to periphery. Top GO enrichment of differentially expressed genes in the RPE/choroid included cell junction organization and epithelial cell development. Expression of transcriptional regulators and various disease-associated genes show distinct location-specific preference and retina-RPE/choroid tissue-tissue interactions.

ConclusionsRegional gene expression changes in the macaque retina and RPE/choroid is greater than that found in previously published transcriptome analysis of the human retina and RPE/choroid. Further, conservation of human macula-specific transcription factor profiles and gene expression in macaque tissues suggest a conservation of programs required for retina and RPE/choroid function and disease susceptibility.

sentences: In vivo selection of nanobodies to target the tumor immune microenvironment for immunotherapy development Cancer immunotherapy has changed the landscape of treatment for some cancers, but variable responses reflect limitations imposed by the tumor microenvironment (TME)1-3. To facilitate the development of novel immunotherapies, we used an unbiased in vivo selection approach using a VHH nanobody library to identify immunologic targets in the TME. Using next generation sequencing (NGS), we demonstrate the parallel enrichment of immune cell-binding nanobodies that target immune cell subsets within the TME. We selected from enriched nanobodies - Cl.1 and Cl.2 - that bind to the CD11b+ and CD11c+ populations and we identified the desmosome complex as the target for Cl.1, suggesting the nanobodies can target key immune populations and may involve unvalidated targets. To demonstrate nanobodies could functionally alter target immune cells in vivo, we showed by single cell RNA sequencing (scRNAseq) that enriched nanobodies can increase activation transcripts in CD8+ T cells and dendritic cells. This approach is being used to develop novel cancer immunotherapies, but the methods can be broadly applicable to biologic discovery experiments in various physiologic or pathologic contexts.

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Early structural connectivity within the sensorimotor network: deviations related to prematurity and association to neurodevelopmental outcome The sensorimotor (SM) network is crucial for optimal neurodevelopment. However, undergoing rapid maturation during the perinatal period, it is particularly vulnerable to preterm birth.

Our work explores the prematurity impact on the microstructure and maturation of primary SM white matter (WM) tracts at term-equivalent age (TEA) and evaluates the relationships between these alterations and neurodevelopmental outcome.

We analyzed diffusion MRI data from the developing Human Connectome Project (dHCP) database: 59 preterm (PT) low-risk infants scanned near TEA, compared to a control group of full-term (FT) neonates paired for age at MRI and sex. We dissected pairwise connections between primary SM cortices and subcortical structures using probabilistic tractography and evaluated their microstructure with diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) models. In addition to tract- specific univariate analyses of diffusion metrics, we computed a maturational distance related to prematurity based on a multi-parametric Mahalanobis distance of each PT infant relative to the FT group. Finally, we evaluated the relationships between this distance and Bayley Scales of Infant and Toddler Development (BSID-III) scaled scores at 18 months corrected age.

Our results confirm important microstructural differences in SM tracts between PT and FT infants, with effects increasing with lower gestational age at birth. Additionally, comparisons of maturational distances highlight that prematurity has a differential effect on SM tracts which follows the established WM caudo-rostral developmental pattern. Our results suggest a particular vulnerability of projections involving the primary sensorimotor cortices (S1) and of the most rostral tracts, with cortico-cortical and S1-Lenticular tracts presenting the highest alterations at TEA. Finally, NODDI-derived maturational distances of specific tracts seem related to fine motor and cognitive scores.

This study expands the understanding of the impact of early WM alterations in the emerging SM network on long-term neurodevelopment. In the future, related approaches have potential to lead to the development of neuroimaging markers for neurodevelopmental disorders, with special interest for subtle neuromotor impairments frequently observed in preterm-born children.

sentences: Easy to interpret Coordinate Based Meta-Analysis of neuroimaging studies: Analysis of Brain Coordinates (ABC) Functional MRI and voxel-based morphometry (VBM) are important approaches to testing hypotheses in neuroscience, helping us to understand neurological disease, and brain function and development. However, they are technically challenging with no one optimal generalisable method, and the multiple popular techniques have been shown to produce different results. Furthermore, results may be sensitive to settings, such as smoothing or statistical thresholding, that can be difficult to optimise per hypothesis. It is useful, therefore, to be able to meta-analyse published results from such studies that tested a similar hypothesis potentially using different analysis methods, scanners, and protocols as well as different subjects. Coordinate based meta-analysis (CBMA) offers this using only commonly reported summary results. It is the aim of CBMA to find those results that indicate replicable effects across studies. However, just like the multiple analysis methods offered for neuroimaging, there are now multiple CBMA algorithms each with specific features and empirical parameters/assumptions. Results derived from CBMA are inevitably conditional on the algorithm used, so conclusions are clearer when the analysis approach is easy to understand. With this in mind a new CBMA method (Analysis of Brain Coordinates; ABC) is presented, with the aim of being easy to interpret by eliminating empirical assumptions where possible and by relating statistical thresholding directly to replication of effect.

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The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties Pectin methylesterases (PMEs) modify homogalacturonans (HG) chemistry and play a key role in regulating primary cell wall mechanical properties. How PME activity can fine-tune pectin structure in the growing plant has remained elusive. Here we report on the Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum suitable for detecting recombinant and native enzyme independent of species source. At neutral pH AtPME2 is preferentially active on pectins with a degree of 55-70% methylesterification and can be inhibited by PME inhibitor protein (PMEI). We show that the mode of action for AtPME2 can switch from full processivity (at pH 8), creating large blocks of unmethylated galacturonic acid, to low processivity (at pH 5) and relate these observations to the differences in electrostatic potential of the protein at acidic and alkaline pH. To assess the role of AtPME2 in development, we characterized two knock-out lines. We show that in the context of acidified apoplast, low-processive demethylesterification by AtPME2 can loosen the cell wall, with consequent increase in cell elongation and etiolated hypocotyl length. Our study brings insights into how the pH-dependent regulation by PME activity could affect pectin structure and associated cell wall mechanical properties in expansion.

One sentence summaryThe processivity of AtPME2, a pectin methylesterase that fine-tunes cell wall pectins is modulated by pH in vitro and impacts the mechanical properties of the wall, affecting development in planta.

sentences: Profiling sensory neuron microenvironment after peripheral and central axon injury reveals key pathways for neural repair Sensory neurons with cell bodies in dorsal root ganglia (DRG) represent a useful model to study axon regeneration. Whereas regeneration and functional recovery occurs after peripheral nerve injury, spinal cord injury or dorsal root injury is not followed by regenerative outcomes. Regeneration of sensory axons in peripheral nerves is not entirely cell autonomous. Whether the DRG microenvironment influences the different regenerative capacities after injury to peripheral or central axons remains largely unknown. To answer this question, we performed a single cell transcriptional profiling of mouse DRG in response to peripheral (sciatic nerve crush) and central axon injuries (dorsal root crush and spinal cord injury). Each cell type responded differently to the three types of injuries. All injuries increased the proportion of a cell type that shares features of both immune cells and glial cells. A distinct subset of satellite glial cells (SGC) appeared specifically in response to peripheral nerve injury. Activation of the PPAR signaling pathway in SGC, which promotes axon regeneration after peripheral nerve injury, failed to occur after central axon injuries. Treatment with the FDA-approved PPAR agonist fenofibrate increased axon regeneration after dorsal root injury. This study provides a map of the distinct DRG microenvironment responses to peripheral and central injuries at the single cell level and highlights that manipulating non-neuronal cells could lead to avenues to promote functional recovery after CNS injuries or disease.

sentences: Deep learning based feature extraction for prediction and interpretation of sharp- wave ripples Local field potential (LFP) deflections and oscillations define hippocampal sharp-wave ripples (SWR), one of the most synchronous events of the brain. SWR reflect firing and synaptic current sequences emerging from cognitively relevant neuronal ensembles. Current spectral methods fail to capture their mechanistic complexity, thus limiting progress. Here, we show how one-dimensional convolutional networks operating over high-density LFP hippocampal recordings allowed for automatic identification of SWR. When applied to ultra-dense hippocampus-wide recordings, we discovered physiologically relevant processes associated to the emergence of SWR, prompting for novel classification criteria. To gain interpretability, we developed a method to interrogate the operation of the artificial network. We found it relied in feature-based specialization, which permit identification of spatially segregated oscillations and deflections, as well as synchronous population firing. Thus, using deep learning based approaches may change the current heuristic for a better mechanistic interpretation of these relevant neurophysiological events.

sentences: Gene regulatory networks controlling temporal patterning, neurogenesis and cell fate specification in the mammalian retina. Gene regulatory networks (GRNs), consisting of transcription factors and their target cis- regulatory sequences, control neurogenesis and cell fate specification in the developing central nervous system, but their organization is poorly characterized. In this study, we performed integrated single-cell RNA- and scATAC-seq analysis in both mouse and human retina to profile dynamic changes in gene expression, chromatin accessibility and transcription factor footprinting during retinal neurogenesis. We identified multiple interconnected, evolutionarily-conserved GRNs consisting of cell type-specific transcription factors that both activate expression of genes within their own network and often inhibit expression of genes in other networks. These GRNs control state transitions within primary retinal progenitors that underlie temporal patterning, regulate the transition from primary to neurogenic progenitors, and drive specification of each major retinal cell type. We confirmed the prediction of this analysis that the NFI transcription factors Nfia, Nfib, and Nfix selectively activate expression of genes that promote late-stage temporal identity in primary retinal progenitors. We also used GRNs to identify additional transcription factors that promote (Insm1/2) and inhibit (Tbx3, Tcf7l1/2) rod photoreceptor specification in postnatal retina. This study provides an inventory of cis- and trans-acting factors that control retinal development, identifies transcription factors that control the temporal identity of retinal progenitors and cell fate specification, and will potentially guide cell-based therapies aimed at replacing retinal neurons lost due to disease.

sentences: The effects of antibiotic combination treatments on Pseudomonas aeruginosa tolerance evolution and coexistence with Stenotrophomonas maltophilia Pseudomonas aeruginosa bacterium is a common pathogen of Cystic Fibrosis (CF) patients due to its ability to evolve resistance to antibiotics during treatments. While P. aeruginosa resistance evolution is well characterised in monocultures, it is less well understood in polymicrobial CF infections. Here, we investigated how exposure to ciprofloxacin, colistin, or tobramycin antibiotics, administered at sub-MIC doses alone and in combination, shaped the tolerance evolution of P. aeruginosa (PAO1 lab and clinical CF LESB58 strains) in the absence and presence of a commonly co-occurring species, Stenotrophomonas maltophilia. Increases in antibiotic tolerances were primarily driven by the presence of that antibiotic in the treatment. We observed a reciprocal cross-tolerance between ciprofloxacin and tobramycin, and when combined these antibiotics selected increased MICs for all antibiotics. Though the presence of S. maltophilia did not affect the tolerance or the MIC evolution, it drove P. aeruginosa into extinction more frequently in the presence of tobramycin due to its relatively greater innate tobramycin tolerance. In contrast, P. aeruginosa dominated and drove S. maltophilia extinct in most other treatments. Together, our findings suggest that besides driving high-level antibiotic tolerance evolution, sub-MIC antibiotic exposure can alter competitive bacterial interactions, leading to target pathogen extinctions in multi-species communities.

sentences: Uncertainty in cell fate decision making: Lessons from potential landscapes of bifurcation systems Cell fate decision making is known to be a complex process and is still far from being understood. The intrinsic complexity, but also features such as molecular noise represent challenges for modelling these systems. Waddingtons epigenetic landscape has become the overriding metaphor for developmental processes: it both serves as pictorial representation, and can be related to mathematical models. In this work we investigate how the landscape is affected by noise in the underlying system. Specifically, we focus on those systems where minor changes in the parameters cause major changes in the stability properties of the system, especially bifurcations. We analyse and quantify the changes in the landscapes shape as the effects of noise increase. We find ample evidence for intricate interplay between noise and dynamics which can lead to qualitative change in a systems dynamics and hence the corresponding landscape. In particular, we find that the effects can be most pronounced in the vicinity of the bifurcation point of the underlying deterministic dynamical systems, which would correspond to the cell fate decision event in cellular differentiation processes.

sentences: Fluorinated oil-surfactant mixtures with the density of water: artificial cells for synthetic biology There is a rising interest in biotechnology for the compartmentalization of biochemical reactions in water droplets. Several applications, such as the widely used digital PCR, seek to encapsulate a single molecule in a droplet to be amplified. Directed evolution, another technology with growing popularity, seeks to replicate what happens in nature by encapsulating a single gene and the protein encoded by this gene, linking genotype with phenotype. Compartmentalizing reactions in droplets also allows the experimentalist to run millions of different reactions in parallel. Compartmentalization requires a fluid that is immiscible with water and a surfactant to stabilize the droplets. While there are fluids and surfactants on the market that have been used to accomplish encapsulation, there are reported concerns with these. Span(R) 80, for example, a commonly used surfactant, has contaminants that interfere with various biochemical reactions. Similarly, synthetic fluids distributed by the cosmetic industry allow some researchers to produce experimental results that can be published, but then other researchers fail to reproduce some of these protocols due to the unreliable nature of these products, which are not manufactured with the intent of being used in biotechnology. The most reliable fluids, immiscible with water and suitable for biochemical reactions, are fluorinated fluids. Fluorinated compounds have the peculiar characteristic of being immiscible with water while at the same time not mixing with hydrophobic molecules. This peculiar characteristic has made fluorinated fluids attractive because it seems to be the basis of their being biologically inert. However, commercially available fluorinated fluids have densities between 1.4 to 1.6 g/mL. The higher-than-water density of fluorinated oils complicates handling of the droplets since these would float on the fluid since the water droplets would be less dense. This can cause aggregation and coalescence of the droplets. Here, we report the synthesis, characterization, and use of fluorinated polysiloxane oils that have densities similar to the one of water at room temperature, and when mixed with non-ionic fluorinated surfactants, can produce droplets encapsulating biochemical reactions. We show how droplets in these emulsions can host many biological processes, including PCR, DNA origami, rolling circle amplification (RCA), and Taqman(R) assays. Some of these use unnatural DNA built from an Artificially Expanded Genetic Information System (AEGIS) with six nucleotide "letters".

sentences: Functional gradients in the human lateral prefrontal cortex revealed by a comprehensive coordinate-based meta-analysis The human lateral prefrontal cortex (LPFC) enables flexible goal-directed behavior. Yet, its organizing principles remain actively debated despite decades of research. Meta-analysis efforts to map the LPFC have either been restricted in scope or suffered from limited expressivity in meta-analysis tools. The latter short-coming hinders the complexity of questions that can be expressed in a meta-analysis and hence limits the specificity of structure-function associations. Here, we adopt NeuroLang, a novel approach to meta-analysis based on first-order probabilistic logic programming, to infer the organizing principles of the LPFC with greater specificity from 14,371 neuroimaging publications. Our results reveal a rostrocaudal and a dorsoventral gradient, respectively explaining the most and second-most variance in whole-brain meta-analytic connectivity in the LPFC. Moreover, we find a cross-study agreement on a spectrum of increasing abstraction from caudal to rostral LPFC both in specific network connectivity and structure-function associations that supports a domain-general role for the mid-LPFC. Furthermore, meta-analyzing inter-hemispheric asymmetries along the rostrocaudal gradient reveals specific associations with topics of language, memory, response inhibition, and error processing. Overall, we provide a comprehensive mapping of the organizing principles of task-dependent activity in the LPFC, grounding future hypothesis generation on a quantitative overview of past findings.

sentences: Dispersal of southern elephant seals (Mirounga leonina) from Davis Base, Antarctica: Combining population genetics and tracking data Marine animals such as the southern elephant seal (Mirounga leonina) rely on a productive marine environment and are vulnerable to oceanic changes that can affect their reproduction and survival rates. Davis Base, Antarctica, acts as a moulting site for southern elephant seals that forage in Prydz Bay, but the genetic diversity and natal source populations of these seals has not been characterized. Determining the genetic diversity of moulting populations like this one provides essential information on seal dispersal, inter-population mixing, and foraging behaviours. In this study, we combined genetic and animal tracking data on these moulting seals to identify levels of genetic diversity, natal source population, and movement behaviours during foraging and haul-out periods. Using mitochondrial sequence data, we identified two major breeding lineages of seals at Davis Base. We found that the majority of the seals originated from breeding stocks within the South Atlantic Ocean and South Indian Ocean. One seal was grouped with the Macquarie Island breeding stock (South Pacific Ocean). The Macquarie Island population, unlike the other two stocks, is decreasing in size. Tracking data revealed long-distance foraging activity of the Macquarie Island seal around Crozet Islands. We speculate that changes to the Antarctic marine environment have resulted in a shift in foraging and dispersal strategies, which subsequently affects seal population growth rates. These findings have implications for conservation management plans aimed at improving the population status of the southern elephant seal.

sentences: Kronos scRT: a uniform framework for single-cell replication timing analysis Mammalian genomes are replicated in a cell-type specific order and in coordination with transcription and chromatin organization. Although the field of replication is also entering the single-cell era, current studies require cell sorting, individual cell processing and have yielded a limited number (<100) of cells. Here, we have developed Kronos scRT (https://github.com/CL-CHEN-Lab/Kronos_scRT), a software for single-cell Replication Timing (scRT) analysis. Kronos scRT does not require a specific platform nor cell sorting, allowing the investigation of large datasets obtained from asynchronous cells. Analysis of published available data and droplet-based scWGS data generated in the current study, allows exploitation of scRT data from thousands of cells for different mouse and human cell lines. Our results demonstrate that, although most cells replicate within a close timing range for a given genomic region, replication can also occur stochastically throughout S phase. Altogether, Kronos scRT allows investigating the RT program at a single-cell resolution for both homogeneous and heterogeneous cell populations in a fast and comprehensive manner.

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Detection of an unstable minor QTL linked to fire blight resistance on linkage group 16 of Malus fusca ObjectiveErwinia amylovora causes fire blight disease in Malus. A strong resistance QTL (Mfu10) was previously detected on linkage group 10 of Malus fusca accession MAL0045, using several strains of the bacterium. As no strain capable of breaking the resistance of MAL0045 has been found, it was hypothesized that a second resistance factor contributes to the fire blight resistance of MAL0045. However, to date, no minor locus has been detected with previously published strains of the bacterium. We detected a minor QTL only on a subset of a population following inoculation with strain Ea1038, which heterologously expresses an effector in a derivative of isolate Ea3049. Two genetic maps of MAL0045, one scarce, the other dense with markers, were used for QTL analyses.

ResultsMfu10 was detected on LG10 with Ea1038, as was previously with Ea3049. Although no other QTLs of significant LOD was previously detected in other linkage groups with Ea3049, a QTL of significant LOD was detected on LG16 (Mfu16) after inoculation of a subset of 76 individuals with Ea1038, but only using the dense genetic map. Mfu16 improved the effect of Mfu10. However, when the number of individuals inoculated with Ea1038 was increased to 121, Mfu16 was no longer detected in the dense genetic map. We hypothesize some factors, which might be responsible for the instability of this QTL.

sentences: Exogenous estradiol and oxytocin modulate sex differences in hippocampal reactivity and episodic memory Considerable evidence supports sex differences in episodic memory, which may translate to heightened vulnerability to stress- and trauma-related disorders in women. The hormones estradiol and oxytocin both affect episodic memory, but possible underlying hormonal interactions have not been systemically tested in humans. To this end, healthy women (n = 111) and men (n = 115) received estradiol gel (2 mg) or placebo before the administration of intranasal oxytocin (24 IU) or placebo in a randomized, placebo-controlled, parallel-group functional magnetic resonance imaging (fMRI) study. In the fMRI session, participants viewed positive, neutral, and negative scenes. A surprise recognition task was conducted three days later. Under placebo, women showed a significantly better recognition memory and increased hippocampal responses to subsequently remembered items independent of the emotional valence compared to men. The separate treatments with either hormone significantly diminished this mnemonic sex difference and reversed the hippocampal activation pattern. However, the combined treatments led to a memory performance comparable to that of the placebo group. Collectively, the results suggest that both hormones play a crucial role in modulating sex differences in episodic memory. Furthermore, possible antagonistic interactions between estradiol and oxytocin could explain previously observed opposing hormonal effects in women and men.

sentences: Profiling of transcribed cis-regulatory elements in single cells Profiling of cis-regulatory elements (CREs, mostly promoters and enhancers) in single cells allows the interrogation of the cell-type and cell-state-specific contexts of gene regulation and genetic predisposition to diseases. Here we demonstrate single-cell RNA-5'end-sequencing (sc-end5-seq) methods can detect transcribed CREs (tCREs), enabling simultaneous quantification of gene expression and enhancer activities in a single assay at no extra cost. We showed enhancer RNAs can be detected using sc-end5-seq methods with either random or oligo(dT) priming. To analyze tCREs in single cells, we developed SCAFE (Single Cell Analysis of Five-prime Ends) to identify genuine tCREs and analyze their activities (https://github.com/chung-lab/scafe). As compared to accessible CRE (aCRE, based on chromatin accessibility), tCREs are more accurate in predicting CRE interactions by co-activity, more sensitive in detecting shifts in alternative promoter usage and more enriched in diseases heritability. Our results highlight additional dimensions within sc-end5-seq data which can be used for interrogating gene regulation and disease heritability.

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Feature-based Quality Assessment of Middle Cerebral Artery Occlusion Using 18F-fluorodeoxyglucose Positron Emission Tomography BackgroundThe association between brain metabolic change and ischemic stroke has attracted a lot of attention in the research community. 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging is widely used to measure the metabolism. In experiments, ischemic stroke is usually induced through middle cerebral artery occlusion (MCAO), and quality assessment of this procedure is of vital importance. However, an assessment method based on FDG PET images is still lacking. Herein, we propose an image feature-based protocol to assess the quality of the procedure.

MethodsWe performed permanent MCAO to a total of 161 Sprague-Dawley rats. FDG micro-PET images were acquired both before and after the MCAO procedure. Triphenyl tetrazolium chloride (TTC) staining was also conducted to obtain ground truth of the infarct volume. After preprocessing of the PET images, a combination of 3D scale invariant feature transform (SIFT) and support vector machine (SVM) was applied to extract features and train a classifier that can assess the quality of the MCAO procedure.

Results106 rats and 212 images were used as training data to construct the classification model. The SVM classifier achieved over 98% accuracy in cross validation. 10 rats with TTC results showing infarction in the ipsilateral brain region served as validation data. Their images were tested by the classifier and all of them were categorized into the correct group. Finally, the remaining 45 rats from a separate experiment were treated as independent test data. The prediction accuracy for these 90 images reached the level of 91%. An online interface was constructed for users to upload their images and obtain the assessment results.

ConclusionThis feature-based protocol provides a convenient, accurate and reliable tool to assess the quality of the MCAO procedure in FDG PET study.

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dCas9 targeted chromatin and histone enrichmentfor mass spectrometry (Catchet-MS) identifies IKZF1as a novel drug-able target for HIV-1 latency reversal A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has not been possible. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to describe the protein composition of the latent and activated HIV-1 5LTR. Catchet-MS identified known and novel latent 5LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs that, by targeting IKZF1 for degradation, reverse HIV-1 latency in CD4+T-cells isolated from virally suppressed people living with HIV-1.

One Sentence SummarydCas9 targeted chromatin and histone enrichment for mass spectrometry (Catchet-MS) led to the identification of IKZF1-targeting thalidomide analogues as novel HIV-1 latency reversal agents

sentences: Empirical Transmit Field Bias Correction of T1w/T2w Myelin Maps T1-weighted divided by T2-weighted (T1w/T2w) myelin maps were initially developed for neuroanatomical analyses such as identifying cortical areas, but they are increasingly used in statistical comparisons across individuals and groups with other variables of interest. Existing T1w/T2w myelin maps contain radiofrequency transmit field (B1+) biases, which may be correlated with these variables of interest, leading to potentially spurious results. Here we propose two empirical methods for correcting these transmit field biases using either explicit measures of the transmit field or alternatively a 'pseudo-transmit' approach that is highly correlated with the transmit field at 3T. We find that the resulting corrected T1w/T2w myelin maps are both better neuroanatomical measures (e.g., for use in cross-species comparisons), and more appropriate for statistical comparisons of relative T1w/T2w differences across individuals and groups (e.g., sex, age, or body-mass-index) within a consistently acquired study at 3T. We recommend that investigators who use the T1w/T2w approach for mapping cortical myelin use these B1+ transmit field corrected myelin maps going forward.

sentences: The evolution of recombination in self-fertilizing organisms Cytological data from flowering plants suggest that the evolution of recombination rates is affected by the mating system of organisms, as higher chiasma frequencies are often observed in self-fertilizing species compared with their outcrossing relatives. Understanding the evolutionary cause of this effect is of particular interest, as it may shed light on the selective forces favoring recombination in natural populations. While previous models showed that inbreeding may have important effects on selection for recombination, existing analytical treatments are restricted to the case of loosely linked loci and weak selfing rates, and ignore the effect of genetic interference (Hill-Robertson effect), known to be an important component of selection for recombination in randomly mating populations. In this article, we derive general expressions quantifying the stochastic and deterministic components of selection acting on a mutation affecting the genetic map length of a whole chromosome along which deleterious mutations occur, valid for arbitrary selfing rates. The results show that selfing generally increases selection for recombination caused by interference among mutations as long as selection against deleterious alleles is sufficiently weak. While interference is often the main driver of selection for recombination under tight linkage or high selfing rates, deterministic effects can play a stronger role under intermediate selfing rates and high recombination, selecting against recombination in the absence of epistasis, but favoring recombination when epistasis is negative. Individual-based simulation results indicate that our analytical model often provides accurate predictions for the strength of selection on recombination under partial selfing.

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Compartment-driven imprinting of intestinal CD4 (regulatory) T cells in inflammatory bowel disease and homeostasis ObjectiveThe mucosal immune system is implicated in the etiology and progression of inflammatory bowel diseases. The lamina propria and epithelium of the gut mucosa constitute two separate compartments, containing distinct T cell populations. Human CD4 T cell programming and regulation of lamina propria and epithelium CD4 T cells, especially during inflammation, remains incompletely understood.

DesignWe performed imaging mass cytometry, flow cytometry, bulk and single-cell RNA-sequencing to profile ileal lamina propria and intraepithelial CD4 T cells (CD4CD8, regulatory T cells (Tregs), CD69- and CD69high Trm T cells) in controls and Crohns disease (CD) patients (paired non-inflamed and inflamed).

ResultsInflammation results in alterations of the CD4 T cell population with a pronounced increase in Tregs and migrating/infiltrating cells. On a transcriptional level, inflammation within the epithelium induced T cell activation, increased IFN{gamma} responses and effector Treg differentiation. Conversely, few transcriptional changes within the lamina propria were observed. Key regulators including the chromatin remodelers ARID4B and SATB1 were found to drive compartment-specific transcriptional programming of CD4 T(reg) cells.

ConclusionInflammation in CD patients primarily induces changes within the epithelium and not the lamina propria. Additionally, there is compartment-specific CD4 T cell imprinting, driven by shared regulators, upon translocation from the lamina propria to the epithelium. The main consequence of epithelial translocation, irrespective of inflammation, seems to be an overall dampening of broad (pro-inflammatory) responses and tight regulation of lifespan. These data suggest differential regulation of the lamina propria and epithelium, with a specific regulatory role in the inflamed epithelium.

sentences: The bacterial amyloids phenol soluble modulins from Staphylococcus aureus catalyze alpha-synuclein aggregation Aggregated -synuclein (-syn) is the main constituent of Lewy bodies, the main pathological hallmark of Parkinsons disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric -syn. Braaks seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role that the microbiome might play in the pathology of PD. Staphylococcus aureus is a bacterium carried by 30-70% of the general population. It has been shown to produce functional amyloids, called Phenol Soluble Modulins (PSMs). Here, we studied the kinetics of -syn aggregation under quiescent conditions in the presence or absence of four different PSM peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure -syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSM peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSM peptides displayed distinct effects on the kinetics of -syn aggregation. Kinetic analyses of the data suggest that while all four peptides catalyze -syn aggregation, the underlying mechanisms might differ with a model of nucleation and elongation fitting the -syn aggregation induced by PSM2 but not PSM1. The results of immunogold TEM imply that the aggregates are fibrillar and composed of -syn. Addition of the co-aggregated materials to HEK cells expressing the A53T -syn variant fused to GFP was found to catalyze -syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.

sentences: Genetic continuity of Indo-Iranian speakers since the Iron Age in southern Central Asia. Since prehistoric times, South Central Asia has been at the crossroads of the movement of people, culture, and goods. Today, the Central Asias populations are divided into two cultural and linguistic groups: the Indo-Iranian and the Turko-Mongolian groups. Previous genetic studies unveiled that migrations from East Asia contributed to the spread of Turko-Mongolian populations in Central Asia and the partial replacement of the Indo-Iranian population. However, little is known about the origin of the latter. To shed light on this, we compare the genetic data on two current-day populations- Yaghnobis and Tajiks - with genome-wide data from published ancient individuals. The present Indo-Iranian populations from Central Asia display a strong genetic continuity with Iron Age samples from Turkmenistan and Tajikistan. We model Yaghnobis as a mixture of 93% Iron Age individual from Turkmenistan and 7% from Baikal. For the Tajiks, we observe a higher Baikal ancestry and an additional admixture event with a South Asian population. Our results, therefore, suggest that in addition to a complex history, Central Asia shows a remarkable genetic continuity since the Iron Age, with only limited gene flow.

sentences: Targeted gene sequencing with Nanopore enables rapid and accurate confirmatory diagnostic of Tilapia lake virus Infectious diseases represent one of the major challenges to sustainable aquaculture production. Rapid, accurate diagnosis and genotyping of emerging pathogens during early-suspected disease cases is critical to facilitate timely response to deploy adequate control measures and prevent or reduce spread. Currently, most laboratories use PCR to amplify partial pathogen genomic regions, occasionally combined with sequencing of PCR amplicon(s) using conventional Sanger sequencing services for confirmatory diagnosis. The main limitation of this approach is the lengthy turnaround time. Here, we report an innovative approach using a previously developed specific PCR assay for pathogen diagnosis combined with a new Oxford Nanopore Technologies (ONT)-based amplicon sequencing method for pathogen genotyping. Using fish clinical samples, we applied this approach for the rapid confirmation of PCR amplicon sequences identity and genotyping of tilapia lake virus (TiLV), a disease-causing virus affecting tilapia aquaculture globally. The consensus sequences obtained after polishing exhibit strikingly high identity to references derived by Illumina and Sanger methods (99.83-100%). This study suggests that ONT-based amplicon sequencing is a promising platform to deploy in regional aquatic animal health diagnostic laboratories in low and medium income countries, for fast identification and genotyping of emerging infectious pathogens from field samples within a single day.

sentences: Plasticity of body axis polarity in Hydra regeneration under constraints One of the major events in animal morphogenesis is the emergence of a polar body axis. Here, we combine classic grafting techniques with live imaging to study the emergence of body axis polarity during whole body regeneration in Hydra. Composite tissues are made by fusing two rings, excised from separate animals, in different configurations that vary in the polarity and original positions of the rings along the body axes of the parent animals. Under frustrating initial configurations, body axis polarity that is otherwise stably inherited from the parent animal, can become labile and even be reversed. Importantly, the site of head regeneration exhibits a strong bias toward the edges of the tissue, even when this involves polarity reversal. In particular, we observe head formation at an originally aboral tissue edge, which is not compatible with models of Hydra regeneration based only on preexisting morphogen gradients or an injury response. Rather, we suggest that the structural bias toward head formation at the doublets edge is reinforced by the presence of a defect in the organization of the supra-cellular actin fibers, which invariably forms at the edge as the tissue heals. In this scenario, the defect supports head formation at the edge, even though a defect is neither required nor sufficient for head formation. Altogether, our results suggest that body axis determination is an integrated process that arises from the dynamic interplay between mechanical feedback and signaling processes.

sentences: Myth-busting the provider-user relationship for digital sequence information The United Nations Convention on Biological Diversity (CBD) formally recognized the sovereign rights of nations over their biological diversity. Implicit within the treaty is the idea that mega-biodiverse countries will provide genetic resources and grant access to them and scientists in high-income countries will use these resources and share back benefits. However, little research has been conducted on how this framework is reflected in real-life scientific practice. Currently, parties to the CBD) are debating whether or not digital sequence information (DSI) should be regulated under a new benefit-sharing framework. At this critical time point in the upcoming international negotiations, we test the fundamental hypothesis of provision and use by looking at the global patterns of access and use in scientific publications. Our data reject the provider-user relationship and suggest far more complex information flow for digital sequence information. Therefore, any new policy decisions on digital sequence information should be aware of the high level of use of DSI across low- and middle-income countries and seek to preserve open access to this crucial common good.

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Making Genomic Surveillance Deliver: A Lineage Classification and Nomenclature System to Inform Rabies Virus Elimination The availability of pathogen sequence data and use of genomic surveillance is rapidly increasing. Genomic tools and classification systems need updating to reflect this. Here, rabies virus is used as an example to showcase the potential value of updated genomic tools to enhance surveillance to better understand epidemiological dynamics and improve disease control. Previous studies have described the evolutionary history of rabies virus; however, the resulting taxonomy lacks the definition necessary to identify incursions, lineage turnover and transmission routes at high resolution. Here we propose a lineage classification system based on the dynamic nomenclature used for SARS-CoV-2, defining a lineage by phylogenetic methods, for tracking virus spread and comparing sequences across geographic areas. We demonstrate this system through application to the globally distributed Cosmopolitan clade of rabies virus, defining 73 total lineages within the clade, beyond the 22 previously reported. We further show how integration of this tool with a new rabies virus sequence data resource (RABV-GLUE) enables rapid application, for example, highlighting lineage dynamics relevant to control and elimination programmes, such as identifying importations and their sources, and areas of persistence and transmission, including transboundary incursions. This system and the tools developed should be useful for coordinating and targeting control programmes and monitoring progress as we work towards eliminating dog-mediated rabies, as well as having potential for broad application to the surveillance of other viruses.

Author SummaryThe importance of the ability to track the diversity and spread of viruses in a universal way that can be clearly communicated has been highlighted during the SARS-CoV-2 pandemic. This, accompanied with the increase in the availability and use of pathogen sequence data, means the development of new genomic tools and classification systems can strengthen outbreak response and disease control. Here, we present an easy-to-use objective and transferable classification tool for tracking viruses at high resolution. We use rabies virus, a neglected zoonotic disease that causes around 59,000 human deaths each year, as an example use case of this tool. Applying our tool to a global clade of rabies virus, we find an over 200% increase in the definition at which we can classify the virus, allowing us to identify areas of persistence and transmission that were not previously apparent, and patterns of virus spread. Insights from the application of this tool should prove valuable in targeting vaccination campaigns and improving surveillance as countries work towards the elimination of dog-mediated rabies.

sentences: Metacognition, cortical thickness, and tauopathy in aging We investigated two aspects of metacognition and their relationship with cortical thickness and Alzheimers Disease (AD) biomarkers, amyloid and tau, in cognitively healthy older adults (N=151). The two metacognition measures were self-appraisal rating of task performance and the difference between self-appraisal rating and actual task performance (appraisal discrepancy). All participants underwent neuropsychological testing and 1.5T structural MRI. A subset (N=66) received amyloid-PET with [11C] PiB and tau-PET with [18F] Flortaucipir. We found that worse performers had lower self-appraisal ratings, but still overestimated their performance, consistent with the Dunning-Kruger effect. Self-appraisal rating and appraisal discrepancy revealed distinct relationships with cortical thickness and AD pathology. Greater appraisal discrepancy, indicating overestimation, was related to thinning of inferior-lateral temporal, fusiform, and rostral anterior cingulate cortices. Lower self-appraisal was associated with higher entorhinal and inferior temporal tau. These results suggest that overestimation could implicate structural atrophy beyond AD pathology, while lower self-appraisal could indicate early behavioral alteration due to AD pathology, supporting the notion of subjective cognitive decline prior to objective deficits.

sentences: Investigating densities of Symbiodiniaceae in two species of Antipatharians (black corals) from Madagascar Here, we report the first methodological approach to investigate the presence and estimate the density of Symbiodiniaceae cells in corals of the order Antipatharia subclass Hexacorallia, known as black corals. Antipatharians are understudied ecosystem engineers of shallow (<30 m depth), mesophotic (30-150 m) and deep-sea (>200 m) reefs. They provide habitat to a vast number of marine fauna, enhancing and supporting coral reefs biodiversity globally. Nonetheless, little biological and ecological information exists on antipatharians, including the extent at which global change disturbances are threatening these corals. The assumption that they were exempted from threats related to climate change was challenged by findings of high density of dinoflagellates within three antipatharian colonies. Further methodical studies were necessary to investigate the regularity of these findings. An integrated design combining microscopy and molecular techniques was used to investigate the presence and estimate density of Symbiodiniaceae cells within two antipatharians species -Cupressopathes abies and Stichopathes maldivensis -from shallow and mesophotic reefs of SW Madagascar. Symbiodiniaceae-like cells were found within the two species from both shallow and mesophotic reefs, although the overall cell density was very low (0-4 cell mm-3). These findings suggest that high abundance of Symbiodiniaceae is not characteristic of antipatharians, which has relevant implications considering disruptions associated to climate change affecting other corals. However, the high densities of dinoflagellates found in antipatharian colonies exposed to higher light irradiance in other studies should be further examined.

sentences: Long-read metagenomics of soil communities reveals phylum-specific secondary metabolite dynamics Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 695 novel, full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night in cyanobacteria but not in other phyla, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts.

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An altered balance of integrated and segregated brain activity is a marker of cognitive deficits following sleep deprivation Sleep deprivation (SD) leads to impairments in cognitive function. Here, we tested the hypothesis that cognitive changes in the sleep-deprived brain can be explained by information processing within and between large-scale cortical networks. We acquired functional magnetic resonance imaging (fMRI) scans of 20 healthy volunteers during attention and executive tasks following a regular night of sleep, a night of sleep deprivation, and a recovery nap containing non-rapid eye movement (NREM) sleep. Overall, sleep deprivation was associated with increased cortex-wide functional integration, driven by a rise of integration within cortical networks. The ratio of within vs between network integration in the cortex increased further in the recovery nap, suggesting that prolonged wakefulness drives the cortex toward a state resembling sleep. This balance of integration and segregation in the sleep-deprived state was tightly associated with deficits in cognitive performance. This was a distinct and better marker of cognitive impairment than conventional indicators of homeostatic sleep pressure, as well as the pronounced thalamo-cortical connectivity changes that occurs towards falling asleep. Importantly, restoration of the balance between segregation and integration of cortical activity was also related to performance recovery after the nap, demonstrating a bi-directional effect. These results demonstrate that intra- and inter-individual differences in cortical network integration and segregation during task performance may play a critical role in vulnerability to cognitive impairment in the sleep deprived state.

Significance StatementSleep deprivation has significant negative consequences for cognitive function. Understanding how changes in brain activity underpin changes in cognition is important not only to discover why performance declines following extended periods of wakefulness, but also for answering the fundamental question of why we require regular and recurrent sleep for optimal performance. Finding neural correlates that predict performance following sleep deprivation also has the potential to understand which individuals are particularly vulnerable to sleep deprivation, and what aspects of brain function may protect them from these negative consequences on performance. Finally, understanding how perturbations to regular (well-rested) brain functioning affect cognitive performance, will provide important insight into how underlying principles of information processing in the brain may support cognition generally.

sentences: Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells Heme oxygenases (HO) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms: inducible HO-1, which is up-regulated in response to various stressors, including excess heme, and constitutive HO-2. While much is known about the regulation and physiological function of HO-1, comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is largely dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or over-expressed HO-2, and various HO-2 mutant alleles, we found that endogenous heme is too limiting to support HO-2 catalyzed heme degradation. Rather, we discovered that a novel role for HO-2 is to bind and buffer labile heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor in control of heme bioavailability. When heme is in excess, HO-1 is induced and both HO-2 and HO-1 can provide protection from heme toxicity by enzymatically degrading it. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with the labile heme pool being oxidized, thereby providing new insights into heme trafficking and signaling.

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Immunogenic potential of neopeptides depends on parent protein subcellular location Antigen presentation via the major histocompatibility complex (MHC) is essential for anti-tumor immunity, however the rules that determine what tumor-derived peptides will be immunogenic are still incompletely understood. Here we investigate whether protein subcellular location driven constraints on accessibility of peptides to the MHC associate with potential for peptide immunogenicity. Analyzing over 380,000 peptides from studies of MHC presentation and peptide immunogenicity, we find clear spatial biases in both eluted and immunogenic peptides. We find that including parent protein location improves prediction of peptide immunogenicity in multiple datasets. In human immunotherapy cohorts, location was associated with response to a neoantigen vaccine, and immune checkpoint blockade responders generally had a higher burden of neopeptides from accessible locations. We conclude that protein subcellular location adds important information for optimizing immunotherapies.

HighlightsO_LIPeptides eluted from class I and II MHC reflect biases in the subcellular location of the parent proteins
C_LIO_LIAn embedding-based indicator of parent protein location improves prediction of neoepitope immunogenicity and immunotherapy response
C_LIO_LINeoepitope location improves estimation of effective neoantigen burden and stratification of potential for immunotherapy response
C_LI

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A CNN model with feature integration for MI EEG subject classification in BMI ObjectiveElectroencephalogram (EEG) based motor imagery (MI) classification is an important aspect in brain-machine interfaces (BMIs) which bridges between neural system and computer devices decoding brain signals into recognizable machine commands. However, the MI classification task is challenging due to inherent complex properties, inter-subject variability, and low signal-to-noise ratio (SNR) of EEG signals. To overcome the above-mentioned issues, the current work proposes an efficient multi-scale convolutional neural network (MS-CNN).

ApproachIn the framework, discriminant user-specific features have been extracted and integrated to improve the accuracy and performance of the CNN classifier. Additionally, different data augmentation methods have been implemented to further improve the accuracy and robustness of the model.

Main resultsThe model achieves an average classification accuracy of 93.74% and Cohens kappa-coefficient of 0.92 on the BCI competition IV2b dataset outperforming several baseline and current state-of-the-art EEG-based MI classification models.

SignificanceThe proposed algorithm effectively addresses the shortcoming of existing CNN-based EEG-MI classification models and significantly improves the classification accuracy.

sentences: Sex differences in parental response to offspring begging are associated with pair bond strength across birds Mothers, fathers, and offspring regularly clash over how much care offspring receive. Offspring beg to solicit for more resources--but how much begging is rewarded can depend on who is listening. While both parents benefit from provisioning offspring, each would benefit from their partner shouldering more of the burden of care, leading to sexual conflict. Additionally, if the costs and benefits of provisioning differ by sex, parent-offspring conflict should vary by sex. How these evolutionary conflicts influence sex differences in parent-offspring communication is unknown. To determine whether the sexes differ in their response to offspring signals, we conducted a meta-analysis on 30 bird species, comparing responsiveness to social and physiological traits affecting conflict. We found that a species typical pair bond strength predicts whether males or females respond more to offspring begging. In species with stable and/or monogamous bonds, and thus lower sexual and paternal-offspring conflict, males provisioning effort is more strongly correlated with offspring begging than females. The opposite holds for species with weak pair bonds: females respond more to begging, perhaps compensating for males lower responsiveness. These results demonstrate that sex differences in parental care can arise via sex differences in parent-offspring communication, driven by evolutionary conflicts.

sentences: Gene regulatory networks underlying human microglia maturation The fetal period is a critical time for brain development, characterized by neurogenesis, neural migration, and synaptogenesis1-3. Microglia, the tissue resident macrophages of the brain, are observed as early as the fourth week of gestation4 and are thought to engage in a variety of processes essential for brain development and homeostasis5-11. Conversely, microglia phenotypes are highly regulated by the brain environment12-14. Mechanisms by which human brain development influences the maturation of microglia and microglia potential contribution to neurodevelopmental disorders remain poorly understood. Here, we performed transcriptomic analysis of human fetal and postnatal microglia and corresponding cortical tissue to define age-specific brain environmental factors that may drive microglia phenotypes. Comparative analysis of open chromatin profiles using bulk and single-cell methods in conjunction with a new computational approach that integrates epigenomic and single-cell RNA-seq data allowed decoding of cellular heterogeneity with inference of subtype- and development stage-specific transcriptional regulators. Interrogation of in vivo and in vitro iPSC-derived microglia models provides evidence for roles of putative instructive signals and downstream gene regulatory networks which establish human-specific fetal and postnatal microglia gene expression programs and potentially contribute to neurodevelopmental disorders.

sentences: Evaluation of efficacy- versus affinity-driven agonism with biased GLP-1R ligands P5 and exendin-F1 The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of glucose homeostasis and has been successfully targeted for the treatment of type 2 diabetes. Recently described biased GLP-1R agonists with selective reductions in {beta}-arrestin versus G protein coupling show improved metabolic actions in vivo. However, two prototypical G protein-favouring GLP-1R agonists, P5 and exendin-F1, are reported to show divergent effects on insulin secretion. In this study we aimed to resolve this discrepancy by performing a side-by-side characterisation of these two ligands across a variety of in vitro and in vivo assays. Exendin-F1 showed reduced acute efficacy versus P5 for several readouts, including recruitment of mini-G proteins, G protein-coupled receptor kinases (GRKs) and {beta}-arrestin-2. Maximal responses were also lower for both GLP-1R internalisation and the presence of active GLP-1R-mini-Gs complexes in early endosomes with exendin-F1 treatment. In contrast, prolonged insulin secretion in vitro and sustained anti-hyperglycaemic efficacy in mice were both greater with exendin-F1 than with P5. We conclude that the particularly low acute efficacy of exendin-F1 and associated reductions in GLP-1R downregulation appear to be more important than preservation of endosomal signalling to allow sustained insulin secretion responses. This has implications for the ongoing development of affinity- versus efficacy-driven biased GLP-1R agonists as treatments for metabolic disease.

sentences: Sustained Ranavirus outbreak causes mass-mortality and morbidity in imperiled amphibians. A persistent two-month long outbreak of Ranavirus in a natural community of amphibians contributed to a mass die-off of gopher frog tadpoles (Lithobates capito) and severe disease in striped newts (Notophthalmus perstriatus) in Florida. Ongoing mortality in L. capito and signs in N. perstriatus continued for five weeks after the first observation. Hemorrhagic disease and necrosis were diagnosed from pathological examination of L. capito tadpoles. We confirmed detection of a Frog Virus 3 (FV3)-like Ranavirus via quantitative PCR in all species. Our findings highlight the susceptibility of these species to Rv and the need for long-term disease surveillance during epizootics.

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Multiple routes of functional diversification of the plant BAHD acyltransferase family revealed by comparative biochemical and genomic analyses Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Here, we addressed this question using BAHD acyltransferases as a model, and identified the routes by which duplication, promiscuity and sequence changes influenced BAHD diversification. This fast-evolving family expanded drastically during land plant evolution from 1-5 copies in algae to [~]100 copies in diploid angiosperm genomes. In vitro characterization of fourteen BAHDs against a substrate panel and compilation of >160 published activities revealed the wide prevalence of promiscuity among BAHDs. Using phylogenetic analysis, we predicted the substrate classes that the ancestral enzymes were likely capable of using prior to land plant origins. While the anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms, in vitro testing of BAHDs from non-seed plant lineages suggested that the ability to acylate anthocyanins likely existed promiscuously millions of years prior to its fixation. Motif enrichment analysis in anthocyanin-acylating BAHDs identified two motifs fixed in the largest anthocyanin acylating clade. Molecular dynamic simulations and enzyme kinetics revealed the important role of an active site tryptophan, whose bulkiness, hydrophobicity and aromaticity are critical for anthocyanin acylation. Our results thus describe the molecular processes in robust, evolvable enzymes that drive emergence of functional diversity in enzyme families.

One sentence summaryUsing a combination of phylogenetics, biochemistry and protein structure analysis, we investigated how the BAHD acyltransferase family evolved to use a structurally diverse array of substrates.

sentences: Combining Stem Cell Rejuvenation and Senescence Targeting to Synergistically Extend Lifespan Why biological age is a major risk factor for many of the most important human diseases remains mysterious. We know that as organisms age, stem cell pools are exhausted while senescent cells progressively accumulate. Independently, induction of pluripotency via expression of Yamanaka factors (Oct4, Klf4, Sox2, c-Myc; OKSM) and clearance of senescent cells have each been shown to ameliorate cellular and physiological aspects of aging, suggesting that both processes are drivers of organismal aging. However, stem cell exhaustion and cellular senescence likely interact in the etiology and progression of age-dependent diseases because both undermine tissue and organ homeostasis in different if not complementary ways. Here, we combine transient cellular reprogramming (stem cell rejuvenation) with targeted removal of senescent cells to test the hypothesis that simultaneously targeting both cell-fate based aging mechanisms will maximize life and health span benefits. We show that these interventions protect the intestinal stem cell pool, lower inflammation, activate pro-stem cell signaling pathways, and synergistically improve health and lifespan. Our findings suggest that a combination therapy, simultaneously replacing lost stem cells and removing senescent cells, shows synergistic potential for anti-aging treatments. Our finding that transient expression of both is the most effective suggests that drug-based treatments in non-genetically tractable organisms will likely be the most translatable.

sentences: Phenotype-Centric Modeling for Rational Metabolic Engineering Phenotype-centric modeling enables a paradigm shift in the analysis of kinetic models. It brings the focus to a networks biochemical phenotypes and their relationship with measurable traits (e.g., product yields, system dynamics, signal amplification factors, etc.) and away from computationally intensive parameter sampling and numerical simulation. Here, we explore applications of this new modeling strategy in the field of Rational Metabolic Engineering using the amorphadiene biosynthetic network as a case study. Our phenotype-centric approach not only identifies known beneficial intervention strategies for this network, but it also provides an understanding of mechanistic context for the validity of these predictions. Additionally, we propose a set of hypothetical strains with the potential to outperform reported production strains and enhance the mechanistic understanding of the amorphadiene biosynthetic network. We believe that phenotype-centric modeling can advance the field of Rational Metabolic Engineering by enabling the development of next generation kinetics-based algorithms and methods that do not rely on a priori knowledge of kinetic parameters but allow a structured, global analysis of the design space of parameter values.

sentences: HIGHER VIRAL VIRULENCE SELECTS FOR FASTER EVOLUTION OF HOST RESISTANCE Parasites and pathogens vary strikingly in their virulence and the resulting selection they impose on their hosts. While the evolution of different virulence levels is well studied, the evolution of host resistance in response to different virulence levels is less understood and as of now mainly based on observations and theoretical predictions with few experimental tests. Increased virulence can increase selection for host resistance evolution if resistance costs are outweighed by the benefits of avoiding infection. To test this, we experimentally evolved the bacterium Vibrio alginolyticus in the presence of two variants of the filamentous phage, VALG{Phi}8, that differ in their virulence. The bacterial host exhibited two alternative defence strategies against future phage infection: (1) super infection exclusion (SIE) whereby phage-infected cells were immune to subsequent infection at a cost of reduced growth, and (2) surface receptor mutations (SRM) in genes encoding the MSHA type-IV pilus providing resistance to infection by preventing phage attachment. While SIE emerged rapidly against both phages, SRM evolved faster against the high virulence compared to the low virulence phage. Using a mathematical model of our system we show that increasing virulence strengthens selection for SRM due to the higher costs of infection suffered by SIE immune hosts. In both the experiments and the model, higher levels of SRM in the host population drove more rapid phage extinction. Thus, by accelerating the evolution of host resistance, more virulent phages caused shorter epidemics.

sentences: Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243A>G mtDNA mutation Mutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are very limited. Most mtDNA diseases show heteroplasmy - tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have carried out extensive bioenergetic, metabolomic and RNAseq studies on heteroplasmic patient derived cells carrying the most prevalent disease related mtDNA mutation, m.3243A>G. These studies reveal that the mutation promotes changes in metabolites which is associated with the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 activated mitophagy, reduced mtDNA mutant load and rescued cellular bioenergetic function. The rescue was prevented by inhibition of mitophagy. The PI3K-Akt-mTORC1 axis thus represents a potential therapeutic target that may benefit people suffering from the consequences of the m.3243A>G mutation.

sentences: Markers of Fungal Translocation Are Elevated During Post-Acute Sequelae of SARS-CoV-2 Infection and Induce NF-κB Triggered Inflammation. Long COVID, a type of Post-Acute Sequelae of SARS CoV-2 infection (PASC), has been associated with sustained elevated levels of immune activation and inflammation. However, the pathophysiological mechanisms that drive this inflammation remain unknown. Inflammation during acute Coronavirus Disease 2019 (COVID-19) could be exacerbated by microbial translocation (from the gut and/or lung) to the blood. Whether microbial translocation contributes to inflammation during PASC is unknown. We found higher levels of fungal translocation - measured as {beta}-glucan, a fungal cell wall polysaccharide - in the plasma of individuals experiencing PASC compared to those without PASC or SARS-CoV-2 negative controls. The higher {beta}-glucan correlated with higher levels of markers of inflammation and elevated levels of host metabolites involved in activating N-Methyl-D-aspartate receptors (such as metabolites within the tryptophan catabolism pathway) with established neuro-toxic properties. Mechanistically, {beta}-glucan can directly induce inflammation by binding to myeloid cells (via the Dectin-1 receptor) and activating Syk/NF-{kappa}B signaling. Using an in vitro Dectin-1/NF-{kappa}B reporter model, we found that plasma from individuals experiencing PASC induced higher NF-{kappa}B signaling compared to plasma from SARS-CoV-2 negative controls. This higher NF-{kappa}B signaling was abrogated by the Syk inhibitor Piceatannol. These data suggest a potential targetable mechanism linking fungal translocation and inflammation during PASC.

sentences: Organellar transcripts dominate the cellular mRNA pool across plants of varying ploidy levels Mitochondrial and plastid functions depend on coordinated expression of proteins encoded by genomic compartments that have radical differences in copy number of organellar and nuclear genomes. In polyploids, doubling of the nuclear genome may add challenges to maintaining balanced expression of proteins involved in cytonuclear interactions. Here, we use ribo-depleted RNA-seq to analyze transcript abundance for nuclear and organellar genomes in leaf tissue from four different polyploid angiosperms and their close diploid relatives. We find that, even though plastid genomes contain <1% of the number of genes in the nuclear genome, they generate the majority (69.9-82.3%) of mRNA transcripts in the cell. Mitochondrial genes are responsible for a much smaller percentage (1.3-3.7%) of the leaf mRNA pool but still produce much higher transcript abundances per gene compared to nuclear genome. Nuclear genes encoding proteins that functionally interact with mitochondrial or plastid gene products exhibit mRNA expression levels that are consistently more than ten-fold lower than their organellar counterparts, indicating an extreme cytonuclear imbalance at the RNA level despite the predominance of equimolar interactions at the protein level. Nevertheless, interacting nuclear and organellar genes show strongly correlated transcript abundances across functional categories, suggesting that the observed mRNA stoichiometric imbalance does not preclude coordination of cytonuclear expression. Finally, we show that nuclear genome doubling does not alter the cytonuclear expression ratios observed in diploid relatives in consistent or systematic ways, indicating that successful polyploid plants are able to compensate for cytonuclear perturbations associated with nuclear genome doubling.

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A new adaptive procedure for estimating perceptual thresholds: the effects of observer bias and its correction. Adaptive threshold estimation procedures sample close to a subjects perceptual threshold by dynamically adapting the stimulation based on the subjects performance. Yet, perceptual thresholds not only depend on the observers sensory capabilities but also on any bias in terms of their expectations and response preferences, thus distorting the precision of the threshold estimates. Using the framework of signal detection theory (SDT), independent estimates of both, an observers sensitivity and internal processing bias can be delineated from threshold estimates. While this approach is commonly available for estimation procedures engaging the method of constant stimuli (MCS), correction procedures for adaptive methods (AM) are only scarcely applied. In this article, we introduce a new AM that takes individual biases into account, and that allows for a bias-corrected assessment of subjects sensitivity. This novel AM is validated with simulations and compared to a typical MCS-procedure, for which the implementation of bias correction has been previously demonstrated.

Comparing AM and MCS demonstrates the viability of the presented AM. Besides its feasibility, the results of the simulation reveal both, advantages, and limitations of the proposed AM. The procedure has considerable practical implications, in particular for the design of shaping procedures in sensory training experiments, in which task difficulty has to be constantly adapted to an observers performance, to improve training efficiency.

sentences: A complex CTCF binding code defines TAD boundary structure and function Topologically Associating Domains (TADs) compartmentalize vertebrate genomes into sub-Megabase functional neighbourhoods for gene regulation, DNA replication, recombination and repair1-10. TADs are formed by Cohesin-mediated loop extrusion, which compacts the DNA within the domain, followed by blocking of loop extrusion by the CTCF insulator protein at their boundaries11-20. CTCF blocks loop extrusion in an orientation dependent manner, with both experimental and in-silico studies assuming that a single site of static CTCF binding is sufficient to create a stable TAD boundary21-24. Here, we report that most TAD boundaries in mouse cells are modular entities where CTCF binding clusters within extended genomic intervals. Optimized ChIP-seq analysis reveals that this clustering of CTCF binding does not only occur among peaks but also frequently within those peaks. Using a newly developed multi-contact Nano-C assay, we confirm that individual CTCF binding sites additively contribute to TAD separation. This clustering of CTCF binding may counter against the dynamic DNA-binding kinetics of CTCF25-27, which urges a re-evaluation of current models for the blocking of loop extrusion21-23. Our work thus reveals an unanticipatedly complex code of CTCF binding at TAD boundaries that expands the regulatory potential for TAD structure and function and can help to explain how distant non-coding structural variation influences gene regulation, DNA replication, recombination and repair5,28-34.

sentences: Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly.

sentences: Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes Early blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here, we identified three major 2 cell (2C) specific endogenous retroviruses (ERVs) as the molecular hallmark of the bi-potential plasticity. Using the LTRs of all three 2C-ERVs, we identified Klf5 as their major upstream regulator. Klf5 is essential for bi-potential cell fate: a single Klf5-overexpressing ESC generated terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces both ICM and TE specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. Altogether, the 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.

sentences: Proteomic profiling identifies co-regulated expression of splicing factors as a characteristic feature of intravenous leiomyomatosis Intravenous leiomyomatosis (IVLM) is a rare benign smooth muscle tumour that is characterised by intravenous growth in the uterine and pelvic veins. Previous DNA copy number and transcriptomic studies have shown that IVLM harbours unique genomic and transcriptomic alterations when compared to uterine leiomyoma (uLM), which may account for their distinct clinical behaviour. Here we undertake the first comparative proteomic analysis of IVLM and other smooth muscle tumours (comprising uLM, soft tissue leiomyoma and benign metastasising leiomyoma) utilising data-independent acquisition mass spectrometry. We show that, at the protein level, IVLM is defined by the unique co-regulated expression of splicing factors. In particular, IVLM is enriched in two clusters composed of co-regulated proteins from the hnRNP, LSm, SR and Sm classes of the spliceosome complex. One of these clusters (Cluster 3) is associated with key biological processes including nascent protein translocation and cell signalling by small GTPases. Taken together, our study provides evidence of co-regulated expression of splicing factors in IVLM compared to other smooth muscle tumours which suggests a possible role for alternative splicing in the pathogenesis of IVLM.

sentences: Effect of Ionic Strength on the Assembly of Simian Vacuolating Virus Capsid Protein Around Poly(Styrene Sulfonate) Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent like RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with combinatorial intermediates, and therefore the mechanism of the reaction is poorly understood. In this paper, we determined the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by solution small-angle X-ray scattering (SAXS) and cryo-TEM. To analyze the data, we performed Brownian dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures that were consistent with the experimental data. We found that close to physiological ionic strength, T=1 VLPs coexisted with VP1 pentamers. At lower or higher ionic strengths, incomplete particles coexisted with pentamers and T=1 particles. Including the simulation, structures were essential to explaining the SAXS data.

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Time dependent proinflammatory responses shape virus interference during coinfections of influenza A virus and influenza D virus Both influenza A virus (IAV) and influenza D virus (IDV) are enzootic in pigs. IAV causes approximately 100% morbidity with low mortality, whereas IDV leads to only mild respiratory diseases in pigs. In this study, we performed a series of coinfection experiments in vitro and in vivo to understand how IAV and IDV interact and cause pathogenesis during coinfection. Results showed that IAV inhibited IDV replication when infecting swine tracheal epithelial cells (STEC) with IAV 24- or 48-hours prior to IDV inoculation, and that IDV suppressed IAV replication when IDV preceded IAV inoculation by 48 hours. Virus interference was not identified during simultaneous IAV/IDV infections or with 6 hours between the two viral infections, regardless of their order. The interference pattern at 24- and 48-hours correlated with proinflammatory responses induced by the first infection, which was about 24-hours slower for IDV than IAV. The viruses did not interfere with each other if both infected the cells before proinflammatory responses were induced. Coinfection in pigs further demonstrated that IAV interfered both viral shedding and virus replication of IDV, especially in the upper respiratory tract. Clinically, coinfection of IDV and IAV did not show significant enhancement of disease pathogenesis, compared with the pigs infected with IAV alone. In summary, this study suggests that interference during coinfection of IAV and IDV is primarily due to the proinflammatory response and is therefore dependent on the time between infection, and the order of infection.

ImportanceBoth IAV and IDV are enzootic in pigs, and feral pigs have a higher risk for both IAV and IDV exposures than IDV exposure alone. This study suggests that in coinfection with IAV and IDV either virus can interfere with the replication of the other virus by stimulating proinflammatory responses; however, the proinflammatory response was 24 hours slower for IDV than IAV. In vitro there was no interference during simultaneous coinfection, regardless of infection order. Coinfection of IDV and IAV in pigs did not show enhanced pathogenesis, compared with those infected only with IAV. This study can facilitate our understanding of virus epidemiology and pathogenesis associated with IAV and IDV coinfection.

sentences: Ligand-mediated structural dynamics of a mammalian pancreatic KATP channel Regulation of pancreatic KATP channels involves orchestrated interactions of channel subunits, Kir6.2 and SUR1, and their ligands. How ligand interactions affect channel conformations and activity is not well understood. To elucidate the structural correlates pertinent to ligand interactions and channel gating, we compared cryo-EM structures of channels in the presence and absence of pharmacological inhibitors and ATP, focusing on channel conformational dynamics. We found pharmacological inhibitors and ATP enrich a channel conformation in which the Kir6.2 cytoplasmic domain is closely associated with the transmembrane domain relative to one where the Kir6.2 cytoplasmic domain is extended away into the cytoplasm. This conformation change remodels a network of intra and inter-subunit interactions as well as both the ATP and PIP2 binding pockets. The structures resolved key contacts between the distal N-terminus of Kir6.2 and SUR1s ABC module involving residues implicated in channel function. A SUR1 residue, K134, is identified to directly contribute to the PIP2 binding pocket. Molecular dynamics simulations revealed two Kir6.2 residues, K39 and R54, that mediate both ATP and PIP2 binding, suggesting a mechanism for competitive gating by ATP and PIP2.

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Differential Regulation of Rice Transcriptome to Rhizoctonia solani infection Sheath Blight (SB) disease in rice crop caused by the infection of the fungal pathogen Rhizoctonia solani (R. solani) is one of the severe rice diseases that can cause up to 50% yield losses. Naturally occurring rice varieties resistant to SB have not been reported yet. We have performed a Time-Series RNA-Seq analysis on a widely cultivated rice variety BPT-5204 for identifying its transcriptomic response signatures to R. solani infection at 1st, 2nd and 5th day post inoculation (dpi). In total, 428, 3225 and 1225 genes were differentially expressed in the treated rice plants post 1, 2 and 5 dpi, respectively. GO and KEGG enrichment analysis identified significant processes and pathways differentially altered in the rice plant after the fungal infection. Machine learning and network based integrative approach was used to construct Transcriptional Regulatory Networks (TRNs) of the rice plant at the three Time Points. Regulatory network analysis identified SUB1B, MYB30 and CCA1 as important regulatory hub Transcription Factors in rice during R. solani infection. Jasmonic acid signaling pathway was activated and in contrast, photosynthesis and carbon fixation processes were significantly compromised. Involvement of MAPK, CYPs, Peroxidases and PAL genes was observed in response to the fungal infection. Circadian clock was also strongly influenced by R. solani infection. Our integrative analysis identified 7 putative SB resistant genes altered in rice after R. solani infection and provided a better understanding of rice plant response to R. solani infection.

One sentence summaryTime series expression analysis of rice variety BPT-5204 identifies key molecular signatures involved in rice plant response to R. solani infection.

sentences: Why do fungicide mixtures delay the evolution of resistance? An experimental evolutionary approach Pesticide resistance poses a critical threat to agriculture, human health and biodiversity. Mixtures of fungicides are recommended and widely used in resistance management strategies. However, the components of the efficiency of such mixtures remain unclear. We performed an experimental evolution study on the fungal pathogen Z. tritici, to determine how mixtures managed resistance. We compared the effect of the continuous use of single active ingredients to that of mixtures, at the minimal dose providing full control of the disease, which we refer to as the "efficient" dose. We found that the performance of efficient-dose mixtures against an initially susceptible population depended strongly on the components of the mixture. Such mixtures were either as durable as the best mixture component used alone, or worse than all components used alone. Moreover, efficient-dose mixture regimes probably select for generalist resistance profiles as a result of the combination of selection pressures exerted by the various components and their lower doses. Our results indicate that mixtures should not be considered a universal strategy. Experimental evaluations of specificities for the pathogens targeted, their interactions with fungicides and the interactions between fungicides are crucial for the design of sustainable resistance management strategies.

sentences: Whole Genome and Exome Sequencing Reference Datasets from A Multi-center and Cross-platform Benchmark Study With the rapid advancement of sequencing technologies in the past decade, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from errors or artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically benchmarking sequencing platforms, library protocols, bioinformatics pipelines and for measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples, a human triple-negative breast cancer cell line and a matched normal cell line derived from B lymphocytes. We generated whole-genome (WGS) and whole-exome sequencing (WES) data using 16 NGS library preparation protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the paired reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

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Intracellular force comparison of pathogenic KIF1A, KIF5, and dynein by fluctuation analysis In mammalian cells, there exist approximately 40 types of microtubule motor proteins that are assigned to specific cargo deliveries. For example, the kinesin-1 family motor KIF5 is the major motor responsible for anterograde mitochondrial transport, whereas the kinesin-3 family motor KIF1A is responsible for synaptic vesicle precursor transport. In contrast, cytoplasmic dynein is responsible for retrograde transport of nearly all cargos. The force and velocity of these microtubule motors have been investigated in in-vitro single-molecule experiments. In the present study, we compared the intracellular force and velocity of various types of motors in the mammalian neuronal axon obtained by non-invasive force measurement (fluctuation analysis) and extreme value analysis with those obtained by previous single-molecule experiments. As we found a high correlation between our results and the previous results, we next investigated synaptic vesicle precursor transport by hereditary spastic paraplegia-associated KIF1A variants (V8M, R350G, and A255V). KIF1A-V8M and KIF1A-A255V exhibited force and velocity impairment in mammalian neuronal axons, whereas the physical property of KIF1A-R350G was similar to that of the wild type. We believe that the development of new analytical techniques for investigating intracellular cargo transports is helpful to elucidate the molecular mechanism of KIF1A-associated neurological disorders.

Statement of SignificanceRecent in-vitro single-molecule experiments have clearly revealed that microtubule motors only fully exert their functions when fully equipped with the proteins associated with cargo vesicle transport. This emphasizes the significance of intracellular physical measurements, in which the motors should fully function. In addition to motor force and velocity, the number of motors transporting a single cargo together is an important physical quantity to characterize cargo transport, but is difficult to estimate using in-vitro single-molecule experiments. In this study, we aimed to extract physical information on microtubule motors in the intracellular environment.

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Amazonian run-of-river dam reservoir impacts underestimated: Evidence from a Before-After Control-Impact study of freshwater turtle nesting areas O_LIConstruction of hydropower dams is associated with negative impacts on biodiversity, yet there remains a lack of robust scientific evidence documenting the magnitude of these impacts particularly across highly biodiverse tropical waterways. Hydropower expansion is an increasing threat to the Endangered yellow-spotted river turtle (Podocnemis unifilis) across its tropical South American range.
C_LIO_LIYellow-spotted river turtle nesting areas were monitored as an indicator of dry season river level changes following run-of-river dam reservoir filling. A Before-After-Control-Impact (BACI) study design was used with multi-year field campaigns monitoring turtle nesting areas upstream of the dam.
C_LIO_LIThe cause and extent of changes in nesting areas were established using Generalized Additive Models. Nesting area density was evaluated in relation to: time (before versus after), treatment (control versus impact), time treatment interaction (BACI), distance to the dam and precipitation. The extent of changes was examined by comparing the proportion of nesting areas remaining during four years after reservoir filling.
C_LIO_LIDam construction generated an immediate and apparently permanent dry season river level rise that extended more than 20 km beyond impact assessment limits. On average the density of nesting areas declined 69% (from 0.48 to 0.15 per km) across 33 km of river directly impacted by the dam. This loss was reflected in a significant BACI interaction. Nesting area density was not explained by seasonal precipitation.
C_LIO_LIStandardized monitoring of freshwater turtle nesting areas provided an effective means to quantify impacts of hydropower developments across biodiverse yet rapidly changing waterways. The negative impacts documented in this study should be preventable by mitigation actions including habitat creation and dry season flow regulation. Such measures would also likely benefit multiple species elsewhere in tropical rivers increasingly impacted by run-of-river dams.
C_LI

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Discovering differential genome sequence activity with interpretable and efficient deep learning Discovering sequence features that differentially direct cells to alternate fates is key to understanding both cellular development and the consequences of disease related mutations. We introduce Expected Pattern Effect and Differential Expected Pattern Effect, two black-box methods that can interpret genome regulatory sequences for cell type-specific or condition specific patterns. We show that these methods identify relevant transcription factor motifs and spacings that are predictive of cell state-specific chromatin accessibility. Finally, we integrate these methods into framework that is readily accessible to non-experts and available for download as a binary or installed via PyPI or bioconda at https://cgs.csail.mit.edu/deepaccess-package/.

Author SummaryWithin the genome are the instructions to build all the cell types that make up the human body. However, understanding these instructions and how and when these instructions go wrong in cancer or genetically inherited disease is an open problem. Deep neural networks provide powerful models to learn the relationship between DNA sequence and functional consequence across many different cell types, such as whether a particular stretch of DNA is accessible and genes in that region can be expressed or is inaccessible and therefore genes are inactive. Despite these advances, a major setback in deep learning is that it is challenging to understand what patterns of DNA sequences a deep learning model has learned to associate with a particular genomic function, whether these patterns are significant, and how to determine whether these patterns are specific to a particular cell type or are general "housekeeping" patterns that function across many cell types. We introduce Expected Pattern Effect and Differential Expected Pattern Effect, two methods which allow us to evaluate the significance of particular patterns of DNA sequence features on models trained to predict function across multiple cell types, and apply this to problems of transcription factor binding and DNA accessibility across multiple cell types.

sentences: Combining genetic resources and elite material populations to improve the accuracy of genomic prediction in apple Genomic selection is an attractive strategy for apple breeding that could reduce the length of breeding cycles. A possible limitation to the practical implementation of this approach lies in the creation of a training set large and diverse enough to ensure accurate predictions. In this study, we investigated the potential of combining two available populations, i.e. genetic resources and elite material, in order to obtain a large training set with a high genetic diversity. We compared the predictive ability of genomic predictions within-population, across-population or when combining both populations, and tested a model accounting for population-specific marker effects in this last case. The obtained predictive abilities were moderate to high according to the studied trait and were always highest when the two populations were combined into a unique training set. We also investigated the potential of such a training set to predict hybrids resulting from crosses between the two populations, with a focus on the method to design the training set and the best proportion of each population to optimize predictions. The measured predictive abilities were very similar for all the proportions, except for the extreme cases where only one of the two populations was used in the training set, in which case predictive abilities could be lower than when using both populations. Using an optimization algorithm to choose the genotypes in the training set also led to higher predictive abilities than when the genotypes were chosen at random. Our results provide guidelines to initiate breeding programs that use genomic selection when the implementation of the training set is a limitation.

sentences: Parallel processing, hierarchical transformations, and sensorimotor associations along the 'where pathway Visually guided behaviors require the brain to transform ambiguous retinal images into object-level spatial representations and implement sensorimotor transformations. These processes are supported by the dorsal  where pathway. However, the specific functional contributions of areas along this pathway remain elusive due in part to methodological differences across studies. We previously showed that macaque caudal intraparietal (CIP) area neurons possess robust three-dimensional (3D) visual representations, carry choice-related and presaccadic activity, and exhibit experience-dependent sensorimotor associations (Chang et al., 2020b). Here, we used a common experimental design to reveal parallel processing, hierarchical transformations, and the formation of sensorimotor associations along the  where pathway by extending the investigation to V3A, a major feedforward input to CIP. Higher-level 3D representations and choice-related activity were more prevalent in CIP than V3A. Both areas contained presaccadic activity that predicted the direction/timing of eye movements. Intriguingly, the time-course of presaccadic activity in CIP aligned with the temporally integrated V3A output. Sensorimotor associations between 3D orientation and saccade direction preferences were strongest in CIP and moderated by choice signals in both areas. Together, the results explicate parallel representations, hierarchical transformations, and functional associations of visual and presaccadic signals at a key juncture in the  where pathway.

sentences: Predicting drug concentrations in PDMS microfluidic organ chips Microfluidic organ-on-a-chip (Organ Chip) cell culture devices are often fabricated using polydimethylsiloxane (PDMS) because it is biocompatible, transparent, elastomeric, and oxygen permeable; however, hydrophobic small molecules can absorb to PDMS, which makes it challenging to predict drug responses. Here, we describe a combined simulation and experimental approach to predict the spatial and temporal concentration profile of a drug under continuous dosing in a PDMS Organ Chip containing two parallel channels separated by a porous membrane that is lined with cultured cells, without prior knowledge of its log P value. First, a three-dimensional finite element simulation of drug loss into the chip was developed that incorporates absorption, adsorption, convection, and diffusion, which predicts changes in drug levels over time and space as a function of potential PDMS diffusion coefficients and log P values. By then experimentally measuring the diffusivity of the compound in PDMS and determining its partition coefficient through mass spectrometric analysis of the drug concentration in the channel outflow, it is possible to estimate the effective log P range of the compound. The diffusion and partition coefficients were experimentally derived for the antimalarial drug and potential SARS-CoV-2 therapeutic, amodiaquine, and incorporated into the model to quantitatively predict the drug-specific concentration profile over time measured in human Lung Airway Chips lined with bronchial epithelium interfaced with pulmonary microvascular endothelium. The same strategy can be applied to any device geometry, surface treatment, or in vitro microfluidic model to predict the spatial and temporal gradient of a drug in 3D without prior knowledge of the partition coefficient or the rate of diffusion in PDMS. Thus, this approach may expand the use of PDMS Organ Chip devices for various forms of drug testing.

sentences: A Biologically Interpretable Graph Convolutional Network to Link Genetic Risk Pathways and Neuroimaging Phenotypes of Disease AO_SCPLOWBSTRACTC_SCPLOWWe propose a novel end-to-end framework for whole-brain and whole-genome imaging-genetics. Our genetics network uses hierarchical graph convolution and pooling operations to embed subject-level data onto a low-dimensional latent space. The hierarchical network implicitly tracks the convergence of genetic risk across well-established biological pathways, while an attention mechanism automatically identifies the salient edges of this network at the subject level. In parallel, our imaging network projects multimodal data onto a set of latent embeddings. For interpretability, we implement a Bayesian feature selection strategy to extract the discriminative imaging biomarkers; these feature weights are optimized alongside the other model parameters. We couple the imaging and genetic embeddings with a predictor network, to ensure that the learned representations are linked to phenotype. We evaluate our framework on a schizophrenia dataset that includes two functional MRI paradigms and gene scores derived from Single Nucleotide Polymorphism data. Using repeated 10-fold cross-validation, we show that our imaging-genetics fusion achieves the better classification performance than state-of-the-art baselines. In an exploratory analysis, we further show that the biomarkers identified by our model are reproducible and closely associated with deficits in schizophrenia.

sentences: A protein microarray-based in vitro transglutaminase assay platform for epitope mapping and peptide discovery: implication for transglutaminase-mediated immunodominant determination Transglutaminases (TGs) are a family of crosslinking enzymes catalyzing the formation of intra- and inter-molecular glutamine-lysine isopeptide bonds in a calcium dependent manner. Activation of transglutaminases is pathogenically associated with severe human diseases including neurodegenerations, cardiovascular diseases, and autoimmune diseases. Although continuous efforts determining the enzymes substrate preference have been witnessed, a high-throughput assay platform with the "omic" efficiency is still missing for the global identification of substrate-specific TG modification sites. In this study we report a protein microarray-based in vitro TG assay platform for rapid and large-scale (up to 30000 reactions per chip) determination of the glutamine (Q)-bearing TG modification motifs. With this platform we identified the Q16 in superoxide dismutase 1 and Q109 in alpha-synuclein as the primary modification sites for tissue transglutaminase (TG2), the most ubiquitous member of the enzyme family. Of particular interest, we found a close match between the modification motifs and published vaccine epitope sequences in alpha-synuclein, implying an essential and intrinsic role transglutaminase might play in the determination of immunodominant epitopes. Our data collectively suggest the glutamine and its follow-up five residues on the C terminal of a protein compose a minimal determinant motif for TG2 modification and the TG2 modification motifs determined by our platform could finally correspond to the substrates epitope sequences in antigen processing. To screen for site-specific interfering peptides and assist gene editing-based protein engineering, we optimized through onchip amino-acid scanning the TG2 modification motif QQIV in the extracellular matrix protein fibronectin and obtained 14 variants with significantly higher TG2 reactivity that might serve as the competitive inhibitor peptides and 1 with lower reactivity. We further confirmed this optimization approach with 12-mer peptides, the longest ones that could be synthesized on the chip. Taken together, our synthetic transglutaminase assay platform might be able to deliver a precise epitope blueprint for immunotherapeutic targeting and provide proof-of-concept and directional studies for TG-based peptide discovery and protein design.

sentences: Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly-discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNAIle and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.

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Phylogenetic analysis of human parainfluenza type 3 virus strains responsible for the outbreak during the COVID-19 pandemic in Seoul, South Korea BackgroundHuman parainfluenza virus 3 (HPIV3) is a major respiratory pathogen that causes acute respiratory infections in infants and children. Since September 2021, an out-of-season HPIV3 rebound has been noted in Korea. The objective of this study was to analyze the molecular characteristics of the HPIV3 strains responsible for the outbreak in Seoul, South Korea.

MethodsA total of 61 HPIV3-positive nasopharyngeal swab specimens were collected between October and November 2021. Using 33 HPIV3-positive specimens, partial nucleotide sequences of the HPIV3 hemagglutinin-neuraminidase (HN) gene were aligned with previously published HN gene sequences for phylogenetic and genetic distance (p-distance) analyses.

ResultsPhylogenetic tree revealed that all Seoul HPIV3 strains grouped within the phylogenetic subcluster C3. However, these strains formed a unique cluster that branched separately from the C3a lineage. This cluster showed 99% bootstrap support with a p-distance < 0.001. Genetic distances within the other C3 lineages ranged from 0.013 (C3a) to 0.023 (C3c). Deduced amino acid sequences of the HN gene revealed four protein substitutions in Seoul HPIV3 strains that have rarely been observed in other reference strains: A22T, K31N, G387S, and E514K.

ConclusionsPhylogenetic analysis of Seoul HPIV3 strains revealed that the strain belonged to a separate cluster within subcluster C3. Genetic distances among strains within subcluster C3 suggest the emergence of a new genetic lineage. The emergence of a new genetic lineage could pose a potential risk of a new epidemic. Further monitoring of the circulating HPIV3 strains is needed to understand the importance of newly discovered mutations.

sentences: Dynamics and heterogeneity of Erk-induced immediate-early gene expression Many canonical signaling pathways exhibit complex time-varying responses, yet how minutes-timescale pulses of signaling interact with the dynamics of transcription and gene expression remains poorly understood. Erk-induced immediate early gene (IEG) expression is a model of this interface, exemplifying both dynamic pathway activity and a rapid, potent transcriptional response. Here, we quantitatively characterize IEG expression downstream of dynamic Erk stimuli in individual cells. We find that IEG expression responds rapidly to acute changes in Erk activity, but only in a sub-population of stimulus-responsive cells. We find that while Erk activity partially predicts IEG expression, a majority of response heterogeneity is independent of Erk and can be rapidly tuned by different mitogenic stimuli and parallel signaling pathways. We extend our findings to an in vivo context, the mouse epidermis, where we observe heterogenous immediate-early gene accumulation in both fixed tissue and single-cell RNA-sequencing data. Our results demonstrate that signaling dynamics can be faithfully transmitted to gene expression and suggest that the signaling-responsive population is an important parameter for interpreting gene expression responses.

sentences: Discovery and Biosynthesis of Persiathiacins: Unusual Polyglycosylated Thiopeptides Active against Multi-drug resistant Tuberculosis Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, in particular their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinobacterial extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2475 and Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus (MRSA) and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 M. In vitro translation assays showed that, like other thiopeptide antibiotics, persiathiacin A targets protein biosynthesis. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster. A cytochrome P450 encoded by this gene cluster catalyses the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyse the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. The discovery of the persiathiacins and their biosynthetic gene cluster thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.

sentences: Chemical inhibition of pathological reactive astrocytes promotes neural protection Disease, injury, and aging induce reactive astrocyte states with pathological functions1-4. In neurodegenerative diseases, inflammatory reactive astrocytes are abundant and contribute to progressive cell loss. Modulating the state or function of these reactive astrocytes thereby represents an attractive therapeutic goal5,6. Leveraging a cellular phenotypic screening platform, we show that chemical inhibitors of HDAC3 effectively block pathological astrocyte reactivity. Inhibition of HDAC3 reduces molecular and functional features of reactive astrocytes in vitro including inflammatory gene expression, cytokine secretion, and antigen presentation. Transcriptional and chromatin mapping studies show that HDAC3 inhibition mediates a switch between pro-inflammatory and anti-inflammatory states, which disarms the pathological functions of reactive astrocytes. Systemic administration of a blood-brain barrier penetrant chemical inhibitor of HDAC3, RGFP966, blocks reactive astrocyte formation and promotes axonal protection in vivo. Collectively, these results establish a platform for discovering chemical modulators of reactive astrocyte states, inform the mechanisms controlling astrocyte reactivity, and demonstrate the therapeutic potential of modulating astrocyte reactivity for neurodegenerative diseases.

sentences: Distributed brain co-processor for tracking electrophysiology and behavior during electrical brain stimulation Early implantable epilepsy therapy devices provided open-loop electrical stimulation without brain sensing, computing, or an interface for synchronized behavioral inputs from patients. Recent epilepsy stimulation devices provide brain sensing but have not yet developed analytics for accurately tracking and quantifying behavior and seizures. Here we describe a distributed brain co-processor providing an intuitive bi-directional interface between patient, implanted neural stimulation and sensing device, and local and distributed computing resources. Automated analysis of continuous streaming electrophysiology is synchronized with patient reports using a hand-held device and integrated with distributed cloud computing resources for quantifying seizures, interictal epileptiform spikes, and patient symptoms during therapeutic electrical brain stimulation. The classification algorithms for interictal epileptiform spikes and seizures were developed and parameterized using long-term ambulatory data from 9 humans and 8 canines with epilepsy, and then implemented prospectively in out-of-sample testing in 2 pet canines and 4 humans with drug resistant epilepsy living in their natural environments.

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Vht hydrogenase is required for hydrogen cycling during nitrogen fixation by the non-hydrogenotrophic methanogen Methanosarcina acetivorans. Methanosarcina acetivorans is the primary model to understand the physiology of methanogens that do not use hydrogenase to consume or produce hydrogen (H2) during methanogenesis. The genome of M. acetivorans encodes putative methanophenazine-reducing hydrogenases (Vht and Vhx), F420-reducing hydrogenase (Frh), and hydrogenase maturation machinery (Hyp), yet cells lack significant hydrogenase activity under all growth conditions tested to date. Thus, the importance of hydrogenase to the physiology of M. acetivorans has remained a mystery. M. acetivorans can fix dinitrogen (N2) using nitrogenase that is documented in bacteria to produce H2 during the reduction of N2 to ammonia. Therefore, we hypothesized that M. acetivorans uses hydrogenase to recycle H2 produced by nitrogenase during N2 fixation. Results demonstrate that hydrogenase expression and activity is higher in N2-grown cells compared to cells grown with fixed nitrogen (NH4Cl). To test the importance of each hydrogenase and the maturation machinery, the CRISPRi-dCas9 system was used to generate separate M. acetivorans strains where transcription of the vht, frh, vhx, or hyp operons is repressed. Repression of vhx and frh does not alter growth with either NH4Cl or N2 and has no effect on H2 metabolism. However, repression of vht or hyp results in impaired growth with N2 but not NH4Cl. Importantly, H2 produced endogenously by nitrogenase is detected in the headspace of culture tubes containing the vht or hyp repression strains. Overall, the results reveal that Vht hydrogenase recycles H2 produced by nitrogenase that is required for optimal growth of M. acetivorans during N2 fixation.

IMPORTANCEThe metabolism of M. acetivorans and closely related Methanosarcinales is thought to not involve H2. Here we show for the first time M. acetivorans is capable of H2 cycling like hydrogenotrophic Methanosarcinales (e.g., Methanosarcina barkeri). However, unlike M. barkeri hydrogenase activity and H2 cycling is tightly regulated in M. acetivorans and is only utilized during N2 fixation to consume H2 production endogenously by nitrogenase. The in vivo production of H2 by nitrogenase during N2 reduction is also demonstrated for the first time in a methanogen. Overall, the results provide new insight into the evolution and diversity of methanogen metabolism and new details about methanogen nitrogenase that could be leveraged for practical applications, such as nitrogenase-dependent production of H2 as a biofuel.

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From partial to whole genome imputation of SARS-CoV-2 for epidemiological surveillance Backgroundthe current SARS-CoV-2 pandemic has emphasized the utility of viral whole genome sequencing in the surveillance and control of the pathogen. An unprecedented ongoing global initiative is increasingly producing hundreds of thousands of sequences worldwide. However, the complex circumstances in which viruses are sequenced, along with the demand of urgent results, causes a high rate of incomplete and therefore useless, sequences. However, viral sequences evolve in the context of a complex phylogeny and therefore different positions along the genome are in linkage disequilibrium. Therefore, an imputation method would be able to predict missing positions from the available sequencing data.

ResultsWe developed impuSARS, an application that includes Minimac, the most widely used strategy for genomic data imputation and, taking advantage of the enormous amount of SARS-CoV-2 whole genome sequences available, a reference panel containing 239,301 sequences was built. The impuSARS application was tested in a wide range of conditions (continuous fragments, amplicons or sparse individual positions missing) showing great fidelity when reconstructing the original sequences. The impuSARS application is also able to impute whole genomes from commercial kits covering less than 20% of the genome or only from the Spike protein with a precision of 0.96. It also recovers the lineage with a 100% precision for almost all the lineages, even in very poorly covered genomes (< 20%)

Conclusionsimputation can improve the pace of SARS-CoV-2 sequencing production by recovering many incomplete or low-quality sequences that would be otherwise discarded. impuSARS can be incorporated in any primary data processing pipeline for SARS-CoV-2 whole genome sequencing.

sentences: EOMES is responsible for WNT memory and can substitute for WNT in mesendoderm specification Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs) called gastruloids. Our previous work demonstrated that during early self-organization of gastruloids, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent Activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening, but rather, acts by inducing the expression of the SMAD2 co-factor, EOMES. Expression of EOMES is sufficient to replace WNT upstream of Activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development.

sentences: Normalization by orientation anisotropy in human V1-V3 An influential account of neuronal responses in primary visual cortex is the normalized energy model. This model is often implemented as a two-stage computation. The first stage is the extraction of contrast energy, whereby a complex cell computes the squared and summed outputs of a pair of linear filters in quadrature phase. The second stage is normalization, in which a local population of complex cells mutually inhibit one another. Because the population includes cells tuned to a range of orientations and spatial frequencies, the result is that the responses are effectively normalized by the local stimulus contrast. Here, using evidence from human functional MRI, we show that the classical model fails to account for the relative responses to two classes of stimuli: straight, parallel, band-passed contours (gratings), and curved, band-passed contours (snakes). The snakes elicit fMRI responses that are about twice as large as the gratings, yet traditional energy models, including normalized energy models, predict responses that are about the same. Here, we propose a computational model, in which responses are normalized not by the sum of the contrast energy, but by the orientation anisotropy, computed as the variance in contrast energy across orientation channels. We first show that this model accounts for differential responses to these two classes of stimuli. We then show that the model successfully generalizes to other band-pass textures, both in V1 and in extrastriate cortex (V2 and V3). We speculate that high anisotropy in the orientation responses leads to larger outputs in downstream areas, which in turn normalizes responses in these later visual areas, as well as in V1 via feedback.

sentences: Functional annotation of human long noncoding RNAs using chromatin conformation data Transcription of the human genome yields mostly long non-coding RNAs (lncRNAs). Systematic functional annotation of lncRNAs is challenging due to their low expression level, cell type-specific occurrence, poor sequence conservation between orthologs, and lack of information about RNA domains. Currently, 95% of human lncRNAs have no functional characterization. Using chromatin conformation and Cap Analysis of Gene Expression (CAGE) data in 18 human cell types, we systematically located genomic regions in spatial proximity to lncRNA genes and identified functional clusters of interacting protein-coding genes, lncRNAs and enhancers. Using these clusters we provide a cell type-specific functional annotation for 7,651 out of 14,198 (53.88%) lncRNAs. LncRNAs tend to have specialized roles in the cell type in which it is first expressed, and to incorporate more general functions as its expression is acquired by multiple cell types during evolution. By analyzing RNA-binding protein and RNA-chromatin interaction data in the context of the spatial genomic interaction map, we explored mechanisms by which these lncRNAs can act.

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Neuronal mechanism of a BK channelopathy in absence epilepsy and movement disorders A growing number of gain-of-function (GOF) BK channelopathy have been identified in patients with epilepsy and paroxysmal movement disorders. Nevertheless, the underlying pathophysiology and corresponding therapeutics remain obscure. Here we utilized a knock-in mouse model carrying human BK-D434G channelopathy to investigate the neuronal mechanism of BK GOF in the pathogenesis of epilepsy and movement disorders. We found that the BK-D434G mice manifest the clinical features of absence epilepsy and exhibit severe motor deficits. BK-D434G mutation causes hyperexcitability of cortical pyramidal neurons and cerebellar Purkinje cells, which contributes to the pathogenesis of absence seizures and the motor defects, respectively. A BK channel blocker paxilline potently suppresses BK-D434G-induced hyperexcitability and effectively mitigates absence seizures in mice. Our study thus uncovered a neuronal mechanism of BK GOF in absence epilepsy and provided the evidence that BK inhibition is a promising therapeutic strategy to mitigate BK GOF-induced neurological disorders.

SignificanceDysfunction of BK channels or BK channelopathy has been increasingly implicated in diverse neurological disorders including epilepsy, movement, cognitive and neurodevelopmental disorders. However, precision medicine to treat BK channelopathy is lacking. Here we characterized a mouse model carrying a gain-of-function BK channelopathy D434G from a large family of patients with absence epilepsy and involuntary movement disorders. The BK-D434G mice resemble the clinical manifestations of absence seizures and exhibit severe motor defects. The hyperexcitability in BK-D434G cortical neurons and cerebellar Purkinje cells underscores the neuronal mechanism of BK gain-of-function induced absence epilepsy and movement disorders. The effectiveness of a BK channel blocker on preventing absence seizures suggests that BK inhibition is a promising strategy to treat gain-of-function BK channelopathy.

sentences: Trick or Treat? Pollinator attraction in Vanilla pompona (Orchidaceae) Natural pollination of species belonging to the pantropical orchid genus Vanilla remains poorly understood. Based on sporadic records, euglossine bees have been observed visiting flowers of Neotropical Vanilla species. Our research aimed at better understanding the pollinator attraction mechanism of the Neotropical species Vanilla pompona, a crop wild relative with valuable traits for vanilla crop improvement programs. Using video footage, we identified floral visitors and examined their behavior. The flowers of V. pompona attracted Eulaema cingulata males, which distinctively displayed two behaviors: floral scent collection and nectar search; with the latter leading to pollen removal. Morphological measurements of floral and visitor traits showed that other Eulaema species may also act as potential pollinators. Additionally, we recorded natural fruit set in three populations and over a period of two years, tested for nectar presence and analyzed floral fragrances through gas chromatography - mass spectrometry. We observed a low natural fruit set (2.42%) and did not detect nectar. Twenty floral volatile compounds were identified, with the dominant compound trans-carvone oxide previously found to attract Eulaema cingulata males. We hypothesize a dual attraction of Eulaema cingulata males to V. pompona flowers, based on floral fragrance reward as the primary long-distance attraction, and food deception for successful pollen removal. Further research confirming this hypothesis is recommended to develop appropriate conservation policies for Vanilla crop wild relatives, which are the primary reserves of this crops genetic variation.

sentences: Superstitious learning of abstract order from random reinforcement Survival depends on identifying learnable features of the environment that predict reward, and avoiding others that are random and unlearnable. However, humans and other animals often infer spurious associations among unrelated events, raising the question of how well they can distinguish learnable patterns from unlearnable events. Here, we tasked monkeys with discovering the serial order of two pictorial sets: a "learnable" set in which the stimuli were implicitly ordered and monkeys were rewarded for choosing the higher-rank stimulus and an "unlearnable" set in which stimuli were unordered and feedback was random regardless of the choice. We replicated prior results that monkeys reliably learned the implicit order of the learnable set. Surprisingly, the monkeys behaved as though some ordering also existed in the unlearnable set, showing consistent choice preference that transferred to novel untrained pairs in this set, even under a preference-discouraging reward schedule that gave rewards more frequently to the stimulus that was selected less often. In simulations, a model-free RL algorithm (Q-learning) displayed a degree of consistent ordering among the unlearnable set but, unlike the monkeys, failed to do so under the preference, discouraging reward schedule. Our results suggest that monkeys infer abstract structures from objectively random events using heuristics that extend beyond stimulus-outcome conditional learning to more cognitive model-based learning mechanisms.

sentences: Structural and biophysical properties of FopA, a major outer membrane protein of Francisella tularensis Francisella tularensis is an extremely infectious pathogen and a category A bioterrorism agent. It causes the highly contagious zoonosis, Tularemia. Currently, FDA approved vaccines against tularemia are unavailable. F. tularensis outer membrane protein A (FopA) is a well-studied virulence determinant and protective antigen against tularemia. It is a major outer membrane protein (Omp) of F. tularensis. However, FopA-based therapeutic intervention is hindered due to lack of complete structural information for membrane localized mature FopA. In our study, we established recombinant expression, monodisperse purification, crystallization and X-ray diffraction ([~]6.5 [A]) of membrane localized mature FopA. Further, we performed bioinformatics and biophysical experiments to unveil its structural organization in the outer membrane. FopA consists of 393 amino acids and has less than 40% sequence identity to known bacterial Omps. Using comprehensive sequence alignments and structure predictions together with existing partial structural information, we propose a two-domain organization for FopA. Circular dichroism spectroscopy and heat modifiability assay confirmed FopA has a {beta}-barrel domain consistent with alphafold2s prediction of an eight stranded {beta}-barrel at the N-terminus. Small angle X-ray scattering (SAXS) and native-polyacrylamide gel electrophoresis revealed FopA purified in detergent micelles is predominantly dimeric. Molecular density derived from SAXS at 31 [A] shows putative dimeric N-terminal {beta}-barrels surrounded by detergent corona and connected to C-terminal domains via flexible linker. Disorder analysis predicts N- and C-terminal domains are interspersed by a long intrinsically disordered region and alphafold2 predicts this region to be largely unstructured. Taken together, we propose a dimeric, two-domain organization of FopA in the outer membrane: the N-terminal {beta}-barrel is membrane embedded, provides dimerization interface and tethers to membrane extrinsic C-terminal domain via long flexible linker. Structure determination of membrane localized mature FopA is essential to understand its role in pathogenesis and develop anti-tularemia therapeutics. Our results pave the way towards it.

sentences: Arid5a mediates an IL-17-dependent pathway that drives autoimmunity but not antifungal host defense IL-17 contributes to the pathogenesis of certain autoimmune diseases, but conversely is essential for host defense against fungi. Antibody-based biologic drugs that neutralize IL-17 are effective in autoimmunity but can be accompanied by adverse side effects. Candida albicans is a commensal fungus that is the primary causative agent of oropharyngeal and disseminated candidiasis. Defects in IL-17 signaling cause susceptibility to candidiasis in mice and humans. A key facet of IL-17 receptor signaling involves RNA binding proteins (RBP), which orchestrate the fate of target mRNA transcripts. In tissue culture models we showed that the RBP AT-rich interacting protein 5a (Arid5a) promotes the stability and/or translation of multiple IL-17-dependent mRNAs. Moreover, during OPC, Arid5a is elevated within the oral mucosa in an IL-17-dependent manner. However, the contribution of Arid5a to IL-17-driven events in vivo is poorly defined. Here, we used CRISPR/Cas9 to generate mice lacking Arid5a. Arid5a-/- mice were fully resistant to experimental autoimmune encephalomyelitis (EAE), an autoimmune setting in which IL-17 signaling drives pathology. Surprisingly, Arid5a-/- mice were resistant to OPC and systemic candidiasis, similar to immunocompetent WT mice and contrasting with mice defective in IL-17 signaling. Therefore, Arid5a-dependent signals mediate pathology in autoimmunity yet are not required for immunity to candidiasis, indicating that selective targeting of IL-17 signaling pathway components may be a viable strategy for development of therapeutics that spare IL-17-driven host defense.

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Self-organization of a doubly asynchronous irregular network state for spikes and bursts Cortical pyramidal cells (PCs) have a specialized dendritic mechanism for the generation of bursts, suggesting that these events play a special role in cortical information processing. In vivo, bursts occur at a low, but consistent rate. Theory suggests that this network state increases the amount of information they convey. However, because burst activity relies on a threshold mechanism, it is rather sensitive to dendritic input levels. In spiking network models, network states in which bursts occur rarely are therefore typically not robust, but require fine-tuning. Here, we show that this issue can be solved by a homeostatic inhibitory plasticity rule in dendrite-targeting interneurons that is consistent with experimental data. The suggested learning rule can be combined with other forms of inhibitory plasticity to self-organize a network state in which both spikes and bursts occur asynchronously and irregularly at low rate. Finally, we show that this network state creates the network conditions for a recently suggested multiplexed code and thereby indeed increases the amount of information encoded in bursts.

Author summaryThe language of the brain consists of sequences of action potentials. These sequences often contain bursts, short "words" consisting of a few action potentials in rapid succession. Bursts appear to play a special role in the brain. They indicate whether a stimulus was perceived or missed, and they are very effective drivers of synaptic plasticity, the neural substrate of learning. Bursts occur rarely, but consistently, a condition that is thought to maximize the amount of information they can communicate. In our article, we argue that this condition is far from self-evident, but requires very special circumstances. We show that these circumstances can be reached by homeostatic inhibitory plasticity in certain inhibitory neuron types. This may sound complicated, but basically works just like a thermostat. When bursts occur too often, inhibition goes up and suppresses them. When they are too rare, inhibition goes down and thereby increases their number. In computer simulations, we show that this simple mechanism can create circumstances akin to those in the brain, and indeed allows bursts to convey information effectively. Whether this mechanism is indeed used by the brain now remains to be tested by our experimental colleagues.

sentences: Single-cell immune repertoire sequencing of B and T cells in murine models of infection and autoimmunity Adaptive immune repertoires are composed by the ensemble of B and T cell receptors (BCR, TCR) within an individual and reflect both past and current immune responses. Recent advances in single-cell sequencing enable recovery of the complete adaptive immune receptor sequences in addition to transcriptional information. Such high-dimensional datasets enable the molecular quantification of clonal selection of B and T cells across a wide variety of conditions such as infection and disease. Due to costs, time required for the analysis and current practices of academic publishing, small-scale sequencing studies are often not made publicly available, despite having informative potential to elucidate immunological principles and guide future-studies. Here, we performed single-cell sequencing of B and T cells to profile clonal selection across murine models of viral infection and autoimmune disease. Specifically, we recovered transcriptome and immune repertoire information for polyclonal T follicular helper cells following acute and chronic viral infection, CD8+ T cells with binding specificity restricted to two distinct peptides of lymphocytic choriomeningitis virus, and B and T cells isolated from the nervous system in the context of experimental autoimmune encephalomyelitis. We could relate repertoire features such as clonal expansion, germline gene usage, and clonal convergence to cell phenotypes spanning activation, memory, naive, antibody secretion, T cell inflation, and regulation. Together, this dataset provides a resource for experimental and computational immunologists that can be integrated with future single-cell immune repertoire and transcriptome sequencing datasets.

sentences: Cryo-EM structure of an active central apparatus Accurately regulated ciliary beating in time and space is critical for diverse cellular activities, which impact the survival and development of nearly all eukaryotic species. An essential beating regulator is the conserved central apparatus (CA) of motile cilia, composed of a pair of microtubules (C1 and C2) associated with hundreds of protein subunits per repeating unit. It is largely unclear how CA plays its regulatory roles in ciliary motility. Here, we present high-resolution structures of Chlamydomonas reinhardtii CA by cryo-electron microscopy (cryo-EM) and its dynamic conformational behavior in multiple scales. The structures show how functionally related projection proteins of CA are clustered onto a spring-shaped scaffold of armadillo-repeat proteins, facilitated by elongated rachis-like proteins. The two halves of CA are brought together by elastic chain-like bridge proteins to achieve coordinated activities. We captured an array of kinesin-like protein (KLP1) in two different stepping states, which are actively correlated with beating wave propagation of cilia. These findings establish a structural framework for understanding the role of CA in cilia.

sentences: Sequence analysis paradigm shift reveals unsuspected semantic properties of species proteomes Alignment-based methods dominate molecular biology. However, by primarily allowing one-to-one comparisons, these methods are focused on a gene-centered viewpoint and lack the broad context essential to analyze how complex biological systems function and evolve. In actuality, a gene is part of genome where more than one sequence contributes to the functional network and evolutionary trajectory of the cell. The need for conservation of established interactions, is arguably more important to the evolutionary success of species than conservation of individual function. To test whether such contextual information exists, a distributional semantics method - Latent Semantic Analysis (LSA), was applied to thousands of species proteomes. Using natural language processing, Latent Taxonomic Signatures (LTSs) were identified that outperformed existing alignment-based BLAST methods when random protein sequences were being mapped to annotated taxonomy according to GenBank. LTSs are a novel proteome distributed feature, suggesting the existence of evolutionary constraints imposed on individual proteins by their proteome context. Even orphan proteins are exhibiting LTSs, which makes their uniqueness linked to a specific taxonomic level questionable. Unlike more simple bias, LTSs represent a self-similarity pattern, where random sets of species proteins show the same statistical properties of a complete proteome at many scales. Natural language processing and machine learning provide insights not easily discernable using alignment based methods suggestive there is more to species related differences than just translational optimization.

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Impact of stimulation location relative to grey and white matter on single pulse electrical stimulation responses in the human brain BackgroundElectrical neuromodulation is an increasingly common therapy for a wide variety of neuropsychiatric diseases. Unfortunately, therapeutic efficacy is inconsistent, possibly a result of our limited understanding of the mechanisms and the massive stimulation parameter space.

Objective/HypothesisTo better understand the role different parameters play in inducing a response, we systematically examined single pulse-induced cortico-cortico evoked potentials (CCEP) as a function of stimulation amplitude, duration and location in the brain and relative to grey and white matter.

MethodsWe measured voltage peak amplitudes and area under the curve of intracranially recorded stimulation responses as a function of distance from the stimulation site, pulse width, current injected, location relative to grey and white matter, and brain region stimulated (N=52, n=719 stimulation sites).

ResultsIncreasing stimulation pulse width increased response values near the stimulation location. Increasing stimulation amplitude (current) increased responses nonlinearly. Locally (<15 mm from the stimulation site), stimulation closer to the grey matter-white matter boundary induced larger responses. In contrast, for distant sites (>15 mm), white matter stimulation consistently produced larger responses than stimulation in or near grey matter. These relationships were different between cingulate, lateral prefrontal, and lateral temporal cortical stimulation.

ConclusionThese results demonstrate the importance of location and stimulation parameters in inducing a specific output and indicate that a stronger local response may require stimulation in the grey-white boundary while stimulation in the white matter may be needed for network activation, suggesting that stimulation location can be tailored for a specific outcome, key to informed neuromodulatory therapy.

HighlightsO_LIIntracranial single pulse electrical stimulation (SPES) response increases with increased pulse duration mostly near the stimulation site
C_LIO_LISPES response varies nonlinearly with injected current with an effect of distance from the stimulation site.
C_LIO_LISPES near the grey-white boundary and 90{degrees} to the nearest cortical axis induces larger local responses, but white matter stimulation produces larger distant responses.
C_LIO_LIThe relationship between SPES location and responses depends on brain region stimulated
C_LI

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A predictive processing model of episodic memory and time perception Human perception and experience of time is strongly influenced by ongoing stimulation, memory of past experiences, and required task context. When paying attention to time, time experience seems to expand; when distracted, it seems to contract. When considering time based on memory, the experience may be different than in the moment, exemplified by sayings like "time flies when youre having fun". Experience of time also depends on the content of perceptual experience - rapidly changing or complex perceptual scenes seem longer in duration than less dynamic ones. The complexity of interactions between attention, memory, and perceptual stimulation is a likely reason that an overarching theory of time perception has been difficult to achieve. Here, we introduce a model of perceptual processing and episodic memory that makes use of hierarchical predictive coding, short-term plasticity, spatio-temporal attention, and episodic memory formation and recall, and apply this model to the problem of human time perception. In an experiment with ~ 13, 000 human participants we investigated the effects of memory, cognitive load, and stimulus content on duration reports of dynamic natural scenes up to ~ 1 minute long. Using our model to generate duration estimates, we compared human and model performance. Model-based estimates replicated key qualitative biases, including differences by cognitive load (attention), scene type (stimulation), and whether the judgement was made based on current or remembered experience (memory). Our work provides a comprehensive model of human time perception and a foundation for exploring the computational basis of episodic memory within a hierarchical predictive coding framework.

Author summaryExperience of the duration of present or past events is a central aspect of human experience, the underlying mechanisms of which are not yet fully understood. In this work, we combine insights from machine learning and neuroscience to propose a combination of mathematical models that replicate human perceptual processing, long-term memory, attention, and duration perception. Our computational implementation of this framework can process information from video clips of ordinary life scenes, record and recall important events, and report the duration of these clips. To assess the validity of our proposal, we conducted an experiment with ~ 13, 000 human participants. Each was shown a video between 1-64 seconds long and reported how long they believed it was. Reports of duration by our computational model qualitatively matched these human reports, made about the exact same videos. This was true regardless of the video content, whether time was actively judged or based on memory of the video, or whether the participants focused on a single task or were distracted - all factors known to influence human time perception. Our work provides the first model of human duration perception to incorporate these diverse and complex factors and provides a basis to probe the deep links between memory and time in human experience.

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Ancestral sequence reconstructions evaluated by extant sequence cross-validation Ancestral sequence reconstruction (ASR) has become widely used to analyze the properties of ancient biomolecules and to elucidate the mechanisms of molecular evolution. By recapitulating the structural, mechanistic, and functional changes of proteins during their evolution, ASR has been able to address many fundamental and challenging evolutionary questions where more traditional methods have failed. Despite the tangible successes of ASR, the accuracy of its reconstructions is currently unknown, because it is generally impossible to compare resurrected proteins to the true ancient ancestors that are now extinct. Which evolutionary models are the best for ASR? How accurate are the resulting inferences? Here we answer these questions by applying cross-validation (CV) to sets of aligned extant sequences.

To assess the adequacy of a chosen evolutionary model for predicting extant sequence data, our column-wise CV method iteratively cross-validates each column in an alignment. Unlike other phylogenetic model selection criteria, this method does not require bias correction and does not make restrictive assumptions commonly violated by phylogenetic data. We find that column-wise CV generally provides a more conservative criterion than the AIC by preferring less complex models.

To validate ASR methods, we also apply cross-validation to each sequence in an alignment by reconstructing the extant sequences using ASR methodology, a method we term "extant sequence reconstruction" (ESR). We can thus quantify the accuracy of ASR methodology by comparing ESR reconstructions to the corresponding true sequences. We find that a common measure of the quality of a reconstructed sequence, the average probability of the sequence, is indeed a good estimate of the fraction of the sequence that is correct when the evolutionary model is accurate or overparameterized. However, the average probability is a poor measure for comparing reconstructions, because more accurate phylogenetic models typically result in reconstructions with lower average probabilities. In contrast, the entropy of the reconstructed distribution is a reliable indicator of the quality of a reconstruction, as the entropy provides an accurate estimate of the log-probability of the true sequence. Both column-wise CV and ESR are useful methods to validate evolutionary models used for ASR and can be applied in practice to any phylogenetic analysis of real biological sequences.

sentences: Is galactose a hormetic sugar? Evidence from rat hippocampal redox regulatory network Galactose is a ubiquitous simple monosaccharide with yet incompletely understood biochemical and physiological role. Most of what we currently know about galactose is based on induction from the research on inherited disorders of galactose metabolism and animal models that exploit galactose-induced oxidative stress to model aging in rodents, however, recent evidence also demonstrates unique properties of galactose to conserve cellular function during the periods of starvation, and prevent and alleviate cognitive deficits in a rat model of sporadic Alzheimers disease. Here, we try to understand the molecular background of both detrimental and beneficial effects of galactose by exploring the acute systemic and hippocampal biochemical changes upon oral administration of galactose solution focusing primarily on the components of the redox regulatory network (RRN). Although orogastric gavage of galactose solution (200 mg/kg) was insufficient to induce systemic RRN disbalance in the first two hours upon administration, analysis of hippocampal RRN revealed a mild pro-oxidative shift accompanied by a paradoxical increase in tissue reductive capacity, suggesting overcompensation of endogenous antioxidant systems in the response to the pro-oxidative stimulus. The more thorough analysis revealed that galactose-induced increment of reductive capacity was accompanied by inflation of the hippocampal pool of nicotinamide adenine dinucleotide phosphates indicating ROS detoxification through disinhibition of the oxidative pentose phosphate pathway flux, reduced neuronal activity, and upregulation of Leloir pathway gatekeeper enzyme galactokinase-1. Based on the observed findings, and in the context of previous work on galactose, we propose a hormetic hypothesis of galactose action suggesting that the protective effects of galactose might be inseparable from its pro-oxidative effects at the biochemical level.

sentences: Tyr is Responsible for the Cctq1a QTL and Links Developmental Environment to Central Corneal Thickness Determination Central corneal thickness is a quantitative trait with important associations to human health. In a phenotype-driven approach studying corneal thickness of congenic derivatives of C57BLKS/J and SJL/J mice, the critical region for a quantitative trait locus influencing corneal thickness, Cctq1a, was delimited to a 10-gene interval. Exome sequencing, RNAseq, and studying independent mutations eliminated multiple candidate genes and confirmed one. Though the causative gene, Tyr, has no obvious direct function in the transparent cornea, studies with multiple alleles on matched genetic backgrounds, both in isolation and genetic complementation crosses, confirmed allelism of Tyr-Cctq1a; albino mice lacking Tyr function had thin corneas. Albino mice also had increased axial length. Because albinism exposes eyes to increased light, the effect of dark-rearing was tested and found to rescue central corneal thickness. In sum, the results point to an epiphenomenon; developmental light exposure interacts with genotype as an important determinate of adult corneal thickness.

sentences: Differences in Ion-RNA binding modes due to charge density variations explain the stability of RNA in monovalent salts The stability of RNA increases as the charge density of the alkali metal cations increases. The molecular mechanism for the stability does not exist. To fill this gap, we performed all-atom molecular dynamics (MD) pulling simulations to dissect the microscopic origin of this phenomenon. We first established that the free energy landscape obtained in the simulations is in excellent agreement with the single-molecule optical tweezer experiments. The origin of the stronger stability in Na+ compared to K+ is found to be due to the differences in the charge-density related binding modes. The smaller hydrated Na+ ion preferentially binds to the highly charged phosphates. In contrast, the larger K+ ions interact with the major grooves. As a result, the electrostatic repulsion between the phosphate groups is reduced more effectively by Na+ ions. Because the proposed mechanism is generic, we predict that the same conclusions are valid for divalent alkaline earth metal cations.

sentences: Transcranial magnetic stimulation of the occipital cortex interferes with foot movements in blind individuals Research in blind individuals has shown that after visual loss, the occipital cortex can be reorganized and repurposed for nonvisual perception and cognitive functions. However, no studies have directly examined the involvement of the visual cortex in motor function. Here, we show that a rhythmic foot movement performed by blind individuals can be disrupted by transcranial magnetic stimulation (TMS) to their primary and secondary visual cortex (V1/V2). This disruptive effect of TMS was absent for sighted participants. Our result suggests that the visual cortex of blind individuals is involved in sensorimotor control. This is the first experimental evidence that functional repurposing of the human visual cortex is not be restricted to perception and cognitive functions, but also extends to motor function.

sentences: Diversity of cave Phlebotomines (Diptera: Psychodidae) from a Colombian cave Sandflies are vector species of Leishmania, among many other pathogens, with a global distribution and a variety of ecological niches. Previous samplings have found that karstic formations (i.e., caves and folds formed by the erosion of limestone) serve as a natural habitat to sandfly species. The majority of samplings of cave sandfly diversity have occurred in Brazil and to date none have studied the species composition in a cave in the Northern Andes. We collected sandflies in the Cave "Los Guacharos", in the state of Antioquia, Colombia. The sampling was carried out during two consecutive nights in September 2019. CDC-type light traps were installed inside the cavern and in other surrounding karst systems (caves and folds). In total, we identified 18 species of sandfly from the cave and surrounding karst systems, including three new records for Colombia (Bichromomyia olmeca nociva, Brumptomyia brumpti, and Warileya leponti), and provide the first karstic reports for four other species (Lutzomyia gomezi, Lutzomyia hartmanni, Pintomyia ovallesi, and Psychodopygus panamensis). We then used the results of our survey and published literature to test two hypotheses. First, that sandfly diversity in Neotropical caves is richest nearer to the equator and second that there is a phylogenetic signal of karstic habitat use in sandflies. Counter to our predictions, we found no evidence that diversity follows a latitudinal gradient. Further, we find no evidence of a phylogenetic signal of karstic habitat use, instead finding that the use of caves likely evolved multiple times across several genera. Our results highlight the importance of a wide sampling to understand the natural habitat of sandflies and other disease vectors.

sentences: Evidence for a conserved queen-worker genetic toolkit across slave-making ants and their ant hosts The ecological success of social Hymenoptera (ants, bees, wasps) depends on the division of labour between the queen and workers. Each caste is highly specialised in its respective function in morphology, behaviour and life-history traits, such as lifespan and fecundity. Despite strong defences against alien intruders, insect societies are vulnerable to social parasites, such as workerless inquilines or slave-making (dulotic) ants. Here, we investigate whether gene expression varies in parallel ways between lifestyles (slave-making versus host ants) across five independent origins of ant slavery in the "Formicoxenus-group" of the ant tribe Crematogastrini. As caste differences are often less pronounced in slave-making ants than non-parasitic ants, we also compare the transcriptomes of queens and workers in these species. We demonstrate a substantial overlap in expression differences between queens and workers across taxa, irrespective of lifestyle. Caste affects the transcriptomes much more profoundly than lifestyle, as indicated by 37 times more genes being linked to caste than to lifestyle and by multiple caste-associated gene modules with strong connectivity. However, several genes and one gene module are linked to the slave-making lifestyle across the independent origins, pointing to some evolutionary convergence. Finally, we do not find evidence for an interaction between caste and lifestyle, indicating that caste differences remain consistent even when species switch to a parasitic lifestyle. Our findings are a strong indication for the existence of a core set of genes whose expression is linked to the queen and worker caste in this ant taxon, supporting the "genetic toolkit" hypothesis.

sentences: Slice-direction geometric distortion evaluation and correction with reversed slice-select gradient acquisitions Accurate spatial alignment of MRI data acquired across multiple contrasts in the same subject is often crucial for data analysis and interpretation, but can be challenging in the presence of geometric distortions that differ between acquisitions. It is well known that single-shot echo-planar imaging (EPI) acquisitions suffer from distortion in the phase-encoding direction due to B0 field inhomogeneities arising from tissue magnetic susceptibility differences and other sources, however there can be distortion in other encoding directions as well in the presence of strong field homogeneities. High-resolution ultrahigh-field MRI typically uses low bandwidth in the slice-encoding direction to acquire thin slices and, when combined with the pronounced B0 inhomogeneities, is prone to an additional geometric distortion in the slice direction as well. Here we demonstrate a presence of this slice distortion in high-resolution 7T EPI acquired with a novel pulse sequence allowing for the reversal of the slice-encoding gradient polarity that enables the acquisition of pairs of images with equal magnitudes of distortion in the slice direction but with opposing polarities. We also show that the slice-direction distortion can be corrected using gradient reversal-based method applying the same software used for conventional corrections of phase-encoding direction distortion.

sentences: Anther cones increase pollen release in buzz-pollinated Solanum flowers The widespread evolution of tube-like anthers releasing pollen from apical pores is associated with buzz pollination, in which bees vibrate flowers to remove pollen. The mechanical connection among anthers in buzz-pollinated species varies from loosely held conformations, to anthers tightly held together with trichomes or bio-adhesives forming a functionally joined conical structure (anther cone). Joined anther cones in buzz-pollinated species have evolved independently across plant families and via different genetic mechanisms, yet their functional significance remains mostly untested. We used experimental manipulations to compare vibrational and functional (pollen release) consequences of joined anther cones in three buzz-pollinated species of Solanum (Solanaceae). We applied bee-like vibrations to focal anthers in flowers with ("joined") and without ("free") experimentally created joined anther cones, and characterised vibrations transmitted to other anthers and the amount of pollen released. We found that joined anther architectures cause non-focal anthers to vibrate at higher amplitudes than free architectures. Moreover, in the two species with naturally loosely held anthers, anther fusion increases pollen release, while in the species with a free but naturally compact architecture it does not. We discuss hypotheses for the adaptive significance of the convergent evolution of joined anther cones.

sentences: Feedback alignment with weight normalization can provide a biologically plausible mechanism for learning The mechanism by which plasticity in millions of synapses in the brain is orchestrated to achieve behavioral and cognitive goals is a fundamental question in neuroscience. In this regard, insights from learning methods in artificial neural networks (ANNs) and in particular the idea of backpropagation (BP) seem inspiring. However, the implementation of BP requires exact matching of forward and backward weights, which is unrealistic given the known connectivity pattern in the brain (known as "weight transport problem"). Notably, it is recently shown that under certain conditions, error BackPropagation Through Random backward Weights (BP-TRW), can lead to partial alignment of forward and backward weights overtime (feedback alignment or FA) and result in surprisingly good accuracies in simple classification tasks using shallow ANNs. In this work, we took a closer look at FA to find out why it occurs when using BP-TRW and explored ways to boost it for deep ANNs. We first show that the gradual alignment of forward and backward weights arises from the successive application of BP-TRW update rule on forward weights regardless of learning or loss function if error signals and outputs of neurons satisfy certain conditions such as when they are autocorrelated. Moreover, we show that FA in deeper networks can be improved significantly by applying a biologically-inspired weight normalization (WN) to the input weights of each neuron. In addition, WN can improve the performance of both BP and BP-TRW when class labels are changed across time, an under-explored phenomenon in ANNs which is crucial for flexible learning in the brain in everyday life. With WN, BP-TRW test accuracy can almost match that of BP following class label changes. Altogether, our results portray a clearer picture of the FA mechanism and provide evidence for how learning can occur using BP-like mechanisms while abiding by biological limits on synaptic weights.

sentences: Mesoscopic oblique plane microscopy (Meso-OPM) - enabling large-scale 4D isotropic cellular resolution imaging with a diffractive light sheet Fundamental understanding of large-scale dynamic connectivity within a living organism requires volumetric imaging over a large field of view (FOV) at biologically relevant speed and resolution. However, most microscopy methods make trade-offs between FOV and resolution or imaging speed, making it challenging to observe highly dynamic processes at cellular resolution across mesoscopic scales (e.g., whole zebrafish larva). To overcome this limitation, we have developed mesoscopic oblique plane microscopy (Meso-OPM) with a diffractive light sheet. By augmenting the illumination angle of the light sheet with a transmission grating, the axial resolution was improved ~7-fold over existing methods and ~2-fold beyond the diffraction limitation of the primary objective lens. We demonstrated an unprecedented FOV up to 5.4 x 3.3 mm with resolution of 2.5x 3 x 6 m, allowing volumetric imaging of cellular structures in an uncleared brain slice with a single scan. Applying Meso-OPM for in vivo imaging of zebrafish larvae, we report here the first whole fish volumetric recordings of neuronal activity at 2 Hz volume rate and the first example of whole fish volumetric recordings of blood flow dynamics at 5 Hz with isotropic cellular resolution.

sentences: EXPERT: Transfer Learning-enabled context-aware microbial source tracking Microbial source tracking quantifies the potential origin of microbial communities, facilitates better understanding of how the taxonomic structure and community functions were formed and maintained. However, previous methods involve a tradeoff between speed and accuracy, and have encountered difficulty in source tracking under many context-dependent settings. Here, we present EXPERT for context-aware microbial source tracking, in which we adopted a Transfer Learning approach to profoundly elevate and expand the applicability of source tracking, enabling biologically informed novel microbial knowledge discovery. We demonstrate that EXPERT can predict microbial sources with performance superior to other methods in efficiency and accuracy. More importantly, we demonstrate EXPERTs context-aware ability on several applications, including tracking the progression of infant gut microbiome development and monitoring the changes of gut microbiome for colorectal cancer patients. Broadly, transfer learning enables accurate and context-aware microbial source tracking and has the potential for novel microbial knowledge discovery.

sentences: AKAP79 enables calcineurin to directly suppress protein kinase A activity Interplay between the second messengers cAMP and Ca2+ is a hallmark of dynamic cellular processes. A common motif is the opposition of the Ca2+-sensitive phosphatase calcineurin and the major cAMP receptor, protein kinase A (PKA). Calcineurin dephosphorylates sites primed by PKA to bring about changes including synaptic long-term depression (LTD). AKAP79 supports signaling of this type by anchoring PKA and calcineurin in tandem. In this study, we discovered that AKAP79 increases the rate of calcineurin dephosphorylation of type II PKA regulatory subunits by an order of magnitude. Fluorescent PKA activity reporter assays, supported by kinetic modeling, show how AKAP79-enhanced calcineurin activity enables suppression of PKA without altering cAMP levels by increasing PKA catalytic subunit capture rate. Experiments with hippocampal neurons indicate that this mechanism contributes towards LTD. This non- canonical mode of PKA regulation may underlie many other cellular processes.

sentences: A synaptic novelty signal in the dentate gyrus supports switching hippocampal attractor networks from generalization to discrimination Episodic memory formation and recall are complementary processes that put conflicting requirements on neuronal computations in the hippocampus. How this challenge is resolved in hippocampal circuits is unclear. To address this question, we obtained in vivo whole-cell patch-clamp recordings from dentate gyrus granule cells in head-fixed mice trained to explore and distinguish between familiar and novel virtual environments. We find that granule cells consistently show a small transient depolarization of their membrane potential upon transition to a novel environment. This synaptic novelty signal is sensitive to local application of atropine, indicating that it depends on metabotropic acetylcholine receptors. A computational model suggests that the observed transient synaptic response to novel environments may lead to a bias in the granule cell population activity, which can in turn drive the downstream attractor networks to a new state, thereby favoring the switch from generalization to discrimination when faced with novelty. Such a novelty-driven cholinergic switch may enable flexible encoding of new memories while preserving stable retrieval of familiar ones.

sentences: Dissecting gating mechanisms of Orai calcium channel paralogs using constitutively active Orai mutants that mimic STIM1-gated state In humans, there are three paralogs of the Orai Ca2+ channel, which lie at the heart of the store-operated calcium entry (SOCE) machinery. While the STIM-mediated gating mechanism of Orai channels is still being actively investigated, several artificial and natural variants are known to cause constitutive activity of the human Orai1 channel. Surprisingly, little is known about the conservation of the gating mechanism among the different human Orai paralogs and orthologs in other species. In our work, we show that the mutation corresponding to the activating mutation H134A in transmembrane helix 2 (TM2) of human Orai1 also activates Orai2 and Orai3, likely via a similar mechanism. However, this cross-paralog conservation does not apply to the "ANSGA" nexus mutations in TM4 of human Orai1 which mimic the STIM1-activated state of the channel. Investigating the mechanistic background of these differences, we identified two positions, H171 and F246 in human Orai1, which directly control the channel activation triggered by the "ANSGA" mutations in Orai1. Our results shed new light on these important gating checkpoints and show that the gating mechanism of the Orai channels is affected by multiple factors that are not necessarily evolutionarily conserved, such as the TM4-TM3 coupling.

sentences: Microbial induced calcite precipitation can consolidate martian and lunar regolith simulants We demonstrate that Microbial Induced Calcite Precipitation (MICP) can be utilized for creation of consolidates of Martian Simulant Soil (MSS) and Lunar Simulant Soil (LSS) in the form of a  brick. A urease producer bacteria, Sporosarcina pasteurii, was used to induce the MICP process for the both simulant soils. An admixture of guar gum as an organic polymer and NiCl2, as bio-catalyst to enhance urease activity, was introduced to increase the compressive strength of the biologically grown bricks. A casting method was utilized for a slurry consisting of the appropriate simulant soil and microbe; the slurry over a few days consolidated in the form of a  brick of the desired shape. In case of MSS, maximum strength of 3.3 MPa was obtained with 10mM NiCl2 and 1% guar gum supplementation whereas in case of LSS maximum strength of 5.65 MPa was obtained with 1% guar gum supplementation and 10mM NiCl2. MICP mediated consolidation of the simulant soil was confirmed with field emission scanning electron microscopy (FESEM) and X-ray diffraction (XRD). Our work demonstrates a biological approach with an explicit casting method towards manufacturing of consolidated structures using extra-terrestrial regolith simulant; this is a promising route for in situ development of structural elements on the extra-terrestrial habitats.

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Microbial loop of Proterozoic ocean analogue Meromictic Lake Cadagno, an ancient ocean analogue, is known for its permanent stratification and persistent anoxygenic microbial bloom within the chemocline. Although the anaerobic microbial ecology of the lake has been extensively studied for at least 25 years, a comprehensive picture of the microbial food web linking the bacterial layer to phytoplankton and viruses, with explicit measures of primary and secondary production, is still missing. This study sought to better understand the abundances and productivity of microbes in the context of nutrient biogeochemical cycling across the stratified zones of Lake Cadagno. Photosynthetic pigments and chloroplast 16S rRNA gene phylogenies suggested the presence of eukaryotic phytoplankton through the water column. Evidence supported high abundances of Ankyra judayi, a high-alpine adapted chlorophyte, in the oxic mixolimnion where oxygenic-primary production peaked. Through the no- and low-oxygen chemocline and monimolimnion, chlorophytes related to Closteriopsis acicularis, a known genus of meromictic lakes, and Parachlorella kessleri were observed. Chromatium, anoxygenic phototrophic sulfur bacteria, dominated the chemocline along with Lentimicrobium, a genus of known fermenters whose abundance was newly reported in Lake Cadagno. Secondary production peaked in the chemocline suggesting primary producers depend on hetrotrophs for nutrient remineralization. As previously observed, sulfur reducing bacteria (SRBs), especially Desulfocapsa and Desulfobulbus, were present in the chemocline and anoxic monimolimnion. Virus-to-microbe ratios (VMR) peaked in the zone of phytoplankton, yet were at a minimum at the peak of Chromatium. These dynamic trends suggest viruses may play a role in the modulation of oxygenic and anoxygenic photo- and chemosynthesis in Lake Cadagno and other permanently stratified systems.

ImportanceAs a window to the past, the study offers insights into the role of microbial guilds of Proterozoic ocean chemoclines in the production and recycling of organic matter of sulfur- and ammonia-containing ancient oceans. The new observations described here suggest that eukaryotic algae were persistent in the low oxygen upper-chemocline in association with purple and green sulfur bacteria in the lower half of the chemocline. Further, this study provides the first insights into Lake Cadagno viral ecology. High viral abundances suggested viruses may be essential components of the chemocline where their activity may result in the release and recycling of organic matter. The framework developed in this study through the integration of diverse geochemical and biological data types lays the foundation for future studies to quantitatively resolve the processes performed by discrete populations comprising the microbial loop in this early anoxic ocean analogue.

sentences: Human blood contains circulating cell-free mitochondria, but are they really functional? Dache et al. (2020, FASEB J. 15, e2002338-15) recently reported the presence of respiratory-competent cell-free mitochondria in human blood (up to 3.7 x 106 per mL of blood), providing exciting perspectives on the potential role of these extra-cellular mitochondria. While their evidence for the presence of cell-free mitochondria in human blood is compelling, their conclusion that these cell-free mitochondria are respiratory-competent or functional has to be re-evaluated. To this end, we evaluated the functionality of cell-free mitochondria in human blood using high-resolution respirometry and mitochondria extracted from platelets of the same blood samples as positive controls. While cell-free mitochondria were present in human plasma (i.e. significant MitoTracker Green Fluorescence and complex IV activity), there was no evidence suggesting that their mitochondrial electron transport system (ETS) was functional (i.e. respiration rate not significantly different from 0; no significant responses to ADP, uncoupler or mitochondrial inhibitors oligomycin and antimycin A). Yet, in vitro complex IV activity was detectable and even slightly higher than levels found in mitochondria extracted from platelets, suggesting that cell-free mitochondria in human blood are likely to only retain a non-functional part of the electron transport system. Despite being unlikely to be fully functional in the narrow-sense (i.e. capable of oxidative phosphorylation), circulating cell-free mitochondria may have significant physiological roles that remain to be elucidated.

sentences: Metapopulations with habitat modification Across the tree of life, organisms modify their local environment, rendering it more or less hospitable for other species. Despite the ubiquity of these processes, simple models that can be used to develop intuitions about the consequences of widespread habitat modification are lacking. Here we extend the classic Levins metapopulation model to a setting where each of n species can colonize patches connected by dispersal, and when patches are vacated via local extinction, they retain a "memory" of the previous occupant--modeling habitat modification. While this model can exhibit a wide range of dynamics, we draw several overarching conclusions about the effects of modification and memory. In particular, we find that any number of species may potentially coexist, provided that each is at a disadvantage when colonizing patches vacated by a conspecific. This notion is made precise through a quantitative stability condition, which provides a way to unify and formalize existing conceptual models. We also show that when patch memory facilitates coexistence, it generically induces a positive relationship between diversity and robustness (tolerance of disturbance). Our simple model provides a portable, tractable framework for studying systems where species modify and react to a shared landscape.

sentences: Colistin resistance in Acinetobacter baumannii is driven by multiple genomic traits: Evaluating the role of ISAba1-driven eptA overexpression among Indian isolates Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrAB two component system and overexpression of eptA due to upstream insertion of ISAba1 play a major role.To characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 224 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrAB genes were characterized by Whole Genome Shotgun sequencing. Twenty eight complete genomes were further characterized for insertional inactivation of lpx genes and the association of ISAba1-eptA using hybrid assembly approach. Non-sysnonymous mutations like M12I in pmrA, A138T and A444V in pmrB and E117K in lpxD were identified. Four of the five colistin resistant A.baumannii isolates had insertion of ISAba1 upstream eptA. No mcr genes were identified.Overall, the present study highlights the diversity of colistin resistance mechanisms in A. baumannii. ISAba1-driven eptA overexpression could be responsible for colistin resistance among Indian isolates of colistin resistant A. baumannii.

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WAPO-A1  is the causal gene of the 7AL QTL for spikelet number per spike in wheat Improving our understanding of the genes regulating grain yield can contribute to the development of more productive wheat varieties. Previously, a highly significant QTL affecting spikelet number per spike (SNS), grain number per spike (GNS) and grain yield was detected on chromosome arm 7AL in multiple genome-wide association studies. Using a high-resolution genetic map, we established that the A-genome homeolog of WHEAT ORTHOLOG OF APO1 (WAPO-A1) was a leading candidate gene for this QTL. Using mutants and transgenic plants, we demonstrate in this study that WAPO-A1 is the causal gene underpinning this QTL. Loss-of-function mutants wapo-A1 and wapo-B1 showed reduced SNS in tetraploid wheat, and the effect was exacerbated in wapo1 combining both mutations. By contrast, spikes of transgenic wheat plants carrying extra copies of WAPO-A1 driven by its native promoter had higher SNS, a more compact spike apical region and a smaller terminal spikelet than the wild type. Taken together, these results indicate that WAPO1 affects SNS by regulating the timing of terminal spikelet formation. Both transgenic and wapo1 mutant plants showed a wide range of floral abnormalities, indicating additional roles of WAPO1 on wheat floral development. Previously, we found three widespread haplotypes in the QTL region (H1, H2 and H3), each associated with particular WAPO-A1 alleles. Integrating results from this study and previous findings, we show that the WAPO-A1 allele in the H1 haplotype (115-bp deletion in the promoter) is expressed at significantly lower levels in the developing spikes than the alleles in the H2 and H3 haplotypes, resulting in reduced SNS. Field experiments also showed that the H2 haplotype is associated with the strongest effects in increasing SNS and GNS (H2>H3>H1). The H2 haplotype is already present in most modern common wheats, so it might be particularly useful in durum wheat where H2 is rare.

AUTHOR SUMMARYA region on wheat chromosome 7A has been previously shown to affect the number of spikelets and grains per spike as well as total grain yield in multiple breeding programs. In this study, we show that loss-of-function mutations in the WAPO1 gene located within this region reduce the number of spikelets per spike and that additional transgenic copies of this gene increase this number. These results demonstrate that WAPO1 is the gene responsible for the differences in grain number and yield associated with the 7A chromosome region. Among the three main variants identified for this gene, we demonstrate in field experiments that the H2 variant is associated with the largest increases in number of spikelets and grains per spike. The H2 WAPO1 variant is frequent in bread wheat breeding programs but is almost absent in modern pasta wheat varieties. Therefore, the introgression of the H2 represents a promising opportunity to improve grain yield in pasta wheat breeding programs.

sentences: Elongated Hypocotyl 5 (HY5) regulates BRUTUS (BTS) to maintain Iron homeostasis in Arabidopsis thaliana. Iron (Fe) is an essential micronutrient for both plants and animals. Fe limitation significantly reduces crop yield and therefore has adverse impacts on human nutrition. Owing to limited bioavailability of Fe, plants have adapted different strategies that regulate Fe uptake and homeostasis. Particularly, modifications of root growth traits are a key for the survival of plants on Fe-deficient soils. Understanding the molecular basis for these root growth responses will have critical implications for plant breeding. Fe uptake is regulated by a cascade of basic helix-loop-helix (bHLH) transcription factors. In our study, we show that HY5 (Elongated Hypocotyl 5), a member of the basic leucine zipper (bZIP) family of transcription factors plays an important role in the Fe deficiency signalling pathway in Arabidopsis thaliana. The hy5 mutant plants failed to mount an optimum Fe deficiency response and showed severe root growth defects under Fe limitation that could be partially reverted by complementation of hy5 mutant. qRT-PCR analysis revealed that the induction of the genes involved in Fe uptake pathway [FER-like iron deficiency-induced transcription factor (FIT), Ferric Reduction Oxidase 2 (FRO2) and Iron-Regulated Transporter1 (IRT1)] is significantly reduced in the hy5 mutants as compared to the wild-type plants under Fe deficiency. Moreover, we also found that HY5 function is critical for activating the expression of coumarin biosynthesis genes (F6H1, S8H, PDR9 and BGLU42) under Fe deficiency. Interestingly, our results showed that HY5 acts as a negative regulator of BRUTUS (BTS) which is known to negatively regulate Fe deficiency response. Chromatin Immunoprecipitation followed by qPCR revealed direct binding of HY5 to the promoter of BTS. Altogether, our results showed that HY5 plays an important role in regulation of Fe deficiency responses in Arabidopsis.

sentences: Attracting pollinators vs escaping herbivores: eco-evolutionary dynamics of plants confronted with an ecological trade-off A large number of plant traits are subject to an ecological trade-off between attracting pollinators and escaping herbivores. The interplay of both plant-animal interactions determines their evolution. Within a plant-pollinator-herbivore community in which interaction strengths depend on trait-matching, eco-evolutionary dynamics are studied using the framework of adaptive dynamics. We characterize the type of selection acting on the plant phenotype and the consequences for multispecies coexistence. We find that pollination favors stabilizing selection and coexistence. In contrast, herbivory fosters runaway selection, which threatens plant-animal coexistence. These contrasting dynamics highlight the key role of ecological trade-offs in structuring ecological communities. In particular, we show that disruptive selection is possible when such trade-offs are strong. While the interplay of pollination and herbivory is known to maintain plant polymorphism in several cases, our work suggests that it might also have fueled the diversification process itself.

sentences: Internalisation and biological activity of nucleic acids delivering cell-penetrating peptide nanoparticles is controlled by the biomolecular corona Nucleic acid molecules can be transferred into cells to alter gene expression and, thus, alleviate certain pathological conditions. Cell-penetrating peptides (CPPs) are vectors that can be used for transfecting nucleic acids as well as many other compounds. CPPs associate nucleic acids non-covalently, forming stable nanoparticles and providing efficient transfection of cells in vitro. However, in vivo, expected efficiency is achieved only in rare cases. One of the reasons for this discrepancy is formation of protein corona around nanoparticles, once they are exposed to a biological environment, e.g. blood stream. In this study, we compared CPP-nucleic acid nanoparticles formed in the presence of bovine, murine and human serum. We used Western blot and mass-spectrometry to identify the major constituents of protein corona forming around nanoparticles, showing that proteins involved in transport, haemostasis and complement system are its major components. We investigated physical features of nanoparticles, and measured their biological efficiency in splice-correction assay. We showed that protein corona constituents might alter the fate of nanoparticles in vivo, e.g. by subjecting them to phagocytosis. We demonstrated that composition of protein corona of nanoparticles is species-specific that leads to dissimilar transfection efficiency and should be taken into account while developing delivery systems for nucleic acids.

sentences: Novel epigenetic-metabolic inhibitor combination treatment blocks platinum-induced ovarian cancer stem cell enrichment High grade serous ovarian cancer (HGSOC) is the most common and aggressive type of ovarian cancer. Platinum resistance is a common occurrence in HGSOC and a main cause of tumor relapse resulting in high patient mortality rates. Recurrent OC is enriched in aldehyde dehydrogenase (ALDH)+ ovarian cancer stem cells (OCSCs), which are resistant to platinum agents. We demonstrated that acute platinum treatment induced a DNA damage-dependent decrease in BRCA1 levels. In a parallel response associated with G2/M arrest, platinum treatment also induced an increase in expression of NAMPT, the rate limiting regulator of NAD+ production from the salvage pathway, and levels of NAD+, the cofactor required for ALDH activity. Concurrent inhibition of DNA methyltransferases (DNMTs) and NAMPT synergistically abrogated the platinum-induced increase in OCSCs. Combining pharmacological inhibitors of DNMT and NAMPT with carboplatin reduced tumorigenesis and OCSC percentage in vivo. We conclude that both epigenetic and metabolic alterations lead to platinum induced OCSC enrichment, providing preclinical evidence that in the neoadjuvant setting, combining DNMT and NAMPT inhibitors with platinum has the potential to reduce OC recurrence and avert the development of platinum resistance.

sentences: Multi-layered transcriptomic analyses reveal an immunological overlap between COVID-19 and hemophagocytic lymphohistiocytosis associated with disease severity Severe COVID-19 patients present a clinical and laboratory overlap with other hyperinflammatory conditions such as hemophagocytic lymphohistiocytosis (HLH). However, the underlying mechanisms of these conditions remain to be explored. Here, we investigated the transcriptome of 1596 individuals, including patients with COVID-19 in comparison to healthy controls, other acute inflammatory states (HLH, multisystem inflammatory syndrome in children [MIS-C], Kawasaky disease [KD]), and different respiratory infections (seasonal coronavirus, influenza, bacterial pneumonia). We observed that COVID-19 and HLH share immunological pathways (cytokine/chemokine signaling and neutrophil-mediated immune responses), including gene signatures that stratify COVID-19 patients admitted to the intensive care unit (ICU) and COVID-19_nonICU patients. Of note, among the common differentially expressed genes (DEG), there is a cluster of neutrophil-associated genes that reflects a generalized hyperinflamatory state since it is also dysregulated in patients with KD and bacterial pneumonia. These genes are dysregulated at protein level across several COVID-19 studies and form an interconnected network with differentially expressed plasma proteins that point to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils. Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention.

sentences: Dopamine Signaling in the Dorsomedial Striatum Promotes Compulsive Behavior Compulsive behavior is a defining feature of disorders such as substance use disorder and obsessive-compulsive disorder. Current evidence suggests that corticostriatal circuits control the expression of established compulsions, but little is known about the mechanisms regulating the development of compulsions. We hypothesized that dopamine, a critical modulator of striatal synaptic plasticity, could control alterations in corticostriatal circuits leading to the development of compulsions (defined as continued reward-seeking in the face of punishment). We used dual-site fiber photometry to measure dopamine axon activity in the dorsomedial striatum (DMS) and the dorsolateral striatum (DLS) as compulsions emerged. Individual variability in the speed with which compulsions emerged was predicted by DMS dopamine axon activity. Amplifying this dopamine signal accelerated animals transitions to compulsion, whereas inhibition led to learning delays. In contrast, amplifying DLS dopamine signaling had no effect on the emergence of compulsions. These results establish DMS dopamine signaling as a key controller of the development of compulsive reward-seeking.

sentences: Identification of a divergent basal body assembly protein involved in land plant spermatogenesis Oogamy is a form of sexual reproduction and evolved independently in animals, fungi, and plants. In streptophyte plants, Charophyceae, Coleochaetophyceae, bryophytes, lycophytes, ferns (monilophytes), and some gymnosperms (Cycads and Ginkgo) utilize spermatozoids as the male gamete. Plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning plant spermatogenesis remain to be elucidated. To identify the genes involved in plant spermatogenesis, we performed computational analyses and successfully found deeply divergent BLD10s by combining multiple methods and omics-data. We then validated the functions of candidate genes in the liverwort Marchantia polymorpha and the moss Physcomitrium patens and found that MpBLD10 and PpBLD10 are required for normal basal body and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. Streptophyte BLD10s are orthologous to BLD10/CEP135 family proteins, which function in basal body assembly, but we found that BLD10s evolved especially fast in land plants and MpBLD10 might obtain additional functions in spermatozoid formation through the fast molecular evolution. This study provides a successful example of combinatorial study from evolutionary and molecular genetic perspectives that elucidated a function of the key protein of the basal body formation that fast evolved in land plants.

sentences: Unravelling microalgal-bacterial interactions in aquatic ecosystems through 16S co-occurrence networks Microalgae and bacteria have a wide spectrum of associations in aquatic environments. Since their interactions can directly influence global carbon and nutrient cycling, understanding these associations help us evaluate their influence on ecosystem productivity. Algal biodiversity is large, and bacterial associations have been characterised for a small fraction of them. While experiments based on algal-bacterial co-culturing are commonly used to infer interactions, deciphering all associations present in nature through such methods is impractical and approaches based on co-occurrence network analysis can help infer associations. In this study, we used microbial co-occurrence networks built from Earth microbiome project 16S metabarcoding data to detect microalgal-bacterial associations in aquatic environments. We analysed marine and freshwater environments to understand what groups of bacteria are tightly co-occurring with different algal groups in both aquatic environments, to see patterns of interactions, and to evaluate the overall use of co-occurrence networks to infer meaningful algal-bacterial interactions. In line with expectations from co-culturing work, our results show that the phyla Proteobacteria and Bacteroidetes are the major bacterial associates of microalgae and the co-occurring bacteria may be specific to the algal host. From the independent analysis of environments, we also show that sample origin may be an important determinant of these interactions. By unravelling previously established microalgal-bacterial links as well as identifying a range of previously unknown interactions, we show that co-occurrence network analysis is a promising hypothesis-generating framework to study microalgal-bacterial interactions that can guide future research into the functional nature of interactions.

sentences: Systematic morphological and morphometric analysis of identified olfactory receptor neurons in Drosophila melanogaster The biophysical properties of sensory neurons are influenced by their morphometric and morphological features, whose precise measurements require high-quality volume electron microscopy (EM). However, systematic surveys of these nanoscale characteristics for identified neurons are scarce. Here, we characterize the morphology of Drosophila olfactory receptor neurons (ORNs) across the majority of genetically identified sensory hairs. By analyzing serial block-face electron microscopy (SBEM) images of cryofixed antennal tissues, we compile an extensive morphometric dataset based on 122 reconstructed 3D models of 33 identified ORN types. In addition, we observe multiple novel features--including extracellular vacuoles within sensillum lumen, intricate dendritic branching, mitochondria enrichment in select ORNs, novel sensillum types, and empty sensilla containing no neurons--which raise new questions pertinent to cell biology and sensory neurobiology. Our systematic survey is critical for future investigations into how the size and shape of sensory neurons influence their responses, sensitivity and circuit function.

sentences: Metagenomic and Metatranscriptomic Analysis Reveals Enrichment for Xenobiotic-Degrading Bacterial Specialists and Xenobiotic-Degrading Genes in a Canadian Prairie Two-Cell Biobed System Biobeds are agriculture-based bioremediation tools used to safely contain and microbially degrade on-farm pesticide waste and rinsate, thereby reducing the negative environmental impacts associated with pesticide use. While these engineered ecosystems demonstrate efficient pesticide removal, their microbiome dynamics remain largely understudied both taxonomically and functionally. As such, further characterization of these parameters may aid in the optimization of biobed management and deployment. This study used metagenomic and metatranscriptomic techniques to characterize the microbial community in a two-cell Canadian biobed system before and after a field season of pesticide application. These culture-independent approaches identified an enrichment of xenobiotic-degrading bacteria, such as Afipia, Sphingopyxis and Pseudomonas, and enrichment and transcription of xenobiotic-degrading genes, such as peroxidases, oxygenases, and hydroxylases, among others; we were able to directly link the transcription of these genes to Pseudomonas, Oligotropha, Mesorhizobium, Rhodopseudomonas, and Stenotrophomonas taxa.

sentences: Timing and magnitude of climate driven range shifts in transboundary fish stocks challenge their management Climate change is shifting the distribution of shared fish stocks between neighboring countries Exclusive Economic Zones (EEZ) and the high seas. The timescale of these transboundary shifts determines how climate change will affect international fisheries governance. Coupling a large ensemble simulation of an Earth system model to a species distribution model, we show that by 2030, 23% of transboundary stocks would have shifted and 78% of the world EEZs will experience at least one shifting stock under a high emission climate change scenario. By the end of this century, 81% of EEZ waters will see at least one shifting stock with a total of 45% of stocks shifting globally, under a high emissions scenario. Importantly, many countries that are highly dependent on fisheries for income, food and nutrition security, as well as livelihoods emerge as hotspots for transboundary shifts showing early, and sometimes past shifts. Existing fisheries agreements need to be assessed for their capacity in addressing transboundary shifts, and strengthened where necessary to limit conflict over these fish stocks while new agreements are urged to considere this problematic in order to be resilient to global change.

sentences: A Neuromuscular Model of Human Locomotion Combines Spinal Reflex Circuits with Voluntary Movements Existing models of human walking use low-level reflexes or neural oscillators to generate movement. While appropriate to generate the stable, rhythmic movement patterns of steady-state walking, these models lack the ability to change their movement patterns or spontaneously generate new movements in the specific, goal-directed way characteristic of voluntary movements. Here we present a neuromuscular model of human locomotion that bridges this gap and combines the ability to execute goal directed movements with the generation of stable, rhythmic movement patterns that are required for robust locomotion. The model represents goals for voluntary movements of the swing leg on the task level of swing leg joint kinematics. Smooth movements plans towards the goal configuration are generated on the task level and transformed into descending motor commands that execute the planned movements, using internal models. The movement goals and plans are updated in real time based on sensory feedback and task constraints. On the spinal level, the descending commands during the swing phase are integrated with a generic stretch reflex for each muscle. Stance leg control solely relies on dedicated spinal reflex pathways. Spinal reflexes stimulate Hill-type muscles that actuate a biomechanical model with eight internal joints and six free-body degrees of freedom. The model is able to generate voluntary, goal-directed reaching movements with the swing leg and combine multiple movements in a rhythmic sequence. During walking, the swing leg is moved in a goal-directed manner to a target that is updated in real-time based on sensory feedback to maintain upright balance, while the stance leg is stabilized by low-level reflexes and a behavioral organization switching between swing and stance control for each leg. With this combination of reflex-based stance leg and voluntary, goal-directed control of the swing leg, the model controller generates rhythmic, stable walking patterns in which the swing leg movement can be flexibly updated in real-time to step over or around obstacles.

sentences: Speech Production in Intracranial Electroencephalography: iBIDS Dataset Speech production is an intricate process involving a large number of muscles and cognitive processes. The neural processes underlying speech production are not completely understood. As speech is a uniquely human ability, it can not be investigated in animal models. High-fidelity human data can only be obtained in clinical settings and is therefore not easily available to all researchers. Here, we provide a dataset of 10 participants reading out individual words while we measured intracranial EEG from a total of 1103 electrodes. The data, with its high temporal resolution and coverage of a large variety of cortical and sub-cortical brain regions, can help in understanding the speech production process better. Simultaneously, the data can be used to test speech decoding and synthesis approaches from neural data to develop speech Brain-Computer Interfaces and speech neuroprostheses.

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A novel fully-spliced accessory gene in equine lentivirus with distinct Rev-responsive element All lentiviruses encode an accessory protein Rev, whose main biological function is to mediate the nuclear export of unspliced and incompletely spliced viral transcripts by binding to a viral cis-acting element (termed the Rev-responsive element, RRE) that is located within env-encoding region. Equine infectious anemia virus (EIAV) is a member of the Lentivirus genus in the Retroviridae family, and is considered an important model for the study of lentivirus pathogenesis. Here, we identified a novel transcript from the EIAV genome that encodes a viral protein, named Mat, with unknown function. The transcript mat is fully spliced and comprises parts of the coding regions of MA and TM. Interestingly, the expression of Mat depends on Rev and the CRM1 pathway. Rev could specifically bind to Mat mRNA to promote its nuclear export. We further identified that the first exon of Mat mRNA, which is located within the Gag-encoding region, acts as an unreported RRE. Altogether, we identified a novel fully spliced transcript mat with an unusual RRE, which interacts with Rev for nuclear export through the CRM1 pathway. Our findings may help to expend the understanding of gene transcription and expression of lentivirus.

Author summaryIn lentiviruses, the nuclear export of viral transcripts is an important step in controlling viral gene expression. Generally, the unspliced and incompletely spliced (that is, intron-containing) transcripts are exported via the CRM1-dependent export pathway in a process mediated by the viral Rev protein by binding to the Rev-responsive element (RRE) located within the Env-coding region. However, the completely spliced (intronless) transcripts are exported via an endogenous cellular pathway which is Rev independent. Here we identified a novel intronless transcript from EIAV and demonstrated that it encodes a viral protein, termed Mat. Interestingly, we have determined that the expression of Mat depends on Rev, and identified that the first exon of Mat mRNA could specifically bind to Rev and be exported to the cytoplasm, which suggests that first exon of Mat mRNA is a second RRE of EIAV. These findings update the EIAV genome structure and highlight the diversification of post-transcriptional regulation patterns in EIAV, and provides important insights into the Rev-dependent nuclear export of completely spliced transcripts in lentiviruses.

sentences: Gene Set Analysis for time-to-event outcome with the Generalized Berk-Jones statistic Gene Set analysis allows to evaluate the impact of groups of genes on an outcome of interest, such as the occurrence of a disease. Through the definition of the gene sets, gene set analysis takes into account biological knowledge and makes it easier to interpret the results, while improving the statistical power compared to a gene-wise analysis. In the time-to-event context, few methods exist, but most of them do not take into account the correlation that occurs inside a gene set, which can be strong. As the Generalized Berk-Jones statistics showed great consistency and includes the correlation inside the test statistic, we adapted this method to the time-to-event context by using a Cox model. We compared our approach to other methods based on the Cox model, and showed that the Generalize Berk-Jones statistic offers great adaptability, meaning that it can be used in all kinds of data structures. We applied the different methods to two different contexts: Gliomas and Breast cancer. In terms of statistical power, we did offer similar results to the other Cox model methods, but with greater accuracy. In the breast cancer framework, we showed better statistical power than methods based on Kernel Machine score.

sentences: Functional genomics analysis identifies impairment of HNF1B function as a cause of Mayer-Rokitansky-Küster-Hauser syndrome. Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome is a congenital condition characterized by aplasia or hypoplasia of the uterus and vagina in women with a typical 46,XX karyotype. This condition can occur as type I when isolated or as type II when associated with extragenital anomalies including kidney and skeletal abnormalities. The genetic basis of MRKH syndrome remains unexplained and several candidate genes have been proposed to play a role in its etiology, including HNF1B, LHX1, and WNT4. Here, we conducted a genomic analysis of 13 women affected by MRKH syndrome, resulting in the identification of candidate genes, including several novel candidates. We focused on HNF1B for further investigation due to its known association with, but unknown etiological role in, MRKH syndrome. We ablated Hnf1b specifically in the epithelium of the Mullerian ducts in mice, and found that this caused hypoplastic development of both the epithelial and stromal compartments of the uterus, as well as kidney anomalies, closely mirroring the MRKH type II phenotype. Using single-cell RNA sequencing of uterine tissue in the Hnf1b-ablated embryos, we analyzed the molecules and pathways downstream of Hnf1b, revealing a dysregulation of processes associated with cell proliferation, migration, and differentiation. Thus, we establish that loss of Hnf1b function leads to an MRKH phenotype, and generate the first mouse model of MRKH syndrome type II. Our results support the diagnostic value of HNF1B in clinical genetic testing for MRKH syndrome, and shed new light on the genetic causes of this poorly understood condition in womens reproductive health.

sentences: Identification of HCN1 as a 14-3-3 client Hyperpolarization activated cyclic nucleotide-gated channel 1 (HCN1) is expressed throughout the nervous system and is critical for regulating neuronal excitability, with mutations being associated with multiple forms of epilepsy. Adaptive modulation of HCN1 has been observed as has pathogenic dysregulation. While the mechanisms underlying this modulation remain incompletely understood, regulation of HCN1 has been shown to include phosphorylation. A candidate phosphorylation-dependent regulator of HCN1 channels is 14-3-3. We used bioinformatics to identify three potential 14-3-3 binding sites in HCN1. Isothermal titration calorimetry demonstrated that recombinant 14-3-3 binds all three phospho-peptides with low micromolar affinity. We confirmed that 14-3-3 could pull down HCN1 from multiple tissue sources and used HEK293 cells to detail the interaction. Two binding sites in the intrinsically disordered C-terminus of HCN1 were necessary and sufficient for a phosphorylation-dependent interaction with 14-3-3. The same region of HCN1 containing the 14-3-3 binding sites is required for phosphorylation-independent protein degradation. We propose a model in which phosphorylation of S810 and S867 (human S789 and S846) recruits 14-3-3 to inhibit a yet unidentified factor signaling for protein degradation, thus increasing the half-life of HCN1.

sentences: Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates1-3. The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle4-7, together with variations in substrate docking to sites on associated cyclin and Cks subunits3, 6, 8-10. Many substrates are modified at multiple sites to provide more complex regulation9, 11-14. Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of genes required for mitotic progression15, 16. As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback17-20. Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that CLB2 expression is inhibited by high levels of Cdk1 activity in a mitotic arrest. Inhibition is accompanied by Ndd1 degradation, and we present evidence that high mitotic Cdk1-Clb2 activity generates phosphodegrons on Ndd1, leading to its degradation. Complete Ndd1 phosphorylation by the Clb2-Cdk1-Cks1 complex requires the phosphothreonine-binding site of Cks1, as well as a novel phosphate-binding pocket on the cyclin Clb221. We therefore propose that initial phosphorylation by Cdk1 primes the protein for secondary phosphorylation at phosphodegrons, resulting in degradation only at high Cdk1 activity. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

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Protein undernutrition reduces the efficacy of praziquantel in a murine model of Schistosoma mansoni infection BackgroundUndernutrition and schistosomiasis are public health problems and often occur in low and middle-income countries. Protein undernutrition can alter the host-parasite environment system and aggravate the course of schistosomiasis. This study aimed to assess the impact of a protein-deficient diet on the efficacy of praziquantel.

Methodology/principal findingsThirty-day-old mice were fed with a protein-deficient diet, and 40 days later, they were individually infected with fifty Schistosoma mansoni cercariae. A 28-day-treatment with praziquantel at 100 mg/kg for five consecutive days followed by distilled water begins at the 36th day post-infection. Mice were sacrificed on the 64th day post-infection. We determined the parasitological burden, liver and intestine histomorphometry, liver injury, and immunomodulation parameters. Praziquantel treatment of infected mice fed with a standard diet (IN-PZQ) resulted in a significant reduction of worm and egg burdens and a normalization of iron and calcium levels. The therapy also improved schistosomiasis-induced hepatopathy and oxidative stress. The anti-inflammatory and immunomodulatory activities of praziquantel were also significant in these mice. When infected mice receiving the protein-deficient diet were treated with praziquantel (ILP-PZQ), the body weight loss and hepatomegaly were not alleviated, and the worm and liver egg burdens were significantly higher than those of IN-PZQ mice (P < 0.001). The treatment did not reduce the increased activities of ALT and {gamma}-GGT, the high malondialdehyde concentration, and the liver granuloma volume. The iron and calcium levels were not ameliorated and differed from those of IN-PZQ mice (P < 0.001 and P < 0.05). Moreover, in these mice, praziquantel treatment did not reverse the high level of IL-5 and the low mRNA expression of CCL3/MIP-1 and CXCL-10/IP-10 induced by S. mansoni infection.

Conclusion/significanceThese results demonstrated that a protein-deficient diet reduced the schistosomicidal, antioxidant, anti-inflammatory, and immunomodulatory activities of praziquantel.

Authors summaryAlmost 90% of people requiring schistosomiasis preventive chemotherapy in 2018 lived in sub-Saharan Africa. Besides, 205.3 million children under five years suffer and die of undernutrition in low- and middle-income countries. The physiopathology of schistosomiasis mansoni involves liver damage, oxidative stress, and perturbation of the immune response. These disturbances are intensified by undernutrition. Praziquantel is used to treat schistosomiasis, but its efficacy on the comorbidity of S. mansoni infection and undernutrition has not been investigated. We conducted this study to assess the effectiveness of praziquantel on S. mansoni infection in mice fed with a protein-deficient diet. We recorded growth retardation, hepatomegaly, and high worm and egg burdens in mice fed with a protein-deficient diet and treated with PZQ. Moreover, the treatment did not reverse the liver function injury, oxidative stress, high iron level, and low calcium level. The proinflammatory cytokine IL-5 was still high, and the gene expression of some macrophage-associated chemokines was reduced. Therefore, this study demonstrated that in a murine model of a protein-deficient diet, the efficacy of praziquantel on S. mansoni infection was reduced. It also underlines the importance of targeting protein deficiency and malnutrition in populations living in schistosomiasis endemic areas for efficient disease control.

sentences: Divalent metal ions boost effect of nucleic acids delivered by cell-penetrating peptides Cell-penetrating peptides (CPPs) are promising tools for transfection of various substances, including nucleic acids, into cells. The aim of current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed of CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed an effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed with addition of the ions, without increasing cytotoxicity. We propose a novel strategy of preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, in the aim to increase CPP-mediated transfection efficiency and likelihood of using CPPs in clinics.

sentences: Individual activator and repressor transcription factors induce global changes in chromatin mobility The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. Yet how nucleus-wide chromatin structures respond dynamically to the activity of specific TFs remains unclear. Here we asked how TFs with opposite effects on modulating local chromatin accessibility impact nucleus-wide chromatin dynamics in living cells. We combine High-resolution Diffusion mapping (Hi-D) and Dense Flow reConstruction and Correlation (DFCC) to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs in live cells. We show that expression of individual activator and repressor TFs increases nucleus-wide chromatin mobility and induces opposite coherent chromatin motions at the micron scale. Our quantitative analysis thus reveals the broad and differential impact of TFs opening and closing chromatin in nucleus-wide chromatin dynamics.

sentences: Unexpected growth of a classic yeast auxotroph In budding yeast, the Met15 enzyme has long been assumed to be the sole homocysteine synthase facilitating de novo synthesis of sulfur-containing organic compounds (organosulfurs) from inorganic precursors. Here we show that an alternative homocysteine synthase encoded by the previously uncharacterized gene YLL058W supports growth of mutants lacking MET15 in the absence of exogenous organosulfurs. This growth is observed specifically when cells are deposited in an automated fashion to seed colonies, but not with traditional cell propagation techniques such as thick patches of cells or liquid cultures. We show that the lack of growth in these contexts, which has historically justified the status of MET15 as a classic auxotrophic marker, is largely due to toxic levels of hydrogen sulfide accumulation rather than an inability to perform de novo homocysteine biosynthesis. These data suggest that cell fate decisions in yeast are influenced by hydrogen sulfide levels in the microenvironment. Our results have broad implications for investigations of sulfur metabolism, including studies of methionine restriction and aging.

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Homeostatic depression shows heightened sensitivity to synaptic calcium Synapses and circuits rely on homeostatic forms of regulation in order to transmit meaningful information. The Drosophila melanogaster neuromuscular junction (NMJ) is a well-studied synapse that shows robust homeostatic control of function. Most prior studies of homeostatic plasticity at the NMJ have centered on presynaptic homeostatic potentiation (PHP). PHP happens when postsynaptic muscle neurotransmitter receptors are impaired, triggering retrograde signaling that causes an increase in presynaptic neurotransmitter release. As a result, normal levels of evoked excitation are maintained. The counterpart to PHP at the NMJ is presynaptic homeostatic depression (PHD). Overexpression of the Drosophila vesicular glutamate transporter (VGlut) causes an increase in the amplitude of spontaneous events. PHD happens when the synapse responds to the challenge by decreasing quantal content during evoked neurotransmission - again, resulting in normal levels of postsynaptic excitation.

We hypothesized that there may exist a class of molecules that affects both PHP and PHD. Impairment of any such molecule could hurt a synapses ability to respond to any significant homeostatic challenge. We conducted an electrophysiology-based screen for blocks of PHD. We did not observe a block of PHD in the genetic conditions screened, but we did find loss-of-function conditions that led to a substantial deficit in evoked amplitude when combined with VGlut overexpression. The conditions causing this phenotype included a double heterozygous loss-of-function condition for genes encoding the inositol trisphosphate receptor (IP3R - itpr) and ryanodine receptor (RyR). IP3Rs and RyRs gate calcium release from intracellular stores. Pharmacological agents targeting IP3R and RyR recapitulated the genetic losses of these factors, as did lowering calcium levels from other sources. Our data are consistent with the idea that the homeostatic signaling process underlying PHD is especially sensitive to levels of calcium at the presynapse.

sentences: Systems-wide Analysis Revealed Shared and Unique Responses to Moderate and Acute High Temperatures in the Green Alga Chlamydomonas reinhardtii Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green alga Chlamydomonas reinhardtii under highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35{degrees}C) and acute (40{degrees}C) high temperatures and subsequent recovery at 25{degrees}C. We identified previously overlooked unique elements in response to moderate high temperature. Heat at 35{degrees}C transiently arrested the cell cycle followed by partial synchronization, up-regulated transcripts/proteins involved in gluconeogenesis/glyoxylate-cycle for carbon uptake, promoted growth, and increased starch accumulation. Heat at 40{degrees}C arrested the cell cycle, inhibited growth, resulting in carbon uptake over usage and increased starch accumulation. Both high temperatures induced photoprotection, while 40{degrees}C decreased photosynthetic efficiency, distorted thylakoid/pyrenoid ultrastructure, and affected the carbon concentrating mechanism. We demonstrated increased transcript/protein correlation during both heat treatments, suggesting reduced post-transcriptional regulation during heat may help coordinate heat tolerance activities efficiently. During recovery after both heat treatments, transcripts/proteins related to DNA synthesis increased while those involved in photosynthetic light reactions decreased. We propose down-regulating photosynthetic light reactions during DNA replication benefits cell cycle resumption by reducing ROS production. Our results provide potential targets to increase thermotolerance in algae and crops.

sentences: An Evolutionarily Conserved Regulatory Pathway of Muscle Mitochondrial Network Organization Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial network-type separately from contractile fiber-type remain unclear. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated independently. Proteomic analyses of indirect flight, jump, and leg muscles together with muscles misexpressing known fiber-type specification factor salm identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization separately from contractile type, mitochondrial content, and mitochondrial size through an evolutionarily conserved pathway involving cut, salm, and H15.

sentences: Host-directed therapies against early-lineage SARS-CoV-2 retain efficacy against B.1.1.7 variant Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths worldwide and massive societal and economic burden. Recently, a new variant of SARS-CoV-2, known as B.1.1.7, was first detected in the United Kingdom and is spreading in several other countries, heightening public health concern and raising questions as to the resulting effectiveness of vaccines and therapeutic interventions. We and others previously identified host-directed therapies with antiviral efficacy against SARS-CoV-2 infection. Less prone to the development of therapy resistance, host-directed drugs represent promising therapeutic options to combat emerging viral variants as host genes possess a lower propensity to mutate compared to viral genes. Here, in the first study of the full-length B.1.1.7 variant virus, we find two host-directed drugs, plitidepsin (aplidin; inhibits translation elongation factor eEF1A) and ralimetinib (inhibits p38 MAP kinase cascade), as well as remdesivir, to possess similar antiviral activity against both the early-lineage SARS-CoV-2 and the B.1.1.7 variant, evaluated in both human gastrointestinal and lung epithelial cell lines. We find that plitidepsin is over an order of magnitude more potent than remdesivir against both viruses. These results highlight the importance of continued development of host-directed therapeutics to combat current and future coronavirus variant outbreaks.

sentences: Revealing the Physiological Origin of Event-Related Potentials using Electrocorticography in Humans The scientific and clinical value of event-related potentials (ERPs) depends on understanding the contributions to them of three possible mechanisms: (1) additivity of time-locked voltage changes; (2) phase resetting of ongoing oscillations; (3) asymmetrical oscillatory activity. Their relative contributions are currently uncertain. This study uses analysis of human electrocorticographic activity to quantify the origins of movement-related potentials (MRPs) and auditory evoked potentials (AEPs). The results show that MRPs are generated primarily by endogenous additivity (88%). In contrast, P1 and N1 components of AEPs are generated almost entirely by exogenous phase reset (93%). Oscillatory asymmetry contributes very little. By clarifying ERP mechanisms, these results enable creation of ERP models; and they enhance the value of ERPs for understanding the genesis of normal and abnormal auditory or sensorimotor behaviors.

sentences: Trait-trait relationships and functional tradeoffs vary with genome size in prokaryotes We report genomic traits that have been associated with the life history of prokaryotes and highlight conflicting findings concerning earlier observed trait correlations and tradeoffs. In order to address possible explanations for these contradictions we examined trait-trait variations of 11 genomic traits from [~] 17,000 sequenced genomes. The studied trait-trait variations suggested: (i) the predominance of two resistance and resilience-related orthogonal axes, (ii) an overlap between a resilience axis and an axis of resource usage efficiency. These findings imply that resistance associated traits of prokaryotes are globally decoupled from resilience and resource use efficiencies associated traits. However, further inspection of pairwise scatterplots showed that resistance and resilience traits tended to be positively related for genomes up to roughly five million base pairs and negatively for larger genomes. This in turn precludes a globally consistent assignment of prokaryote genomic traits to the competitor - stress-tolerator -ruderal (CSR) schema that sorts species depending on their location along disturbance and productivity gradients into three ecological strategies and may serve as an explanation for conflicting findings from earlier studies. All reviewed genomic traits featured significant phylogenetic signals and we propose that our trait table can be applied to extrapolate genomic traits from taxonomic marker genes. This will enable to empirically evaluate the assembly of these genomic traits in prokaryotic communities from different habitats and under different productivity and disturbance scenarios as predicted via the resistance-resilience framework formulated here.

sentences: Acorde: unraveling functionally-interpretable networks of isoform co-usage from single cell data Alternative splicing (AS) is a highly-regulated post-transcriptional mechanism known to modulate isoform expression within genes and contribute to cell-type identity. However, the extent to which alternative isoforms establish co-expression networks that may relevant in cellular function has not been explored yet. Here, we present acorde, a pipeline that successfully leverages bulk long reads and single-cell data to confidently detect alternative isoform co-expression relationships. To achieve this, we developed and validated percentile correlations, a novel approach that overcomes data sparsity and yields accurate co-expression estimates from single-cell data. Next, acorde uses correlations to cluster co-expressed isoforms into a network, unraveling cell type-specific alternative isoform usage patterns. By selecting same-gene isoforms between these clusters, we subsequently detect and characterize genes with co-differential isoform usage (coDIU) across neural cell types. Finally, we predict functional elements from long read-defined isoforms and provide insight into biological processes, motifs and domains potentially controlled by the coordination of post-transcriptional regulation.

sentences: On-chip light diffraction imaging of nano structures in the guanine platelet Light projection over short distances can minimize the size of photonic devices, e.g., head-mounted displays and lens-free microscopes. Small lenses or light condensers without typical lenses are essential for light control in micron-scale spaces. In this work, micro-platelets floating in water are used for light projection near the image sensor. These platelets, which are made from guanine, have nanohole gratings and demonstrate light diffraction toward specific directions. By setting a thin water layer on the image sensors cover glass, each platelet in water forms column-or bar-code-shaped images on the screen. The projected image shapes and colors are inferred to contain information about nano-structures present in the guanine platelet. The proposed down-sized imaging technique can realize extremely compact and portable imagers for nanoscale object detection.

sentences: Engineering Antioxidant and Oxygen-Releasing Lignin Composites to Accelerate Wound Healing The application of engineered biomaterials for wound healing has been pursued since the beginning of tissue engineering. Here, we attempt to apply functionalized lignin to confer antioxidation to wound extracellular microenvironments and to deliver oxygen via calcium peroxide for enhanced vascularization and healing responses without eliciting inflammatory responses. We demonstrated in vitro that thiolated lignosulfonate in alkene-incorporated gelatin effectively promoted the expression of angiogenesis and extracellular matrix production genes and attenuated the expression of oxidative stress genes of human dermal fibroblasts. Elemental analysis of oxygen releasing nanoparticles showed the positive incorporation of calcium peroxide. Lignin composites including nanoparticles of sodium lignosulfonate and calcium peroxide released around 0.05% oxygen per day at least for 7 days. By modulating the concentration the alkene-incorporated gelatin, we were able to maintain the injectability of lignin composite precursors and the stiffness of lignin composites after photo-crosslinking. In situ formation of lignin composites with oxygen generation nanoparticles enhanced the formation of blood vessels and promoted infiltration of a-smooth muscle actin+ fibroblasts into the wounds over 7 days. At 30 days after surgery, the lignin composite with oxygen generating nanoparticles remodeled the collagen architecture resembling to the reticular pattern of normal collagen with minimal scar formation. Thus, our study shows the potential of functionalized lignosulfonate for wound healing applications requiring balanced antioxidation and controlled release of oxygen for enhanced vascularization and maturation of collagens.

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Complex landscapes partially mitigate negative pesticide effects on tropical pollinator communities Land-use change and pesticides have been identified as two of the main causes behind pollinator decline. Understanding how these factors affect crop pollinator communities is crucial to inform practices that generate optimal pollination and ensure sustainable food production. In this study, we investigated the effects of landscape composition and pesticide residues on bee communities and their pollination services in Solanum quitoense "lulo" crops in Colombia. On 10 farms, located along a gradient of landscape complexity that varied from 0.15 to 0.62 in their natural habitat proportion, we characterized the bee community visiting the crop, and carried out pollination experiments with bagged and open inflorescences to later estimate fruit set, weight, and diameter at every site. Additionally, we performed pesticide analysis on collected anthers through liquid chromatography to estimate pesticide risk coming from the crop fields using hazard quotients (HQ). Bee abundance and species richness decreased with increased HQ, but these negative pesticide effects were less detrimental in farms with higher natural habitat proportions. However, this buffer effect was lost at sites with very high HQs. Imidacloprid was frequently found in the anthers and there were extremely high concentrations in some farms (0.6 to 13063 g/kg), representing the molecule of greater risk for bees in this context. Pollinators importance to crop yield was demonstrated in the exclusion experiments, where we found a reduction in fruit set (51%), weight (39%), and diameter (25%). We found a significant effect of bee richness on fruit set, while landscape composition and HQ had no significant effect on fruit set, suggesting that the last two factors do not affect yield directly, but indirectly through a decrease in pollinator diversity. Our results provide novel evidence that the natural habitat loss due to the expansion of pastures for cattle ranching and pesticide residues in anthers reduce bee diversity and abundance in this Andean cropping system, but strategies to protect and restore natural habitat can help to buffer, until certain levels, these negative effects.

Highlights- We explored how landscape composition and pesticide residues impact bee communities and pollination services in Solanum quitoense crops.
- As the proportion of natural habitat in the landscape increased, bee richness also increased. While as pesticide hazard quotients in S. quitoense anthers increased, bee diversity and abundance decreased.
- The natural habitat surrounding farms mitigates the negative pesticide effects on bees when hazard quotients are low/medium, but not when they are high.
- S. quitoense crops are highly dependent on bees for optimum yield.

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Interaction between BID and VDAC1 is required for mitochondrial demise and cell death in neurons Mitochondrial damage is a key feature of regulated cell death in neurons. In particular, mitochondrial outer membrane permeabilization (MOMP) has been proposed as a starting point for mitochondrial demise upon cellular stress. Potential mechanisms for MOMP presented in the literature include membrane pore formation by Bcl2-family proteins such as BID and BAX, oligomerization of voltage-dependent anion channels (VDACs) and hetero-oligomer formation of these proteins. In our study, we demonstrate a direct interaction between the voltage-dependent anion channel VDAC1 and the pro-apoptotic protein BID in dying neurons both in vitro and in vivo. Binding of BID to VDAC1 affects anion conductance through VDAC1 and is associated with glutamate-induced cell death in cultured neurons and ischemic brain injury. In cultured neurons, reducing VDAC1 expression significantly attenuates BID-induced hallmarks of mitochondrial damage such as mitochondrial fission, declined mitochondrial respiration, increased ROS production, and mitochondrial membrane potential breakdown. Our data highlight a critical role for VDAC1 as a mitochondrial receptor for activated BID, thereby serving as a key decision point between life and death in neurons.

One Sentence SummaryVDAC1 interacts with BID to mediate mitochondrial membrane permeabilization and neuronal cell death.

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Pseudomonas aeruginosa C-terminal processing protease CtpA assembles into a hexameric structure that requires activation by a spiral-shaped lipoprotein binding partner Pseudomonas aeruginosa CtpA is a carboxyl terminal-processing protease that partners with the outer membrane lipoprotein LbcA to degrade at least five cell wall-associated proteins, four of which are cell wall hydrolases. This activity plays an important role in supporting P. aeruginosa virulence in a mouse model of acute pneumonia. However, almost nothing is known about the molecular mechanisms underlying CtpA and LbcA function. Here, we used structural analysis to show that CtpA alone assembles into an inactive hexamer comprising a trimer of dimers, which limits its substrate access and prevents nonspecific degradation. The adaptor protein LbcA is a right-handed open spiral with 11 tetratricopeptide repeats, which might wrap around a substrate to deliver it to CtpA for degradation. By structure-guided mutagenesis and functional assays, we also showed that the interfaces of the CtpA trimer-of-dimers, and an N-terminal helix of LbcA, are important for LbcA-mediated substrate degradation by CtpA both in vitro and in vivo. This work improves our understanding of the molecular mechanism of a CTP within the C-terminal processing peptidase-3 group.

IMPORTANCECarboxyl-terminal processing proteases (CTPs) are found in all three domains of life. In bacteria, some CTPs have been associated with virulence, raising the possibility that they could be theraputic targets. However, relatively little is known about their molecular mechanisms of action. In Pseudomonas aeruginosa, CtpA supports virulence by working in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. Here, we report structure-function analyses of CtpA and LbcA, which reveals that CtpA assembles into an inactive hexamer comprising a trimer of dimers. LbcA is monomeric, with an N-terminal region important for binding to and activating CtpA, followed by a spiral structure composed of 11 tetratricopetide repeats, which could wrap around a substrate for delivery to CtpA. This work provides the first structure of a CTP-3 group member, revealing a unique mutimeric arrangement and insight into how this important proteolytic system functions.

sentences: Phyllotaxis Models: from the Inhibition Potential to the Real Plant Previous phyllotaxis models allowed the initiation of new primordia when a threshold of inhibition potential is reached on the meristem front: their adequacy to botanical reality is only qualitative. We formulated the hypothesis that it is not the value of the inhibition threshold that remains constant as the meristem develops, but the difference of the inhibition thresholds during the initiation of two successive primordia. We were thus able to model with accuracy the sequence of plastochron ratios observed by Williams (1975) on the leaf meristem of flax: an outstanding result. More generally, we have shown that the evolution trajectories of the phyllotaxis modes as a function of the plastochron ratios follow the minima of the potential under decreasing plastochron ratios constraint and bifurcate when the number of these minima increases, thus giving physicochemical foundations to the famous van Iterson diagram. This historical representation of rising phyllotaxis shows the trajectories, but doesnt give the velocity of the movement: our plastochron ratio sequence adds this major dynamical information.

sentences: The Role of Extended Range of Interactions in the Dynamics of Interacting Molecular Motors Motor proteins, also known as biological molecular motors, play important roles in various intracellular processes. Experimental investigations suggest that molecular motors interact with each other during the cellular transport, but the nature of such interactions remains not well understood. Stimulated by these observations, we present a theoretical study aimed to understand the effect of the range of interactions on dynamics of interacting molecular motors. For this purpose, we develop a new version of the totally asymmetric simple exclusion processes in which nearest-neighbor as well as the next nearest-neighbor interactions are taken into account in a thermodynamically consistent way. A theoretical framework based on a cluster mean-field approximation, which partially takes correlations into account, is developed to evaluate the stationary properties of the system. It is found that fundamental current-density relations in the system strongly depend on the strength and the sign of interactions, as well as on the range of interactions. For repulsive interactions stronger than some critical value, increasing the range of interactions leads to a change from unimodal to trimodal dependence in the flux-density fundamental diagram. Theoretical calculations are tested with extensive Monte Carlo computer simulations. Although in most ranges of parameters excellent agreement between theoretical predictions and computer simulations is observed, there are situations when the cluster mean-field approach fails to describe properly the dynamics in the system. Theoretical arguments to explain these observations are presented. Our theoretical analysis clarifies the microscopic picture of how the range of interactions influences the dynamics of interacting molecular motors.

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SOX9 acts as a dynamic pioneer factor inducing stable changes in the chromatin landscape to reprogram endothelial cells. The transcription factor SOX9 is expressed in multiple tissues during embryogenesis and directs developmental processes. SOX9 is activated upon endothelial-to-mesenchymal transition (EndMT) in the developing heart, but its role in regulating this process is less clear. Using human umbilical vein endothelial cells as an EndMT model, we show that SOX9 expression alone is sufficient to activate mesenchymal enhancers and steer endothelial cells towards a mesenchymal fate. By genome-wide mapping of the chromatin landscape, we show that SOX9 acts as a pioneer transcription factor, having the ability to open chromatin and lead to deposition of active histone marks at a specific subset of previously silent enhancers, guided by SOX motifs and H2A.Z enrichment. This leads to a switch in enhancer activity states resulting in activation of mesenchymal genes and concurrent suppression of endothelial genes to drive EndMT. Moreover, we show that SOX9 chromatin binding is dynamic, but induces stable changes in the chromatin landscape. Our data also show widespread SOX9 chromatin scanning in silent chromatin that is not associated with SOX motifs or H2A.Z enrichment. Our study highlights the crucial developmental role of SOX9 and provides new insight into key molecular functions of SOX9 in the chromatin landscape and mechanisms of EndMT.

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sentences: Urban-adapted mammal species have more known pathogens The world is rapidly urbanising, inviting mounting concern that urban environments will experience increased zoonotic disease risk. Urban animals could have more frequent contact with humans, and therefore may transmit more zoonotic parasites; however, these animals have a specific set of underlying traits that may determine their parasite burdens while predisposing them to urban living, and they may be subject to more intense research effort, both of which could complicate our ability to reliably identify the role of urbanisation in driving zoonotic risk. Here, we test whether urban mammal species host more known zoonotic parasites, investigating the potential underlying drivers while accounting for a correlated suite of phenotypic, taxonomic, and geographic predictors. We found that urban-adapted mammals have more documented parasites, and more zoonotic parasites specifically: despite comprising only 157 of the 2792 investigated species (6%), urban mammals provided 39% of known host-parasite combinations and showed consistently higher viral discovery rates throughout the last century. However, contrary to predictions, much of the observed effect was attributable to research effort rather than to urban adaptation status itself, and urban-adapted species in fact hosted fewer zoonoses than expected given their total observed parasite richness. We conclude that extended historical contact with humans has had a limited impact on the number of observed zoonotic parasites in urban-adapted mammals; instead, their greater observed zoonotic richness likely reflects sampling bias arising from proximity to humans, which supports a near-universal underlying pattern of conflation between zoonotic risk, research effort, and synanthropy. These findings underscore the need to resolve the ecological mechanisms underlying links between anthropogenic change, sampling bias, and observed wildlife disease dynamics.

sentences: FKBP5 expression is related to HPA flexibility and the capacity to cope with stressors in the house sparrow The hypothalamic-pituitary-adrenal (HPA) axis and its end products, the glucocorticoids, are critical to responding appropriately to stressors. Subsequently, many studies have sought relationships between glucocorticoids and measures of health or fitness, but such relationships are at best highly context dependent. Recently, some endocrinologists have started to suggest that a focus on HPA flexibility, the ability of an individual to mount appropriate responses to different stressors, could be useful. Here, we tested the hypothesis that expression of FKBP5, a cochaperone in the glucocorticoid receptor complex, is a simple and reliable proxy of HPA flexibility in a wild songbird, the house sparrow (Passer domesticus). We quantified HPA flexibility in a novel way, using guidance from research on heart rhythm regulation. As predicted, we found that adult sparrows with low stress-induced FKBP5 expression in the hypothalamus exhibited high HPA flexibility. Moreover, low FKBP5 expression was associated with better stress coping capacities in terms of exploratory disposition and body mass maintenance. Altogether, these results suggest that FKBP5 may be important in the regulation HPA flexibility, potentially affecting how individuals cope with natural and anthropogenic adversity.

sentences: Multi-state Modeling of G-protein Coupled Receptors at Experimental Accuracy The family of G-protein coupled receptors (GPCRs) is one of the largest protein families in the human genome. GPCRs transduct chemical signals from extracellular to intracellular regions via a conformational switch between active and inactive states upon ligand binding. While experimental structures of GPCRs remain limited, high-accuracy computational predictions are now possible with AlphaFold2. However, AlphaFold2 only predicts one state and is biased towards either the active or inactive conformation depending on the GPCR class. Here, a multi-state prediction protocol is introduced that extends AlphaFold2 to predict either active or inactive states at very high accuracy using state-annotated templated GPCR databases. The predicted models accurately capture the main structural changes upon activation of the GPCR at the atomic level. For most of the benchmarked GPCRs (10 out of 15), models in the active and inactive states were closer to their corresponding activation state structures. Median RMSDs of the transmembrane regions were 1.12 [A] and 1.41 [A] for the active and inactive state models, respectively. The models were more suitable for protein-ligand docking than the original AlphaFold2 models and template-based models. Finally, our prediction protocol predicted accurate GPCR structures and GPCR-peptide complex structures in GPCR Dock 2021, a blind GPCR-ligand complex modeling competition. We expect that high accuracy GPCR models in both activation states will promote understanding in GPCR activation mechanisms and drug discovery for GPCRs. At the time, the new protocol paves the way towards capturing the dynamics of proteins at high-accuracy via machine-learning methods.

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Transcriptomic Changes During Stage Progression of Mycosis Fungoides BackgroundMycosis fungoides (MF) is the most common cutaneous T cell lymphoma, which in the early patch/plaque stages runs an indolent course. However, ~25% of MF patients develop skin tumors, a hallmark of progression to the advanced stage and associated with high mortality. The mechanisms involved in stage progression are poorly elucidated.

MethodsWe performed whole-transcriptome and whole-exome sequencing of malignant MF cells from skin biopsies obtained by laser-capture microdissection. We compared three types of MF lesions: early-stage plaques (ESP, n=12), and plaques and tumors from patients in late-stage disease (late-stage plaques, LSP, n=10, and tumors, TMR, n=15). Gene Ontology (GO) and KEGG analysis were used to determine pathway changes specific for different lesions which we linked to the recurrent somatic mutations overrepresented in MF tumors.

ResultsThe key upregulated pathways during stage progression were those related to cell proliferation and survival (MEK/ERK, Akt-mTOR), Th2/Th9 signaling (IL4, STAT3, STAT5, STAT6), meiomitosis (CT45A1, CT45A3, STAG3, GTSF1, and REC8) and DNA repair (PARP1, MYCN, OGG1). Principal coordinate clustering of the transcriptome revealed extensive gene expression differences between early (ESP) and advanced-stage lesions (LSP and TMR). LSP and TMR showed remarkable similarities at the level of the transcriptome, which we interpreted as evidence of cell percolation between lesions via hematogenous self-seeding.

ConclusionStage progression in MF is associated with Th2/Th9 polarization of malignant cells, activation of proliferation, survival, as well as increased genomic instability. Global transcriptomic changes in multiple lesions are probably caused by hematogenous cell percolation between discrete skin lesions.

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Context-dependent triggering of STING-interferon signaling by CD11b agonists supports anti-tumor immunity in mouse models and human cancer patients Chronic activation of inflammatory pathways and suppressed interferon signaling are hallmarks of myeloid cells in immunosuppressive tumors that drive poor responsiveness to conventional and immune therapies. Previous studies have identified agonistic activation of the CD11b integrin as a potential strategy to enhance anti-tumor immunity. However, the mechanisms by which CD11b-agonism reprogram tumor immunity are poorly understood, and this may impair patient selection and identification of effective treatment combinations. Herein we used a combination of in vitro systems, animal models, and samples from first in human clinical trials of the CD11b-agonist GB1275 to identify the mechanism of action of this approach and identify combinations for further testing. We found that CD11b agonism altered tumor-associated macrophage (TAM) phenotypes by simultaneously repressing NF{kappa}B/IL1-signaling and activating interferon (IFN) gene expression. Repression of NF{kappa}B/IL-1 signaling was due to rapid degradation of p65 protein by the proteosome and was not context dependent. In contrast, CD11b agonism triggered mitochondrial dysfunction to stimulate STING-induced, STAT1-mediated interferon signaling. The magnitude of CD11b agonist induction of STING/IFN signaling was dependent on the tumor microenvironment and was significantly amplified by cytotoxic therapies. Using tissues from phase I clinical trials, we demonstrated that GB1275 treatment activated STING and STAT1 signaling in TAMs in human tumors. Together, these mechanisms allowed macrophages to augment anti-tumor T cell immunity. These studies identified potential mechanism-based therapeutic strategies for CD11b agonist use and identified potential patient populations more likely to benefit from them.

Statement of significanceCD11b agonists are a novel approach to reprogram myeloid cells in solid tumors. We show that GB1275, a CD11b-agonist, amplified STING/IFN signaling in TAMs to support anti-tumor immunity and this signaling is amplified further by cytotoxic therapy. These studies support new treatment strategies for advanced solid tumors with myeloid immunosuppression.

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The archaeal triphosphate tunnel metalloenzyme SaTTM defines structural determinants for the diverse activities in the CYTH protein family CYTH is a large protein superfamily that is conserved in all three domains of life with its unique triphosphate tunnel metalloenzyme (TTM) fold. Besides phosphatase functions, e.g. as RNA triphosphatase, inorganic polyphosphatase or thiamine triphosphatase, some CYTH orthologs cyclize nucleotide triphosphates to 3,5-cyclic nucleotides. So far, archaeal CYTH proteins are annotated as adenylyl cyclases although experimental evidence is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our initial in silico studies suggested a close relationship between archaeal CYTH enzymes and class IV adenylyl cyclases compared to the other CYTH-subclasses, but biochemical data showed no cyclic nucleotide production. Instead, our structural and functional analyses show a classical TTM behavior. The Ca2+-inhibited Michaelis complex indicates a two-metal ion reaction mechanism analogous to other TTMs. Different co-crystal structures of SaTTM further reveal conformational dynamics in SaTTM, let us to assume feedback inhibition in TTMs due to tunnel closure in the product state. Combining our structural insights with sequence-similarity network based in silico analysis, we further set out a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases.

Major highlights- CyaB-like class IV adenylyl cyclase homologs in archaea are triphosphatases.
- The co-crystal structure of SaTTM in sulfate and triphosphate bound state revealed conformational transition of the TTM tunnel during catalysis.
- Atomic insights into TTM inhibition by calcium and pyrophosphate.
- In silico and structure-function analysis revealed the molecular determinant for functional diversification among CYTH proteins.

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Identification of a discrete neuronal circuit that relays insulin signaling into the brain to regulate glucose homeostasis 26RFa (QRFP) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localized in the hypothalamus. In the present study, we have investigated whether the 26RFa neurons may be involved in the hypothalamic regulation of glucose homeostasis. Our data indicate that 26RFa, i.c.v. injected, induces a robust antihyperglycemic effect associated with an increase of insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26RFa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26RFa in the lateral hypothalamic area and the ventromedial hypothalamic nucleus also express the insulin receptor and that insulin induces the expression of 26RFa in these neurons. Concurrently, we show that the central antihyperglycemic effect of insulin is abolished in presence of a 26RFa receptor (GPR103) antagonist as well as in mice deficient for 26RFa. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production.

To conclude, in the present study we have identified a novel actor of the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system and we provide the evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.

sentences: Epigenetic signals that direct cell type specific interferon beta response in mouse cells The antiviral response induced by type I interferon (IFN) via the JAK-STAT signaling cascade activates hundreds of IFN-stimulated genes (ISGs). While this response occurs essentially in all human and mouse tissues it varies between different cell types. However, the linkage between the underlying epigenetic features and the ISG pattern of a given cell is not well understood. We mapped ISGs, binding sites of the STAT1 and STAT2 transcription factors and chromatin features in three different mouse cell types (embryonic stem cells, neural progenitor cells and embryonic fibroblasts) before and after treatment with IFN{beta}. The analysis included gene expression, chromatin accessibility and histone H3 lysine modification by acetylation (ac) and mono-/tri-methylation (me1, me3). A large fraction of ISGs and STAT binding sites were cell type specific with promoter binding of a STAT1-STAT2 complex (STAT1/2) being a key driver of ISG induction. Furthermore, STAT1/2 binding to putative enhancers at intergenic and intronic sites induced ISG expression as inferred from a chromatin co-accessibility analysis. STAT1/2 binding was dependent on the chromatin context and positively correlated with pre-existing H3K4me1 and H3K27ac marks in an open chromatin state while the presence of H3K27me3 had an inhibitory effect. Thus, chromatin features present before stimulation represent an additional regulatory layer for the cell type specific antiviral response.

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Emergent myxobacterial behaviors arise from reversal suppression induced by kin contacts A wide range of biological systems - from microbial swarms to bird flocks, display emergent behaviors driven by coordinated movement of individuals. To this end, individual organisms interact by recognizing their kin and adjusting their motility based on others around them. However, even in the best-studied systems, the mechanistic basis of the interplay between kin recognition and motility coordination is not understood. Here, using a combination of experiments and mathematical modeling, we uncover the mechanism of an emergent social behavior in Myxococcus xanthus. By overexpressing cell surface adhesins, TraA and TraB, involved in kin recognition, large numbers of cells adhere to one another and form organized macroscopic circular aggregates that spin clockwise or counterclockwise. Mechanistically, TraAB adhesion results in sustained cell-cell contacts that trigger cells to suppress cell reversals, and circular aggregates form as the result of cells ability to follow their own cellular slime trails. Furthermore, our in-silico simulations demonstrate a remarkable ability to predict self-organization patterns when phenotypically distinct strains are mixed. For example, defying naive expectations, both models and experiments found that strains engineered to overexpress different and incompatible traAB allelles nevertheless form mixed circular aggregates. Therefore, this work provides key mechanistic insights into M. xanthus social interactions and demonstrates how local cell contacts induce emergent collective behaviors by millions of cells.

ImportanceIn many species, large populations exhibit emergent behaviors whereby all related individuals move in unison. For example, fish in schools can all dart in one direction simultaneously to avoid a predator. Currently, it is impossible to explain how such animals recognize kin through brain cognition and elicit such behaviors at a molecular level. However, microbes also recognize kin and exhibit emergent collective behaviors that are experimentally tractable. Here, using a model social bacterium, we engineer dispersed individuals to organize into synchronized collectives that create emergent patterns. With experimental and mathematical approaches we explain how this occurs at both molecular and population levels. The results demonstrate how the combination of local physical interactions triggers intracellular signaling, which in turn leads to emergent behavior on a population scale.

sentences: DeepDRIM: a deep neural network to reconstruct cell-type-specific gene regulatory network using single-cell RNA-seq data AO_SCPLOWBSTRACTC_SCPLOWSingle-cell RNA sequencing is used to capture cell-specific gene expression, thus allowing reconstruction of gene regulatory networks. The existing algorithms struggle to deal with dropouts and cellular heterogeneity, and commonly require pseudotime-ordered cells. Here, we describe DeepDRIM a supervised deep neural network that represents gene pair joint expression as images and considers the neighborhood context to eliminate the transitive interactions. Deep-DRIM yields significantly better performance than the other nine algorithms used on the eight cell lines tested, and can be used to successfully discriminate key functional modules between patients with mild and severe symptoms of coronavirus disease 2019 (COVID-19).

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Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency Alpha-1 antitrypsin deficiency is a life-threatening condition caused by inheritance of the SERPINA1 gene  Z variant. This single base pair mutation leads to protein misfolding, ER entrapment and gain of toxic function. Despite the significant unmet medical need presented by this disorder, there remain no approved medicines and the only curative option is liver transplantation. We hypothesized that an unbiased screen of human hepatocytes harbouring the Z mutation (ATZ) using small molecules targeted against protein degradation pathways would uncover novel biological insights of therapeutic relevance. Here we report the results of that screen performed in a patient-derived iPSC model of ATZ. Starting from 1,041 compounds we identified 14 targets capable of reducing polymer burden, including Leucine-rich repeat kinase-2 (LRRK2), a well-studied target in Parkinsons. Genetic deletion of LRRK2 in ATZ mice reduced polymers and associated fibrotic liver disease leading us to test a library of commercially available LRRK2 kinase inhibitors in both patient iPSC and CHO cell models. One of the molecules tested, CZC-25146, reduced polymer load, increased normal AAT secretion and reduced inflammatory cytokines with pharmacokinetic properties supporting its potential use for treating liver diseases. We therefore tested CZC-25146 in the ATZ mouse model and confirmed its efficacy for polymer reduction without signs of toxicity. Mechanistically, in both human and mouse models, our data show CZC-25146 inhibits LRRK2 kinase activity and induces autophagy. Cumulatively, these findings support the use of CZC-25146 and LRRK2 inhibitors in general in hepatic proteopathy disease research and as potential new treatment approaches for patients.

One Sentence SummaryA small molecule screen in patient iPSCs with in vivo validation in mice identifies LRRK2 as a new therapeutic target for Alpha-1 Antitrypsin Deficiency.

sentences: Clustering and visualizing the distribution of overlapping reading frames in virus genomes Gene overlap occurs when two or more genes are encoded by the same nucleotides. This phenomenon is found in all taxonomic domains, but is particularly common in viruses, where it may increase the information content of compact genomes or influence the creation of new genes. Here we report a global comparative study of overlapping reading frames (OvRFs) of 12,609 virus reference genomes in the NCBI database. We retrieved metadata associated with all annotated reading frames in each genome record to calculate the number, length, and frameshift of OvRFs. Our results show that while the number of OvRFs increases with genome length, they tend to be shorter in longer genomes. The majority of overlaps involve +2 frameshifts, predominantly found in ds-DNA viruses. However, the longest overlaps involve no shift in reading frame (+0), increasing the selective burden of the same nucleotide positions within codons, instead of exposing additional sites to purifying selection. Next, we develop a new graph-based representation of the distribution of OvRFs among the reading frames of genomes in a given virus family. In the absence of an unambiguous partition of reading frames by homology at this taxonomic level, we used an alignment-free k-mer based approach to cluster protein coding sequences by similarity. We connect these clusters with two types of directed edges to indicate (1) that constituent reading frames are adjacent in one or more genomes, and (2) that the reading frames overlap. These adjacency graphs not only provide a natural visualization scheme, but also a novel statistical framework for analyzing the effects of gene- and genome-level attributes on the frequencies of overlaps.

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A cell wall-associated gene network shapes leaf boundary domains Boundary domains delimit and organize organ growth throughout plant development almost relentlessly building plant architecture and morphogenesis. Boundary domains display reduced growth and orchestrate development of adjacent tissues in a non-cell autonomous manner. How these two functions are achieved remains elusive despite the identification of several boundary-specific genes. Here, we show using morphometrics at the organ and cellular levels that leaf boundary domain development requires SPINDLY (SPY), an O-fucosyltransferase, to act as cell growth repressor. Further we show that SPY acts redundantly with the CUP-SHAPED COTYLEDON transcription factors (CUC2 and CUC3), which are major determinants of boundaries development. Accordingly at the molecular level, CUC2 and SPY repress a common set of genes involved in cell wall loosening providing a molecular framework for the growth repression associated with boundary domains. Atomic force microscopy (AFM) confirmed that young leaf boundary domain cells have stiffer cell walls than marginal outgrowth. This differential cell wall stiffness was reduced in spy mutant. Taken together our data reveal a concealed CUC2 cell wall associated gene network linking tissue patterning with cell growth and mechanics.

Summary statementDecreased cell-wall loosening gene expression contributes to the coordination of cell growth and mechanics with tissue patterning thus driving boundary development.

sentences: The petrosal and basicranial morphology of Protoceras celer Protoceratids are an extinct family of endemic North American artiodactyls. The phylogenetic position of protoceratids in relation to camelids and ruminants has been contentious for over a century. The petrosal morphology of basal (Leptotragulus) and derived (Syndyoceras) protoceratids has suggested that protoceratids are closely related to ruminants, whereas a prior description of a disarticulated intermediate protoceratid petrosal (Protoceras celer) indicated that protoceratids were closely related to camelids. This contradictory evidence implied that there were several character reversals within the protoceratid lineage and brought into question the utility of basicranial characters in artiodactyl phylogenetics. Here, we provide descriptions of an additional P. celer petrosal. The descriptions are based on data produced by computed tomography scans, which allowed us to image the petrosal in situ in the skull. Our results indicate that the petrosal morphology of P. celer is similar to that of other protoceratids, implying that, contrary to previous evidence, petrosal morphology is conserved within the Protoceratidae.

sentences: The predicted bZIP transcription factor ZIP-1 promotes resistance to intracellular infection in Caenorhabditis elegans Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts. For example, almost nothing is known about the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans. Two types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is a positive-sense RNA virus, and microsporidia, which are fungal pathogens. Surprisingly, these molecularly distinct pathogens induce a common host transcriptional response called the Intracellular Pathogen Response (IPR). Here we describe zip-1 as an IPR regulator that functions downstream of all known IPR activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor of previously unknown function, and we show how zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for all triggers of the IPR, and that this transcription factor plays a protective role against intracellular pathogen infection in C. elegans.

sentences: The extended Price equation: migration and sex The Price equation provides a general partition of evolutionary change into two components. The first is usually thought to represent natural selection and the second, transmission bias. Here, we provide a new derivation of the generalised equation, which contains a largely ignored third term. Unlike the original Price equation, this extension can account for migration and mixed asexual and sexual reproduction. The notation used here expresses the generalised equation explicitly in terms of fitness, rendering this otherwise difficult third term more open to biological interpretation and use. This re-derivation also permits fundamental results, derived from the Price equation, to be more easily generalised. We take Hamiltons rule as a case study, and provide an exact, total expression that allows for population structures like haplodiploidy. Our analysis, more generally, makes clear the previously hidden assumptions in similar fundamental results, highlighting the caution that must be taken when interpreting them.

sentences: A critical role of stable grain filling rate in maximizing rice yield revealed by whole plant carbon nitrogen interaction modeling Crop yield is co-determined by potential size of the harvest organ, photosynthetic potential of source organs, and pattern of partitioning and use of photosynthates among sink organs. Given a sufficient potential size of the harvest organ at flowering, how to fully fill them remains a central challenge in crop breeding for high yields. Here, we develop a kinetic model of rice grain filling, scaling primary biochemical and biophysical processes to whole-plant carbon and nitrogen dynamics. Predicted post-anthesis physiological and agronomic behaviors validate experimental observations under six endogenous and external perturbations. By large scale in silico screening, we show here that a stable grain filling rate from flowering to harvest is required to maximize grain yield, which is validated here in two independent super-high yielding rice cultivars (~21 t ha-1 rough rice yield at 14% moisture). On the other hand, we show grain yields in an elite rice cultivar may increase by about 30-40% by stabilizing its grain filling rate. Intriguingly, we have found that the sum of grain filling rates around 15 and 38 days after flowering largely determines grain yield, and have further developed a novel in situ approach quantifying grain filling rates and grain yield precisely with the measurements of ear respiratory rates (r>0.93). Potential post-anthesis molecular targets to maximize rice yield include delaying leaf senescence, enhancing leaf sucrose synthesis and export, limiting root growth, strengthening stem starch synthesis, accelerating endosperm starch synthesis, and moderating endosperm cell division. Our study provides an effective computational framework for post-anthesis crop physiology research and ideotype design.

sentences: Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential DRACH motif methylation among variants The causative agent of COVID-19 pandemic, the SARS-CoV-2 coronavirus, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 100 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced so far are in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because incorporation of dNTPs hides RNA base modifications. Here, in this perspective paper, we present an initial exploration of the Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3-untranslated region. We identified 15 m6A methylated positions, of which, 6 are in ORF N. Also, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants we show that variants Beta and Eta have a fourth position C>U change in DRACH at 28,884b that could affect methylation. The Nanopore technology offers a unique opportunity for the study of viral epitranscriptomics. This technique is PCR-free and is not sequencing-by-synthesis, therefore, no PCR bias and synthesis errors are introduced. The modified bases are preserved and assessed directly with no need for chemical treatments or antibodies. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.

sentences: Evolutionary Dynamics, Evolutionary Forces, and Robustness: A Nonequilibrium Statistical Mechanics Perspective Any realistic evolutionary theory has to consider: (i) the dynamics of organisms that reproduce and possess heritable traits; (ii) the appearance of stochastic variations in these traits; and (iii) the selection of those organisms that better survive and reproduce. These elements shape the "evolutionary forces" that characterize the evolutionary dynamics. Here, we introduce a general model of reproduction-variation-selection dynamics. By treating these dynamics as a non-equilibrium thermodynamic process, we make precise the notion of the forces that characterize evolution. One of these forces, in particular, can be associated with the robustness of reproduction to variations. The emergence of this trait in our model--without any explicit selection for it--is an example of a general phenomenon, which can be called enaptation, distinct from the well-known and studied phenomena of adaptation and exaptation. Some of the detailed predictions of our model can be tested by quantitative laboratory experiments, similar to those performed in the past on evolving populations of proteins or viruses.

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Origin and evolutionary landscape of Nr2f transcription factors across Metazoa BackgroundNuclear Receptor Subfamily 2 Group F (Nr2f) orphan nuclear hormone transcription factors (TFs) are fundamental regulators of many developmental processes in invertebrates and vertebrates. Despite the importance of these TFs throughout metazoan development, previous work has not clearly outlined their evolutionary history.

ResultsWe integrated molecular phylogeny with comparisons of intron/exon structure, domain architecture, and syntenic conservation to define critical evolutionary events that distinguish the Nr2f gene family in Metazoa. Our data indicate that a single ancestral pre-metazoan Nr2f gene, we have termed Nr2f1/2/5/6, predated six main Bilateria subfamilies, which include a single Nr2f1/2/5 homolog that is present throughout protostomes and invertebrate deuterostomes, Nr2f1/2 homologs in agnathans, and Nr2f1, Nr2f2, Nr2f5, Nr2f6 orthologs that are found in gnathostomes. The three Nr2f1/2 members in agnathans are due to independent expansions not found in gnathostomes, while the vertebrate Nr2f1, Nr2f2, Nr2f5 members arose from whole-genome duplications (WGDs). However, Nr2f6 members are the most divergent subfamily, likely originating from an ancient duplication, and are only retained by gnathostomes. Interestingly, Nr2f5 TFs have been independently lost in both cartilaginous fish and amniotes, such as humans. Furthermore, our analysis shows there are differential expansions and losses of Nr2f genes in teleosts following their additional rounds of WGDs.

ConclusionOverall, our evolutionary genomic analysis of Nr2f proteins helps to reveal the origins and previously unrecognized relationships of this ancient transcription factor family, which may allow for greater insights into the conservation of Nr2f functions that shape Metazoan body plans.

sentences: Modeling the C. elegans Germline Stem Cell Genetic Network using Automated Reasoning Computational methods and tools are a powerful complementary approach to experimental work for studying regulatory interactions in living cells and systems. We demonstrate the use of formal reasoning methods as applied to the Caenorhabditis elegans germ line, which is an accessible model system for stem cell research. The dynamics of the underlying genetic networks and their potential regulatory interactions are key for understanding mechanisms that control cellular decision-making between stem cells and differentiation. We model the "stem cell fate" versus entry into the "meiotic development" pathway decision circuit in the young adult germ line based on an extensive study of published experimental data and known/hypothesized genetic interactions. We apply a formal reasoning framework to derive predictive networks for control of differentiation. Using this approach we simultaneously specify many possible scenarios and experiments together with potential genetic interactions, and synthesize genetic networks consistent with all encoded experimental observations. In silico analysis of knock-down and overexpression experiments within our model recapitulate published phenotypes of mutant animals and can be applied to make predictions on cellular decision-making. This work lays a foundation for developing realistic whole tissue models of the C. elegans germ line where each cell in the model will execute a synthesized genetic network.

sentences: Exosomal transmission of α-synuclein initiates Parkinson's disease-like pathology The intercellular transmission of -synuclein is proposed to be involved in the pathogenesis of Parkinsons disease (PD)1. Supporting this transmission hypothesis, -synuclein has been identified to act as a prion2 and has been found in cerebrospinal fluid (CSF), blood, and saliva3. However, the mechanism required to initiate the pathogenic spread of -synuclein in the body that results in PD remains unclear. Here we identify a mechanism for the loading of -synuclein into exosomes, resulting in prion-like transmission that is mediated by the ubiquitin ligase activator, Ndfip1. Risk factors associated with PD, including metal toxicity, lysosome dysfunction and pesticide exposure,4 stimulate the upregulation of Ndfip1 and increase the loading of -synuclein into exosomes. We used this pathway to promote the loading of endogenous -synuclein into exosomes, that when intranasally delivered to either wild type or M83 transgenic mice, resulted in Parkinsons-like pathology including motor impairments and brain amyloids. Recipient cells require -synuclein for pathogenesis, as delivery of exosomes containing -synuclein to Snca knockout mice did not generate brain amyloids or Parkinsons-like motor impairments. Our results demonstrate a novel mechanism to initiate the prion-like spread of -synuclein in exosomes, following exposure to risk factors for PD.

sentences: Causal inference in environmental sound recognition Sound is caused by physical events in the world. Do humans infer these causes when recognizing sound sources? We tested whether the recognition of common environmental sounds depends on the inference of a basic physical variable - the source intensity (i.e., the power that produces a sound). A sources intensity can be inferred from the intensity it produces at the ear and its distance, which is normally conveyed by reverberation. Listeners could thus use intensity at the ear and reverberation to constrain recognition by inferring the underlying source intensity. Alternatively, listeners might separate these acoustic cues from their representation of a sounds identity in the interest of invariant recognition. We compared these two hypotheses by measuring recognition accuracy for sounds with typically low or high source intensity (e.g., pepper grinders vs. trucks) that were presented across a range of intensities at the ear or with reverberation cues to distance. The recognition of low-intensity sources (e.g., pepper grinders) was impaired by high presentation intensities or reverberation that conveyed distance, either of which imply high source intensity. Neither effect occurred for high-intensity sources. The results suggest that listeners implicitly use the intensity at the ear along with distance cues to infer a sources power and constrain its identity. The recognition of real-world sounds thus appears to depend upon the inference of their physical generative parameters, even generative parameters whose cues might otherwise be separated from the representation of a sounds identity.

sentences: AutoCoEv -- a high-throughput in silico pipeline for predicting inter-protein co-evolution Protein-protein communications govern cellular processes via complex regulatory networks, that are still far from being understood. Thus, identifying novel interactions between proteins can significantly facilitate our comprehension of the mechanistic principles of protein functions. Co-evolution between proteins is a sign of functional communication and, as such, provides a powerful approach to search for novel direct or indirect molecular partners. However, evolutionary analysis of large arrays of proteins, in silico, is a highly time-consuming effort, which has limited the usage of this method to protein pairs or small protein groups. Here, we developed AutoCoEv, a user-friendly computational pipeline for the search of co-evolution between a large number of proteins. By driving 15 individual programs, culminating in CAPS2 as the software for detecting co-evolution, AutoCoEv achieves seamless automation and parallelization of the workflow. Importantly, we provide a patch to CAPS2 source code to strengthen its statistical output, allowing for multiple comparisons correction and enhanced analysis of the results. We apply the pipeline to inspect co-evolution among 324 proteins identified to locate at the vicinity of the lipid rafts of B lymphocytes. We successfully detected multiple strong coevolutionary relations between the proteins, predicting many novel partners and previously unidentified clusters of functionally related molecules. We conclude that AutoCoEv, available at https://github.com/mattilalab/autocoev, can be used to predict functional interactions from large datasets in a time and cost-efficient manner.

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Out of the blue; Phototropins of the leaf vascular bundle sheath mediate the blue light regulation of the leaf hydraulic conductance The Arabidopsis leaf veins bundle sheath cells (BSCs) - a selective barrier to water and solutes entering the mesophyll - increase the leaf radial hydraulic conductance (Kleaf) by acidifying the xylem sap by their plasma membrane H+-ATPase, AHA2. Based on this and on the BSCs expression of PHOT1 and PHOT2, and the known blue-light (BL)-induced Kleaf increase, we hypothesized that, resembling the guard cells, BL perception by the BSCs phots activates its H+-ATPase, which, consequently, upregulates Kleaf. Indeed, under BL, the Kleaf of the knockout mutant lines phot1-5, phot2-1, phot1-5phot2-1, and aha2-4 was lower than that of the WT. BSC-only-directed complementation of phot1-5 or aha2-4 by PHOT1 or AHA2, respectively, restored the BL-induced Kleaf increase. BSC-specific silencing of PHOT1 or PHOT2 prevented such Kleaf increase. A xylem-fed kinase inhibitor (tyrphostin 9) replicated this also in WT plants. White light - ineffective in the phot1-5 mutant - acidified the xylem sap (relative to darkness) in WT and in the PHOT1-complemented phot1-5. These results, supported by BL increase of BSC protoplasts water permeability and cytosolic pH and their hyperpolarization by BL, identify the BSCs as a second phot-controlled water conductance element in leaves, in series with stomatal conductance. Through both, BL regulates the leaf water balance.

One-Sentence summaryBlue light regulates the leaf hydraulic conductance via the bundle-sheath cells blue light PHOT receptors which, via an autonomous signaling pathway, activate the BSCs AHA2 H+-pump.

sentences: Euchromatin activity enhances segregation and compaction of heterochromatin in the cell nucleus The large-scale organization of the genome inside the cell nucleus is critical for the cells function. Chromatin - the functional form of DNA in cells - serves as a substrate for active nuclear processes such as transcription, replication and DNA repair. Chromatins spatial organization directly affects its accessibility by ATP-powered enzymes, e.g., RNA polymerase II in the case of transcription. In differentiated cells, chromatin is spatially segregated into compartments - euchromatin and heterochromatin - the former being largely transcriptionally active and loosely packed, the latter containing mostly silent genes and densely compacted. The euchromatin/heterochromatin segregation is crucial for proper genomic function, yet the physical principles behind it are far from understood. Here, we model the nucleus as filled with hydrodynamically interacting active Zimm chains - chromosomes - and investigate how large heterochromatic regions form and segregate from euchromatin through their complex interactions. Each chromosome presents a block copolymer composed of heterochromatic blocks, capable of crosslinking that increases chromatins local compaction, and euchromatic blocks, subjected to stochastic force dipoles that capture the microscopic stresses exerted by nuclear ATPases. These active stresses lead to a dynamic self-organization of the genome, with its coherent motions driving the mixing of chromosome territories as well as large-scale heterochromatic segregation through crosslinking of distant genomic regions. We study the stresses and flows that arise in the nucleus during the heterochromatic segregation, and identify their signatures in Hi-C proximity maps. Our results reveal the fundamental role of active mechanical processes and hydrodynamic interactions in the kinetics of chromatin compartmentalization and in the emergent large-scale organization of the nucleus.

sentences: The Fate of DIET Consortium exposed to continuous and intermittent electrochemical stimulation Electromethanogenesis is the electrochemical conversion of carbon dioxide (CO2) to methane (CH4) with microorganisms as catalysts, and it is a promising approach for biogas upgrading. Many studies have shown increased methane production by electrochemical stimulation, allegedly due to the enhancement of direct interspecies electron transfer (DIET) between electrogenic bacteria and methanogens. However, these studies were done with mixed cultures where the interspecies interactions were undefined, so the impact of an electrochemical stimulus on these interactions remains unresolved. Here we report that an electrochemical stimulus diminished the survival and activity of a defined DIET consortium. We investigated the impact of electrochemical stimulation on a co-culture of Geobacter metallireducens and Methanosarcina barkeri, where the two partners interact syntrophically via DIET. The electrochemical stimulus was a cathode poised at - 700 mV (versus the standard hydrogen electrode, SHE). Electrochemical stimulation was provided either continuously or intermittently, and so was the food substrate. Compared to the untreated DIET co-culture, all treatment strategies relying on cathodic current additions resulted in acetate accumulation, lower methane production and lower cell numbers for both partners, indicating a detrimental impact of electrochemical stimulation to a DIET consortium. These findings suggest that future bioelectrochemical technologies must guarantee the survival of the syntrophic partners during current additions.

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

SIGNIFICANCE STATEMENTMyelin debris from degenerating axons along with reactive astrocytes in the glial scar inhibit CNS axon regeneration. However, even with the recently developed experimental approaches which activate axons to regenerate passed the glial scar, almost all axons still stall growth before reaching their post-synaptic targets. Here, we show that post-injury born oligodendrocytes integrate into the glial scar, and that other than myelin debris, live oligodendrocytes prematurely myelinating the regenerating axons inhibit growth, even if the axons have already regenerated passed the glial scar.

sentences: miR-17~92 exerts stage-specific effects in adult V-SVZ neural stem cell lineages In the adult mouse brain, neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ) generate neurons and glia throughout life. microRNAs are important regulators of cell states, frequently acting in a stage- or context-dependent manner. Here, miRNA profiling of FACS-purified populations identified miR-17[~]92 as highly upregulated in activated NSCs and transit amplifying cells (TACs) in comparison to quiescent NSCs. Conditional deletion of miR-17[~]92 in NSCs reduced stem cell proliferation both in vitro and in vivo. In contrast, in TACs, miR-17[~]92 deletion caused a selective shift from neurogenic DLX2+ TACs towards oligodendrogenic OLIG2+ TACs, resulting in increased oligodendrogenesis to the corpus callosum. miR-17[~]92 deletion also decreased proliferation and maturation of intraventricular oligodendrocyte progenitor cells. Together, these findings reveal stage- and cell-type-specific functions of the miR-17[~]92 cluster within adult V-SVZ neural stem cell lineages.

sentences: Effects of habitual dart training on hemispheric lateralization in older adults: a cross-sectional study using functional near-infrared spectroscopy Exercise training is one of the most effective ways of preventing dementia. In this study, we focus on exercise training using a dart game called Wellness Darts. Previous studies have shown that Wellness Darts is effective in preventing dementia through cognitive memory testing. However, the effects of continual Wellness Dart training on brain function are unclear. The purpose of this study was to elucidate the effects of sustained Wellness Darts practice on brain function in older adults. Here, we cross-sectionally compared the brain activity patterns between an expert older group who practiced darts continually against control groups of a non-expert older group and a younger group who had no experience with darts. Functional near-infrared spectroscopy (fNIRS) was used to measure brain activity, and their patterns were quantified by lateralization index (LI). The results showed that the younger group and the expert older group had significantly higher LI values than the non-expert older group and that these values did not differ between the expert older and younger groups. In addition, the accuracy of the dart-throwing test in the expert older group was positively correlated with the LI value. These results suggest that the Wellness Dart promotes hemispheric lateralization and has potential as exercise training in the prevention of dementia.

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Enterotoxigenic Escherichia coli display a distinct growth phase before entry into stationary phase with shifts in tryptophan- fucose- and putrescine metabolism and degradation of neurotransmitter precursors Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and adults in endemic areas. Gene regulation of ETEC during growth in vitro and in vivo needs to be further evaluated, and here we describe the full transcriptome and metabolome of ETEC during growth from mid-logarithmic growth to stationary phase in rich medium (LB medium). We identified specific genes and pathways subjected to rapid transient alterations in gene expression and metabolite production during the transition between logarithmic to stationary growth. The transient phase during late exponential growth is different from the subsequent induction of stationary phase-induced genes, including stress and survival responses as described earlier. The transient phase was characterized by the repression of genes and metabolites involved in organic substance transport. Genes involved in fucose and putrescine metabolism were upregulated, and genes involved in iron transport were repressed. Expression of toxins and colonization factors were not changed, suggesting retained virulence. Metabolomic analyses showed that the transient phase was characterized by a drop of intracellular amino acids, e.g., L-tyrosine, L-tryptophan, L-phenylalanine, L-leucine, and L-glutamic acid, followed by increased levels at induction of stationary phase. A pathway enrichment analysis of the entire transcriptome and metabolome showed activation of pathways involved in the degradation of neurotransmitters aminobutyrate (GABA) and precursors of 5-hydroxytryptamine (serotonin). This work provides a comprehensive framework for further studies on transcriptional and metabolic regulation in pathogenic E. coli.

ImportanceWe show that E. coli, exemplified by the pathogenic subspecies enterotoxigenic E. coli (ETEC), undergoes a stepwise transcriptional and metabolic transition into the stationary phase. At a specific entry point, E. coli induces activation and repression of specific pathways. This leads to a rapid decrease of intracellular levels of L-tyrosine, L-tryptophan, L-phenylalanine, L-leucine, and L-glutamic acid due to metabolism into secondary compounds. The resulting metabolic activity leads to an intense but short peak of indole production, suggesting that this is the previously described "indole peak," rapid decrease of intermediate molecules of bacterial neurotransmitters, increased putrescine and fucose uptake, increased glutathione levels, and decreased iron uptake. This specific transient shift in gene expression and metabolomics is short-lived and disappears when bacteria enter the stationary phase. We suggest it mainly prepares bacteria for ceased growth, but the pathways involved suggest that this transient phase substantially influences survival and virulence.

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The Central Importance of Hub Proteins in a Disease-Gene Network Model: A New Paradigm of Chronic Myeloid Leukemia Disease Study BackgroundThe network biology of disease-gene association provides a holistic framework to decipher the intrinsic complexity of disease signaling pathways into cellular communication level. Different types of studies including large-scale genome-wide association, multifactor dimensional reduction analysis, whole genome, or exome-based sequencing strategies of diseases are striving to connect genes to diseases. Indeed, these approaches have had some accomplishments, but the cellular communication level needs a more streamlining outcome to understand the mechanistic impact of context. The higher-order combination of disease-gene interaction has a great potential to decipher the intricateness of diseases. The molecular interaction pattern of diseases at the genomic and proteomic level offers a revolutionized platform not only to understand the complexity of particular disease modules and pathways but also leading towards design novel therapeutics.

ResultsThe enrichment and topology analysis was performed by JEPETTO a plugin of Cytoscape software. We identified the chronic myeloid leukemia (CML) disease signaling pathways that appeared first in the ranking order based on XD-score among the bone, breast, and colon genes set and second at kidney and liver. This result validates the highest proximity between CML and five cancerous tissue gene set clusters. The topology analysis also supports the results while (p<0.0001) is considered to be extremely significant between CML and fives cancerous tissues genes set. Enrichment analysis identified that abl-gene acts as an overlapping node which is the major gene for inducing various mutations in CML. Amazingly, we identified 56 common path expansion/added genes among these five cancerous tissues which can be considered the direct cofactors of CML disease. By relative node degree, resolution, possible ligand, stoichiometry, Q-mean, and Z-score analysis we found 11 hubs proteins like SMAD3, GRB2, TP53, SMAD4, RB1, HDAC1, RAF1, ABL1, SHC1, TGFBR1, RELA which can be regarded for further drug target identification.

ConclusionsOur proposed network analysis reflects on the gene set interaction pattern of disease signaling pathways of humans. The integrated multidrug computational and experimental approaches boost up to improve the novel drug target approach. Besides, such a trove can yield unprecedented insights to lead to an enhanced understanding of potential application both in drug target optimization and for drug dislodging.

sentences: DBPOM: a comprehensive database of pharmaco-omics for cancer precision medicine During the course of cancer treatment, both efficacy and the adverse effects of drugs on patients should be taken into account. Although some public databases and modeling frameworks have been developed through studies on drug response, the negative effects of drugs are always neglected. Furthermore, most of them only considered the ramifications of the drug on the cell line, but the effects on the patient still require a huge amount of work to integrate data from various databases and calculations, especially in relation to precision treatment. In order to address these issues, we developed the DBPOM (http://www.dbpom.net/, a comprehensive database of pharmaco-omics for cancer precision medicine), which explores various drugs efficacy levels by calculating their potency in reverse, or enhancing cancer-associated gene expression changes. When compared with existing databases, the DBPOM could estimate the effectiveness of a drug on individual patients through the mapping of various cell lines to each person according to their genetic mutation similarities. The DBPOM is an easy-to-use and one-stop database for clinicians and drug researchers to search and analyze the overall effect of a drug or a drug combination on cancer patients as well as the biological functions that they target. We anticipate that DBPOM will become an important resource and analysis platform for drug development, drug mechanism studies and the discovery of new therapies.

sentences: Experience-dependent plasticity modulates ongoing activity in the antennal lobe and enhances odor representations Ongoing neural activity has been observed across several brain regions and thought to reflect the internal state of the brain. Yet, it is not fully understood how ongoing brain activity interacts with sensory experience and shape sensory representations. Here, we show that projection neurons of the fruit fly antennal lobe exhibit spatiotemporally organized ongoing activity in the absence of odor stimulation. Upon repeated exposure to odors, we observe a gradual and long-lasting decrease in the amplitude and frequency of spontaneous calcium events, as well as a reorganization of correlations between olfactory glomeruli during ongoing activity. Accompanying these plastic changes, we find that repeated odor experience reduces trial-to-trial variability and enhances the specificity of odor representations. Our results reveal a previously undescribed experience-dependent plasticity of ongoing and sensory driven activity at peripheral levels of the fruit fly olfactory system.

sentences: The Most Widespread Phage in Animals: Genomics and Taxonomic Classification of Phage WO Wolbachia are the most common obligate, intracellular bacteria in animals. They exist worldwide in arthropod and nematode hosts in which they commonly act as reproductive parasites or mutualists, respectively. Bacteriophage WO, the largest of Wolbachias mobile elements, includes reproductive parasitism genes, serves as a hotspot for genetic divergence and genomic rearrangement of the bacterial chromosome, and uniquely encodes a Eukaryotic Association Module with eukaryotic-like genes and an ensemble of putative host interaction genes. Despite WOs relevance to genome evolution, selfish genetics, and symbiotic applications, relatively little is known about its origin, host range, diversification, and taxonomic classification. Here we analyze the most comprehensive set of 150 Wolbachia and phage WO assemblies to provide a framework for discretely organizing and naming integrated phage WO genomes. We demonstrate that WO is principally in arthropod Wolbachia with relatives in diverse endosymbionts and metagenomes, organized into four variants related by gene synteny, often oriented opposite the origin of replication in the Wolbachia chromosome, and the large serine recombinase is an ideal typing tool to assign taxonomic classification of the four variants. We identify a novel, putative lytic cassette and WOs association with a conserved eleven gene island, termed Undecim Cluster, that is enriched with virulence-like genes. Finally, we evaluate WO-like Islands in the Wolbachia genome and discuss a new model in which Octomom, a notable WO-like Island, arose from a split with WO. Together, these findings establish the first comprehensive Linnaean taxonomic classification of endosymbiont phages that includes distinguishable genera of phage WO, a family of non-Wolbachia phages from aquatic environments, and an order that captures the collective relatedness of these viruses.

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Drosophila fabp  is a retinoid-inducible gene required for Rhodopsin-1 homeostasis and photoreceptor survival Retinoids act as chromophore co-factors for light-detecting rhodopsin proteins. In vertebrates, retinoids also actively regulate gene expression. Whether retinoids regulate gene expression in Drosophila for a specific biological function remains unclear. Here, we report that Drosophila fatty acid binding protein (fabp) is a retinoid-inducible gene required for Rhodopsin-1 (Rh1) protein homeostasis and photoreceptor survival. Specifically, we performed a photoreceptor-specific gene expression profiling study in flies bearing a misfolding-prone Rhodopsin-1 (Rh1) mutant, ninaEG69D, which serves as a Drosophila model for Retinitis Pigmentosa. ninaEG69D photoreceptors showed increased expression of genes that control Rh1 protein levels, along with a poorly characterized gene, fabp. We found that in vivo fabp expression was reduced when the retinoids were deprived through independent methods. Conversely, fabp mRNA was induced when we challenged cultured Drosophila cells with retinoic acid. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-activated Rh1 degradation. These observations indicate that a retinoid-inducible gene expression program regulates fabp that is required for Rh1 proteostasis and photoreceptor survival.

Author SummaryRhodopsins are light-detecting proteins that use retinoids as chromophore co-factors. In vertebrates, retinoids also actively regulate gene expression. Whether retinoids regulate Rhodopsin function aside from its role as a chromophore remains unclear. Here, we report that Drosophila fatty acid binding protein (fabp) is a retinoid-inducible gene required for Rhodopsin-1 (Rh1) protein homeostasis and photoreceptor survival. Specifically, we found that fabp is among the genes induced by a misfolding-prone Rhodopsin-1 (Rh1) mutant, ninaEG69D, which serves as a Drosophila model for Retinitis Pigmentosa. We further found that fabp induction in ninaEG69D photoreceptors required retinoids. fabp was required in photoreceptors to help degrade light-activated Rh1. In the absence of fabp, Rh1 accumulated in cytoplasmic vesicles in a light-dependent manner, and exhibited light-dependent retinal degeneration. These observations indicate that a retinoid-inducible gene expression program regulates fabp that is required for Rh1 proteostasis and photoreceptor survival.

sentences: An epitope specific patient-derived LGI1-autoantibody enhances neuronal excitability by modulating the Kv1.1 channel. Leucine-rich Glioma Inactivated protein 1 (LGI1) is expressed in the central nervous and genetic loss of function is associated with epileptic disorders. Also, patients with LGI1-directed autoantibodies have frequent focal seizures as a key feature of their disease. LGI1 is composed of a Leucine Rich Repeat (LRR) and an Epitempin (EPTP) domain. These domains are reported to interact with different aspects of the transsynaptic complex formed by LGI1 at excitatory synapses, including presynaptic Kv1 potassium channels. Patient-derived monoclonal antibodies (mAbs) are ideal reagents to study whether domain-specific LGI1-autoantibodies induce epileptiform activities in neurons, and their downstream mechanisms. To address this question, we measured the intrinsic excitability of CA3 pyramidal neurons in organotypic cultures from rat hippocampus treated with either a LRR- or an EPTP-reactive patient-derived mAb. The antibodies induced changes in neuronal intrinsic excitability which led us to measure their effects on Kv1-type potassium currents. We found an increase of intrinsic excitability correlated with a reduction of the sensitivity to a selective Kv1.1-channel blocker in neurons treated with the LRR mAb compared to the control, but not in neurons treated with the EPTP mAb. Our findings suggest LRR mAbs are able to modulate neuronal excitability that could account for epileptiform activities observed in patients.

sentences: Reliability of Mouse Behavioural Tests of Anxiety: a Systematic Review and Meta-Analysis on the Effects of Anxiolytics. Animal research on anxiety and anxiety disorders relies on valid animal models of anxiety. However, the validity of widely used rodent behavioural tests of anxiety has repeatedly been questioned, as they often fail to produce consistent results across independent replicate studies using different study populations or different anxiolytic compounds. In this study, we assessed the sensitivity of behavioural tests of anxiety in mice to detect anxiolytic effects of drugs prescribed to treat anxiety in humans. To this end, we conducted a pre-registered systematic review of studies reporting tests of anxiolytic compounds against a control treatment using common behavioural tests of anxiety in mice. PubMed and EMBASE were searched on August 21st 2019 for studies published in English and 814 papers were identified for inclusion. Risk of bias was assessed based on Syrcles risk of bias tool and the Camarades study quality checklist on a randomly selected subsample of 180 papers. Meta-analyses on effect sizes of treatments using standardized mean differences (Hedges g) showed that only two of 17 test measures reliably detected effects of anxiolytic compounds other than diazepam. Further, we report considerable variation in both direction and size of effects of most anxiolytics on most outcome variables, indicating poor replicability of test results. This was corroborated by high heterogeneity in most test measures. Finally, we found an overall high risk of bias. Our findings indicate a general lack of sensitivity of common behavioural tests of anxiety in mice to anxiolytic compounds and cast serious doubt on both construct and predictive validity of most of those tests. The use of animals to model human conditions can be justified only if the expected results are informative, reproducible, and translatable. In view of scientifically valid and ethically responsible research, we call for a revision of behavioural tests of anxiety in mice and the development of more predictive tests.

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Pseudomonas aeruginosa synthesizes the autoinducers of its oxylipin-dependent quorum sensing system extracellularly The oxylipin-dependent quorum sensing system (ODS) of Pseudomonas aeruginosa relies on the production and sensing of two oxylipin autoinducers, 10S-hydroxy-(8E)-octadecenoic acid (10-HOME) and 7S,10S dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Here, and contrary to the prevailing notion that bacterial autoinducers are synthesized intracellularly, we show that 10-HOME and 7,10-DiHOME biosynthesis occurs extracellularly, and this requires the secretion of the oxylipin synthases. We implemented a genetic screen of P. aeruginosa strain PAO1, which identified fourteen genes required for the synthesis of oxylipins. Among the identified genes, four encoded components of the ODS system and the other ten were part of the Xcp type II secretion system (T2SS). We created a deletion mutant of xcpQ, which encodes the outer membrane component of Xcp, and found it recapitulated the impaired functionality of the transposon mutants. Upon further examination, the lack of ODS function was demonstrated to be caused by the blocking of the DS enzymes secretion. Notably, the xcpQ mutant activated the ODS system when exposed to 10-HOME and 7,10-DiHOME, indicating that the sensing component of this quorum sensing system remains fully functional. In contrast with the detrimental effect previously described for T2SS in biofilm formation, here we observed that T2SS was required for robust in vitro and in vivo biofilm formation in an ODS dependent manner. To the best of our knowledge, this study is the first to find QS autoinducers that are synthetized in the extracellular space and provides new evidence for the role of the T2SS for biofilm formation in P. aeruginosa.

IMPORTANCEWe previously showed that the ODS quorum sensing system of P. aeruginosa produces and responds to oxylipins derived from host oleic acid by enhancing biofilm formation and virulence. Herein, we developed a genetic screen strategy to explore the molecular basis for oxylipins synthesis and detection. Unexpectedly, we found that the ODS autoinducer synthases cross the outer membrane using the Xcp Type 2 secretion system of P. aeruginosa and thus, the biosynthesis of oxylipins occur extracellularly. Biofilm formation, which was thought to be impaired as result of Xcp activity, was found to be enhanced as result of ODS activation. This is a unique QS system strategy and reveals a new way by which P. aeruginosa interacts with the host environment.

sentences: Highly accurate local functional fingerprints and their stability Individual subjects can be accurately identified in a database based on their functional connectome (FC), i.e. the whole-brain patterns of synchrony in fluctuations of brain activation as measured with functional MRI (fMRI). Accurate identification requires a representation of brain function to be simultaneously reliable (i.e. low intraindividual variability) and idiosyncratic (i.e. high interindividual variability), hence calling it a "fingerprint". Importantly, several studies have shown lower test-retest reliability (i.e. higher intraindividual variability) of the global FC compared to local representations of brain function such as regional homogeneity (ReHo) and (fractional) amplitude of low-frequency fluctuations ((f)ALFF). Therefore, here, with resting-state and task fMRI-data from the Human Connectome Project and the enhanced Nathan Kline Institute, we demonstrate that the local functional fingerprint, and especially regional homogeneity (ReHo), achieves near-perfect identification accuracies using four different ways to quantify identification. This finding is replicable across various parcellations as well as resilient to confounding effects. Moreover, using a machine-learning setup, we show that the small intraindividual ReHo fingerprint variability across sessions is meaningful for explaining individual-level intelligence. Further analyses reveal that the attention networks and the Default Mode Network contributed the most to the individual "uniqueness" of the ReHo fingerprint. Last, with another publicly available dataset, we show that ReHo is also stable in individuals with schizophrenia and that its (in)stability across sessions relates to intelligence scores. Altogether, our findings suggest that the ReHo fingerprint is a good candidate for further exploration of applicability in precision medicine.

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Endoglin deficiency elicits hypoxia-driven congestive heart failure in zebrafish. Hereditary hemorrhagic Telangiectasia (HHT) is a rare genetic disease relying on mutations affecting components of Bone Morphogenetic Protein and Transforming Growth Factor-{beta} (BMP/TGF-{beta}) signaling pathway in endothelial cells. This disorder is characterized by arterio-venous malformations prone to rupture. and ensuing hemorrhages are responsible for iron deficiency anemia. Along with Activin receptor-like kinase ALK1, Endoglin is involved in the vast majority of HHT cases. In this report, we characterized zebrafish endoglin locus and demonstrated that it produces two phylogenetically conserved protein isoforms using a distinctive alternative splicing mechanism. Functional analysis of a Crispr/Cas9 zebrafish Endoglin mutant revealed that Endoglin deficiency results in massive death during the course from juvenile stage to adulthood. Endoglin deficient fish develop a cardiomegaly resulting in heart failure and hypochromic anemia which both stem from chronic hypoxia. Histological analysis and confocal imaging evidenced structural alterations of the developing gill and its underlying vascular network that tally with hypoxia. Finally, phenylhydrazine treatment demonstrated that lowering hematocrit/blood viscosity alleviates heart failure and enhances survival of Endoglin deficient fish. Altogether, our data indicate that Endoglin is crucial for gill vascular development and that further studies using zebrafish in general and this endoglin mutant in particular will provide crucial hints regarding the molecular and cellular events altered in HHT for the development of new therapeutic strategies.

Summary StatementEndoglin deficiency in zebrafish recapitulates critical aspects of Hereditary Hemorrhagic Telangiectasia (HHT) and will thus constitute a valuable model in large scale screens for HHT-active drugs.

sentences: Affordable C. elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system.

sentences: Acute inflammation, mediated by lung neutrophils, confers enhanced protection against Mycobacterium tuberculosis infection in mice Inflammation plays a crucial role in the control of Mycobacterium tuberculosis (M.tb) infection. In this study, we demonstrate that an inflammatory pulmonary environment at the time of infection mediated by liposaccharide (LPS) treatment in mice confers enhanced protection against M.tb for up to 6 months post infection. This transient protective inflammatory environment was associated with a neutrophil and monocyte/macrophage influx as well as increased inflammatory cytokines. In vitro infection of neutrophils from LPS treated mice demonstrated that LPS neutrophils exhibited increased recognition of M.tb, and had a greater innate capacity for killing M.tb. Finally, partial depletion of neutrophils in LPS treated mice showed an increase in M.tb burden, suggesting neutrophils conferred the enhanced protection observed in LPS treated mice. These results indicate a positive role of an inflammatory environment during initial M.tb infection, and suggests that acute inflammation at the time of M.tb infection can positively alter disease outcome.

sentences: Predictability and parallelism in the contemporary evolution of hybrid genomes Hybridization between species is widespread across the tree of life. As a result, many species, including our own, harbor regions of their genome derived from hybridization. Despite the recognition that this process is widespread, we understand little about how the genome stabilizes following hybridization, and whether the mechanisms driving this stabilization tend to be shared across species. Here, we dissect the drivers of variation in local ancestry across the genome in replicated hybridization events between two species pairs of swordtail fish: Xiphophorus birchmanni x X. cortezi and X. birchmanni x X. malinche. We find unexpectedly high levels of repeatability in local ancestry across the two types of hybrid populations. This repeatability is attributable in part to the fact that the recombination landscape and locations of functionally important elements play a major role in driving variation in local ancestry in both types of hybrid populations. Beyond these broad scale patterns, we identify dozens of regions of the genome where minor parent ancestry is unusually low or high across species pairs. Analysis of these regions points to shared sites under selection across species pairs, and in some cases, shared mechanisms of selection. We show that one such region is a previously unknown hybrid incompatibility that is shared across X. birchmanni x X. cortezi and X. birchmanni x X. malinche hybrid populations.

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Cancer type classification in liquid biopsies based on sparse mutational profiles enabled through data augmentation and integration Identifying the cell of origin of cancer is important to guide treatment decisions. However, in patients with  cancer of unknown primary (CUP), standard diagnostic tools often fail to identify the primary tumor. As an alternative, machine learning approaches have been proposed to classify the cell of origin based on somatic mutation profiles in the genome of solid tissue biopsies. However, solid biopsies can cause complications and certain tumors are not accessible. A promising alternative would be liquid biopsies, which contain ctDNA originating from the tumor. Problematically, somatic mutation profiles of tumors obtained from liquid biopsies are inherently extremely sparse and current machine learning models fail to perform in this setting.

Here we propose an improved machine learning method to deal with the sparse nature of liquid biopsy data. Firstly, we downsample the SNVs in the samples in order to mimic sparse data conditions. Then extensive data augmentation is performed to artificially increase the number of training samples in order to enhance model robustness under sparse data conditions. Finally, we employ data integration to merge information from i) somatic single nucleotide variant (SNV) density across the genome, ii) somatic SNVs in driver genes and iii) trinucleotide motifs. Our adapted method achieves an average accuracy of 0.88 on the data where only 70% of SNVs are retained, which is comparable to an average accuracy of 0.87 with the original model on the full SNV data. Even when only 2% of the data is retained, the average accuracy is 0.65 compared to 0.41 with the original model. The method and results presented here open the way for application of machine learning in the detection of the cell of origin of cancer from sparse liquid biopsy data.

Author SummaryThe identification of the  cell of origin of cancer is an important step towards more personalized cancer care, but this remains a challenge for patients with  cancer of unknown primary (CUP) where the source of the malignancy cannot be identified even after extensive clinical assessment with standard diagnostic methods. Somatic mutation profile-based  cell of origin classification has emerged in recent years as a promising alternative diagnostic tool that could circumvent the issues of standard CUP diagnostic. In this approach the somatic mutations are obtained from whole genome sequencing (WGS) of solid tissue biopsies from the tumor. However, needle biopsies from tumor tissue can be challenging, as accessibility to the tumor can be limited and taking a biopsy can cause further complications. For these reasons, liquid biopsies have been proposed as a safer alternative to solid tissue biopsies. Problematically, the circulating tumor DNA fragments available in e.g. blood typically represent a much scarcer tumor source than conventional solid tissue biopsies and therefore liquid biopsies give rise to sparse somatic mutation profiles. Therefore it is crucial to investigate the applicability of sparse somatic mutation profiles in the identification of  cell of origin and explore potential improvements of the data analysis and prediction models to overcome sparsity.

sentences: E-Cadherin mediated AMIS localisation Individual cells within de novo polarising tubes and cavities must integrate their forming apical domains into a centralised apical membrane initiation site (AMIS). This is necessary to enable organised lumen formation within multi-cellular tissue. Despite the well documented importance of cell division in localising the AMIS, we have found a division-independent mechanism of AMIS localisation that relies instead on Cadherin-mediated cell-cell adhesion. Our study of de novo polarising mouse embryonic stem cells (mESCs) cultured in 3D suggest that cell-cell adhesion directs the localisation of apical proteins such as PAR-6 to a centralised AMIS. Unexpectedly, we also found that mESC cell clusters lacking functional E-Cadherin were still able to form a lumen-like cavity in the absence of AMIS localisation and did so at a later stage of development via a  closure mechanism, instead of via hollowing. This work suggests that there are two, interrelated mechanisms of apical polarity localisation: cell adhesion and cell division. Alignment of these mechanisms in space allows for redundancy in the system and ensures the development of a coherent epithelial structure within a growing organ.

sentences: Host PDZ-containing proteins targeted by SARS-Cov-2 Small linear motif targeting protein interacting domains called PDZ have been identified at the C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A and N showing significant interactions with dissociation constants values ranging from 3 M to 82 M. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Seven of the PDZ-containing proteins among binders of the SARS-CoV-2 proteins E, 3a or N affect significantly viral replication under knock-down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.

sentences: Young Infants Process Prediction Errors at the Theta Rhythm Examining how young infants respond to unexpected events is key to our understanding of their emerging concepts about the world around them. From a predictive processing perspective, it is intriguing to investigate how the infant brain responds to unexpected events (i.e., prediction errors), because they require infants to refine their predictions about the environment. Here, to better understand prediction error processes in the infant brain, we presented 9-month-olds (N = 36) a variety of physical and social events with unexpected versus expected outcomes, while recording their electroencephalogram (EEG). We found a pronounced response in the ongoing 4 - 5 Hz theta rhythm for the processing of unexpected (in contrast to expected) events, for a prolonged time window (2 s) and across all scalp-recorded electrodes. The condition difference in the theta rhythm was not related to the condition difference in infants event-related activity to unexpected (versus expected) events in the negative central (Nc) component (.4 - .6 s), a component, which is commonly analyzed in infant violation of expectation studies using EEG. These findings constitute critical evidence that the theta rhythm is involved in the processing of prediction errors from very early in human brain development. We discuss how the theta rhythm may support infants refinement of basic concepts about the physical and social environment.

sentences: Small Molecule Stabilization of PINK-1/PINK1 Improves Neurodegenerative Disease Macroautophagic recycling of dysfunctional mitochondria, known as mitophagy, is essential for mitochondrial homeostasis and cell viability. Accumulation of defective mitochondria and impaired mitophagy have been widely implicated in many neurodegenerative diseases, and loss-of-function mutations of two regulators of mitophagy, PINK1 and Parkin, are amongst the most common causes of recessive Parkinsons disease. Activation of mitophagy via pharmacological treatments may be a feasible approach for combating neurodegeneration. In this effort, we screened [~]45,000 small molecules for the ability to activate mitophagy. A high-throughput, whole-organism, phenotypic screen was conducted by monitoring stabilization of PINK-1/PINK1, a key event in mitophagy activation, in a Caenorhabditis elegans strain carrying a Ppink-1::PINK-1::GFP reporter. We obtained eight hits that induced mitophagy, as evidenced by increased mitochondrial fragmentation and autophagosome formation. Several of the compounds also reduced ATP production, oxygen consumption, mitochondrial mass, and/or mitochondrial membrane potential. Importantly, we found that treatment with two compounds, which we named PS83 and PS106 (more commonly known as sertraline) reduced neurodegenerative disease phenotypes (including delayed paralysis in a C. elegans Alzheimers model) in a PINK-1/PINK1-dependent manner. This report presents a promising step toward the identification of compounds that will stimulate mitochondrial turnover.

sentences: Effective Ensemble of Deep Neural Networks Predicts Neural Responses to Naturalistic Videos This report provides a review of our submissions to the Algonauts Challenge 2021. In this challenge, neural responses in the visual cortex were recorded using functional neuroimaging when participants were watching naturalistic videos. The goal of the challenge is to develop voxel-wise encoding models which predict such neural signals based on the input videos. Here we built an ensemble of models that extract representations based on the input videos from 4 perspectives: image streams, motion, edges, and audio. We showed that adding new modules into the ensemble consistently improved our prediction performance. Our methods achieved state-of-the-art performance on both the mini track and the full track tasks.

sentences: Screening of cell-virus, cell-cell, gene-gene interactions among kingdoms of life at single cell resolution The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) issued a significant and urgent threat to global health. The exact animal origin of SARS-CoV-2 remains obscure and understanding its host range is vital for preventing interspecies transmission. Previously, we have assessed the target cell profiles of SARS-CoV-2 in pets, livestock, poultry and wild animals. Herein, we expand this investigation to a wider range of animal species and viruses to provide a comprehensive source for large-scale screening of potential virus hosts. Single cell atlas for several mammalian species (alpaca, hamster, hedgehog, chinchilla etc.), as well as comparative atlas for lung, brain and peripheral blood mononuclear cells (PBMC) for various lineages of animals were constructed, from which we systemically analyzed the virus entry factors for 113 viruses over 20 species from mammalians, birds, reptiles, amphibians and invertebrates. Conserved cellular connectomes and regulomes were also identified, revealing the fundamental cell-cell and gene-gene cross-talks between these species. Overall, our study could help identify the potential host range and tissue tropism of SARS-CoV-2 and a diverse set of viruses and reveal the host-virus co-evolution footprints.

sentences: A linear response theory based method for prediction of large scale protein conformational changes upon ligand binding Prediction of ligand-induced protein conformational transitions is a challenging task due to a large and rugged conformational space, and limited knowledge of probable direction(s) of structure change. These transitions can involve a large scale, global (at the level of entire protein molecule) structural change and occur on a timescale of milliseconds to seconds, rendering application of conventional molecular dynamics simulations prohibitive even for small proteins. We have developed a computational protocol to efficiently and accurately predict these ligand-induced structure transitions solely from the knowledge of protein apo structure and ligand binding site. Our method involves a series of small scale conformational change steps, where at each step linear response theory is used to predict the direction of small scale global response to ligand binding in the protein conformational space (dLRT) followed by construction of a linear combination of slow (low frequency) normal modes (calculated for the structure from the previous step) that best overlaps with dLRT. Protein structure is evolved along this direction using molecular dynamics with excited normal modes (MDeNM) wherein excitation energy along each normal mode is determined by excitation temperature, mode frequency, and its overlap with dLRT. We show that excitation temperature ({Delta}T) is a very important parameter that allows limiting the extent of structural change in any one step and develop a protocol for automated determination of its optimal value at each step. We have tested our protocol for three protein-ligand systems, namely, adenylate Kinase - di(adenosine-5)pentaphosphate, ribose binding protein - {beta}-D-ribopyranose, and DNA {beta}-glucosyltransferase - uridine-5-diphosphate, that incorporate important differences in type and range of structural changes upon ligand binding. We obtain very accurate prediction for not only the structure of final protein-ligand complex (holo-structure) having a large scale conformational change, but also for biologically relevant intermediates between the apo and the holo structures. Moreover, most relevant set of normal modes for conformational change at each step are an output from our method, which can be used as collective variables for determination of free energy barriers and transition timescales along the identified pathway.

sentences: Tree species and genetic diversity increase productivity via functional diversity and trophic feedbacks Addressing global biodiversity loss requires an expanded focus on multiple dimensions of biodiversity. While most studies have focused on the consequences of plant interspecific diversity, our mechanistic understanding of how the diversity within a given plant species (genetic diversity) affects plant productivity remains limited. Here, we use a tree species x genetic diversity experiment to disentangle the effects of species diversity and genetic diversity, and how they are related to tree functional diversity and trophic feedbacks. Tree species as well as genetic diversity increased tree productivity via increased tree functional diversity, reduced soil fungal diversity and marginally reduced herbivory. The effect of tree genetic diversity on productivity was partly different between tree species monocultures and mixtures: the functional diversity effect resulting from tree genetic diversity was only found in tree species monocultures, but the trophic feedbacks via herbivory were similar in species monocultures and mixtures. Given the complexity of interactions between tree species and genetic diversity, tree functional diversity and trophic feedbacks on productivity, we suggest that both tree species and genetic diversity should be considered in reforestation.

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Lateral Orbitofrontal Cortex and Basolateral Amygdala Regulate Sensitivity to Delayed Punishment during Decision-making In real-world decision-making scenarios, negative consequences do not always occur immediately after a choice. This delay between action and outcome drives the underestimation, or "delay discounting", of punishment. While the neural substrates underlying sensitivity to immediate punishment have been well-studied, there has been minimal investigation of delayed consequences. Here, we assessed the role of lateral orbitofrontal cortex (LOFC) and basolateral amygdala (BLA), two regions implicated in cost/benefit decision-making, in sensitivity to delayed vs immediate punishment. The delayed punishment decision-making task (DPDT) was used to measure delay discounting of punishment in rodents. During DPDT, rats choose between a small, single pellet reward and a large, three pellet reward accompanied by a mild foot shock. As the task progresses, the shock is preceded by a delay that systematically increases or decreases throughout the session. We observed that rats avoid choices associated with immediate punishment, then shift preference toward these options when the punishment is delayed. LOFC inactivation did not influence choice of rewards with immediate punishment, but decreased choice of delayed punishment. We also found that BLA inactivation reduced choice of delayed punishment, but this was only evident when punishment was initially delivered immediately after a choice, then preceded by a delay as the task progressed. Therefore, both LOFC and BLA contribute to the delay discounting of punishment, and may serve as promising therapeutic targets to improve sensitivity to delayed punishment during decision-making.

Significance StatementNegative consequences occurring after a delay are often underestimated, which can lead to maladaptive decision-making. While sensitivity to immediate punishment during reward-seeking has been well-studied, the neural substrates underlying sensitivity to delayed punishment remain unclear. Here, we used the Delayed Punishment Decision-making Task to determine that lateral orbitofrontal cortex and basolateral amygdala both regulate the discounting of delayed punishment, suggesting that these regions may be potential targets to improve decision-making in psychopathology.

sentences: Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here we demonstrate a new method to visualize endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps. First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously visualize the dynamics of the two loci in single living cells. This reveals close association between the two loci in the majority of cells, but the loci markedly separate upon the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof-of-principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

sentences: Comprehensive generation, visualization, and reporting of quality control metrics for single-cell RNA sequencing data Single-cell RNA sequencing (scRNA-seq) can be used to gain insights into cellular heterogeneity within complex tissues. However, a variety of technical artifacts can be present in scRNA-seq data and need to be assessed before downstream analyses can be performed. While several algorithms and tools have been developed to perform individual quality control (QC) tasks, they are scattered in different packages across several programming environments. Comprehensive pipelines to streamline the process of generating and visualizing QC metrics are lacking. To address this need, we built the SCTK-QC pipeline within the singleCellTK R package (https://github.com/compbiomed/singleCellTK). Features in this pipeline include the ability to import data from 11 different preprocessing tools or file formats, perform empty droplet detection with 2 different algorithms, generate standard quality control metrics such as number of UMIs per cell or the percentage of mitochondrial counts, predict doublets using 6 different algorithms, and estimate ambient RNA. QC data can be exported to R and/or Python objects used in popular down-stream workflows. Results are visualized in an easy-to-read HTML report. This pipeline can also be used by non-computational users with an interactive graphical user interface developed with R/Shiny. Overall, the SCTK-QC pipeline will streamline and standardize QC analysis for scRNA-seq data across a variety of different single-cell transcriptomic platforms and preprocessing tools.

sentences: Telomere transcription in MLL-rearranged acute leukemia: increased levels of TERRA associate with lymphoid lineage and are independent of telomere length and ploidy Telomere transcription into telomeric repeat-containing RNA (TERRA) is an integral component of all aspects of chromosome end protection consisting of telomerase- or recombination-dependent telomere elongation, telomere capping, and preservation of (sub)telomeric heterochromatin structure. The chromatin modifier and transcriptional regulator MLL associates with telomeres and regulates TERRA transcription in telomere length homeostasis and response to telomere dysfunction. MLL fusion proteins (MLL-FPs), the product of MLL rearrangements in leukemia, are also associated with telomeric chromatin. However, an effect on telomere transcription in MLL-rearranged (MLL-r) leukemia has not yet been evaluated. Here, we show increased UUAGGG repeat-containing RNA levels in MLL-r acute lymphoblastic leukemia (ALL) when compared to myeloid and non-MLL-r ALL leukemia. MLL rearrangements do not affect telomere length and increased levels of UUAGGG repeatcontaining RNA correlate with mean telomere length and reflect increased levels of TERRA. Also, increased TERRA levels in MLL-r ALL occur in the presence of telomerase activity and are independent of ploidy, an underestimated source of variation on the overall transcriptome size in a cell. This MLL rearrangement-dependent and lymphoid lineage-associated increase in levels of TERRA supports a sustained telomere transcription by MLL-FPs that correlates with marked genomic stability previously reported in pediatric MLL-r ALL.

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Investigation of the Effects of N-Linked Glycans on the Stability of the Spike Protein in SARS-CoV-2 by Molecular Dynamics Simulations We perform all-atom molecular dynamics simulations to study the effects of the N-linked glycans on the stability of the spike glycoprotein in SARS-CoV-2. After a 100 ns of simulation on the spike proteins without and with the N-linked glycans, we found that the presence of glycans increases the local stability in their vicinity; even though their effect on the full structure is negligible.

Graphical Abstract

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sentences: Life history strategies explain bacterial activity in the soil carbon cycle Soil microorganisms determine the fate of soil organic matter (SOM), and their activities comprise a major component of the global carbon (C) cycle. We employed a multi-substrate DNA-stable isotope probing experiment to track bacterial assimilation of C derived from distinct sources that varied in bioavailability. This approach allowed us to measure microbial contributions to SOM processing by measuring the C assimilation dynamics of diverse microorganisms as they interact within soil. We identified and tracked 1,286 bacterial taxa that assimilated 13C in an agricultural soil over a period of 48 days. Overall 13C-assimilation dynamics of bacterial taxa, defined by the source and timing of the 13C they assimilated, exhibited low phylogenetic conservation. We identified bacterial guilds comprised of taxa that had similar 13C assimilation dynamics. We show that C source bioavailability explained significant variation in both C mineralization dynamics and guild structure. In addition, guild structure explained significant variation in bacterial growth dynamics. We demonstrate that the observed guild structure is consistent with predictions made by bacterial life history theory. We also demonstrate that the guild structure explains significant variation in the biogeographical distribution of bacteria at continental and global scales. We interpret these findings in the context of bacterial life history strategies and their relationship to terrestrial C-cycling.

sentences: Capped nascent RNA sequencing reveals novel therapy-responsive enhancers in prostate cancer Mounting evidence suggests that enhancer RNA (eRNA) transcription start sites (TSSs) provide higher sensitivity and specificity for enhancer identification than histone modifications and chromatin accessibility. The extent to which changes in eRNA transcription correspond to changes in enhancer activity, however, remains unclear. Here, we used precision run-on and capped RNA sequencing (PRO-cap) to assess changes in enhancer activity in response to treatment with the androgen receptor signaling inhibitor, enzalutamide (ENZ). We identified 6,189 high-confidence candidate enhancers in the human prostate cancer cell line, LNCaP; 853 of which demonstrated significant changes in activity in response to drug treatment. Notably, we found that 67% and 54% of drug-responsive enhancers did not show similar changes in activity in previous studies that utilized ChIP-seq and ATAC-seq, respectively. Strikingly, 79% of regions with increased eRNA transcription showed no other biochemical alterations, implying that PRO-cap can capture a set of precise changes in enhancer activity that classical approaches lack the sensitivity to detect. We performed in vivo functional validations of candidate enhancers and found that CRISPRi targeting of PRO-cap-specific drug-responsive enhancers impaired ENZ regulation of downstream target genes, suggesting that changes in eRNA TSSs mark true biological changes in enhancer activity with high sensitivity. Our study highlights the utility of using PRO-cap as a complementary approach to canonical biochemical methods for detecting precise changes in enhancer activity and, in particular, for better understanding disease progression and responses to treatment.

sentences: Spatially heterogeneous microstructural development within subcortical regions from 9-13 years During late childhood behavioral changes, such as increased risk-taking and emotional reactivity, have been associated with the maturation of cortico-subcortical circuits. Understanding microstructural changes in subcortical regions may aid our understanding of how individual differences in these behaviors emerge. Restriction spectrum imaging (RSI) is a framework for modelling diffusion-weighted imaging that decomposes the diffusion signal from a voxel into hindered and restricted compartments. This yields greater specificity than conventional methods of characterizing intracellular diffusion. Using RSI, we modelled voxelwise restricted isotropic, N0, and anisotropic, ND, diffusion across the brain and measured cross-sectional and longitudinal age associations in a large sample (n=8,039) from the Adolescent Brain and Cognitive Development (ABCD) study aged 9-13 years. Older participants had higher N0 and ND across subcortical regions. The largest associations for N0 were within the basal ganglia and for ND within the ventral diencephalon. Importantly, age associations varied with respect to the internal cytoarchitecture within subcortical structures, for example age associations differed across thalamic nuclei. This suggests that developmental effects may map onto specific cell populations or circuits and highlights the utility of voxelwise compared to ROI-wise analyses. Future analyses will aim to understand the relevance of this subcortical microstructural developmental for behavioral outcomes.

sentences: An early Shh-H2O2 feedback loop controls the progression of the regenerative program during adult zebrafish fin regeneration Reactive oxygen species (ROS), originally classified as toxic molecules, have attracted increasing interest given their actions in cell signaling. Among these molecules, Hydrogen peroxide (H2O2) is the major ROS produced by cells and acts as a second messenger to modify redox-sensitive proteins or lipids. After amputation, tight spatiotemporal regulation of ROS is required first for wound healing and later to initiate the regenerative program. However, the mechanisms carrying out this sustained ROS production and their integration with signaling pathways are still poorly understood. We focused on the early dialog between H2O2 and Sonic Hedgehog (Shh) during fin regeneration. We demonstrate that H2O2 controls Shh expression and that Shh in turn regulates the H2O2 level via a canonical pathway. Moreover, this tightly controlled feedback loop changes during the successive phases of the regenerative program. Dysregulation of the Hedgehog pathway has been implicated in several developmental syndromes, diabetes and cancer. These data support the existence of a very early feedback loop between Shh and H2O2 that might be more generally involved in various physiological or pathological processes. These new findings pave the way to improve regenerative processes, particularly in vertebrates.

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The winding roads to adulthood: a twin study In the 21st century, emerging adulthood has stretched from the late teens through the twenties. Although this extended transition to adulthood can create stress, it can also offer opportunities to explore vocations and relationships that provide a better fit to individuals proclivities, including their genetic propensities.

Here we report the results of the first systematic investigation of genetic and environmental influences on 57 psychological traits covering major issues in emerging adulthood such as aspirations, thoughts and attitudes, relationships and personality. We also investigate how these traits relate to physical and mental health, educational attainment and wellbeing using a sample of nearly 5000 pairs of UK twins aged 21-25 from the Twins Early Development Study.

All 57 traits showed significant genetic influence, with an average heritability of 34% (SNP heritability [~]10%). Most of the variance (59% on average) was explained by non-shared environmental influences. These diverse traits were associated with mental health (average correlation .20), wellbeing (.16), physical health (.12) and educational attainment (.06). Shared genetic factors explained the majority of these correlations ([~]50%). Together, these emerging adulthood traits explained on average 30% of the variance in the outcomes (range = 8 to 69%), suggesting that these traits relate to the outcomes additively.

We conclude that the environmental uncertainties of emerging adulthood in the 21st century do not diminish the importance of genetics. As adolescents travel down long and winding roads to adulthood, their trip is substantially influenced by genetic proclivities that nudge them down different paths leading to different destinations.

sentences: Compressive forces stabilise microtubules in living cells Cell mechano-sensation and adaptation are supported by the actin network. The microtubule network is not considered to be directly sensitive to mechanical forces acting on a cell. However, recent studies on isolated microtubules in vitro have shown that bending forces have an impact on their structure, composition and lifespan, suggesting that, in a cellular context, microtubules may react to mechanical forces. We tested this hypothesis in living cells by subjecting them to cycles of compressive forces and found that microtubules became distorted, less dynamic and more stable. This mechano-stabilisation depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. These results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected.

sentences: Monosomies, trisomies and segmental aneuploidies differentially affect chromosomal stability Aneuploidy and chromosomal instability are both commonly found in cancer. Chromosomal instability leads to karyotype heterogeneity in tumors and is associated with therapy resistance, metastasis and poor prognosis. It has been hypothesized that aneuploidy per se is sufficient to drive CIN, however due to limited models and heterogenous results, it has remained controversial which aspects of aneuploidy can drive CIN. In this study we systematically tested the impact of different types of aneuploidies on the induction of CIN. We generated a plethora of isogenic aneuploid clones harboring whole chromosome or segmental aneuploidies in human p53-deficient RPE-1 cells. We observed increased segregation errors in cells harboring trisomies that strongly correlated to the number of gained genes. Strikingly, we found that clones harboring only monosomies do not induce a CIN phenotype. Finally, we found that an initial chromosome breakage event and subsequent fusion can instigate breakage-fusion-bridge cycles in segmental aneuploidies. This suggests that monosomies, trisomies and segmental aneuploidies have fundamentally different effects on chromosomal instability and these results help us to decipher the complex relationship between aneuploidy and CIN.

sentences: Population-level survey of loss-of-function mutations revealed that background dependent fitness genes are rare and functionally related in yeast In natural populations, the same mutation can lead to different phenotypic outcomes due to the genetic variation that exists among individuals. Such genetic background effects are commonly observed, including in the context of many human diseases. However, systematic characterization of these effects at the species level is still lacking to date. Here, we sought to comprehensively survey background-dependent traits associated with gene loss-of-function (LoF) mutations in 39 natural isolates of Saccharomyces cerevisiae using a transposon saturation strategy. By analyzing the modeled fitness variability of a total of 4,469 genes, we found that 15% of them, when impacted by a LoF mutation, exhibited a significant gain- or loss-of-fitness phenotype in certain natural isolates compared to the reference strain S288C. Out of these 632 genetic background-dependent fitness genes identified, a total of 2/3 show a continuous variation across the population while 1/3 are specific to a single genetic background. Genes related to mitochondrial function are significantly overrepresented in the set of genes showing a continuous variation and display a potential functional rewiring with other genes involved in transcription and chromatin remodeling as well as in nuclear-cytoplasmic transport. Such rewiring effects are likely modulated by both the genetic background and the environment. While background-specific cases are rare and span diverse cellular processes, they can be functionally related at the individual level. All background-dependent fitness genes tend to have an intermediate connectivity in the global genetic interaction network and have shown relaxed selection pressure at the population level, highlighting their potential evolutionary characteristics.

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A call to rethink the cognitive benefits of physical exercise: An umbrella review of randomized controlled trials BackgroundMany meta-analytic reviews have examined the cognitive benefits of regular physical exercise across the lifespan, with a wide range of inclusion criteria and disparate analytic approaches. In fact, most global health organizations incorporate the purported cognitive benefits as part of their policies to promote regular physical exercise. We assess whether those claims are well supported by scientific evidence.

MethodsWe conducted a pre-registered (PROSPERO: CRD42020191357) umbrella review of meta-analyses limited to randomized controlled trials (RCTs) on exercise-cognition. Outcomes of each meta-analysis were reanalyzed following a multilevel model approach. Publication bias was also analyzed to correct final effect sizes.

FindingsMost of the 12 reviewed meta-analyses reported a positive small effect (d = 0.21, [0.07-0.40]) of physical exercise across all cognitive domains in all life stages. However, a critical assessment of the primary studies included in these reviews and the meta-analytic approaches used indicates that the evidence might not be reliable and therefore no strong conclusions should be drawn based on the existing evidence. We found that the literature has undergone an unusually rapid growth upon underpowered RCTs and potentially biased meta-analytic reviews.

InterpretationThe exponential accumulation of low-quality evidence on the exercise-cognition topic has led to stagnation in the field, hindering the discernment of the potential impact of exercise on cognition. We urge worldwide organizations committed to public health to reconsider their recommendations on the promotion of regular physical exercise to boost cognitive functions in the healthy population until high-quality empirical evidence confirms these exercise-induced cognitive benefits.

FundingSpanish Ministry of Science and Innovation (PID2019-105635GB-I00 and PID2020-118583GB-I00).

sentences: Network analysis after epigenome-wide methylation study revealed JUP as a regulator of co-methylated risk-module for T2DM Controlling the global Type 2 diabetes mellitus (T2DM) pandemic requires a better understanding of its risk factors across different populations, and needs markers that can precisely predict the individual risk to the disease. DNA methylation due to environmental factors is a known mechanism for conferring risk to T2DM. To identify such methylation signatures and associated risk to the disease, we performed an epigenome-wide-association study (EWAS) in 844 individuals of Indo-European origin. Within the Indian population, we identified and validated 6 novel differentially methylated CpG sites in PDCD6IP, MIR1287, 5S_rRNA, HDAC9, KCNK16, and RTN1 genes associated with T2DM risk at the epigenome-wide-significance-level (p<1.2x10-7). Further, we also replicated the association of 3 known CpG sites in TXNIP, SREBF1 and CPT1A in the Indian population. Using methylation-based-network analysis, we identified 4 co-methylated modules, which we named as turquoise, yellow, brown, and blue, among differentially methylated CpG sites in discovery phase samples. We observed that methylation of the brown module with 28 CpG sites, associated with T2DM risk factors (e.g., BMI, insulin, C-peptide). Upon further analysis, we noted that these methylation signatures at 14 of the brown modules CpG can be used as marker to segregate T2DM patients with good glycemic control (e.g., low HbA1c) but poor lipid profile (low HDL and high TG) from the other patients. Additionally, we discovered that rs6503650 in the JUP gene regulates methylation at all the 14 hub CpG sites of the brown modules as methQTL. Our network-assisted epigenome-wide association study is the first to systematically explore DNA methylations conferring risks to T2DM. In addition, the study shows the potential use of identified risk CpG sites for patient segregation with different clinical outcomes. These findings can be useful for better stratification of patient in order to improve the clinical management and treatment outcomes.

sentences: The probability of joint monophyly of all species in an arbitrary species tree. Monophyly is a feature of a set of genetic lineages in which every lineage in the set is more closely related to all other members of the set than it is to any lineage outside the set. Multiple sets of lineages that are separately monophyletic are said to be reciprocally monophyletic, or jointly monophyletic. The prevalence of reciprocal monophyly, or joint monophyly, has been used to evaluate phylogenetic and phylogeographic hypotheses, as well as to delimit species. These applications often make use of a probability of joint monophyly under models of gene lineage evolution. Studies in coalescent theory have computed this joint monophyly probability for small numbers of separate groups in arbitrary species trees, and for arbitrary numbers of separate groups in trivial species trees. Here, generalizing existing results on monophyly probabilities under the multispecies coalescent, we derive the probability of joint monophyly for arbitrary numbers of separate groups in arbitrary species trees. We illustrate how our result collapses to previously examined cases. We also study the effect of tree height, sample size, and number of species on the probability of joint monophyly. The result also enables computation of relatively simple lower and upper bounds on the joint monophyly probability. Our results expand the scope of joint monophyly calculations beyond small numbers of species, subsuming past formulas that have been used in simpler cases.

sentences: Dimensionality reduction and data integration for scRNA-seq data based on integrative hierarchical Poisson factorisation Single-cell RNA sequencing (scRNA-seq) data sets consist of high-dimensional, sparse and noisy feature vectors, and pose a challenge for classic methods for dimensionality reduction. We show that application of Hierarchical Poisson Factorisation (HPF) to scRNA-seq data produces robust factors, and outperforms other popular methods. To account for batch variability in composite data sets, we introduce Integrative Hierarchical Poisson Factorisation (IHPF), an extension of HPF that makes use of a noise ratio hyper-parameter to tune the variability attributed to technical (batches) vs. biological (cell phenotypes) sources. We exemplify the advantageous application of IHPF under data integration scenarios with varying alignments of technical noise and cell diversity, and show that IHPF produces latent factors with a dual block structure in both cell and gene spaces for enhanced biological interpretability.

sentences: Rigid DNA nanotube tethers suppress high frequency noise in dual-trap optical tweezers systems Dual trap optical tweezers are a powerful tool to trap and characterize the biophysical properties of single biomolecules such as the folding pathways of proteins and nucleic acids, and the chemomechanical activity of molecular motors. Despite its vastly successful application, noise from drift and fluctuation of the optics, and Brownian motion of the trapped beads still hinder the techniques ability to directly visualize folding of small biomolecules or the single nucleotide stepping of polymerases, especially at low forces (<10 pN) and sub-millisecond timescales. Rigid DNA nanotubes have been used to replace the conventional dsDNA linker to reduce optical tweezers noise in the low force range. However, optical tweezers are used to study a wide range of biophysical events, with timescales ranging from microseconds to seconds, and length changes ranging from sub nanometers to tens of nanometers. In this study, we systematically evaluate how noise is distributed across different frequencies in dual trap optical tweezers systems-and show that rigid DNA nanotube tethers suppress only high frequency noise (kHz), while the low frequency noise remains the same when compared to that of dsDNA tethers.

sentences: Modulation of cutaneous responses in the cuneate nucleus of macaques during active movement AO_SCPLOWBSTRACTC_SCPLOWTo achieve stable and precise movement execution, the sensorimotor system integrates exafferent sensory signals originating from interactions with the external world and reafferent signals caused by our own movements. This barrage of sensory information is regulated such that behaviorally relevant signals are boosted at the expense of irrelevant ones. For example, sensitivity to touch is reduced during movement - when cutaneous signals caused by skin stretch are expected and uninteresting - a phenomenon reflected in a decreased cutaneous responsiveness in thalamus and cortex. Some evidence suggests that movement gating of touch may originate from the cuneate nucleus (CN), the first recipient of signals from tactile nerve fibers along the dorsal column-medial lemniscus pathway. To test this possibility, we intermittently delivered mechanical pulses to the receptive fields (RFs) of identified cutaneous CN neurons as monkeys performed a reach-to-grasp task. As predicted, we found that the cutaneous responses of individual CN neurons were reduced during movement. In contrast, cutaneous signals from neurons with RFs on the hand were enhanced during reach. We conclude that tactile signals are already modulated in the first processing stage along the somatosensory neuraxis. Furthermore, the sign and magnitude of this modulation depends on the behavioral relevance of the sensory signals.

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Differential regulation of degradation and immune pathways underlies adaptation of the symbiotic nematode Laxus oneistus to oxic-anoxic interfaces Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also upregulated when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worms Toll- like innate immunity pathway and several immune effectors (e.g., Bacterial Permeability Increasing proteins, fungicides).

In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires oxidative phosphorylation and by reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.

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Jaagsiekte sheep retrovirus infection induces changes in microRNA expression in the ovine lung Ovine pulmonary adenocarcinoma (OPA) is an infectious neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus. OPA is an important veterinary problem and is also a valuable large animal model for human lung adenocarcinoma. JSRV infects type 2 alveolar epithelial cells in the lung and induces the growth of tumors, but little is known about the molecular events that lead to the activation of oncogenic pathways in infected cells. MicroRNAs (miRNAs) are small RNA molecules of approximately 22 nucleotides with important roles in regulating gene expression in eukaryotes and with well-established roles in cancer. Here we used small-RNA sequencing to investigate the changes in miRNA expression that occur in JSRV-infected ovine lung. After filtering out low abundance miRNAs, we identified expression of 405 miRNAs, 32 of which were differentially expressed in JSRV-infected lung compared to mock-inoculated control lung. Highly upregulated miRNAs included miR-182, miR-183, miR-96 and miR-135b, which have also been associated with oncogenic changes in human lung cancer. Network analysis of genes potentially targeted by the deregulated miRNAs identified their involvement in pathways known to be dysregulated in OPA. We found no evidence to support the existence of miRNAs encoded by JSRV. This study provides the first information on miRNA expression in OPA and identifies a number of targets for future studies into the role of these molecules in the pathogenesis of this unique veterinary model for human lung adenocarcinoma.

IMPORTANCEOvine pulmonary adenocarcinoma is a neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant welfare and economic concern for sheep producers and is a valuable large animal model for human lung adenocarcinoma. MicroRNAs are small RNA molecules of approximately 22 nucleotides with important functions in regulating gene expression in eukaryotes and with well-established roles in cancer. In this study, we examined the changes in microRNA expression that occur in the lung in response to JSRV infection. We identified differential expression of a number of host-encoded microRNAs in infected tissue, including microRNAs with roles in human cancer. We found no evidence that JSRV encodes a microRNA. This study provides new insights on the cellular response to JSRV infection in the ovine lung, which will inform future studies into the pathogenesis of OPA in sheep and its use as a model for human lung adenocarcinoma.

sentences: Application of Parallel Reaction Monitoring in 15N labeled Samples for Quantification Accurate relative quantification is critical in proteomic studies. The incorporation of stable isotope 15N to plant-expressed proteins in vivo is a powerful tool for accurate quantification with a major advantage of reducing preparative and analytical variabilities. However, 15N labeling quantification has several challenges. Less identifications are often observed in the heavy labeled samples because of incomplete labeling, resulting in missing values in reciprocal labeling experiments. Inaccurate quantification can happen when there is contamination from co-eluting peptides or chemical noise in the MS1 survey scan. These drawbacks in quantification can be more pronounced in less abundant but biologically interesting proteins, which often have very few identified peptides. Here we demonstrate the application of parallel reaction monitoring (PRM) to 15N labeled samples on a high resolution, high mass accuracy Orbitrap mass spectrometer to achieve reliable quantification even of low abundance proteins in samples.

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Selective prefrontal-amygdala circuit interactions underlie social and nonsocial valuation in rhesus macaques Lesion studies in macaques suggest dissociable functions of the orbitofrontal cortex (OFC) and medial frontal cortex (MFC), with OFC being essential for goal-directed decision making and MFC supporting social cognition. Bilateral amygdala damage results in impairments in both of these domains. There are extensive reciprocal connections between these prefrontal areas and the amygdala; however, it is not known whether the dissociable roles of OFC and MFC depend on functional interactions with the amygdala. To test this possibility, we compared the performance of male rhesus macaques (Macaca mulatta) with crossed surgical disconnection of the amygdala and either MFC (MFC x AMY, n=4) or OFC (OFC x AMY, n=4) to a group of unoperated controls (CON, n=5). All monkeys were assessed for their performance on two tasks to measure: (1) food-retrieval latencies while viewing videos of social and nonsocial stimuli in a test of social interest, and (2) object choices based on current food value using reinforcer devaluation in a test of goal-directed decision making. Compared to the CON group, the MFC x AMY group, but not the OFC x AMY group, showed significantly reduced food-retrieval latencies while viewing videos of conspecifics, indicating reduced social valuation and/or interest. By contrast, on the devaluation task, group OFC x AMY, but not group MFC x AMY, displayed deficits on object choices following changes in food value. These data indicate that the MFC and OFC must functionally interact with the amygdala to support normative social and nonsocial valuation, respectively.

Significance StatementAscribing value to conspecifics (social) vs. objects (nonsocial) may be supported by distinct but overlapping brain networks. Here we test whether two nonoverlapping regions of the prefrontal cortex, the medial frontal cortex and the orbitofrontal cortex, must causally interact with the amygdala to sustain social valuation and goal-directed decision making, respectively. We found that these prefrontal-amygdala circuits are functionally dissociable, lending support for the idea that medial frontal and orbital frontal cortex make independent contributions to cognitive appraisals of the environment. These data provide a neural framework for distinct value assignment processes and may enhance our understanding of the cognitive deficits observed following brain injury or in the development of mental health disorders.

sentences: Whokaryote: distinguishing eukaryotic and prokaryotic contigs in metagenomes based on gene structure Metagenomics has become a prominent technology to study the functional potential of all organisms in a microbial community. Most studies focus on the bacterial content of these communities, while ignoring eukaryotic microbes. Indeed, many metagenomics analysis pipelines silently assume that all contigs in a metagenome are prokaryotic. However, because of marked differences in gene structure, prokaryotic gene prediction tools fail to accurately predict eukaryotic genes. Here, we developed a classifier that distinguishes eukaryotic from prokaryotic contigs based on foundational differences between these taxa in gene structure. We first developed a random forest classifier that uses intergenic distance, gene density and gene length as the most important features. We show that, with an estimated accuracy of 97%, this classifier with principled features grounded in biology can perform almost as well as the classifiers EukRep and Tiara, which use k-mer frequencies as features. By re-training our classifier with Tiara predictions as additional feature, weaknesses of both types of classifiers are compensated; the result is an enhanced classifier that outperforms all individual classifiers, with an F1-score of 1.00 on precision, recall and accuracy for both eukaryotes and prokaryotes, while still being fast. In a reanalysis of metagenome data from a disease-suppressive plant endosphere microbial community, we show how using Whokaryote to select contigs for eukaryotic gene prediction facilitates the discovery of several biosynthetic gene clusters that were missed in the original study. Our enhanced classifier, which we call  Whokaryote, is wrapped in an easily installable package and is freely available from https://git.wageningenur.nl/lotte.pronk/whokaryote.

sentences: The mouse bone metabolome is sexually dimorphic and is associated with whole bone strength Cortical bone quality, which is sexually dimorphic, depends on bone turnover and therefore the activities of remodeling bone cells. However, sex differences in cortical bone metabolism are not yet defined. Adding to the uncertainty about cortical bone metabolism, the metabolomes of whole bone, isolated cortical bone without marrow, and bone marrow have not been compared. We hypothesized that the metabolome of isolated cortical bone would be distinct from that of bone marrow and would reveal sex differences. Metabolite profiles from LC-MS of whole bone, isolated cortical bone, and bone marrow were generated from humeri from 20-week-old female C57Bl/6J mice. The cortical bone metabolomes were then compared for 20-week-old female and male C57Bl/6J mice. Femurs from male and female mice were evaluated for flexural material properties and were then categorized into bone strength groups. The metabolome of isolated cortical bone was distinct from both whole bone and bone marrow. We also found sex differences in the isolated cortical bone metabolome. Based on metabolite pathway analysis, females had higher lipid metabolism, and males had higher amino acid metabolism. High-strength bones, regardless of sex, had greater tryptophan and purine metabolism. For males, high strength bones had upregulated nucleotide metabolism, whereas lower strength bones had greater pentose phosphate pathway metabolism. Since the higher strength groups (females compared with males, high strength males compared with lower strength males) had higher serum CTX1/P1NP, we estimate that the metabolomic signature of bone strength in our study at least partially reflects differences in bone turnover. These data provide novel insight into bone bioenergetics and the sexual dimorphic nature of bone material properties in C57Bl/6 mice.

sentences: Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display increased Cystathionine-{beta}-synthase (CBS) expression and consequent activation of the transsulfuration pathway controlling hydrogen sulfide (H2S) and glutathione (GSH) metabolism. Activated transsulfuration pathway during AIS maintenance enhances the antioxidant capacity, protecting senescent cells from ROS-induced cell death via GSH and H2S. Importantly, CBS depletion allows cells that have undergone AIS to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production and increasing GSH metabolism. These findings implicate a potential tumor-suppressive role for CBS in cells with inappropriately activated PI3K/AKT signaling. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

sentences: Azole resistance associated regulatory motifs within the promoter of cyp51A in Aspergillus fumigatus Aspergillus fumigatus is one of the deadliest fungal species causing hundreds of thousands of deaths each year. As azoles provide the preferred first-line option for treatment of Aspergillosis, the increase in rates of resistance and the poor therapeutic outcomes for those infected with a resistant isolate constitutes a serious global health threat. Azole resistance is frequently associated with specific tandem repeat duplications of a promoter element upstream of cyp51A, the gene which encodes the target for this drug class in A. fumigatus. This promoter element is recognized by the activating transcription factors SrbA and AtrR. This region also provides a docking platform for the CCAAT-binding-complex (CBC) and HapX that cooperate in the regulation of genes involved in iron-consuming pathways including cyp51A. Here, we studied the regulatory contribution of SrbA, AtrR, CBC and HapX binding sites on cyp51A expression and azole resistance during different iron availability employing promoter mutational analysis and protein/DNA interaction analysis. This strategy revealed iron status-dependent and -independent roles of these regulatory elements. We show that promoter occupation by both AtrR and SrbA is required for iron-independent steady-state transcriptional activation of cyp51A and its induction during short-term iron exposure relies on HapX binding. We further uncover the HapX binding site as repressor element the disruption of which elevates cyp51A expression and azole resistance regardless of iron availability.

sentences: Generation of chimeric respiratory viruses with altered tropism by coinfection of influenza A virus and respiratory syncytial virus Interactions between co-circulating respiratory viruses are recognized for their impact on viral transmission and clinical outcomes. However, the consequences of these virus-virus interactions at the cellular level are unclear. We coinfected human lung cells with influenza A virus (IAV) and respiratory syncytial virus (RSV). Super-resolution microscopy combined with live-cell imaging and scanning electron microscopy identified extracellular and membrane-associated filamentous structures, likely composed of elements of both IAV and RSV virions. Cryo-electron tomography confirmed the presence of chimeric virus particles exhibiting glycoproteins and ribonucleoproteins of both parental viruses. Functional assays revealed chimeric particles facilitate IAV infection in cells depleted of IAV receptors, demonstrating expanded tropism. Our observations define a previously unknown interaction that is likely to affect virus pathogenesis and have profound implications for infection biology.

sentences: Human cells contain myriad excised linear introns with potential functions in gene regulation and as RNA biomarkers By using TGIRT-seq, we identified >8,500 short full-length excised linear intron (FLEXI) RNAs in human cells. Subsets of FLEXIs accumulated in a cell-type specific manner, and [~]200 corresponded to agotrons or mirtrons or encoded snoRNAs. Analysis of CLIP-seq datasets identified potential interactions between FLEXIs and >100 different RNA-binding proteins (RBPs), 53 of which had binding sites in [≥]30 different FLEXIs. In addition to proteins that function in RNA splicing, these 53 RBPs included transcription factors, chromatin remodeling proteins, and cellular growth regulators that impacted FLEXI host gene alternative splicing and/or mRNA levels in knockdown datasets. We computationally identified six groups of RBPs whose binding sites were enriched in different subsets of FLEXIs: AGO1-4 and DICER associated with agotrons and mirtrons; AATF, DKC1, NOLCI, and SMNDC1 associated with snoRNA-encoding FLEXIs; two different combinations of alternative splicing factors found in stress granules; and two novel RBP-intron combinations, one including LARP4 and PABC4, which function together in the cytoplasm to regulate ribosomal protein translation. Our results suggest a model in which proteins involved in transcriptional regulation, alternative splicing, or post-splicing secondary functions bind and stabilize cell-type specific subsets of FLEXIs that perform different biological functions and have potential utility as biomarkers.

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{-}Cellular and biochemical response to chaperone versus substrate reduction therapies in neuropathic Gaucher disease{-} Gaucher disease (GD) is caused by the deficiency of the lysosomal membrane enzyme glucocerebrosidase (GCase), and the subsequent accumulation of its substrate, glucosylceramide substrate (GC). Mostly missense mutations of the glucocerebrosidase gene (GBA) lead to GCase misfolding and inhibiting the lysosomes proper trafficking. The accumulated GC leads to lysosomal dysfunction and impairs the autophagy pathway.

GD types 2 and 3 (GD2-3), or the neuronopathic forms, affect not only the Central Nervous System (CNS) but also have severe systemic involvement and progressive bone disease. Enzyme replacement therapy (ERT) successfully treats the hematologic manifestations; however, due to the lack of equal distribution of the recombinant enzyme in different organs, it has no impact on the nervous system and has minimal effect on bone involvement. Small molecules have the potential for better tissue distribution. Ambroxol (AMB) is a pharmacologic chaperone that partially recovers the mutated GCase activity and crosses the blood-brain barrier. Eliglustat (EGT) works by inhibiting UDP-glucosylceramide synthase, an enzyme that catalyzes the GC biosynthesis, reducing a GC influx load into the lysosome. Substrate reduction therapy (SRT) using EGT is associated with improvement in GD bone marrow burden score and bone mineral density.

The effects of EGT and ABX on GCase activity and autophagy-lysosomal pathway (ALP) were assessed in primary cell lines derived from patients with GD2-3 and compared to cell lines from healthy controls. While both compounds enhanced GCase activity in control cells, an individualized response was observed in cells from patients with GD2-3 that varied with GBA mutations. EGT and AMB enhanced the formation of lysosomal/ late endosomal compartments and autophagy, and this effect was independent of GBA mutations. Both AMB and EGT increased mitochondrial mass and density in GD2-3 fibroblasts, suggesting enhancement of the mitochondrial function by activating the mitochondrial membrane potential.

These results suggest that EGT and ABX may have different molecular mechanisms of action, but both enhance GCase activity, improve autophagy-lysosome dynamics and mitochondrial functions.

sentences: Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability Having its own genome makes mitochondria a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is double-stranded closed circular molecule of about 16 kb coding 37 genes. Mutations, including deletions in the mitochondrial genome can culminate in different human diseases. Mapping of the deletion junctions suggests that the breakpoints are generally seen at hotspots.  9-bp deletion (8271-8281), seen in the intergenic region of cytochrome c oxidase II/tRNALys, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with  9-bp deletion, when it is present on a plasmid or in the mitochondrial genome. Through a series of in vitro and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Besides, we reconstitute the whole process of  9-bp deletion using purified Endonuclease G to induce breaks in mtDNA, followed by mitochondrial extract-mediated DSB repair and establish that microhomology-mediated end joining is responsible for the generation of mtDNA deletion. Finally, we show that the whole process is regulated by different stress conditions, which may modulate release of Endonuclease G to the mitochondrial matrix. Therefore, we uncover a new role for Endonuclease G in generating deletions, which is dependent on the formation of G4 DNA within the mitochondrial genome. Thus, in this study we identify a novel property of Endonuclease G, besides its role in apoptosis, and the recently described  elimination of paternal mitochondria during fertilization.

sentences: Allicin inhibits SARS-CoV-2 replication and abrogates the antiviral host response in the Calu-3 proteome The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is a major health burden. Volatile garlic organosulfur compounds, such as the thiol-reactive allicin (diallyl thiosulfinate) exert strong antimicrobial activity against various respiratory pathogens. Here, we investigated the antiviral activity of allicin against SARS-CoV-2 in infected Vero E6 and Calu-3 lung cells. Calu-3 cells showed greater allicin tolerance due >4-fold increased GSH levels compared to Vero E6. However, biocompatible allicin doses efficiently inhibited viral replication in both cell lines. Proteome analyses of SARS-CoV-2 infected Calu-3 cells revealed a strong induction of the antiviral interferon-stimulated gene (ISG) signature (e.g. cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, 25OAS and ISG15), pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTC1, ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin abrogated the ISG host response and reverted the host cellular pathways to levels of uninfected Calu-3 cells, confirming the antiviral and immunomodulatory activity of allicin in the host proteome. Thus, biocompatible doses of allicin could be promising for protection of lung cells against SARS-CoV-2.

sentences: Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant associated membrane patch (RAMP) once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of lipid collective assembly at the plasma membrane spatiotemporally as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.

sentences: Land Use Cover changes in the western escarpment of Rift Valley in the Gamo Zone, Southern Ethiopia LULC changes are caused by natural and human alterations of the landscape that could largely affect forest biodiversity and the environment. The aim of the study was to analyzed LULC change dynamics in the western escarpment of the rift valley of the Gamo Zone, Southern Ethiopia. Digital satellite images downloaded from USGS were analyzed using ERDAS Imagine (14) and Arc GIS 10.2 software and supervised image classification was used to generate LULC classification, accuracy assessment and Normalized Difference Vegetation Index (NDVI). Drivers of LULC change were identified and analyzed. Four land classes were identified such as forest, farmland, settlement and water-wetland. Settlement and farmlands have increased by 7.83% and 5.88%, respectively. On the other hand, both forest and water bodies and wetland decreased by aerial coverage of 11.03% and 2.68%, respectively. The overall accuracy of the study area was 92.86%, 94.22% and 94.3% with a kappa value of 0.902, 0.92 and 0.922, respectively. NDVI values ranged between -0.42 to 0.73. Agricultural expansion (31.4%), expansion of settlement (25.7%) and Fuelwood collection and Charcoal production (22.9%) were the main driving forces that jeopardize forest biodiversity of the study area. Integrated land use and policy to protect biodiversity loss, forest degradation and climate changes are deemed necessary.

sentences: Reduced myofibroblast transdifferentiation and fibrotic scarring in ischemic stroke after imatinib treatment The tyrosine kinase inhibitor imatinib has been reported to improve outcome in patients following ischemic stroke but the exact mechanism remains elusive. Here, utilizing a photothrombotic murine model of middle cerebral artery occlusion (MCAO), we show that imatinib-mediated inhibition of stroke-induced blood-brain barrier (BBB) dysfunction coincided with decreased expression of genes associated with inflammation and fibrosis in the cerebrovasculature. We found that imatinib effectively dampened stroke-induced reactive gliosis and myofibroblast transdifferentiation, whilst having very limited effect on the rest of the glia scar and on peripheral leukocyte infiltration. Further, our data suggest that consolidation of the PDGFR+ portion of the fibrotic scar in imatinib-treated mice contributes to the improvement in functional outcome compared to the vehicle controls, where the PDGFR+ scar is expanding. Comparison with human stroke transcriptome databases revealed significant overlap with imatinib-regulated genes, suggesting imatinib may modulate reactive gliosis and fibrotic scarring also in human stroke.

sentences: Identification of a Golgi-localized peptide encoded by the CENP-R transcript Prior work has identified signal sequences and motifs that are necessary and sufficient to target proteins to specific subcellular regions and organelles such as the plasma membrane, nucleus, Endoplasmic Reticulum, and mitochondria. In contrast, minimal sequence motifs that are sufficient for Golgi localization remain largely elusive. In this work, we identified a 37 amino acid alternative open reading frame (altORF) within the mRNA of the centromere protein, CENP-R. This altORF peptide localizes specifically to the cytoplasmic surface of the Golgi apparatus. Through mutational analysis, we identify a minimal 10 amino acid sequence and a critical cysteine residue that are necessary and sufficient for Golgi localization. Pharmacological perturbations suggest that this peptide is modified through palmitoylation to promote its localization. Together, our work defines a minimal sequence that is sufficient for Golgi targeting and provide a valuable Golgi marker for live cell imaging.

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Distinct mechanisms underlie H2O2 sensing in C. elegans head and tail Environmental oxidative stress threatens cellular integrity and should therefore be avoided by living organisms. Yet, relatively little is known about environmental oxidative stress perception. Here, using microfluidics, we showed that like I2 pharyngeal neurons, the tail phasmid PHA neurons function as oxidative stress sensing neurons in C. elegans, but display different responses to H2O2 and light. We uncovered that different but related receptors, GUR-3 and LITE-1, mediate H2O2 signaling in I2 and PHA neurons. Still, the peroxiredoxin PRDX-2 is essential for both, and might promote H2O2-mediated receptor activation. Our work demonstrates that C. elegans can sense a broad range of oxidative stressors using partially distinct H2O2 signaling pathways in head and tail sensillae, and paves the way for further understanding of how the integration of these inputs translates into the appropriate behavior.

Graphical abstract

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sentences: Temporary prey storage along swarm columns of army ants: an adaptive strategy for successful raiding? While pillaging the brood of other ant colonies, Eciton army ants accumulate prey in piles, or caches, along their foraging trails. Widely documented, these structures have historically been considered as byproducts of heavy traffic or aborted relocations of the ants temporary nest, or bivouac. However, we recently observed that caches of the hook-jawed army ant, Eciton hamatum, appeared independently from heavy traffic or bivouac relocations. In addition, the flow of prey through caches varied based on the quantity of prey items workers transported. As this suggested a potential adaptive function, we developed agent-based simulations to compare raids of caching and non-caching virtual army ants. We found that caches increased the amount of prey that relatively low numbers of raiders were able to retrieve. However, this advantage became less conspicuous - and generally disappeared - as the number of raiders increased. Based on these results, we hypothesize that caches maximize the amount of prey that limited amounts of raiders can retrieve, especially as prey colonies coordinately evacuate their brood. In principle, caches also allow workers to safely collect multiple prey items and efficiently transport them to the bivouac. Further field observations are needed to test this and other hypotheses emerging from our study.

sentences: Biophysical, Molecular and Proteomic profiling of Human Retinal Organoids derived Exosomes Extracellular vesicles (EVs) are phospholipid bilayer-bound particles released by cells that play a role in cell-cell communication, signal transduction, and extracellular matrix remodeling. There is a growing interest in EVs for ocular applications as therapeutics, biomarkers, and drug delivery vehicles. EVs secreted from mesenchymal stem cells (MSCs) have shown to provide therapeutic benefits in ocular conditions. However, very little is known about the properties of bioreactors cultured-3D human retinal organoids secreted EVs. This study provides a comprehensive morphological, nanomechanical, molecular, and proteomic characterization of retinal organoid EVs and compares it with human umbilical cord (hUC) MSCs. Nanoparticle tracking analysis indicated the average size of EV as 100-250 nm. Atomic force microscopy showed that retinal organoid EVs are softer and rougher than the hUCMSC EVs. Gene expression analysis by qPCR showed a high expression of exosome biogenesis genes in late retinal organoids derived EVs (>120 days). Immunoblot analysis showed highly expressed exosomal markers Alix, CD63, Flotillin-2, HRS and Hsp70 in late retinal organoids compared to early retinal organoids EVs (<120 days). Protein profiling of retinal organoid EVs displayed a higher differential expression of retinal function-related proteins and EV biogenesis/marker proteins than hUCMSC EVs, implicating that the use of retinal organoid EVs may have a superior therapeutic effect on retinal disorders. This study adds supplementary knowledge on the properties of EVs secreted by retinal organoids and suggests their potential use in the diagnostic and therapeutic treatments for ocular diseases.

sentences: Dual Drug Targeting to Kill Colon Cancer Cells Colorectal cancer (CRC) is driven by a small set of oncogenic and tumor suppressor mutations. However, different combinations of mutations often lead to poor tumor responses to individual anti-cancer drugs. We have investigated the anti-proliferative and in vitro cytotoxic activity of pair-wise combinations of inhibitors which target specific signalling pathways. Colon cancer cells in non-adherent cultures were killed more effectively by combinations of pyrvinium pamoate (a Wnt pathway inhibitor) and ABT263 (a pro-apoptotic Bcl-2 family inhibitor) or Ly29004 (a PI3kinase inhibitor). However, in a mouse xenograft model, the formulation and toxicity of the ABT737/PP combination prevent the use of these drugs for treatment of tumors. Fortunately, oral analogues of PP (pyrvinium phosphate, PPh) and ABT737(ABT263) have equivalent activity and can be used for treatment of mice carrying SW620 colorectal cancer xenografts. The PPh/ABT263 induced SW620 tumor cell apoptosis and reduced the rate of SW620 tumor growth. Combinations of Wnt signaling inhibitors and specific inhibitor of pro-survival proteins should be considered for the treatment of precancerous colon adenomas and advanced colorectal cancers with APC mutations.

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A dietary vitamin B12 deficiency impairs motor function and changes neuronal survival and choline metabolism after ischemic stroke to the sensorimotor cortex in middle aged male and female mice BackgroundNutrition is a modifiable risk factor for ischemic stroke. As people age their ability to absorb some nutrients decreases, an example is vitamin B12. Older individuals with a vitamin B12 deficiency are at a higher risk for stroke and have worse outcome. However, the mechanisms through which these occur remain unknown.

ObjectiveThe aim of the study was to investigate the role of vitamin B12 deficiency in ischemic stroke outcome in a mouse model.

MethodsAt 10-weeks of age male and female C57Bl/6J mice were put on vitamin B12 deficient (0 mg/kg) or control (0.025 mg/kg vitamin B12) diets for 4-weeks (n = 10/group). Using the photothrombosis model the sensorimotor cortex was damaged and at 18 weeks of age, motor outcome was measured. At the end of experiments, brain, blood, and liver tissue were collected to measure homocysteine levels, ischemic damage volume, apoptosis, or one-carbon metabolite levels.

ResultsAll animals maintained on the vitamin B12 deficient diet had increased levels of total homocysteine in plasma and liver tissue. In ischemic brain tissue no difference between groups in lesion volume were detected. There were changes in levels of one-carbon metabolites in ischemic and non-ischemic brain tissue because of a vitamin B12 deficiency or sex. There was an increase in total apoptosis in ischemic brain tissue of male vitamin B12 deficient animals. There was more neuronal survival within ischemic brain tissue of the vitamin B12 deficient animals compared to controls. After ischemic stroke, vitamin B12 deficient male and female mice had impaired motor function compared to control animals.

ConclusionsThe data presented in this study confirm that a dietary vitamin B12 deficiency impacts motor function in older adult male mice after ischemic stroke. The mechanisms driving this change may be a result of neuronal survival and compensation in one-carbon metabolism within the damaged brain tissue.

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ACPA-IgG variable domain glycosylation increases before the onset of rheumatoid arthritis and stabilizes thereafter; a cross-sectional study encompassing over 1500 samples ObjectiveThe autoimmune response in rheumatoid arthritis (RA) is marked by anti-citrullinated protein antibodies (ACPA). A remarkable feature of ACPA-IgG is the abundant expression of N-linked glycans in the variable domain. Nonetheless, the presence of ACPA variable domain glycans (VDG) across disease stages and its response to therapy is poorly described. To understand its dynamics, we investigated the abundance of ACPA-IgG VDG in 1574 samples from individuals in different clinical disease stages.

MethodsUsing liquid chromatography, we analyzed ACPA-IgG VDG profiles of 7 different cohorts from Japan, Canada, the Netherlands and Sweden. We assessed 184 healthy, 228 pre-symptomatic, 277 arthralgia, 305 patients at RA-onset and 117 RA-patients 4, 8 and 12 months after disease onset. Additionally, we measured VDG of 234 samples from RA-patients that did or did not achieve long-term drug-free remission (DFR) during up to 16 years follow-up.

ResultsOur data show that ACPA-IgG VDG significantly increases (p<0.0001) towards disease-onset and associates with ACPA-levels and epitope spreading pre-diagnosis. A slight increase in VDG was observed in established RA and a moderate influence of treatment. Individuals who later achieved DFR displayed reduced ACPA-IgG VDG already at RA-onset.

ConclusionThe abundance of ACPA-IgG VDG rises towards RA-onset and correlates with maturation of the ACPA-response. Although, ACPA-IgG VDG levels are rather stable in established disease, a lower degree at RA-onset correlates with DFR. Even though the underlying biological mechanisms are still elusive, our data support the concept that VDG relates to an expansion of the ACPA-response pre-disease and contributes to disease-development.

sentences: High-throughput tandem-microwell assay for ammonia repositions FDA-Approved drugs to Helicobacter pylori infection To date, little attempt has been made to develop new treatments for Helicobacter pylori (H. pylori), although the community is aware of the shortage of treatments for H. pylori. In this study, we developed a 192-tandem-microwell-based high-throughput-assay for ammonia that is a known virulence factor of H. pylori and a product of urease. We could identify few drugs, i.e. panobinostat, dacinostat, ebselen, captan and disulfiram, to potently inhibit the activity of ureases from bacterial or plant species. These inhibitors suppress the activity of urease via substrate-competitive or covalent-allosteric mechanism, but all except captan prevent the antibiotic-resistant H. pylori strain from infecting human gastric cells, with a more pronounced effect than acetohydroxamic acid, a well-known urease inhibitor and clinically used drug for the treatment of bacterial infection. This study offers several bases for the development of new treatments for urease-containing pathogens and to study the mechanism responsible for the regulation of urease activity.

sentences: Freshwater dissolved oxygen dynamics: changes due to glyphosate, 2,4-D and their mixture, both under clear and turbid-organic conditions. We performed two independent outdoor mesocosm experiments where we measured the variation of DO saturation (DO%) in freshwater after a single input of Roundup Max(R) (G) (glyphosate-based formulation), AsiMax 50(R) (2,4-D) (2,4-D-based formulation) and their mixture (M). Two concentration levels were tested; 0.3 mg/L G and 0.135 mg/L 2,4-D (Low; L) and 3 mg/L G and 1.35 mg/L 2,4-D (High; H). We assayed consolidated microbial communities coming from a system in organic turbid eutrophic status and a system in clear mesotrophic status during 21 and 23 days, respectively. A sample of phytoplankton (micro+nano, pico-eukaryotes, pico-cyanobacteria), mixotrophic algae and heterotrophic bacteria was collected to determine abundances at each of four sampling dates. The clear and turbid systems showed similar, but not synchronized, patterns of daily DO% changes in relation to the controls (DO%v), after exposure to both single and combined formulations. Under glyphosate scenarios (GL, GH, ML and MH), the two types of systems showed similar DO%v but different microbial abundances, being associated to an increase in the micro+nano and pico-eukaryotic phytoplankton fractions for the clear system. In contrast, in the turbid system changes were associated with increased pico-cyanobacteria and decreased mixotrophic algae. Effects of 2,4-D were only observed in the turbid system, leading to decreased micro+nano phytoplankton abundances. Under the turbid scenario, the herbicide mixture at high concentration had a synergistic effect on DO%v and recovery was not detected by the end of the experiment. Our results revealed that herbicides inputs induced changes in phytoplankton abundances that leads to measurable DO variations.

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Introgression between highly divergent sea squirt genomes: an adaptive breakthrough? Human-mediated introductions are reshuffling species distribution on a global scale. Consequently, an increasing number of allopatric taxa are now brought into contact, promoting introgressive hybridization between incompletely isolated species and new adaptive gene transfer. The broadcast spawning marine species, Ciona robusta, has been recently introduced in the native range of its sister taxa, Ciona intestinalis, in the English Channel and North-East Atlantic. These sea squirts are highly divergent, yet hybridization has been reported by crossing experiments and genetic studies in the wild. Here, we examined the consequences of secondary contact between C. intestinalis and C. robusta in the English Channel. We produced genomes phased by transmission to infer the history of divergence and gene flow, and analyzed introgressed genomic tracts. Demographic inference revealed a history of secondary contact with a low overall rate of introgression. Introgressed tracts were short, segregating at low frequency, and scattered throughout the genome, suggesting traces of past contacts during the last 30 ky. However, we also uncovered a hotspot of introgression on chromosome 5, characterized by several hundred Kb-long C. robusta haplotypes segregating in C. intestinalis, that introgressed during contemporary times the last 75 years. Although locally more frequent than the baseline level of introgression, C. robusta alleles are not fixed, even in the core region of the introgression hotspot. Still, LD patterns and haplotype-based tests suggest this genomic region is under recent positive selection. We further detected in the hotspot an over-representation of candidate SNPs lying on a cytochrome P450 gene with a high copy number of tandem repeats in the introgressed alleles. Cytochromes P450 are a superfamily of enzymes involved in detoxifying exogenous compounds, constituting a promising avenue for functional studies. These findings support that introgression of an adaptive allele is possible between very divergent genomes and that anthropogenic hybridization can provide the raw material for adaptation of native lineages in the Anthropocene.

Author summaryIntrogression, the transfer of genetic material by hybridization between taxa, is increasingly recognized to sometimes persist for long during species divergence. However, the evolutionary consequences of human-induced introgression remain largely unknown, especially in the marine realm. While some argue it poses a threat to the genome integrity of native species, others consider it has a great potential to fuel adaptation. In this work, we quantify the magnitude and genomic distribution of introgression after secondary contact between a native sea squirt and its divergently related sister species recently introduced in the English Channel. The genome-wide pattern suggests introgression is mostly impeded between these two incompatible genomes that only exchanged a few very short chromosome tracts sparsely distributed in the native genome. We nonetheless found a hotspot of long tracts that recently introgressed in a single region of the genome, with a clear footprint of recent positive selection. In the center of the hotspot, we further detected a promising candidate gene for adaptive introgression: a cytochrome P450 detoxifying enzyme with a high copy number in the introgressed allele. Therefore, our results support that adaptive introgression can remain possible between very divergent genomes and that anthropogenic hybridization can provide the raw material for the adaptation of native lineages in the Anthropocene.

sentences: Breeding responses to environmental variation are age- and trait-dependent in female Nazca boobies Age and environment are important determinants of reproductive parameters in long-lived organisms. These factors may interact to determine breeding responses to environmental change, yet few studies have examined the environmental-dependence of aging patterns across the entire lifespan. We do so, using a 20-year longitudinal dataset of reproductive phenotypes in long-lived female Nazca boobies (Sula granti), a monogamous seabird breeding in the eastern tropical Pacific. Young and old females may suffer from inexperience and senescence, respectively, and/or practice reproductive restraint. Breeding performance (for breeding participation, breeding date, clutch size, egg volume, and offspring production) was expected to be lower in these age classes, particularly under environmental challenge, in comparison with middle-aged breeders. Sea surface temperature anomalies (SSTA) represented interannual variation in the El Nino-Southern Oscillation (ENSO) and were one proxy for environmental quality (a population count of clutch initiations was a second). Although only females lay eggs, both sexes care for eggs and nestlings, and the male partners age, alone or in interaction with female age, was evaluated as a predictor of breeding performance. Middle-aged females performed better than young and old birds for all reproductive traits. Pairing with a young male delayed breeding (particularly for old females) and reduced clutch size, and pairing with an old male reduced offspring production. Challenging environments increased age effects on breeding probability and breeding date across young to middle ages and for offspring production across middle to old ages. However, important exceptions to the predicted patterns for clutch size and fledging success across young to middle ages suggested trade-offs between fitness components may complicate patterns of trait expression across the lifespan. Relationships between breeding participation, environment, and individual quality and/or experience in young females may also contribute to unexpected patterns for clutch size and fledging success, traits expressed only in breeders. Finally, independent of age, breeding responses of female Nazca boobies to the ENSO did not follow expectations derived from oceanic forcing of primary productivity. During El Nino-like conditions, egg-laying traits (clutch size, breeding date) improved but offspring production declined, while La Nina-like conditions were "poor" environments throughout the breeding cycle.

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Glutamate-Weighted Magnetic Resonance Imaging (GluCEST) Detects Effects of Transcranial Magnetic Stimulation to the Motor Cortex Transcranial magnetic stimulation (TMS) is used in several FDA-approved treatments and, increasingly, to treat neurological disorders in off-label uses. However, the mechanism by which TMS causes physiological change is unclear, as are the origins of response variability in the general population. Ideally, objective in vivo biomarkers could shed light on these unknowns and eventually inform personalized interventions. Continuous theta burst stimulation (cTBS) is a form of TMS which has been observed to reduce motor evoked potentials (MEPs) for 60 minutes or longer post-stimulation, although the consistency of this effect and its mechanism continue to be under debate. Here, we use glutamate-weighted chemical exchange saturation transfer (gluCEST) magnetic resonance imaging (MRI) at ultra-high magnetic field (7T) to measure changes in glutamate concentration at the site of cTBS. We find that gluCEST signal in the ipsilateral hemisphere of the brain generally decreases in response to cTBS, whereas consistent changes were not detected in the contralateral or in subjects receiving a sham stimulation.

One Sentence SummaryWe used glutamate-weighted Chemical Exchange Saturation Transfer (GluCEST) imaging to detect changes in glutamate contrast in the brains of young, healthy adults undergoing transcranial magnetic stimulation (TMS) to the motor cortex.

sentences: West Nile virus in crocodiles and mosquitoes in Zimbabwe We detected, for the first time, West Nile virus lineages 1 and 2 in Zimbabwe in mosquitoes and crocodile tissue samples, including fluid from egg waste. Our results provide evidence of WNV circulation in Zimbabwe, suggesting that an evaluation of the risk to humans and susceptible animals should be considered.

sentences: Physiological noise modeling in fMRI based on the pulsatile component of photoplethysmograph The blood oxygenation level-dependent (BOLD) contrast mechanism allows the noninvasive monitoring of changes in deoxyhemoglobin content. As such, it is commonly used in functional magnetic resonance imaging (fMRI) to study brain activity since levels of deoxyhemoglobin are indirectly related to local neuronal activity through neurovascular coupling mechanisms. However, the BOLD signal is severely affected by physiological processes as well as motion. Due to this, several noise correction techniques have been developed to correct for the associated confounds. The present study focuses on cardiac pulsatility fMRI confounds, aiming to refine model-based techniques that utilize the photoplethysmograph (PPG) signal. Specifically, we propose a new technique based on convolution filtering, termed cardiac pulsatility model (CPM) and compare its performance with the cardiac-related RETROICOR (Card-RETROICOR), which is a technique commonly used to model fMRI fluctuations due to cardiac pulsatility. Further, we investigate whether variations in the amplitude of the PPG pulses (PPG-Amp) covary with variations in amplitude of pulse-related fMRI fluctuations, as well as with the systemic low frequency oscillations (SLFOs) component of the fMRI global signal (GS - defined as the mean signal across all gray matter voxels). Capitalizing on 3T fMRI data from the Human Connectome Project, CPM was found to explain a significantly larger fraction of the fMRI signal variance compared to Card-RETROICOR, particularly for subjects with larger heart rate variability during the scan. The amplitude of the fMRI pulse-related fluctuations did not covary with PPG-Amp; however, PPG-Amp explained significant variance in the GS that was not attributed to variations in heart rate or breathing patterns. Our results suggest that the proposed approach can model high-frequency fluctuations due to pulsation as well as low-frequency physiological fluctuations more accurately compared to model-based techniques commonly employed in fMRI studies.

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AAV-Mediated In Vivo CAR Gene Therapy for Targeting Human T Cell Leukemia Chimeric antigen receptor (CAR) T cell therapy is the most active field in immuno-oncology and brings substantial benefit to patients with B cell malignancies. However, the complex procedure for CAR T cell generation hampers its widespread applications. Here, we describe a novel approach in which human CAR T cells can be generated within the host upon injecting an Adeno-associated virus (AAV)vector carrying the CAR gene, which we call AAV delivering CAR gene therapy (ACG). Upon single infusion into a humanized NCG tumor mouse model of human T cell leukemia, AAV generates sufficient numbers of potent in vivo CAR cells, resulting in tumor regression; these in vivo generated CAR cells produce antitumor immunological characteristics. This instantaneous generation of in vivo CAR T cells may bypass the need for patient lymphodepletion, as well as the ex vivo processes of traditional CAR T cell production, which may make CAR therapy simpler and less expensive. It may allow the development of intricate, individualized treatments in the form of on-demand and diverse therapies.

Significance StatementAAV can generate enough CAR cells within the host. That act as a living drug, distributed throughout the body, and persist for weeks, with the ability to recognize and destroy tumor cells.

sentences: Lipid Droplets and Ferritin Heavy Chain: a Devilish Liaison in Cancer Radioresistance Although much progress has been made in cancer treatment, the molecular mechanisms underlying cancer radioresistance (RR) as well as the biological characteristic of radioresistant cancer cells still need to be clarified. In this regard, we discovered that breast, bladder, lung, neuroglioma and prostate 6 Gy X-ray resistant cells were characterized by an increase of Lipid Droplet (LD) number and that the cells containing highest LDs showed the highest clonogenic potential after irradiation. Moreover, we observed that LD content was tightly connected with the iron metabolism and in particular with the presence of the ferritin heavy chain (FTH1). In fact, breast and lung cancer cells silenced for the FTH1 gene showed a reduction in the LD numbers and, by consequence, became radiosensitive. FTH1 restoration as well as iron-chelating treatment by Deferoxamine were able to restore the LD amount and RR. Overall, these results provide evidence of a novel molecular mechanism behind RR in which LDs and FTH1 are tightly connected to each other, a synergistic effect which might be worth deeply investigating in order to make cancer cells more radiosensitive and improve the efficacy of radiation treatments.

sentences: An enhanced inverted encoding model for neural reconstructions Here we present a more interpretable and flexible approach for reconstructing the contents of perception, attention, and memory from neuroimaging data. Our enhanced inverted encoding model (eIEM) incorporates methodological improvements including proper accounting of population-level tuning functions and a trial-by-trial prediction error-based metric where reconstruction quality is measured in meaningful units. Improved flexibility is further gained via eIEMs novel goodness-of-fit feature: for trial-by-trial reconstructions, goodness-of-fits are obtained independently (non-circularly) to prediction error and can be applied to any IEM procedure or decoding metric, resulting in improved reconstruction quality and brain-behavior correlations. We validate eIEM from methodological principles, simulated neuroimaging datasets, and three pre-existing fMRI datasets spanning perception, attention, and working memory. Notably, eIEM is easy to apply and broadly accessible - our Python package (https://pypi.org/project/inverted-encoding) implements eIEM in one line of code - and is easily modifiable to compare performance metrics and/or scale up to more complex models.

sentences: Evolutionarily conserved regions of THAP9 transposase reveal new motifs for subcellular localization and post-translational modification. THAP9 is a transposable element-derived gene that encodes the THAP9 protein, which is homologous to the Drosophila P-element transposase (DmTNP) and can cut and paste DNA. However, the exact functional role of THAP9 is unknown. Here, we perform evolutionary analysis and extensive in silico characterization of THAP9, including predicting domains and putative post-translational modification sites. We predict previously unreported mammalian-specific post-translational modification sites that may play a role in the subcellular localization of THAP9. We also observe that although THAP9 has evolved under a strong pervasive purifying selection, yielding high conservation of THAP9, there are distinct class-specific conservation patterns of key functional residues in certain domains. Furthermore, investigation of THAP9 expression profiles in various cancer and matched normal datasets demonstrated underexpression and overexpression in testicular cancers and thymic epithelial tumors, respectively, thus suggesting a possible role of THAP9 in cancer.

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Does the brain care about averages? A simple test. Trial-averaged metrics, e.g. in the form of tuning curves and population response vectors, are a basic and widely accepted way of characterizing neuronal activity. But how relevant are such trial-averaged responses to neuronal computation itself? Here we present a simple test to estimate whether average responses reflect aspects of neuronal activity that contribute to neuronal processing in a specific context. The test probes two assumptions inherent in the usage of average neuronal metrics: O_LIReliability: Neuronal responses repeat consistently enough across single trial that their average response template remains recognizable to downstream regions.
C_LIO_LIBehavioural relevance: If a single-trial response is more similar to the average template, it will be easier for the animal to identify the correct stimulus or action.
C_LI

We apply this test to a large publicly available data set featuring electrophysiological recordings from 71 brain areas in behaving mice. We show that single-trial responses were less correlated to the average response template than one would expect if they represented discrete versions of the template, down-sampled to the number of spikes in the single trial. Moreover, single-trial responses were barely stimulus-specific - they could not be clearly assigned to the average response template of one stimulus. Most importantly, better-matched single-trial responses did not more accurately predict behaviour for any of the recorded brain areas. We conclude that in this data set, average responses do not seem particularly relevant to neuronal computation in a majority of brain areas, and we encourage other researchers to apply similar tests when using trial-averaged neuronal metrics.

sentences: CRISPR/Cas9 screen for functional MYC binding sites reveals MYC-dependent vulnerabilities in K562 cells The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate expression of adjacent genes in a cell type- and developmental stage-specific manner. To date, a comprehensive analysis of direct MYC targets with essential roles in different types of cancer is missing. To enable identification of functional MYC binding sites and corresponding target genes, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. As a proof of principle, using this library we identified several known and novel MYC targets critical for K562 chronic myelogenous leukemia cells and uncovered specific features of essential E-boxes. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in any cancer type.

sentences: Polarized Dishevelled dissolution and condensation drives embryonic axis specification in oocytes The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes [1]. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled is a cytoplasmic Wnt pathway effector required for axis development in diverse species [2-4], but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dishevelled localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes, but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-condensation mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially-regulated Dishevelled dynamics.

sentences: Using marine cargo traffic to identify countries in Africa with greatest risk of invasion by Anopheles stephensi Anopheles stephensi is an efficient malaria vector commonly found in South Asia and the Arabian Peninsula, but in recent years it has established as an invasive species in the Horn of Africa (HoA). In this region, An. stephensi was first detected in a livestock quarantine station near a major seaport in Djibouti in 2012, in Ethiopia in 2016, in Sudan in 2018 and Somalia in 2019. Anopheles stephensi often uses artificial containers as larval habitats, which may facilitate introduction through maritime trade as has been seen with other invasive container breeding mosquitoes. If An. stephensi is being introduced through maritime traffic, prioritization exercises are needed to identify locations at greatest risk of An. stephensi introduction for early detection and rapid response, limiting further invasion opportunities. Here, we use UNCTAD maritime trade data to 1) identify coastal African countries which were most highly connected to select An. stephensi endemic countries in 2011, prior to initial detection in Africa, 2) develop a ranked prioritization list of countries based on likelihood of An. stephensi introduction for 2016 and 2020 based on maritime trade alone and maritime trade and habitat suitability, and 3) use network analysis to describe intracontinental maritime trade and eigenvector centrality to determine likely paths of further introduction on the continent if An. stephensi is detected in a new location. Our results show that in 2011, Sudan and Djibouti were ranked as the top two countries with likelihood of An. stephensi introduction based on maritime trade alone, and these were indeed the first two coastal countries in the HoA where An. stephensi was detected. Trade data from 2020 with Djibouti and Sudan included as source populations identify Egypt, Kenya, Mauritius, Tanzania, and Morocco as the top five countries with likelihood of An. stephensi introduction. When factoring in habitat suitability, Egypt, Kenya, Tanzania, Morocco, and Libya are ranked highest. Network analysis revealed that the countries with the highest eigenvector centrality scores, and therefore highest degrees of connectivity with other coastal African nations were South Africa (0.175), Mauritius (0.159), Ghana (0.159), Togo (0.157), and Morocco (0.044) and therefore detection of An. stephensi in any one of these locations has a higher potential to cascade further across the continent via maritime trade than those with lower eigenvector centrality scores. Taken together, these data could serve as tools to prioritize efforts for An. stephensi surveillance and control in Africa. Surveillance in seaports of countries at greatest risk of introduction may serve as an early warning system for the detection of An. stephensi, providing opportunities to limit further introduction and expansion of this invasive malaria vector in Africa.

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SAIBR: A simple, platform-independent method for spectral autofluorescence correction Biological systems are increasingly viewed through the lens of mathematics, physics, and systems approaches that demand accurate quantification of gene expression and local protein concentrations. Such approaches have benefited greatly from the revolution in genetic engineering sparked by CRISPR/Cas9. By facilitating the tagging of genes at their genomic loci, CRISPR/Cas9 allows us to use fluorescence to monitor proteins that are expressed at or near endogenous levels under native regulatory control. However, due to their typically lower expression levels, quantitative experiments using endogenously-tagged genes can run into limits imposed by autofluorescence (AF). AF is often a particular challenge in the illumination bands occupied by the most efficient fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image correction By Regression (SAIBR), a simple, platform-independent protocol, and associated GUI-based FIJI plugin to correct for autofluorescence using standard filter sets and illumination conditions. Fully validated for use in C. elegans embryos and tested in diverse systems, including starfish oocytes and fission yeast, SAIBR achieves accurate quantitation of fluorophore signal and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible, low barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

Summary StatementImplemented as an easy-to-use Fiji Plugin, SAIBR provides effective autofluorescence correction for cells and tissues using standard imaging conditions.

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Enhancing Polysaccharide Immunity through Intranasal Immunizations ObjectiveA model conjugate vaccine incorporating a pneumococcal polysaccharide with EcoCRM197 was used to determine vaccine efficacy when administered with our clinical-stage mucosal stimulating adjuvant, NE01. This was the first attempt at combining NE01 with a polysaccharide conjugate vaccine to elicit mucosal immunity against respiratory bacterial infection through intranasal immunizations.

ResultsIntranasal immunizations using the NE01 adjuvant incorporating a polysaccharide conjugate resulted in the generation of a robust IgG and IgA responses in both serum and mucosa. Our data also demonstrated the active homing of immunological memory cells to the lower respiratory tract as evidenced by an increase of IgG- and IgA-producing memory B-cells in the lungs. Further, intranasal immunization enhanced the induction of a balanced Th1/Th2/ Th17 immune response with clear homing of memory T-cells to the lungs. Serum antibodies generated by this formulation and route of administration demonstrated excellent efficacy in killing S. pneumonia in an in vitro OPK assay, a biomarker of vaccine efficacy. Our data suggest that further optimization of the dose and schedule as well as evaluation of the efficacy of this new formulation in animal models of colonization and infection are warranted.

sentences: Genetic patterns in Montipora capitata across an environmental mosaic in Kaneohe Bay Spatial genetic structure (SGS) is important to a populations ability to adapt to environmental change. For species that reproduce both sexually and asexually, the relative contribution of each reproductive mode has important ecological and evolutionary implications because asexual reproduction can have a strong effect on SGS. Reef building corals reproduce sexually, but many species also propagate asexually under certain conditions. In order to understand SGS and the relative importance of reproductive mode across environmental gradients, we evaluated genetic relatedness in almost 600 colonies of Montipora capitata across 30 environmentally characterized sites in K[a]neohe Bay, Oahu, Hawaii using low-depth restriction digest associated sequencing. Clonal colonies were relatively rare overall but influenced SGS. Clones were located significantly closer to one another spatially than average colonies and were more frequent on sites where wave energy was relatively high, suggesting a strong role of mechanical breakage in their formation. Excluding clones, we found no evidence of isolation by distance within sites or across the bay. Several environmental characteristics were significant predictors of the underlying genetic variation (including degree heating weeks, time spent above 30{degrees}C, depth, sedimentation rate and wave height); however, they only explained 5% of this genetic variation. Our results show that colony fragmentation contributes to the ecology of M. capitata at local scales and that genetic diversity is maintained despite strong environmental gradients in a highly impacted ecosystem, suggesting potential for broad adaptation or acclimatization in this population.

sentences: Two Genomic Loci Control Three Eye Colors in the Domestic Pigeon (Columba livia) The iris of the eye shows striking color variation across vertebrate species, and may play important roles in crypsis and communication. The domestic pigeon (Columba livia) has three common iris colors, orange, pearl (white), and bull (dark brown), segregating in a single species, thereby providing a unique opportunity to identify the genetic basis of iris coloration. We used comparative genomics and genetic mapping in laboratory crosses to identify two candidate genes that control variation in iris color in domestic pigeons. We identified a nonsense mutation in the solute carrier SLC2A11B that is shared among all pigeons with pearl eye color, and a locus associated with bull eye color that includes EDNRB2, a gene involved in neural crest migration and pigment development. However, bull eye is likely controlled by a heterogeneous collection of alleles across pigeon breeds. We also found that the EDNRB2 region is associated with regionalized plumage depigmentation (piebalding). Our results establish a genetic link between iris and plumage color, two traits that were long known by pigeon breeders to co-occur, and demonstrate the importance of gene duplicates in establishing possibilities and constraints in the evolution of color and color pattern among vertebrates.

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Oxidative stress profiles of lymphoblasts from Amyotrophic Lateral Sclerosis patients with or without known SOD1 Mutations Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disease that affects motor neurons. This disease is associated with oxidative stress especially in mutant superoxide dismutase 1 (mutSOD1) patients. However, less is known for the most prevalent sporadic ALS due to a lack of disease models. Here, we studied oxidative the stress profiles in lymphoblasts from ALS patients with mutSOD1 or unknown (undSOD1) mutations.

mutSOD1 and undSOD1 lymphoblasts, as well as sex/age-matched controls (3/group) were obtained from Coriell and divided in 46 years-old-men (C1), 46 years-old-women (C2) or 26/27 years-old-men (C3) cohorts. Growth curves were performed, and several parameters associated with redox homeostasis were evaluated, including SOD activity and expression, general oxidative stress levels, lipid peroxidation, response to oxidative stimulus, glutathione redox cycle, catalase expression, and activity, and Nrf2 transcripts. Pooled (all cohorts) and paired (intra-cohort) statistical analyses were performed, followed by clustering and principal component analyses (PCA).

Although a high heterogeneity among lymphoblast redox profiles was found between cohorts, clustering analysis based on 7 parameters with high chi-square ranking (total SOD activity, oxidative stress levels, catalase transcripts, SOD1 protein levels, metabolic response to mM concentrations of tert-butyl hydroperoxide, glutathione reductase activity, and Nrf2 transcript levels) provided a perfect separation between samples from healthy controls and ALS (undSOD1 and mutSOD1), also visualized in the PCA analysis.

Our results show distinct redox signatures in lymphoblasts from mutSOD1, undSOD1, and healthy controls that can be used as therapeutic targets for ALS drug development.

Highlights Lymphoblasts from ALS patients present altered redox properties
 Redox profiling evidenced total SOD and glutathione reductase activities, SOD1 protein levels, DCF fluorescence, catalase and Nrf2 transcripts, and tert-butyl hydroperoxide cytotoxicity, as discriminant features for experimental groups.
 Lymphoblast redox profiles may be helpful for patient stratification and precision medicine

sentences: Mapping Interactome Networks of FOSL1 and FOSL2 in Human Th17 Cells Dysregulated function of Th17 cells has implications in immunodeficiencies and autoimmune disorders. Th17 cell-differentiation is orchestrated by a complex network of transcription factors, including several members of the activator protein (AP-1) family. Among these, FOSL1 and FOSL2 influence the effector responses of Th17 cells. However, the molecular mechanisms underlying these functions are unclear, owing to the poorly characterized protein interaction networks of these factors. Here, we establish the first interactomes of FOSL1 and FOSL2 in human Th17 cells, using affinity purification-mass spectrometry analysis. In addition to the known JUN proteins, we identified several novel binding partners of FOSL1 and FOSL2. Gene ontology analysis found a major fraction of these interactors to be associated with RNA binding activity, which suggests new mechanistic links. Intriguingly, 29 proteins were found to share interactions with FOSL1 and FOSL2, and these included key regulators of Th17-fate. We further validated the binding partners identified in this study by using parallel reaction monitoring targeted mass-spectrometry and other methods. Our study provides key insights into the interaction-based signaling mechanisms of FOSL factors that potentially govern Th17 cell-differentiation and associated pathologies.

sentences: Zebrafish pigment cells develop directly from persistent highly multipotent progenitors. Neural crest cells (NCCs) are highly multipotent stem cells. A long-standing controversy exists over the mechanism of NCC fate specification, specifically regarding the presence and potency of intermediate progenitors. The direct fate restriction (DFR) model, based on early in vivo clonal studies, hypothesised that intermediates are absent and that migrating cells maintain full multipotency1-6. However, most authors favour progressive fate restriction (PFR) models, with fully multipotent early NCCs (ENCCs) transitioning to partially-restricted intermediates before committing to individual fates7-12. Here, single cell transcriptional profiling of zebrafish pigment cell development leads to us proposing a Cyclical Fate Restriction mechanism of NCC development that reconciles the DFR and PFR models. Our clustering of single NCC Nanostring transcriptional profiles identifies only broadly multipotent intermediate states between ENCCs and differentiated melanocytes and iridophores. Leukocyte tyrosine kinase (Ltk) marks the multipotent progenitor and iridophores, consistent with biphasic ltk expression13-15. Ltk inhibitor and constitutive activation studies support expression at an early multipotent stage, whilst lineage-tracing of ltk-expressing cells reveals their multipotency extends beyond pigment cell-types to neural fates. We conclude that pigment cell development does not involve a conventional PFR mechanism, but instead occurs directly and more dynamically from a broadly multipotent intermediate state.

sentences: Genome-wide screens identify calcium signaling as a key regulator of IgE+ plasma celldifferentiation and survival IgE antibodies protect against toxins and parasites, however, they also mediate allergic reactions. In contrast to other antibody isotypes, B cells switched to IgE respond transiently and do not give rise to long-lived plasma cells (PCs) or memory B cells. Although the intrinsic differences of IgE+ B cells have been linked to signaling by the IgE-B cell receptor (BCR), the molecular pathways controlling their behavior remain poorly understood. Here we employ whole-genome CRISPR screening to identify genes regulating IgE+ B cell proliferation, survival and differentiation into PCs. We show that IgE+ B cells are selectively suppressed by the IgE-BCR signaling to intracellular calcium, which inhibits PC differentiation and limits their lifespan after differentiation. Consequently, manipulation of calcium signaling in vivo enhances IgE+ PC responses. Insights from this pathway shed new light on the self-limiting character of IgE responses and open new avenues to eliminate IgE+ PCs in allergy.

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Motor cortical influence relies on task-specific activity covariation During limb movement, spinal circuits facilitate the alternating activation of antagonistic flexor and extensor muscles. Yet antagonist cocontraction is often required to stabilize joints, like when loads are handled. Previous results suggest that these different muscle activation patterns are mediated by separate flexion- and extension-related motor cortical output populations, while others suggest recruitment of task-specific populations. To distinguish between hypotheses, we developed a paradigm in which mice toggle between forelimb tasks requiring antagonist alternation or cocontraction and measured activity in motor cortical layer 5b. Our results conformed to neither hypothesis: consistent flexion- and extension-related activity was not observed across tasks, and no task-specific populations were observed. Instead, activity covariation among motor cortical neurons dramatically changed between tasks, thereby altering the relation between neural and muscle activity. This was also observed specifically for corticospinal neurons. Collectively, our findings indicate that motor cortex drives different muscle activation patterns via task-specific activity covariation.

HIGHLIGHTSMice perform two forelimb tasks involving distinct antagonist muscle activity in a novel paradigm

L5b motor cortical neurons are not organized by task-specific activity

L5b motor cortical neurons do not encode muscle activity consistently across tasks

Task-specific muscle activity is driven by a change in motor cortical activity covariation

eTOC BLURBWarriner et al. simultaneously measured muscle and motor cortical activity in mouse during antagonist forelimb muscle alternation and cocontraction to reveal that these distinct muscle activation patterns are not driven through consistent flexion and extension programs nor through the activity of discrete, task-specific neuronal subsets. Instead, distinct patterns involve task-specific changes in firing pattern covariation among layer 5b neurons, and corticospinal neurons in particular, which change their relationship to muscle activity across tasks.

sentences: The human cerebellum is essential for modulating perceptual sensitivity based on temporal expectations A functional benefit of attention is to proactively enhance perceptual sensitivity in space and time. Although attentional orienting has traditionally been associated with cortico-thalamic networks, recent evidence has shown that individuals with cerebellar degeneration (CD) show a reduced reaction time benefit from cues that enable temporal anticipation. While this deficit may reflect impairment in anticipatory motor preparation, it could also arise from cerebellar contribution to attentional modulation in time of perceptual sensitivity. To examine this, we tested CD participants on a non-speeded, challenging perceptual discrimination task, asking if they benefit from temporal cues. Strikingly, the CD group showed no duration-specific perceptual sensitivity benefit when cued by repeated but aperiodic presentation of the target interval. In contrast, they performed similar to controls when cued by a rhythmic stream. This dissociation further specifies the functional domain of the cerebellum and establishes its role in the attentional adjustment of perceptual sensitivity in time.

sentences: Doors and corners of variance partitioning in statistical ecology Variance partitioning is a common tool for statistical analysis and interpretation in both observational and experimental studies in ecology. Its popularity has led to a proliferation of methods with sometimes confusing or contradicting interpretations. Here, we present variance partitioning as a general tool in a model based Bayesian framework for summarizing and interpreting regression-like models. To demonstrate our approach we present a case study comprising of a simple occupancy model for a metapopulation of the Glanville fritillary butterfly. We pay special attention to the thorny issue of correlated covariates and random effects, and highlight uncertainty in variance partitioning. We recommend several alternative measures of variance, which jointly can be used to better interpret variance partitions. Additionally, we extend the general approach to encompass partitioning of variance within and between groups of observations, an approach very similar to analysis of variance. While noting that many troublesome issues relating to variance partitioning, such as uncertainty quantification, have been neglected in the literature, we likewise feel that the rather general applicability of the methods as an extension of statistical model-based analyses has not been fully utilized by the ecological research community either.

sentences: PARP7 mono-ADP-ribosylates the Agonist Conformation of the Androgen Receptor in the Nucleus We recently described a signal transduction pathway that contributes to AR regulation based on sitespecific ADP-ribosylation by PARP7, a mono-ADP-ribosyltransferase implicated in several human cancers. ADP-ribosylated AR is specifically recognized by PARP9/DTX3L, a heterodimeric complex that contains an ADP-ribose reader (PARP9) and a ubiquitin E3 ligase (DTX3L). Here, we have characterized the cellular and biochemical requirements for AR ADP-ribosylation by PARP7. We found that the reaction requires nuclear localization of PARP7 and an agonist-induced conformation of AR. PARP7 contains a Cys3His1-type zinc finger (ZF), which we found is critical for AR ADP-ribosylation. The Parp7 ZF is required for efficient nuclear localization by the nuclear localization signal (NLS) encoded in PARP7, but rescue experiments indicate the ZF makes a contribution to AR ADP-ribosylation that is transport-independent. ZF structure appears to be dispensable for PARP7 catalytic activity and for PARP7 binding to AR. Androgen induction of the MYBPC1 gene is regulated by AR and PARP7, and we determined that the ZF is required for the PARP7 transcriptional effect on MYBPC1. Our data indicate the PARP7 ZF plays an important role in modulating the subcellular localization of PARP7 and its capacity to ADP-ribosylate and promote AR-dependent transcription.

sentences: Nonsense Mediated RNA Decay Is a Unique Vulnerability of Cancer Cells with SF3B1 and U2AF1 Mutations Nonsense-mediated RNA decay (NMD) is well recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTCs) for degradation; however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system that can accurately measure NMD activity in individual cells. By carrying out a genome-wide CRISPR/Cas9 knockout screen using this reporter system, we identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, we also found that cells with mutations in the U2 spliceosome genes SF3B1 and U2AF1--which are commonly found in myelodysplastic syndrome (MDS) and cancers--have overall attenuated NMD activity. Furthermore, we found that compared to wild type cells, SF3B1 and U2AF1 mutant cells are more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition could be rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, our findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel strategy for the treatment of MDS and cancers with spliceosome mutations.

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Cognitive dysfunction and anxiety resulted from synaptic downscaling, hippocampal atrophy and ventricular enlargement in i.c.v. streptozotocin injected male Wistar rats Intracerebroventricular streptozotocin (ICV-STZ) injection is among the best animal models to simulate sporadic Alzheimers disease (AD) (Hoyer, 2002, 2000, 1998; Salkovic-Petrisic and Hoyer, 2007). Brain insulin signaling abnormality (Grunblatt et al., 2007), neurodegeneration (Kamat, 2015), neuroinflammation (Liu et al., 2022), cholinergic damage (Blokland and Jolles, 1993; Roy et al., 2021), mitochondrial dysfunction (C. Correia et al., 2013), genetic abnormality (Chen et al., 2012), respiratory problem (Ebel et al., 2017), oxidative stress (Sharma and Gupta, 2001), gliosis (Rostami et al., 2017), sleep disturbances (Song et al., 2018) associated with cognitive abnormalities seen in ICV-STZ injected rats. Available experimental evidences has used varying doses of STZ (<1 to 3mg/kg) and studied its effect for different study durations, ranging from 14-21 (short), 30-42 (mild), 90-105 (moderate) and 250-270 (long) days (Chen et al., 2013, 2012; Grunblatt et al., 2007; Knezovic et al., 2015; Kraska et al., 2012; Mehla et al., 2013; Rostami et al., 2017; Salkovic-Petrisic et al., 2006). These studies indicated that 3mg/kg of body weight is the optimum dose for inducing sporadic AD in the rodents. However studies on the pathological process with relating the morphological and functional abnormalities reported were illusive.

Therefore in the present study, we have investigated the effect of 3mg/kg ICV-STZ on three cognitive domains viz. spatial, episodic and avoidance and even neuropsychiatric abnormality i.e. anxiety with extensive morphological examination in the layers associated with hippocampus proper. Further, we have observed the onset of diffuse amyloid plaques, in association with enlargement of lateral ventricles and down scaling of spines in granular cells of dentate gyrus. Finally, to dissect the functional changes in spatial and anxiety variables we had examined the probability of fraction of time spent in Morris water maze and changes in ethological patterns of STZ injected rats in elevated plus maze.

sentences: Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (pooled CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells and present a framework to power biological discovery with the resulting genotype-phenotype map. We use transcriptional phenotypes to predict the function of poorly-characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena - from RNA processing to differentiation. We leverage this ability to systematically identify the genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene function and cellular behavior.

sentences: Structure of cyanobacterial photosystem I complexed with Cytochrome c6 and Ferredoxin at 1.97 A resolution Photosystem I (PSI) is a light driven electron pump transferring electrons from Cytochrome c6 (Cyt c6) to Ferredoxin (Fd). An understanding of this electron transfer process is hampered by a paucity of structural detail concerning the binding of Fd and the bound Cyt c6. Here we describe the high resolution cryo-EM structure of Thermosynechococcus elongatus BP-1 PSI in complex with Fd and Cyt c6. In our structure Cyt c6 is loosely bound in a non-productive site on the periphery of the complex. Side chain interactions at the PSI:Fd interface are visualized in detail. The structure explains the properties of mutants of PsaE and PsaC and suggests a molecular switch for the dissociation of Fd upon reduction. Calorimetry-based thermodynamic analyses confirms a single binding site for Fd and demonstrates that PSI:Fd complexation is purely driven by entropy. A reaction cycle for the efficient transfer of electrons from Cyt c6 to Fd via PSI is proposed.

sentences: GLH/VASA helicases promote germ granule formation to ensure the fidelity of piRNA-mediated transcriptome surveillance The ability to distinguish non-self from self is the key characteristic for any defense system. piRNAs function as guardians of the genome by silencing non- self nucleic acids and transposable elements in animals. Many piRNA factors are enriched in perinuclear germ granules, but whether their localization is required for piRNA biogenesis or function is not known. Here we show that GLH/VASA helicase mutants exhibit defects in forming perinuclear condensates containing PIWI and other small RNA cofactors. These mutant animals produce largely normal levels of piRNA but are defective in triggering piRNA silencing. Strikingly, while many piRNA targets are activated in GLH mutants, we observed that hundreds of endogenous genes are aberrantly silenced by piRNAs. This defect in self versus non-self recognition was also observed in other mutants where perinuclear P granules are disrupted. Together, our results argue that perinuclear germ granules function critically to promote the fidelity of piRNA- based transcriptome surveillance in C. elegans and preserve self versus non-self distinction.

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Identification of a novel AP2 transcription factor in zygotes with an essential role in Plasmodium ookinete development. The sexual phase of Plasmodium represents a crucial step in malaria transmission, during which these parasites fertilize and form ookinetes to infect mosquitoes. Plasmodium development after fertilization is thought to proceed with female-stored mRNAs until the formation of a retort-form ookinete; thus, transcriptional activity in zygotes has previously been considered quiescent. In this study, we reveal the essential role of transcriptional activity in zygotes by investigating the function of a newly identified AP2 transcription factor, AP2-Z. ap2-z was previously reported as a female transcriptional regulator gene whose disruption resulted in developmental arrest at the retort stage of ookinetes. In this study, although ap2-z was transcribed in females, we show that it was translationally repressed by the DOZI complex and translated after fertilization with peak expression at the zygote stage. ChIP-seq analysis of AP2-Z shows that it binds on specific DNA motifs, targeting the majority of genes known as an essential component of ookinetes, which largely overlap with the AP2-O targets, as well as genes that are unique among the targets of other sexual transcription factors. The results of this study also indicate the existence of a cascade of transcription factors, beginning with AP2-G, that proceeds from gametocytogenesis to ookinete formation.

Author summarySexual development in Plasmodium parasites, a causative agent of malaria, is essential for their transmission from vertebrate hosts to mosquitoes. This important developmental process proceeds as follows: formation of a gametocyte/gamete, fertilization and conversion of the zygote into the mosquito midgut invasive stage, called the ookinete. As a target of transmission blocking strategies, it is important to understand the mechanisms regulating Plasmodium sexual development. In this study, we assessed transcriptional regulation after fertilization by investigating the function of a novel transcription factor, AP2-Z. The results revealed the essential role of de novo transcription activated by AP2-Z in zygotes for promoting ookinete development. As transcriptional activity during the zygote stage has previously been considered silent in Plasmodium, novel genes important for ookinete formation can now be explored in the target genes of AP2-Z. Investigating the functions of these genes can help us understand the mechanisms of Plasmodium zygote/ookinete development and identify new targets for transmission blocking vaccines.

sentences: Antibiotic treatment increases yellowness of carotenoid feather coloration in greenfinches (Chloris chloris) Carotenoid plumage coloration is an important signal of quality, and plays an important role in mate choice in many bird species. However, it remains unclear what mechanism makes carotenoids an honest signal. Here, we test the hypothesis that carotenoid plumage coloration might indicate gut health. Parasitic and microbial infections can affect nutrient absorption due to decreased gut surface or by altered gut microbiome. We took an advantage of a naturally occurring coinfection of parasites inhabiting the upper and lower portions of the digestive track to distinguish between the direct and indirect effects of parasites on carotenoid acquisition. Protozoan coccidian intestinal parasites are widespread in greenfinches (Chloris chloris) and the majority of greenfinches are infected in nature. Trichomonosis is an emerging disease of the upper digestive track that causes high mortality among greenfinches. We captured wild greenfinches (N=71) and administered anticoccidial medication toltrazuril (TOLTRA) to one group, antibiotic metronidazole (METRO) that is also effective for treating Trichomonas gallinea, to the second group, and third group received no medication. In the METRO group, feathers grown during the experiment had significantly higher chroma of yellow parts, but there was no effect of TOLTRA on feather chroma. These results suggest that METRO increased the efficiency of carotenoid modification or deposition to the feathers rather than nutrient acquisition, and/or freed energy resources that could be invested in coloration. Alternatively, in accordance with shared pathway hypothesis, increase in efficiency of vital cellular processes might have occurred, as many microbial metabolites can modulate mitochondrial and immune function.

sentences: Mass spectrometry-based sequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme Antibody sequence information is crucial to understanding the structural basis for antigen binding and enables the use of antibodies as therapeutics and research tools. Here we demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by LC-MS/MS in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron transfer high-energy collision dissociation (EThcD) on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody Herceptin, with an accuracy of 99% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.

sentences: Activation Mechanism of Strigolactone Receptors And Its Impact On Ligand Selectivity Between Host And Parasitic Plants Parastic weeds such as Striga have led to significant losses in agricultural productivity worldwide. These weeds use the plant hormone strigolactone as a germination stimulant. Strigolactone signaling involves substrate binding and hydrolysis followed by a large conformational change of the receptor to a "closed" or "active" state that is able to associate with a downstream signaling partner MAX2/D3. The crystal structure of the active and inactive AtD14 receptor have helped in elucidating the structural changes involved in activation. However, the mechanism by which the receptor activates remains unknown. The ligand dependence of AtD14 activation has been disputed by mutagenesis studies showing that enzymatically inactive receptors are able to form a complex with MAX2 proteins. Furthermore, activation differences between strigolactone receptor in Striga, ShHTL7 and textitAtD14 could contribute to the high sensitivity to strigolactones exhibited by parasitic plants. Using molecular dynamics simulations, we demonstrate that both AtD14 and ShHTL7 could adopt an active conformation in absence of ligand. However, the ShHTL7 receptor exhibits higher population in the inactive apo state as compared to the AtD14 receptor. We demonstrate that this difference in inactive state population is caused by sequence differences between their D-loops and its interactions with the catalytic histidine that prevents full binding pocket closure in ShHTL7. These results indicate that hydrolysis of a strigolactone ligand would enhance the active state population by destabilizing the inactive state in ShHTL7 as compared to AtD14. We also show that the mechanism of activation is more concerted in AtD14 than in ShHTL7 and that the main barrier to activation in ShHTL7 is closing of the binding pocket.

sentences: Predicting biomass of resident kokopu (Galaxias) populations using local habitat composition With the global decline of freshwater fishes, quantifying the body size-specific habitat use of vulnerable species is crucial for accurately evaluating population health, identifying the effects of anthropogenic stressors, and directing effective habitat restoration. Populations of New Zealands endemic k[o]kopu species (Galaxias fasciatus, G. argenteus, and G. postvectis) have declined substantially over the last century in response to anthropogenic stressors, including habitat loss and fragmentation, invasive species, and over-exploitation. Despite well-understood habitat associations, key within-habitat features driving the reach-scale biomass of small and large k[o]kopu remain unclear. Here, we investigated whether the total biomass of small ([≤] 90 mm) and large (> 90 mm) k[o]kopu was associated with total pool area, average pool depth, total bank cover, average substrate size, and average forest canopy cover across fifty-seven 50 m reaches. These features were selected because generally pool habitats are productive feeding areas, bank cover and substrate interstices are important refuges, and forest cover provides greater food availability. Because k[o]kopu are nocturnal, populations were sampled with removal at night using headlamps and hand-nets until reaches were visually depleted. Using Akaikes information criterion, it was found that increases in large k[o]kopu biomass were most parsimoniously explained by greater pool area and bank cover, whereas increases in small k[o]kopu biomass were best explained by low bank cover and greater average forest cover. This study demonstrated the importance of considering the ontogenetic shift in species habitat use and provided an effective modelling approach for quantifying the size-specific habitat use of these stream-dwelling fish.

sentences: Single-Cell Analysis Identifies Distinct Immune and Smooth Muscle Cell Populations that Contribute to Chronic Thromboembolic Pulmonary Hypertension Chronic thromboembolic pulmonary hypertension (CTEPH) is a sequelae of acute pulmonary embolism (PE) in which the PE remodels into a chronic scar in the pulmonary arteries. This results in vascular obstruction, small vessel arteriopathy and pulmonary hypertension. Our current understanding of CTEPH pathobiology is primarily derived from cell-based studies limited by the use of specific cell markers or phenotypic modulation in cell culture. Here we used single cell RNA sequencing (scRNAseq) of tissue removed at the time of pulmonary thromboendarterectomy (PTE) surgery to identify the multiple cell types, including macrophages, T cells, and smooth muscle cells, that comprise CTEPH thrombus. Notably, multiple macrophage subclusters were identified but broadly split into two categories, with the larger group characterized by an upregulation of inflammatory signaling predicted to promote pulmonary vascular remodeling. Both CD4+ and CD8+ T cells were identified and likely contribute to chronic inflammation in CTEPH. Smooth muscle cells were a heterogeneous population, with a cluster of myofibroblasts that express markers of fibrosis and are predicted to arise from other smooth muscle cell clusters based on pseudotime analysis. Additionally, cultured endothelial, smooth muscle and myofibroblast cells isolated from CTEPH thrombus have distinct phenotypes from control cells with regards to angiogenic potential and rates of proliferation and apoptosis. Lastly, our analysis identified protease-activated receptor 1 (PAR1) as a potential therapeutic target that links thrombosis to chronic PE in CTEPH, with PAR1 inhibition decreasing smooth muscle cell and myofibroblast proliferation and migration. These findings suggest a model for CTEPH similar to atherosclerosis, with chronic inflammation promoted by macrophages and T cells driving vascular remodeling through smooth muscle cell modulation, and suggest new approaches for pharmacologically targeting this disease.

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Automatic decomposition of electrophysiological data into distinct non-sinusoidal oscillatory modes Neurophysiological signals are often noisy, non-sinusoidal, and consist of transient bursts. Extraction and analysis of oscillatory features (such as waveform shape and cross-frequency coupling) in such datasets remains difficult. This limits our understanding of brain dynamics and its functional importance. Here, we develop Iterated Masking Empirical Mode Decomposition (itEMD), a method designed to decompose noisy and transient single channel data into relevant oscillatory modes in a flexible, fully data-driven way without the need for manual tuning. Based on Empirical Mode Decomposition (EMD), this technique can extract single-cycle waveform dynamics through phase-aligned instantaneous frequency. We test our method by extensive simulations across different noise, sparsity, and non-sinusoidality conditions. We find itEMD significantly improves the separation of data into distinct non-sinusoidal oscillatory components and robustly reproduces waveform shape across a wide range of relevant parameters. We further validate the technique on multi-modal, multi-species electrophysiological data. Our itEMD extracts known rat hippocampal theta waveform asymmetry and identifies subject-specific human occipital alpha without any prior assumptions about the frequencies contained in the signal. Notably, it does so with significantly less mode mixing compared to existing EMD-based methods. By reducing mode mixing and simplifying interpretation of EMD results, itEMD will enable new analyses into functional roles of neural signals in behaviour and disease.

New & NoteworthyWe introduce a novel, data-driven method to identify oscillations in neural recordings. This approach is based on Empirical Mode Decomposition and reduces mixing of components, one of its main problems. The technique is validated and compared with existing methods using simulations and real data. We show our method better extracts oscillations and their properties in highly noisy and non-sinusoidal datasets.

Running HeadDecomposition of data into non-sinusoidal oscillatory modes

sentences: Broad and Long-lasting Immune Response against SARS-CoV-2 Omicron and Other Variants by PIKA-Adjuvanted Recombinant SARS-CoV-2 Spike (S) Protein Subunit Vaccine (YS-SC2-010) Recently SARS-CoV-2 Omicron (B.1.1.529) variant was identified in South Africa with numerous mutations in spike protein, and numerous community infections have been reported and raised grave concern around the world. Some studies found that the neutralization effects of several licensed vaccines against Omicron were dramatically reduced, which significantly affected antibody mediated protection, especially for individuals whose immunization were completed after extended period. In this regard, we studied the persistence and neutralization activity toward mutant strains in animal serum immunized with PIKA-adjuvanted recombinant SARS-CoV-2 spike protein subunit vaccine (YS-SC2-010). Here we are reporting that animal serum collected at 596 days after immunization with YS-SC2-010 still retains high and persistent neutralizing activity against all the Variant of Concern (VOC) variants, including Omicron variant. Although it is a blessed event to achieve 20 months long neutralization against Omicron variant after immunization with YS-SC2-010, it was also founded that the neutralization effect of immune serum on Omicron decreased by 6.29 folds as compared to D614G, more significantly when compared with other mutant strains.

sentences: CNTF enhances aerobic glycolysis and anabolic activities in degenerating retinas Ciliary neurotrophic factor (CNTF) has potent neuroprotective activity in retinal degeneration animal models, yet the cellular mechanisms underlying its broad neuronal survival effects remain unclear. Here, we investigated the impact of CNTF on retinal metabolism in a mouse model of human retinitis pigmentosa. CNTF treatment resulted in improved mitochondrial morphology in mutant rod photoreceptors, but also led to reduced oxygen consumption and suppression of respiratory chain complex activities. Metabolomics analyses detected significantly higher levels of ATP and the energy currency phospho-creatine post CNTF exposure. In addition, CNTF-treated retinas contained elevated glycolytic metabolites and showed increased expression of genes and active enzymes of the glycolytic pathway. Metabolomics analyses also revealed increased TCA cycle products, lipid biosynthetic pathway intermediates, nucleotides, and amino acids, indicating an overall CNTF-dependent augmentation of anabolic activities. Moreover, CNTF treatment restored the key antioxidant glutathione to the wild type level in the degenerating retina. Taken together, these results demonstrate that CNTF profoundly impacts the metabolic status of degenerating retinas by promoting aerobics glycolysis and anabolism, enhancing energy supply, and restoring redox homeostasis. Our study thus reveals important cellular mechanisms underlying CNTF-mediated neuroprotection and provides novel insight for the on-going CNTF clinical trials treating blinding diseases.

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Prediction of cancer mutation states using multiple data modalities reveals the utility and consistency of gene expression and DNA methylation In studies of cellular function in cancer, researchers are increasingly able to choose from many -omics assays as functional readouts. Choosing the correct readout for a given study can be difficult, and which layer of cellular function is most suitable to capture the relevant signal may be unclear. In this study, we consider prediction of cancer mutation status (presence or absence) from functional -omics data as a representative problem. Since functional signatures of cancer mutation have been identified across many data types, this problem presents an opportunity to quantify and compare the ability of different -omics readouts to capture signals of dysregulation in cancer. The TCGA Pan-Cancer Atlas contains genetic alteration data including somatic mutations and copy number variants (CNVs), as well as several -omics data types. From TCGA, we focus on RNA sequencing, DNA methylation arrays, reverse phase protein arrays (RPPA), microRNA, and somatic mutational signatures as -omics readouts.

Across a collection of genes recurrently mutated in cancer, RNA sequencing tends to be the most effective predictor of mutation state. Surprisingly, we found that for many of the genes we considered, one or more other data types are approximately equally effective predictors. Performance was more variable between mutations than it was between data types for the same mutation, and there was often little difference between the top data types. We also found that combining data types into a single multi-omics model provided little or no improvement in predictive ability over the best individual data type. Based on our results, for the design of studies focused on the functional outcomes of cancer mutations, there are often multiple -omics types that can serve as effective readouts, although gene expression seems to be a reasonable default option.

sentences: Investigating the interaction between inter-locus and intra-locus sexual conflict using hemiclonal analysis in Drosophila melanogaster Divergence in the evolutionary interests of males and females leads to sexual conflict. Traditionally, sexual conflict has been classified into two types: inter-locus sexual conflict (IeSC) and intra-locus sexual conflict (IaSC). IeSC is modeled as a conflict over outcomes of intersexual reproductive interactions mediated by loci that are sex-limited in their effects. IaSC is thought to be a product of selection acting in opposite directions in males and females on traits with a common underlying genetic basis. While in their canonical formalisms IaSC and IeSC are mutually exclusive, there is growing support for the idea that the two may interact. Empirical evidence for such interactions, however, is limited. Here, we investigated the interaction between IeSC and IaSC in Drosophila melanogaster. Using hemiclonal analysis, we sampled 39 hemigenomes from a laboratory-adapted population of D. melanogaster. We measured the contribution of each hemigenome to adult male and female fitness at three different intensities of IeSC, obtained by varying the operational sex-ratio. Subsequently, we estimated the intensity of IaSC at each sex-ratio by calculating the intersexual genetic correlation for fitness and the proportion of sexually antagonistic fitness-variation. Our results indicate a statistically non-significant trend suggesting that increasing the strength of IeSC ameliorates IaSC in the population.

sentences: Comparison of endogenously expressed fluorescent protein fusions behaviour for protein quality control and cellular aging research The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing endogenously expressed Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. Overall, such protein fusions used in molecular and microbiology research are often sparsely characterised. To address this issue, we performed a comparative assessment of Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on the fluorophore within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects and points may play a role in other eukaryotic systems.

sentences: Paradigm shift in eukaryotic biocrystallization Despite the widespread occurrence of crystalline inclusions in unicellular eukaryotes, scant attention has been paid to their composition, functions, and evolutionary origins, assuming just their inorganic contents. The advent of Raman microscopy, still scarcely used for biological samples, allowed chemical characterization of cellular inclusions in vivo. Using this method, herein we provide a substantial revision of the cellular crystalline inclusions across the broad diversity of eukaryotes examining all major supergroups. Surprisingly, here we show that 80 % of these crystalline inclusions contain purines, mostly anhydrous guanine (62 %), guanine monohydrate (2 %), uric acid (12 %) and xanthine (4 %). Hence, our findings indicate that purine biocrystallization is a very general and an ancestral eukaryotic process operating by an as-yet-unknown mechanism. Purine crystalline inclusions are high-capacity and rapid-turnover reserves of nitrogen of a great metabolic importance, as well as optically active elements, e.g., present in the light sensing eyespots of flagellates, possessing even more hypothetical functions. Thus, we anticipate our work to be a starting point for more in-depth studies of this phenomenon on the detailed level spanning from cell biology to global ecology, with further potential applications in biotechnologies, bio-optics or in human medicine.

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Common neural bases for processing speech prosody and music: An integrated model It is shared notion that speech and music processing share some commonalities. Brain bioelectrical activity was recorded in healthy participants listening to music obtained by digitally transforming real speech into melodies played by viola. Sentences were originally pronounced with a positive or negative affective prosody. The researchs aim was to investigate if the emotional content of music was extracted similarly to how the affective prosody of speech is processed.

EEG was recorded from 128 electrodes in 20 healthy students. Participants had to detect rare neutral piano sounds while ignoring viola melodies. Stimulus negative valence increased the amplitude of frontal P300 and N400 ERP components while a late inferior frontal positivity was enhanced in response to positive melodies. Similar ERP markers were previously found for processing positive and negative music, vocalizations and speech. Source reconstruction applied to N400 showed that negative melodies engaged the right superior temporal gyrus and right anterior cingulate cortex, while positive melodies engaged the left middle and inferior temporal gyrus and the inferior frontal cortex. An integrated model is proposed depicting a possible common circuit for processing the emotional content of music, vocalizations and speech, which might explain some universal and relatively innate brain reaction to music.

sentences: Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos Mosquito-borne diseases present a worldwide public health burden. Genome-scale screening tools that could inform our understanding of mosquitos and their control are lacking. Here, we adapt a recombination-mediated cassette exchange system for delivery of CRISPR sgRNA libraries into cell lines from several mosquito species and perform pooled CRISPR screens in an Anopheles cell line. To implement this method, we engineered modified mosquito cell lines, validated promoters and developed bioinformatics tools for multiple mosquito species.

sentences: The geometry of domain-general performance monitoring in the human medial frontal cortex Controlling behavior to flexibly achieve desired goals depends on the ability to monitor ones own performance. It is unknown how performance monitoring can be both flexible to support different tasks and specialized to perform well on each. We recorded single neurons in the human medial frontal cortex while subjects performed two tasks that involve three types of cognitive conflict. Neurons encoding predicted conflict, conflict, and error in one or both tasks were intermixed, forming a representational geometry that simultaneously allowed task specialization and generalization. Neurons encoding conflict retrospectively served to update internal estimates of control demand. Population representations of conflict were compositional. These findings reveal how representations of evaluative signals can be both abstract and task-specific and suggest a neuronal mechanism for estimating control demand.

sentences: Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yields viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

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Durability of SARS-CoV-2-specific T cell responses at 12-months post-infection BackgroundCharacterizing the longevity and quality of cellular immune responses to SARS-CoV-2 is critical to understanding immunologic approaches to protection against COVID-19. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Further analysis of the function, durability, and diversity of the cellular response long after natural infection, over a wider range of ages and disease phenotypes, is needed to further identify preventative and therapeutic interventions.

MethodsWe identified participants in our multi-site longitudinal, prospective cohort study 12-months post SARS-CoV-2 infection representing a range of disease severity. We investigated the function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry. In parallel, the magnitude of SARS-CoV-2-specific antibodies was compared.

ResultsSARS-CoV-2-specific antibodies and T cells were detected at 12-months post-infection. Severity of acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12-months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity.

ConclusionsOur data show that SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12-months post-infection, with higher frequency noted in those who originally experienced severe disease.

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Alcohol-sourced acetate reduces T cell filamentous actin branching and migration Alcohol is among the most widely consumed dietary substances. While excessive alcohol consumption damages the liver, heart and brain, clinical observations suggest that alcohol has strong immunoregulatory properties. Mechanisms by which alcohol undermines immune cell functions during autoimmunity and vaccination are currently elusive. Herein, we show that acetate, the main metabolite of alcohol, effectively inhibits the migratory capacity of T cells through acetylation of cortactin, a protein which stabilizes and promotes branching of filamentous actin. Acetate-induced cytoskeletal changes effectively inhibited activation, proliferation, and immune synapse formation in T cells at tissue concentrations reached by alcohol consumption. In summary, these data show that acetate inhibits T cell-mediated immune responses by modulating the T cell cytoskeletal biomechanics.

One-Sentence SummaryExposure to elevated acetate concentrations change T cell biomechanics, reducing cell migration and immune synapse formation.

sentences: Genetic variation in ALDH4A1 predicts muscle health over the lifespan and across species Environmental stress can negatively impact organismal aging, however, the long-term impact of endogenously derived reactive oxygen species from normal cellular metabolism remains less clear. Here we define the evolutionarily conserved mitochondrial enzyme ALH-6/ALDH4A1 as a biomarker for age-related changes in muscle health by combining C. elegans genetics and a gene-wide association study (GeneWAS) from aged human participants of the US Health and Retirement Study (HRS)1-4. In a screen for mutations that activate SKN-1-dependent oxidative stress responses in the muscle of C. elegans5-7, we identified 96 independent genetic mutants harboring loss-of-function alleles of alh-6, exclusively. These genetic mutations map across the ALH-6 polypeptide, which lead to age-dependent loss of muscle health. Intriguingly, genetic variants in ALDH4A1 differentially impact age-related muscle function in humans. Taken together, our work uncovers mitochondrial alh-6/ALDH4A1 as a critical component of normal muscle aging across species and a predictive biomarker for muscle health over the lifespan.

sentences: Semantic scene-object consistency modulates N300/400 EEG components, but does not automatically facilitate object representations During natural vision, objects rarely appear in isolation, but often within a semantically related scene context. Previous studies reported that semantic consistency between objects and scenes facilitates object perception, and that scene-object consistency is reflected in changes in the N300 and N400 components in EEG recordings. Here, we investigate whether these N300/400 differences are indicative of changes in the cortical representation of objects. In two experiments, we recorded EEG signals while participants viewed semantically consistent or inconsistent objects within a scene; in Experiment 1, these objects were task-irrelevant, while in Experiment 2, they were directly relevant for behavior. In both experiments, we found reliable and comparable N300/400 differences between consistent and inconsistent scene-object combinations. To probe the quality of object representations, we performed multivariate classification analyses, in which we decoded the category of the objects contained in the scene. In Experiment 1, in which the objects were not task-relevant, object category could be decoded from around 100 ms after the object presentation, but no difference in decoding performance was found between consistent and inconsistent objects. By contrast, when the objects were task-relevant in Experiment 2, we found enhanced decoding of semantically consistent, compared to semantically inconsistent, objects. These results show that differences in N300/400 components related to scene-object consistency do not index changes in cortical object representations, but rather reflect a generic marker of semantic violations. Further, our findings suggest that facilitatory effects between objects and scenes are task-dependent rather than automatic.

sentences: Drought stress increases the expression of barley defence genes with negative consequences for infesting cereal aphids Crops are exposed to myriad abiotic and biotic stressors with negative consequences. Two stressors that are expected to increase under climate change are drought and infestation with herbivorous insects, including important aphid species. Expanding our understanding of the impact drought has on the plant-aphid relationship will become increasingly important under future climate scenarios. Here we use a previously characterised plant-aphid system comprising a susceptible variety of barley, a wild relative of barley with partial-aphid resistance, and the bird cherry-oat aphid to examine the drought-plant-aphid relationship. We show that drought has a negative effect on plant physiology and aphid fitness and provide evidence to suggest that plant resistance influences aphid responses to drought stress, with the expression of aphid detoxification genes increasing under drought when feeding on the susceptible plant but decreasing on the partially-resistant plant. Furthermore, we show that the expression of thionin genes, plant defensive compounds that contribute aphid resistance, increase ten-fold in susceptible plants exposed to drought stress but remain at constant levels in the partially-resistant plant, suggesting they play an important role in modulating aphid populations. This study highlights the role of plant defensive processes in mediating the interactions between the environment, plants, and herbivorous insects.

sentences: Domain adaptive neural networks improvecross-species prediction of transcription factor binding The intrinsic DNA sequence preferences and cell-type specific cooperative partners of transcription factors (TFs) are typically highly conserved. Hence, despite the rapid evolutionary turnover of individual TF binding sites, predictive sequence models of cell-type specific genomic occupancy of a TF in one species should generalize to closely matched cell types in a related species. To assess the viability of cross-species TF binding prediction, we train neural networks to discriminate ChIP-seq peak locations from genomic background and evaluate their performance within and across species. Cross-species predictive performance is consistently worse than within-species performance, which we show is caused in part by species-specific repeats. To account for this domain shift, we use an augmented network architecture to automatically discourage learning of training species-specific sequence features. This domain adaptation approach corrects for prediction errors on species-specific repeats and improves overall cross-species model performance. Our results demonstrate that cross-species TF binding prediction is feasible when models account for domain shifts driven by species-specific repeats.

sentences: Profiling of open chromatin in developing pig (Sus scrofa) muscle to identify regulatory regions There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programmes. In order to address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We have optimised the  Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq) to profile regions of open chromatin in flash frozen pig muscle tissue samples. This protocol has allowed us to identify putative regulatory regions in semitendinosus muscle from 24 male piglets. We collected samples from the smallest, average, and largest sized male piglets from each litter through five developmental time points. The ATAC-Seq data were mapped to Sscrofa11.1 using Bowtie2 and Genrich was used for post-alignment peak-calling. Of the 4,661 ATAC-Seq peaks identified that represent regions of open chromatin, >50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with down regulation of genes involved in muscle development that were present in small sized foetal piglets but absent in large foetal piglets at day 90 of gestation. The dataset that we have generated provides: i) a resource for studies of genome regulation in pigs, and ii) contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programmes.

sentences: Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells Variation in lung alveolar development is strongly linked to disease susceptibility. However, the cellular and molecular mechanisms underlying alveolar development are difficult to study in humans. Using primary human fetal lungs we have characterized a tip progenitor cell population with alveolar fate potential. These data allowed us to benchmark a self-organising organoid system which captures key aspects of lung lineage commitment and can be efficiently differentiated to alveolar type 2 cell fate. Our data show that Wnt and FGF signalling, and the downstream transcription factors NKX2.1 and TFAP2C, promote human alveolar or airway fate respectively. Moreover, we have functionally validated cell-cell interactions in human lung alveolar patterning. We show that Wnt signalling from differentiating fibroblasts promotes alveolar type 2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. Our organoid system recapitulates key aspects of human lung development allowing mechanistic experiments to determine the underpinning molecular regulation.

sentences: Deep learning methods for designing proteins scaffolding functional sites Current approaches to de novo design of proteins harboring a desired binding or catalytic motif require pre-specification of an overall fold or secondary structure composition, and hence considerable trial and error can be required to identify protein structures capable of scaffolding an arbitrary functional site. Here we describe two complementary approaches to the general functional site design problem that employ the RosettaFold and AlphaFold neural networks which map input sequences to predicted structures. In the first "constrained hallucination" approach, we carry out gradient descent in sequence space to optimize a loss function which simultaneously rewards recapitulation of the desired functional site and the ideality of the surrounding scaffold, supplemented with problem-specific interaction terms, to design candidate immunogens presenting epitopes recognized by neutralizing antibodies, receptor traps for escape-resistant viral inhibition, metalloproteins and enzymes, and target binding proteins with designed interfaces expanding around known binding motifs. In the second "missing information recovery" approach, we start from the desired functional site and jointly fill in the missing sequence and structure information needed to complete the protein in a single forward pass through an updated RoseTTAFold trained to recover sequence from structure in addition to structure from sequence. We show that the two approaches have considerable synergy, and AlphaFold2 structure prediction calculations suggest that the approaches can accurately generate proteins containing a very wide array of functional sites.

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Closed-loop optimization of transcranial magnetic stimulation with electroencephalography feedback BackgroundTranscranial magnetic stimulation (TMS) is widely used in brain research and treatment of various brain dysfunctions. However, the optimal way to target stimulation and administer TMS therapies, for example, where and in which electric field direction the stimuli should be given, is yet to be determined.

ObjectiveTo develop an automated closed-loop system for adjusting TMS parameters (in this work, the stimulus orientation) online based on TMS-evoked brain activity measured with electroencephalography (EEG).

MethodsWe developed an automated closed-loop TMS-EEG set-up. In this set-up, the stimulus parameters are electronically adjusted with multi-locus TMS. As a proof of concept, we developed an algorithm that automatically optimizes the stimulation orientation based on single-trial EEG responses. We applied the algorithm to determine the electric field orientation that maximizes the amplitude of the TMS- EEG responses. The validation of the algorithm was performed with six healthy volunteers, repeating the search twenty times for each subject.

ResultsThe validation demonstrated that the closed-loop control worked as desired despite the large variation in the single-trial EEG responses. We were often able to get close to the orientation that maximizes the EEG amplitude with only a few tens of pulses.

ConclusionOptimizing stimulation with EEG feedback in a closed-loop manner is feasible and enables effective coupling to brain activity.

sentences: Beat Perception in Polyrhythms: Time is Structured in Binary Units In everyday life, we group and subdivide time to understand the sensory environment surrounding us. Our perceptual system establishes hierarchical structures by nesting different groups of time intervals. Organizing time in units such as diurnal rhythms, phrases, and beat patterns, is fundamental to everyday behavior, speech, and music. When listening to music, we extract rhythmic regularities to create a hierarchical metrical structure that enables us to predict the timing of the next events. Foot tapping and head bobbing to musical rhythms are observable evidence of this process. In the special case of polyrhythms, at least two metrical structures compete to become the reference for these temporal regularities, rendering several possible beats with which we can synchronize our movements. While there is general agreement that tempo, pitch, and loudness influence beat perception in polyrhythms, we focused on the yet neglected influence of beat subdivisions. In three online experiments, 300 participants listened to a range of polyrhythms and tapped their index fingers in time with the perceived beat. The polyrhythms consisted of two simultaneously presented isochronous pulse trains with different ratios (2:3, 2:5, 3:4, 3:5, 4:5, 5:6) and different tempi. For ratios 2:3 and 3:4, we additionally manipulated the pitch of the pulse trains. Results showed a highly robust influence of subdivision grouping on beat perception manifested as a propensity towards beats that are subdivided into two or four equally spaced units, as opposed to beats with three or more complex groupings of subdivisions. Additionally, lower pitched pulse trains were more likely to be perceived as the beat. Our findings suggest that subdivisions, not beats, are the basic unit of beat perception, and that the principle underlying the binary grouping of subdivisions reflects a propensity towards simplicity. This preference for simple grouping is widely applicable to human perception and cognition of time.

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POTATO: An automated pipeline for batch analysis of optical tweezers data Optical tweezers is a single-molecule technique that allows probing of intra- and intermolecular interactions that govern complex biological processes involving molecular motors, protein-nucleic acid interactions and protein/RNA folding. Recent developments in instrumentation eased and accelerated optical tweezers data acquisition, but analysis of the data remains challenging. Here, to enable high-throughput data analysis, we developed an automated python-based analysis pipeline called POTATO (Practical Optical Tweezers Analysis TOol). POTATO automatically processes the high-frequency raw data generated by force-ramp experiments and identifies (un)folding events using predefined parameters. After segmentation of the force-distance trajectories at the identified (un)folding events, sections of the curve can be fitted independently to worm-like chain and freely-jointed chain models, and the work applied on the molecule can be calculated by numerical integration. Furthermore, the tool allows plotting of constant force data and fitting of the Gaussian distance distribution over time. All these features are wrapped in a user-friendly graphical interface (https://github.com/REMI-HIRI/POTATO), which allows researchers without programming knowledge to perform sophisticated data analysis.

SIGNIFICANCEStudying (un)folding of biopolymer structures with optical tweezers under different conditions generates very large datasets for statistical data analysis. Recent technical improvements accelerated data acquisition by coupling modern instruments with microfluidic systems, at the same time creating the need for a high-throughput, and unbiased data analysis. We developed Practical Optical Tweezers Analysis TOol (POTATO); an open-source python-based tool that can process data gathered by any OT force-ramp experiment in an automated fashion. POTATO is principally designed for data preprocessing, identification of (un)folding events and the fitting of the force-distance curves. In addition, all parameters for preprocessing, statistical analysis and fitting of the curves can be adapted to suit the dataset under analysis in an easy-to-use graphical user interface.

sentences: Thiophenesulfonamides are specific inhibitors of quorum sensing in pathogenic Vibrios Vibrio bacteria are pathogens of fish, shellfish, coral, and humans due to contaminated seafood consumption. Vibrio virulence factors are controlled by the cell-to-cell communication called quorum sensing, thus this signaling system is a promising target for therapeutic design. We screened a compound library and identified nine compounds, including several 2-thiophenesulfonamides, that inhibit the master quorum sensing transcription factor LuxR in Vibrio campbellii but do not affect cell growth. We synthesized a panel of 50 thiophenesulfonamide compounds to examine the structure-activity relationship effects on quorum sensing in vivo. The most potent molecule identified, PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole), specifically inhibits LuxR homologs in multiple strains of Vibrio vulnificus, Vibrio parahaemolyticus, and V. campbellii with sub-micromolar concentrations. PTSP efficacy is driven by amino acid conservation in the binding pocket, which is accurately predicted using in silico modeling of inhibitors. Our results underscore the potential for developing thiophenesulfonamides as specific quorum sensing-directed treatments for Vibrio infections.

sentences: Ubiquity of the Symbiont Serratia symbiotica in the Aphid Natural Environment: Distribution, Diversity and Evolution at a Multitrophic Level Bacterial symbioses are significant drivers of insect evolutionary ecology. However, despite recent findings that these associations can emerge from environmentally derived bacterial precursors, there is still little information on how these potential progenitors of insect symbionts circulates in the trophic systems. The aphid symbiont Serratia symbiotica represents a valuable model for deciphering evolutionary scenarios of bacterial acquisition by insects, as its diversity includes intracellular host-dependent strains as well as gut-associated strains that have retained some ability to live independently of their hosts and circulate in plant phloem sap. These strains represent a potential reservoir for the emergence of new and more intimate symbioses. Here, we conducted a field study to examine the distribution and diversity of S. symbiotica found in aphid populations, as well as in different compartments of their surrounding environment. A total of 250 aphid colonies, 203 associated insects, and 161 plant samples associated with aphid colonies were screened for S. symbiotica. Twenty percent of aphids were infected with S. symbiotica, and the symbiont includes a wide diversity of strains with varied tissue tropism corresponding to different lifestyle. We also showed that the prevalence of S. symbiotica is influenced by seasonal temperatures. For the first time, we found that S. symbiotica was present in non aphid species and in host plants, and that the prevalence of the bacterium in these samples was higher when associated aphid colonies were infected. Furthermore, phylogenetic analyses suggest the existence of horizontal transfers between the different trophic levels examined. These results provide a completely new picture of the ubiquity of an insect symbiont in nature. They suggest that ecological interactions promote the dissemination of strains that are still free-living and poorly specialized, and for which plants are a proabable reservoir for the acquisition of new bacterial partners in insects.

sentences: Background selection under evolving recombination rates Background selection (BGS), the effect that purifying selection exerts on sites linked to deleterious alleles, is expected to be ubiquitous across eukaryotic genomes. The effects of BGS reflect the interplay of the rates and fitness effects of deleterious mutations with recombination. A fundamental assumption of BGS models is that recombination rates are invariant over time. However, in some lineages recombination rates evolve rapidly, violating this central assumption. Here, we investigate how recombination rate evolution affects genetic variation under BGS. We show that recombination rate evolution modifies the effects of BGS in a manner similar to a localised change in the effective population size, potentially leading to an underestimation of the genome-wide effects of selection. Furthermore, we find evidence that recombination rate evolution in the ancestors of modern house mice may have impacted inferences of the genome-wide effects of selection in that species.

sentences: Estimating the Inter- and Intra-Rater Reliability for NASH Fibrosis Staging in the Presence of Bridge Ordinal Ratings with Hierarchical Bridge Category Models The public health burden of non-alcoholic steatohepatitis (NASH), a liver condition characterized by excessive lipid accumulation and subsequent tissue inflammation and fibrosis, has burgeoned with the spread of western lifestyle habits. Progression of fibrosis into cirrhosis is assessed using histological staging scales (e.g., NASH Clinical Research Network (NASH CRN)). These scales are used to monitor disease progression as well as to evaluate the effectiveness of therapies. However, clinical drug trials for NASH are typically underpowered due to lower than expected inter-/intra-rater reliability, which impacts measurements at screening, baseline, and endpoint. Bridge ratings represent a phenomenon where pathologists assign two adjacent stages simultaneously during assessment and may further complicate these analyses when ad hoc procedures are applied. Statistical techniques, dubbed Bridge Category Models, have been developed to account for bridge ratings, but not for the scenario where multiple pathologists assess biopsies across time points. Here, we develop hierarchical Bayesian extensions for these statistical methods to account for repeat observations and use these methods to assess the impact of bridge ratings on the inter-/intra-rater reliability of the NASH CRN staging scale. We also report on how pathologists may differ in their assignment of bridge ratings to highlight different staging practices. Our findings suggest that Bridge Category Models can capture additional fibrosis staging heterogeneity with greater precision, which translates to potentially higher reliability estimates in contrast to the information lost through ad hoc approaches.

sentences: Identification of BAHD acyltransferases associated with acylinositol biosynthesis in Solanum quitoense (naranjilla) Plants make a variety of specialized metabolites that can mediate interactions with animals, microbes and competitor plants. Understanding how plants synthesize these compounds enables studies of their biological roles by manipulating their synthesis in vivo as well as producing them in vitro. Acylsugars are a group of protective metabolites that accumulate in the trichomes of many Solanaceae family plants. Acylinositol biosynthesis is of interest because it appears to be restricted to a subgroup of species within the Solanum genus. Previous work characterized a triacylinositol acetyltransferase involved in acylinositol biosynthesis in the Andean fruit plant Solanum quitoense (lulo or naranjillo). We characterized three additional S. quitoense trichome expressed enzymes, and found that virus induced gene silencing of each caused changes in acylinositol accumulation. Surprisingly, the in vitro triacylinositol products of these enzymes are distinct from those that accumulate in planta. These enzymes, nonetheless, provide an opportunities to test the biological impact and properties of these triacylinositols in vitro.

sentences: Analysis of the Human Connectome Data Supports the Notion of a "Common Model of Cognition" for Human and Human-Like Intelligence The Common Model of Cognition (CMC) is a recently proposed, consensus architecture intended to capture decades of progress in cognitive science on modeling human and human-like intelligence. Because of the broad agreement around it and preliminary mappings of its components to specific brain areas, we hypothesized that the CMC could be a candidate model of the large-scale functional architecture of the human brain. To test this hypothesis, we analyzed functional MRI data from 200 participants and seven different tasks that cover a broad range of cognitive domains. The CMC components were identified with functionally homologous brain regions through canonical fMRI analysis, and their communication pathways were translated into predicted patterns of effective connectivity between regions. The resulting dynamic linear model was implemented and fitted using Dynamic Causal Modeling, and compared against six alternative brain architectures that had been previously proposed in the field of neuroscience (three hierarchical architectures and three hub-and-spoke architectures) using a Bayesian approach. The results show that, in all cases, the CMC vastly outperforms all other architectures, both within each domain and across all tasks. These findings suggest that a common set of architectural principles that could be used for artificial intelligence also underpins human brain function across multiple cognitive domains.

sentences: Plasmid stability in fluctuating environments:population genetics of multi-copy plasmids Plasmids are extra-chromosomal genetic elements that encode a wide variety of phenotypes and can be maintained in bacterial populations through vertical and horizontal transmission, thus increasing bacterial adaptation to hostile environmental conditions like those imposed by antimicrobial substances. To circumvent the segregational instability resulting from randomly distributing plasmids between daughter cells upon division, non-transmissible plasmids tend to be carried in multiple copies per cell, with the added benefit of exhibiting increased gene dosage. But carrying multiple copies also results in a high metabolic burden to the bacterial host, therefore reducing the overall fitness of the population. This trade-off poses an existential question for plasmids: What is the optimal plasmid copy number? In this manuscript, we address this question using a combination of population genetics modeling with microbiology experiments consisting of Escherichia coli K12 bearing a multi-copy plasmid encoding for blaTEM-1, a gene conferring resistance to {beta}-lactam antibiotics. We postulate and analyze a Wright-Fisher model to evaluate the interaction between selective pressure, the number of plasmid copies carried by each cell, and the energetic cost associated with plasmid bearing. By numerically determining the optimal plasmid copy number for constant and fluctuating selection regimes, we show that the stability of multi-copy plasmids is maximized at intermediate plasmid copy numbers. We conclude by arguing that plasmid copy number is a highly optimized evolutionary trait that depends on the rate of environmental fluctuation and balances the benefit between increased stability in the absence of selection with the burden associated with carrying multiple copies of the plasmid.

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Synergy of chemotherapy and macrophage depletion leads to B cell-mediated T cell memory activation and durable triple negative breast cancer regression Immunosuppressive elements within the tumor microenvironment such as Tumor Associated Macrophages (TAMs) can present a barrier to successful anti-tumor responses by cytolytic T cells. We employed preclinical syngeneic p53 null mouse models of triple negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was used to successfully treat several highly aggressive TNBC murine mammary tumors and lung metastasis. Using this regimen and single cell RNA sequencing we characterized tumor infiltrating lymphocytes (TILs) including helper T cells and antigen-presenting B cells that were highly enriched in good responders to combination therapy. Using high dimensional imaging techniques, we identified the close spatial localization of B220+ CD86+ activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks post-treatment in one model that also exhibited long-term tumor regression post-treatment. We also characterized the transcriptional and metabolic heterogeneity of TAMS in these two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model is highly expressed and conserved in human claudin-low breast cancers, and high expression of the T12 signature correlated with reduced overall survival. This T12 tumor TAM signature may help identify human claudin-low breast cancer patients that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rationale combinations of immunotherapy.

SignificanceA treatment regimen that harnessed the immunostimulatory effects of cyclophosphamide coupled with the inhibition of CSF1R was used to successfully treat several highly aggressive claudin-low TNBC murine mammary tumors and lung metastasis.

sentences: Movement patterns and activity levels are shaped by the neonatal environment in Antarctic fur seal pups Tracking studies of juveniles are rare compared to those of adults, and consequently little is known about the influence of intrinsic and extrinsic factors on activity during this critical life stage. We used hourly GPS data, collected from 66 Antarctic fur seal pups from birth until moulting, to investigate the explanatory power of multiple individual-based and environmental variables on activity levels. Pups were sampled from two nearby breeding colonies of contrasting density during two subsequent years, and a two-state hidden Markov model was used to identify modalities in their movement behaviour, specifically  active and  inactive states. We found that movement was typified by central place exploration, with active movement away from and subsequent return to a location of inactivity. The probability of such directed exploration was unaffected by several factors known to influence marine mammal movement including sex, body condition, and temperature. Compared to pups born at the high-density colony, pups at low-density were more active, increased their activity with age, and transitioned earlier into the tussock grass, which offers protection from predators and extreme weather. Our study illustrates the importance of extrinsic factors, such as colony of birth, to early-life activity patterns and highlights the adaptive potential of movement.

sentences: Detecting natural selection in trait-trait coevolution No phenotypic trait evolves independently of all other traits, but the cause of trait-trait coevolution is poorly understood. While the coevolution could arise simply from pleiotropic mutations that simultaneously affect the traits concerned, it could also result from multivariate natural selection favoring certain trait relationships. To gain a general mechanistic understanding of trait-trait coevolution, we examine the evolution of 220 cell morphology traits across 16 natural strains of the yeast Saccharomyces cerevisiae and the evolution of 24 wing morphology traits across 110 fly species of the family Drosophilidae, along with the variations of these traits among gene deletion or mutation accumulation lines (a.k.a. mutants). For numerous trait pairs, the phenotypic correlation among evolutionary lineages differs significantly from that among mutants. Specifically, we find hundreds of cases where the evolutionary correlation between traits is strengthened or reversed relative to the mutational correlation, which, according to our population genetic simulation, is likely caused by multivariate selection. Furthermore, we detect selection for enhanced modularity of the yeast traits analyzed. Together, these results demonstrate that trait-trait coevolution is shaped by natural selection and suggest that the pleiotropic structure of mutation is not optimal. Because the morphological traits analyzed here are chosen largely because of their measurability, our conclusion is likely general.

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Screening of Chimeric GAA Variants in a Preclinical Study of Pompe Disease Results in Candidate Vector for Hematopoietic Stem Cell Gene Therapy Pompe disease is a rare genetic neuromuscular disorder caused by acid alpha-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy (ERT) is the current standard of care, which prolongs the quality of life for Pompe patients. However, ERT has limitations due to lack of enzyme penetration into the central nervous system (CNS) and skeletal muscles, immunogenicity against the recombinant enzyme, and requires life-long biweekly infusions. In a preclinical mouse model, a clinically relevant promoter to drive lentiviral vector-mediated expression of engineered GAA in autologous hematopoietic stem and progenitor cells (HSPC) was tested with nine unique human chimeric GAA coding sequences incorporating distinct peptide tags and codon-optimization iterations. Vectors including glycosylation independent lysosomal targeting (GILT) tags resulted in effective GAA enzyme delivery into key disease tissues with enhanced reduction of glycogen, myofiber and CNS vacuolation, compared to non-tagged GAA in Gaa knockout mice, a model of Pompe disease. Genetically modified microglial cells in brains were detected at low levels, but provided robust correction. Furthermore, an aminoacid substitution in the tag added to reduced capacity to induce insulin signaling and there was no evidence of off-target effects. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model providing a promising vector candidate for further investigation.

One Sentence SummaryA candidate vector for hematopoietic stem cell gene therapy of Pompe disease.

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An ex vivo human bone marrow milieu identifies CDH2 as a driver of niche-mediated treatment resistance in leukaemia Being able to clinically target the interaction of leukaemic cells with their microenvironment has remained a key therapeutic obstacle. Leukaemia cells re-program their microenvironment to provide support and protection from standard chemotherapy, molecularly targeted therapies as well as immunotherapy. To address this challenge, we have developed experimentally accessible human induced pluripotent stem cell engineered (iPSC) niches ex vivo to reveal insights into druggable cancer-niche interactions. We show that mesenchymal (iMSC) and vascular niche-like (iANG) cells support ex vivo proliferation of patient-derived leukaemia cells, impact dormancy and mediate therapy resistance. Mesenchymal stem cells protected both cycling and non- cycling blasts against dexamethasone treatment while vascular niche-like cells only protected dormant cells. Growth support and protection from dexamethasone induced- apoptosis was dependent on direct cell-cell contact and was mediated by N-cadherin (CDH2). To explore the therapeutic potential of disrupting this cell-cell interaction, we tested the CDH2 antagonist ADH-1 (previously in phase I / II for solid tumours) in a very aggressive patient-derived xenograft acute lymphoblastic leukaemia mouse model. ADH-1 showed high in vivo efficacy and combination therapy with ADH-1 and dexamethasone was superior compared with dexamethasone alone. These findings provide a proof-of-concept starting point to develop novel and potentially safer anti- cancer therapeutics that target niche-mediated cancer cell dependencies in haematological malignancies.

One Sentence SummaryCDH2 drives niche-dependent treatment resistance targetable via ADH-1, a low toxic potential candidate for future leukaemia clinical trials.

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Gut-testis axis: microbiota-(n-3) PUFA improving semen quality in type 1 diabetes Gut dysbiosis and type 1 diabetes (T1D) are closely related, and gut dysbiosis and male infertility are correlated, too. Moreover, most male T1D patients are of active reproductive age. Therefore, it is crucial to explore possible means for improving their semen quality. Here, we found that fecal microbiota transplantation (FMT) from alginate oligosaccharide (AOS) improved gut microbiota (A10-FMT) significantly decreased blood glucose and glycogen, and increased semen quality in streptozotocin-induced T1D subjects. A10-FMT improved T1D-disturbed gut microbiota, especially the increase in small intestinal lactobacillus, and blood and testicular metabolome to produce n-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to ameliorate spermatogenesis and semen quality. Moreover, A10-FMT can improve spleen and liver function to strengthen the systemic environment for sperm development. FMT from gut microbiota of control animals (Con-FMT) produced some beneficial effects; however, to a smaller extent. Thus, AOS improved gut microbiota may be a useful protocol for improving semen quality and male fertility in T1D patients.

ImportanceClinical data suggest that male reproductive dysfunction especially infertility is a critical issue for type 1 diabetic patient (T1D) because most of them are at the reproductive age. Gut dysbiosis is involved in T1D related male infertility. However, improved gut microbiota can be used to improve spermatogenesis and male fertility in T1D remains incompletely understood. We discovered that alginate oligosaccharide-improved gut microbiota (A10-FMT) significantly ameliorated spermatogenesis and semen quality. AOS-improved gut microbiota (specific microbes) may serve as a novel, promising therapeutic approach for the improvement of semen quality and male fertility in T1D patients.

sentences: Anticipatory regulation of cardiovascular system on emergence of auditory-motor interaction in young infants Humans develop auditory-motor interaction to produce a variety of rhythmic sounds using body movements, which are often produced and amplified with tools, such as drumming. The extended production of sounds allows us to express a wide range of emotions, accompanied by physiological changes. According to previous studies, even young infants enhance movements in response to auditory feedback. However, their exhibition of physiological adaptation on emergence of auditory-motor interaction is unclear. We investigated the movement and cardiac changes associated with auditory feedback to spontaneous limb movements in 3-month-old infants. The results showed that infants increased the frequency of limb movements inducing auditory feedback, while they exhibited the more regular rhythm of the limb movements. Furthermore, heart rate increase associated with the limb movement was first inhibited immediately after the timing of the auditory feedback, which may reflect sustained attention to the auditory stimuli. Then, through auditory-motor experience, the heart rate increase was inhibited even prior to the auditory feedback, leading to suppression on the peak intensity of the heart rate increase. These findings suggest that young infants regulate the cardiovascular system as well as limb movements in anticipation of the auditory feedback. The anticipatory regulation associated with movement and attentional changes may contribute to reduced cardiovascular stress in auditory-motor interaction, and provide a developmental basis for more sophisticated goal-directed behavior of producing rhythmic sounds.

sentences: Maternal and zygotic factors sequentially shape the tissue regionalization of chromatin landscapes in early vertebrate embryos One of the first steps in cellular differentiation of vertebrate embryos is the formation of the three germ layers. Maternal pioneer transcription factors (TFs) bind to the regulatory regions of the embryonic genome prior to zygotic genome activation and initiate germ layer specification. While the involvement of maternal TFs in establishing epigenetic marks in whole embryos was addressed previously, how early pluripotent cells acquire spatially restricted epigenetic identity in embryos remain unknown. Here, we report that the H3K4me1 enhancer mark in each germ layer becomes distinct in germ layer specific regulatory regions, forming super-enhancers (SEs), by early gastrula stage. Distinct SEs are established in these germ layers near robustly regulated germ layer identity genes, suggesting that SEs are important for the canalization of development. Establishment of these enhancers requires a sequential function of maternal and zygotic TFs. By knocking down the expression of a critical set of maternal endodermal TFs, an overwhelming majority of the endodermal H3K4me1 marks are lost. Interestingly, this disappearance of endodermal marking coincides with the appearance of ectodermal and mesodermal H3K4me1 marks in the endoderm, suggesting a transformation in the chromatin state of these nuclei towards a more ecto-mesodermal state. De novo motif analysis to identify TFs responsible for the transformation recovers a profile for endodermal maternal TFs as well as their downstream target TFs. We demonstrate the importance of coordinated activities of maternal and zygotic TFs in defining a spatially resolved dynamic process of chromatin state establishment.

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STING activation promotes robust immune response and NK cell-mediated tumor regression in glioblastoma models Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The cGAS-STING cytoplasmic double stranded DNA (dsDNA) sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties.

Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils and NK populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants the further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, CAR T cells, NK therapies or immune checkpoint blockade.

Significance statementModulation of the immune microenvironment is critical for immunosuppressive and therapy refractory tumors like glioblastoma. Activation of the STING pathway deeply remodels the brain tumor environment and attracts innate immune cells and natural killer cell populations, producing a robust antitumor effect with long-term immune memory. We further show that human glioblastoma tissue can respond to the therapy and lay the foundations for combined intracranial immunotherapies by using crosslinked biodegradable brain implants.

sentences: Predicting Individual Task Contrasts From Resting-state Functional Connectivity using a Surface-based Convolutional Network Task-based and resting-state represent the two most common experimental paradigms of functional neuroimaging. While resting-state offers a flexible and scalable approach for characterizing brain function, task-based techniques provide superior localization. In this paper, we build on recent deep learning methods to create a model that predicts task-based contrast maps from resting-state fMRI scans. Specifically, we propose BrainSurfCNN, a surface-based fully-convolutional neural network model that works with a representation of the brains cortical sheet. Our model achieves state of the art predictive accuracy on independent test data from the Human Connectome Project and yields individual-level predicted maps that are on par with the target-repeat reliability of the measured contrast maps. We also demonstrate that BrainSurfCNN can generalize remarkably well to novel domains with limited training data.

sentences: An image reconstruction framework for characterizing initial visual encoding We developed an image-computable observer model of the initial visual encoding that operates on natural image input, based on the framework of Bayesian image reconstruction from the excitations of the retinal cone mosaic. Our model extends previous work on ideal observer analysis and evaluation of performance beyond psychophysical discrimination, takes into account the statistical regularities of the visual environment, and provides a unifying framework for answering a wide range of questions regarding the visual front end. Using the error in the reconstructions as a metric, we analyzed variations of the number of different photoreceptor types on human retina as an optimal design problem. In addition, the reconstructions allow both visualization and quantification of information loss due to physiological optics and cone mosaic sampling, and how these vary with eccentricity. Furthermore, in simulations of color deficiencies and interferometric experiments, we found that the reconstructed images provide a reasonable proxy for modeling subjects percepts. Lastly, we used the reconstruction-based observer for the analysis of psychophysical threshold, and found notable interactions between spatial frequency and chromatic direction in the resulting spatial contrast sensitivity function. Our method is widely applicable to experiments and applications in which the initial visual encoding plays an important role.

sentences: A distinct assembly pathway of the human 39S late pre-mitoribosome Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. However, little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solved cryo-EM structures of the human 39S large subunit pre-ribosomes, representing four distinct late state intermediates. Besides the MALSU1 complex, we identified several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involved rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we found deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.

sentences: Defective subviral particles modify ecological equilibria and enhance viral coexistence Cooperation is a main driver of biological complexity at all levels. In the viral world, gene sharing among viral genomes, complementation between genomes or interactions within quasispecies are frequently observed. In this contribution, we explore the advantages that flexible associations between fully fledged viruses and subviral entities, such as virus satellites, might yield. We devise a mathematical model to compare different situations of competition between two viruses and to quantify how the association with a satellite qualitatively modifies dynamical equilibria. The relevant parameter is the invasion fitness of each virus or of the virus-satellite tandem, which in the model depends on the transmission rate of viruses and on their effect on host survival. While in a virus-virus competition one of the viruses becomes eventually extinct, an association with a satellite might change the outcome of the competition to favor the less competitive virus (regardless of whether it is the helper virus or not) or to allow for the stable coexistence of the two viruses and the satellite. We hypothesize that the latter scenario, in particular, constitutes a parsimonious evolutionary pathway towards more stable cooperative associations, such as bipartite viral forms.

sentences: Seeing the future: predictive control in neural models of ocular accommodation Ocular accommodation is the process of adjusting the eyes crystalline lens so as to bring the retinal image into sharp focus. The major stimulus to accommodation is therefore retinal defocus, and in essence, the job of accommodative control is to send a signal to the ciliary muscle which will minimize the magnitude of defocus. In this paper, we first provide a tutorial introduction to control theory to aid vision scientists without this background. We then present a unified model of accommodative control that explains properties of the accommodative response for a wide range of accommodative stimuli. Following previous work, we conclude that most aspects of accommodation are well explained by dual integral control, with a "fast" or "phasic" integrator enabling response to rapid changes in demand, which hands over control to a "slow" or "tonic" integrator which maintains the response to steady demand. Control is complicated by the sensorimotor latencies within the system, which delay both information about defocus and the accommodation changes made in response, and by the sluggish response of the motor plant. These can be overcome by incorporating a Smith predictor, whereby the system predicts the delayed sensory consequences of its own motor actions. For the first time, we show that critically-damped dual integral control with a Smith predictor accounts for adaptation effects as well as for the gain and phase for sinusoidal oscillations in demand. In addition, we propose a novel proportional-control signal to account for the power spectrum of accommodative microfluctuations during steady fixation, which may be important in hunting for optimal focus, and for the nonlinear resonance observed for low-amplitude, high-frequency input. Complete Matlab/Simulink code implementing the model is provided at https://doi.org/10.25405/data.ncl.14945550.

sentences: The sleep EEG envelope: a novel, neuronal firing-based human biomarker Sleep EEG reflects instantaneous voltage differences relative to a reference, while its spectrum reflects the degree to which it is comprised of oscillations at various frequencies. In contrast, the envelope of the sleep EEG reflects the instantaneous amplitude of oscillations at specific frequencies, and its spectrum reflects the rhythmicity of the occurrence of these oscillations. The ordinary sleep EEG and its spectrum have been extensively studied and its individual stability and relationship to various demographic characteristics, psychological traits and pathologies is well known. In contrast, the envelope spectrum has not been extensively studied before. In two studies, we explored the generating mechanisms and utility of studying the envelope of the sleep EEG. First, we used human invasive data from cortex-penetrating microelectrodes and subdural grids to demonstrate that the sleep EEG envelope spectrum reflects local neuronal firing. Second, we used a large database of healthy volunteers to demonstrate that the scalp EEG envelope spectrum is highly stable within individuals, especially in NREM sleep, and that it is affected by age and sex. Multivariate models based on a learning algorithm could predict both age (r=0.6) and sex (r=0.5) with considerable accuracy from the EEG envelope spectrum. With age, oscillations characteristically shifted from a 4-5 second rhythm to higher rhythms. The envelope spectrum was not associated with general cognitive ability (IQ). Our results demonstrate that the sleep envelope spectrum is a promising, neuronal firing-based biomarker of various demographic and disease-related phenotypes.

sentences: A behavioral syndrome linking boldness and flexibility facilitates invasion success in sticklebacks For a species to expand its range, it needs to be good at dispersing and also capable of exploiting resources and adapting to different environments. Therefore, behavioral and cognitive traits could play key roles in facilitating invasion success. Here, we show that dispersing sticklebacks are bold, while sticklebacks that have recently established in a new region are flexible. Moreover, boldness and flexibility are negatively correlated with one another at the individual, family and population levels. Multiple lines of evidence suggest that the divergence in boldness and flexibility is likely to be evolutionary in origin. If boldness is favored in invaders during the initial dispersal stage, while flexibility is favored in recent immigrants during the establishment stage, then the link between boldness and flexibility could generate positive correlations between successes during both dispersal and establishment, and therefore play a key role in facilitating colonization success in sticklebacks and other organisms.

sentences: Rabbit hindlimb kinematics and ground contact pressure during the stance phase of hopping gait Though the rabbit is a common animal model in musculoskeletal research, there is very limited data reported on healthy rabbit biomechanics. Our objective was to quantify the normative hindlimb biomechanics of six New Zealand White rabbits (3 male, 3 female) during the stance phase of hopping gait. We measured biomechanics by synchronously recording sagittal plane motion and ground contact pressure using a video camera and pressure mat, respectively. Both foot angle (i.e., angle between foot and ground) and ankle angle curves were unimodal. The peak ankle dorsiflexion angle was 65.9{degrees}{+/-}12.7{degrees} and occurred at 39% stance, while the peak ankle plantarflexion angle was 136.9{degrees}{+/-}7.8{degrees} at toe-off. Minimum and maximum foot angles were 16.6{degrees}{+/-}6.4{degrees} at 12% stance and 125.4{degrees}{+/-}4.0{degrees} at toe-off, respectively. During stance, the knee joint center gradually progressed 4.7 cm downward and 18.1 cm forward, on average. The maximum vertical ground reaction force and contact area, both averaged across rabbits, were 42.5 {+/-} 11.4 %BW and 7.5 {+/-} 1.8 cm2, respectively. Our study confirmed that rabbits exhibit a plantigrade gait pattern, similar to humans. Future studies can reference our data to quantify the extent to which orthopedic interventions affect rabbit biomechanics.

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gmxapi: a Gromacs-native Python interface for molecular dynamics with ensemble and plugin support Gmxapi provides an integrated, native Python API for both standard and advanced molecular dynamics simulations in GROMACS. The Python interface permits multiple levels of integration with the core GROMACS libraries, and legacy support is provided via an interface that mimics the command-line syntax, so that all GROMACS commands are fully available. Gmxapi has been officially supported since the GROMACS 2019 release and is enabled by default in current versions of the software. Here we describe gmxapi 0.3 and later. Beyond simply wrapping GROMACS library operations, the API permits several advanced operations that are not feasible using the prior command-line interface. First, the API allows custom user plugin code within the molecular dynamics force calculations, so users can execute custom algorithms without modifying the GROMACS source. Second, the Python interface allows tasks to be dynamically defined, so high-level algorithms for molecular dynamics simulation and analysis can be coordinated with loop and conditional operations. Gmxapi makes GROMACS more accessible to custom Python scripting while also providing support for high-level data-flow simulation algorithms that were previously feasible only in external packages.

Author SummaryThe gmxapi software provides a Python interface for molecular dynamics simulations in GROMACS. In addition to simply wrapping GROMACS commands, it supports custom user plugin code, ensemble simulation, and data-flow chaining of commands. As such, gmxapi enables the writing and execution of high-level simulation algorithms. The software ships with GROMACS and is freely available under an LGPL2 license.

sentences: Different temporal requirements for the LRR transmembrane receptors Tartan and Toll-2 in the formation of contractile interfaces at compartmental boundaries Compartmental boundaries physically separate groups of epithelial cells, a property fundamental for the organization of the body plan in both insects and vertebrates. In many examples, this physical separation has been shown to be the consequence of a regulated increase in contractility of the actomyosin cortex at boundary cell-cell interfaces, a property important in developmental morphogenesis beyond compartmental boundary formation. In this study, we took an unbiased screening approach to identify cell surface receptors required for actomyosin enrichment and polarisation at parasegmental boundaries (PSBs) in early Drosophila embryos, leading us to uncover different temporal requirements for two LRR receptors, Tartan and Toll-2. First, we find that Tartan is required during germband extension for actomyosin enrichment at PSBs, confirming an earlier report. Next, by following in real time the dynamics of loss of boundary straightness in tartan mutant embryos compared to wildtype and ftz mutant embryos, we show that Tartan is not required beyond germband extension. At this stage, actomyosin enrichment at PSBs becomes regulated by Wingless signalling. We find that Wingless signalling regulates Toll-2 expression and we show that Toll-2 is required for planar polarization of actomyosin after the completion of germ-band extension. Thus the formation of contractile interfaces at PSBs depends on a dynamic set of LRR receptors cues. Our study also suggests that the number of receptor cues is small and that the receptors are interchangeable.

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Photoprotection is regulated by light-independent CO2 availability Photosynthetic algae cope with suboptimal levels of light and CO2. In low CO2 and excess light, the green alga Chlamydomonas reinhardtii activates a CO2 Concentrating Mechanism (CCM) and photoprotection; the latter is mediated by LHCSR1/3 and PSBS. How light and CO2 signals converge to regulate photoprotective responses remains unclear. Here we show that excess light activates expression of photoprotection- and CCM-related genes and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3 and CCM genes while stabilizing the LHCSR1 protein. We also show that CIA5, which controls CCM genes, is a major regulator of photoprotection, elevating LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 accumulation. Our work emphasizes the importance of CO2 in regulating photoprotection and the CCM, demonstrating that the impact of light on photoprotection is often indirect and reflects intracellular CO2 levels.

TeaserPhotoprotection- and CCM-related genes can be activated by changes in CO2 availability even in the absence of light.

sentences: CRISPR/Cas9 targeting of MCPyV T antigen in Merkel tumors Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCPyV) T antigen gene expression, post integration ([~]80% cases), or by UV mediated DNA damage. Viral-positive Merkel tumors are not only caused by but also oncogenically addicted to tumor antigen expression. In this study we used CRISPR-Cas9 based gene-editing to develop a potential therapeutic tool for MCPyV positive MCC. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a genome editing technology whereby a guide RNA (gRNA) molecule, targets a Cas endonuclease to a specific genomic site, using sequence homology, and induces a double strand break. To target MCPyV T antigens, we designed a strategy using 2 gRNAs targeting the T antigen genomic region that would cut off a substantial portion of the gene thereby rendering it dysfunctional. We validated the MCPYV T antigen targeting efficiency of our gRNAs, both individually and together by in vitro cleavage assays. Finally, to translate this finding, we delivered this CRISPR system in patient-derived MCC cell lines and show reduction in T antigen gene expression. Our proof-of-concept study shows that 2 MCPyV targeting CRISPR/Cas gRNAs in combination can knock out MCPyV T antigen, thus, being of therapeutic importance. We hope that this CRISPR system can be potentially delivered in vivo for advancing MCPyV positive MCC treatment in the future.

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Italian universities do not recruit according to proficiency in scientific production We analyzed the files regarding recruitment competitions won by 186 professors of selected bibliometric disciplines in Florence between January 2014 and 30 June 2021. An equal number of professors recruited at other Italian universities and of researchers who never attained professorship in Italy were randomly chosen in the same disciplines as each Florentine professor among individuals possessing National Scientific Qualification, a prerequisite for professorship. The H-indexes at the time of qualification (T1), of the Florence call (T2), and the current (T3) time were obtained from Scopus.

Non-recruited researchers were more likely (Chi-square test) to show a higher H-index than both Florentine (T1 p=0.0005, T2 p=0.0015, T3 p=0.0095) and non-Florentine professors (T1 p=0.0078, T2 p=0.0245, T3 p=0.0500). Fifty-four non-recruited scientists serve in foreign universities, 100 at national/international research centers. The remaining 32 scientists (25 who keep producing despite precarious employment, and seven who have stopped publishing) were, at any rate, as likely as Florentine (T3 p=0.69) and non-Florentine professors (T3 p=0.14) to show a higher H-index.

This study suggests that Italian academia does not recruit professors according to their qualitative/quantitative ability to publish, a detriment to knowledge for the nationwide system and on a global scale.

sentences: Localized delivery of β-NGF via injectable microrods accelerates endochondral fracture repair Currently, there are no biological approaches to accelerate bone fracture repair. Osteobiologics that promote endochondral ossification are an exciting alternative to surgically implanted bone grafts, however, the translation of osteobiologics remains elusive because of the need for localized and sustained delivery that is both safe and effective. In this regard, an injectable system composed of hydrogel-based microparticles designed to release osteobiologics in a controlled and localized manner is ideal in the context of bone fracture repair. Here, we describe poly (ethylene glycol) dimethacrylate (PEGDMA)-based microparticles, in the form of microrods, engineered to be loaded with beta nerve growth factor ({beta}-NGF) for use in a murine tibial fracture model. In-vitro studies demonstrated that protein-loading efficiency is readily altered by varying PEGDMA macromer concentration and that {beta}-NGF loaded onto PEGDMA microrods exhibited sustained release over a period of 7 days. In-vitro bioactivity of {beta}-NGF was confirmed using a tyrosine receptor kinase A (Trk-A) expressing cell line, TF-1. Moreover, TF-1 cell proliferation significantly increased when incubated with {beta}-NGF loaded PEGDMA microrods versus {beta}-NGF in media. In-vivo studies show that PEGDMA microrods injected into the fracture calluses of mice remained in the callus for over 7 days. Importantly, a single injection of {beta}-NGF-loaded PEGDMA microrods resulted in significantly improved fracture healing as indicated by significant increases in bone volume, trabecular connective density, and bone mineral density and a significant decrease in cartilage despite a remarkably lower dose ([~]111 fold) than the {beta}-NGF in media. In conclusion, we demonstrate a novel and translational method of delivering {beta}-NGF via injectable PEGDMA microrods to improve bone fracture repair.

sentences: Tongue immune compartment analysis reveals spatial macrophage heterogeneity The tongue is a unique muscular organ situated in the oral cavity where it is involved in taste sensation, mastication and articulation. As a barrier organ, which is constantly exposed to environmental pathogens, the tongue is expected to host an immune cell network ensuring local immune defence. However, the composition and the transcriptional landscape of the tongue immune system are currently not completely defined. Here we characterised the tissue-resident immune compartment of the murine tongue during development, health and disease, combining single cell RNA-sequencing with in situ immunophenotyping. We identified distinct local immune cell populations and described two specific subsets of tongue-resident macrophages occupying discrete anatomical niches. Cx3cr1+ macrophages were located specifically in the highly innervated lamina propria beneath the tongue epidermis and at times in close proximity to fungiform papillae. Folr2+ macrophages were detected in deeper muscular tissue. The two macrophage subsets originate from a common proliferative precursor during early postnatal development and responded differently to systemic LPS in vivo. Our description of the under-investigated tongue immune system sets a starting point to facilitate research on tongue immune-physiology and pathology including cancer and taste disorders.

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The SARS-CoV-2 receptor-binding domain facilitates neutrophil transepithelial migration and nanoparticle uptake in the mice airways SARS-CoV-2-induced infection is still dangerous. Mouse models are convenient to the investigation of virus-activated immune response mechanisms. However, mice are not proper model organisms to study COVID-19 due to decreased interaction affinity between the SARS-CoV-2 receptor-binding domain (RBD) and mouse angiotensin-converting enzyme 2 (ACE2) compared with human ACE2. In the present study, we propose a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system. To mimic the effects of RBD- ACE2 high-affinity interaction, mice received the ACE2 inhibitor MLN-4760. To simulate virus loading, we applied 100 nm particles suspended in the solution of RBD via the oropharyngeal route to mice. In this model, MLN-4760 application enhanced neutrophil egress from the bone marrow to the bloodstream and RBD attracted neutrophils to the luminal side of the conducting airway epithelium. By contrast, inert 100 nm particles were not potent to stimulate neutrophil recruitment to the conducting airway mucosa. Using this model, and by altering the dosage of the ACE2 inhibitor, nanoparticles, and RBD, one can adapt it to investigate different COVID-19 states characterized with mild or severe airway inflammation.

StatementThis study presents a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system and investigates immune cell-model virus particle interactions in the conducting airway mucosa.

sentences: A genealogical estimate of genetic relationships The application of genetic relationships among individuals, characterized by a genetic relationship matrix (GRM), has far-reaching effects in human genetics. However, the current standard to calculate the GRM generally does not take advantage of linkage information and does not reflect the underlying genealogical history of the study sample. Here, we propose a coalescent-informed framework to infer the expected relatedness between pairs of individuals given an ancestral recombination graph (ARG) of the sample. Through extensive simulations we show that the eGRM is an unbiased estimate of latent pairwise genome-wide relatedness and is robust when computed using genealogies inferred from incomplete genetic data. As a result, the eGRM better captures the structure of a population than the canonical GRM, even when using the same genetic information. More importantly, our framework allows a principled approach to estimate the eGRM at different time depths of the ARG, thereby revealing the time-varying nature of population structure in a sample. When applied to genotyping data from a population sample from Northern and Eastern Finland, we find that clustering analysis using the eGRM reveals population structure driven by subpopulations that would not be apparent using the canonical GRM, and that temporally the population model is consistent with recent divergence and expansion. Taken together, our proposed eGRM provides a robust tree-centric estimate of relatedness with wide application to genetic studies.

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Intraneuronal chloride levels encode tiredness in cortex Continuous periods of wakefulness are associated with reduced performance levels due to the build-up of sleep pressure in active regions of the brain. These effects manifest as use-dependent changes in cortical network activity and the mechanisms underlying these changes represent targets for overcoming the cognitive effects of tiredness. Here we reveal a central role for intraneuronal chloride levels, which increase in a use-dependent manner during waking, and reduce the strength of local synaptic inhibition in mouse cortex. Activity-dependent increases in chloride account for spatial and temporal features of sleep pressure, they underlie cortical network oscillations in the sleep-deprived state, and targeting chloride regulation in cortex can rescue performance levels when tired. These findings provide a missing link between sleep-wake history, synaptic transmission and cortical dynamics.

One-Sentence SummaryThe effects of sleep pressure on cortical function are caused by use-dependent changes in chloride-mediated synaptic inhibition.

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Habitat deterioration despite protection: long-term declines of littoral area of fishponds in Czech nature reserves Fishponds play a key role in current pondscapes in many developed countries. Their littoral areas, supporting multiple ecosystem functions including the maintenance of aquatic and riparian biodiversity, have been adversely affected by the move shift towards more intensive aquaculture and widespread eutrophication in the middle 20th century. To counteract these changes, many fishponds received some protection, but its long-term efficiency has not been studied. Here we focus on the role of conservation status in protecting the area of littoral areas of fishponds in Czechia between the years 1950 and 2019. We found that the conservation status of these fishponds did not prevent habitat deterioration in most of the fishponds, especially during the second half of the 20th century. Moreover, we detected no significant effects of the reserve establishment year, fishpond area and conservation target on the littoral areas. This suggests that the conservation measures are insufficient across fishpond reserve types. We attribute the negative trends to persisting high fish stocks, especially of common carp, and eutrophication resulting from additional feeding, pond manuring, and ongoing nutrient inputs from the pond catchments. Sediment dredging and high grazing pressure by waterfowl in some reserves can further aggravate the situation. We conclude that effective protection of the littoral areas requires a paradigm shift towards less intensive fish stock management, more frequent summer drainage, and effective reduction of all nutrient inputs to increase the water quality. Such measures can help recover the littoral areas and the associated biota.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/473897v1_ufig1.gif" ALT="Figure 1">
View larger version (27K):
org.highwire.dtl.DTLVardef@8e9b31org.highwire.dtl.DTLVardef@6ecaf9org.highwire.dtl.DTLVardef@1cfaefdorg.highwire.dtl.DTLVardef@1e9952f_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LILittoral vegetation in ponds supports high biodiversity but often lacks protection.
C_LIO_LIWe evaluated long-term changes in the littoral areas of fishponds in Czechia.
C_LIO_LIThe areas decreased markedly in both protected and unprotected ponds since 1950.
C_LIO_LIReserve duration, fishpond area and conservation target did not affect the trends.
C_LIO_LIChanges in fishpond management are recommended for littoral area recovery.
C_LI

sentences: NOCICEPTRA: Gene and microRNA signatures and their trajectories characterizing human iPSC-derived nociceptor maturation Nociceptors are primary afferent neurons serving the reception of acute pain but also the transit into maladaptive pain disorders. Since native human nociceptors are hardly available for mechanistic functional research, and rodent models do not necessarily mirror human pathologies in all aspects, human iPSC-derived nociceptors (iDN) offer superior advantages as a human model system. Unbiased mRNA::microRNA co-sequencing, immunofluorescence staining and qPCR validations, revealed expression trajectories as well as miRNA target spaces throughout the transition of pluripotent cells into iDNs. mRNA and miRNA candidates emerged as regulatory hubs for neurite outgrowth, synapse development and ion channel expression. The exploratory data analysis tool NOCICEPTRA is provided as a containerized platform to retrieve experimentally determined expression trajectories, and to query custom gene sets for pathway and disease enrichments. Querying NOCICEPTRA for marker genes of cortical neurogenesis revealed distinct similarities and differences for cortical and peripheral neurons. The platform provides a public domain neuroresource to exploit the entire data sets and explore miRNA and mRNA as hubs regulating human nociceptor differentiation and function.

sentences: Human-specific enrichment of schizophrenia risk-genes in callosal neurons of the developing neocortex Human genetic studies have provided a wealth of information on genetic risk factors associated with neuropsychiatric diseases. However, whether different brain cell types are differentially affected in disease states and when in their development and maturation alterations occur is still poorly understood. Here we generated a longitudinal transcriptional map of excitatory projection neuron (PN) and inhibitory interneuron (IN) subtypes of the cerebral cortex, across a timeline of mouse embryonic and postnatal development, as well as fetal human cortex and human cortical organoids. We found that three types of gene signatures uniquely defined each cortical neuronal subtype: dynamic (developmental), adult (terminal), and constitutive (stable), with individual neuronal subtypes varying in the degree of similarity of their signatures between species. In particular, human callosal projection neurons (CPN) displayed the greatest species divergence, with molecular signatures highly enriched for non-coding, human-specific RNAs. Evaluating the association of neuronal class-specific signatures with neuropsychiatric disease risk genes using linkage disequilibrium score regression showed that schizophrenia risk genes were enriched in CPN identity signatures from human but not mouse cortex. Human cortical organoids confirmed the association with excitatory projection neurons. The data indicate that risk gene enrichment is both species- and cell type-specific. Our study reveals molecular determinants of cortical neuron diversification and identifies human callosal projection neurons as the most species-divergent population and a potentially vulnerable neuronal class in schizophrenia.

sentences: A distributed and efficient population code of mixed selectivity neurons for flexible navigation decisions Decision-making requires flexibility to rapidly switch sensorimotor associations depending on behavioral goals stored in memory. We identified cortical areas and neural activity patterns that mediate this flexibility during virtual-navigation, where mice switched navigation toward or away from a visual cue depending on its match to a remembered cue. An optogenetics screen identified V1, posterior parietal cortex (PPC), and retrosplenial cortex (RSC) as necessary for accurate decisions. Calcium imaging revealed neurons that can mediate rapid sensorimotor switching by encoding a conjunction of a current and remembered visual cue that predicted the mouses navigational choice from trial-to-trial. Their activity formed efficient population codes before correct, but not incorrect, choices. These neurons were distributed across posterior cortex, even V1, but were densest in RSC and sparsest in PPC. We propose the flexibility of navigation decisions arises from neurons that mix visual and memory information within a visual-parietal-retrosplenial network, centered in RSC.

sentences: Comparing genome-based estimates of relatedness for use in pedigree-based conservation management Researchers have long debated which estimator of relatedness best captures the degree of relationship between two individuals. In the genomics era, this debate continues, with relatedness estimates being sensitive to the methods used to generate markers, marker quality, and levels of diversity in sampled individuals. Here, we compare six commonly used genome-based relatedness estimators (kinship genetic distance (KGD), Wang Maximum Likelihood (TrioML), Queller and Goodnight (Rxy), Kinship INference for Genome-wide association studies (KING-robust), and Pairwise Relatedness (RAB), allele-sharing co-ancestry (AS)) across five species bred in captivity-including three birds and two mammals-with varying degrees of reliable pedigree data, using reduced-representation and whole genome resequencing data. Genome-based relatedness estimates varied widely across estimators, sequencing methods, and species, yet the most consistent results for known first order relationships were found using Rxy, RAB, and AS. However, AS was found to be less consistently correlated with known pedigree relatedness than either Rxy or RAB. Our combined results indicate there is not a single genome-based estimator that is ideal across different species and data types. To determine the most appropriate genome-based relatedness estimator for each new dataset, we recommend assessing the relative: (1) correlation of candidate estimators with known relationships in the pedigree and (2) precision of candidate estimators with known first-order relationships. These recommendations are broadly applicable to conservation breeding programs, particularly where genome-based estimates of relatedness can complement and complete poorly pedigreed populations. Given a growing interest in the application of wild pedigrees, our results and are also applicable to in-situ wildlife management.

sentences: Molecular transport and packing underlie increasing ribosome productivity in faster growing cells Faster growing cells must make proteins more quickly. Increasing ribosome abundance only partly accounts for increased protein production; the productivity of individual ribosomes must also increase, almost doubling via an unknown mechanism. To investigate, we model translation elongation in Escherichia coli by explicitly representing spatially-resolved transport and reactions among individual molecules each occupying finite volume. We predict that physical transport of individual ternary complexes accounts for ~80% of elongation latency. We find crowding increases at faster growth, which predictably decreases diffusion of ternary complexes, but that crowding also improves proximity between ternary complexes and ribosomes. Despite slowed diffusion, we predict that improved proximity wins out, offering a simple mechanism for how individual elongating ribosomes become more productive. We speculate how crowding imposes a physical limit on growth rate and likely undergirds cellular behavior more broadly. Our work reveals a fundamental mechanism arising from physical transport at the colloidal scale that cannot be obtained via kinetics-only modeling.

sentences: Water levels primarily drive variation in photosynthesis and nutrient use of scrub Red Mangroves in the southeastern Florida Everglades We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades.

sentences: Laboratory Yeast Strains Rely On Oxidative Phosphorylation For Efficient ATP Production Even though it is a well-accepted fact that the energy metabolism of yeast is likely to impact all cellular activities, surprising little is known about the ATP homeostasis of particular yeast strains that are commonly used in cell biological studies. Therefore, we determined key parameters such as oxygen consumption and fermentation rates of the lab strain SEY6210. Our data indicated that even at high glucose concentrations, SEY6210 produces 30-50% of cellular ATP from oxidative phosphorylation. Loss of respiration, either by disrupting ATP synthase function or by growth in anaerobic conditions, was not fully compensated by fermentation and as a result affected energy intensive processes such as the maintenance of the plasma membrane proton gradient and the associated import of nutrients.

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Distinct corticostriatal compartments drive competition between adaptive and automatized behavior Cortical and basal ganglia circuits play a crucial role in the formation of goal-directed and habitual behaviors. In this study, we investigate the cortico-striatal circuitry involved in learning and the role of this circuitry in the emergence of inflexible behaviors such as those observed in addiction. Specifically, we develop a computational model of cortico-striatal interactions that performs concurrent goal-directed and habit learning. The model accomplishes this by distinguishing learning processes in the dorsomedial striatum (DMS) that rely on reward prediction error signals as distinct from the dorsolateral striatum (DLS) where learning is supported by salience signals. These striatal subregions each operate on unique cortical input: the DMS receives input from the prefrontal cortex (PFC) which represents outcomes, and the DLS receives input from the premotor cortex which determines action selection. Following an initial learning of a two-alternative forced choice task, we subjected the model to reversal learning, reward devaluation, and learning a punished outcome. Behavior driven by stimulus-response associations in the DLS resisted goal-directed learning of new reward feedback rules despite devaluation or punishment, indicating the expression of habit. We repeated these simulations after the impairment of executive control, which was implemented as poor outcome representation in the PFC. The degraded executive control reduced the efficacy of goal-directed learning, and stimulus-response associations in the DLS were even more resistant to the learning of new reward feedback rules. In summary, this model describes how circuits of the dorsal striatum are dynamically engaged to control behavior and how the impairment of executive control by the PFC enhances inflexible behavior.

Author SummarySubstance abuse changes an animals brain so that it is not able to appropriately respond to new environmental cues such as negative consequences for drug use. It is thought that the transition from adaptive to inflexible responding involves the inappropriate engagement of neural circuitry responsible for habit. In this study, we model neural circuitry that is critical for adaptive responding to changes in reward feedback to animal behavior. We extended previous computational models of the basal ganglia to incorporate detailed biophysical representations of the dorsomedial striatum, which is critical for goal-directed behavior, and the dorsolateral striatum, which is critical for habitual behavior. The model exhibited learning in simulations of reward reversal, devaluation, and punished outcome tasks. Recent work from our group has quantified the impairment of executive control in cortical activity of compulsively drinking rats. Here, we challenged the model in additional simulations by implementing the impairment of executive control as a reduction in the fidelity of prefrontal cortical outcome representations. Our results illustrate how impaired executive control reduces the performance of goal-directed learning and, hence, reduces the models ability to overcome an established habitual response.

sentences: Growth and adaptation mechanisms of tumour spheroids with time-dependent oxygen availability Tumours are subject to external environmental variability. However, in vitro tumour spheroid experiments, used to understand cancer progression and develop cancer therapies, have been routinely performed for the past fifty years in constant external environments. Furthermore, spheroids are typically grown in ambient atmospheric oxygen (normoxia), whereas most in vivo tumours exist in hypoxic environments. Therefore, there are clear discrepancies between in vitro and in vivo conditions. We explore these discrepancies by combining tools from experimental biology, mathematical modelling, and statistical uncertainty quantification. Focusing on oxygen variability to develop our framework, we reveal key biological mechanisms governing tumour spheroid growth. Growing spheroids in time-dependent conditions, we identify and quantify novel biological adaptation mechanisms, including unexpected necrotic core removal, and transient reversal of the tumour spheroid growth phases.

sentences: TwinEQTL: Ultra Fast and Powerful Association Analysis for eQTL and GWAS in Twin Studies We develop a computationally efficient alternative, TwinEQTL, to a linear mixed-effects model (LMM) for twin genome-wide association study (GWAS) data. Instead of analyzing all twin samples together with LMM, TwinEQTL first splits twin samples into two independent groups on which multiple linear regression analysis can be validly performed separately, followed by an appropriate meta-analysis-like approach to combine the two non-independent test results. Through mathematical derivations, we prove the validity of TwinEQTL algorithm and show that the correlation between two dependent test statistics at each single-nucleotide polymorphism (SNP) are independent of its minor allele frequency (MAF). Thus the correlation is constant across all SNPs. Through simulations, we show empirically that TwinEQTL has well controlled type I error with negligible power loss compared to the gold-standard linear mixed effects models. To accommodate eQTL analysis with twin subjects, we further implement TwinEQTL into a R package with much improved computational efficiency. Our approaches provide a significant leap in terms of computing speed for GWAS and eQTL analysis with twin samples.

sentences:
Can double PPO mutations exist in the same allele and are such mutants functional? BackgroundResistance to PPO-inhibiting herbicides is primarily endowed by target site mutations at the PPO2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus ssp. PPO2, the most prevalent target mutations are deletion of the G210 codon and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same ppo2 allele.

ResultsPlants carrying R128G+/+ {Delta}G210+/-, where + indicates presence of the mutation, were crossed with each other. The ppo2 of the offspring was subjected to pyrosequencing and E. coli- based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G {Delta}G210 can occur only in one allele; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying {Delta}G210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions.

ConclusionsHomozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele.

NomenclaturePalmer amaranth, Amaranthus palmeri S. Wats.; protoporphyrinogen oxidase, PPO; tall waterhemp, Amaranthus tuberculatus (Moq.) J.D.Sauer

sentences: Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway. SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.

sentences: Single-cell transcriptome lineage tracing of human pancreatic development identifies distinct developmental trajectories of alpha and beta cells In comparison to mouse, the developmental process of human islets has not been properly elucidated. The advancement of single cell RNA-seq technology enables us to study the properties of alpha and beta cells at single cell resolution. By using mitochondrial genome variants as endogenous lineage-tracing markers, we found that human alpha and beta cells have different lineage features. This finding suggests specific endocrine progenitors for alpha and beta cells, which is different from mouse islet cells. This strategy was also applied to a study of chemically-induced islet cell reprogramming and was used to help identify artemether-induced alpha-to-beta trans-differentiation in human islets. The computational results of this study will inspire future studies to establish, maintain, and expand beta cell-specific progenitors in vitro and in vivo.

sentences: House sparrows do not exhibit a preference for the scent of potential partners with different MHC-I allele numbers and genetic distances MHC genes play a fundamental role in immune recognition of pathogens and parasites. Therefore, females may increase offspring heterozygosity and genetic diversity by selecting MHC genetically compatible or heterozygous males. In birds, several studies suggest that MHC genes play a role in mate choice, and recent evidence suggest that olfaction may play a role in such discrimination. Previous studies indicated that house sparrow females with low allelic diversity prefer males with higher diversity in MHC-I alleles. Here, we directly explored whether both house sparrow females and males could estimate by scent the number in MHC amino acid and functional variants as well as the level of MHC-I similarity or dissimilarity of potential partners. Our results show that neither females nor males exhibit a preference related to the number of MHC-I amino acid variants or functional variants or in relation to MHC amino acid or functional similarity of potential partners, suggesting that MHC-I is not detected through olfaction. Further studies are needed to understand the mechanisms responsible for MHC-I based mate discrimination in birds.

sentences: Cellular Mechanisms Underlying Central Sensitization in a Mouse Model of Chronic Muscle Pain Chronic pain disorders are often associated with psychiatric symptoms. The central nucleus of the amygdala (CeA) has emerged as an integrative hub for nociceptive and affective components during the development of central pain. Although the exact cause for this process remains unknown, prior adverse injuries are precipitating factors and thought to transform nociceptors into a primed state for chronic pain. However, the cellular basis underlying the primed state and the subsequent pain chronification remains unknown. Here, we investigated cellular and synaptic alterations of the CeA in a mouse model of chronic muscle pain. In these mice, local infusion of pregabalin, a clinically approved drug for fibromyalgia and other chronic pain disorders, into the CeA or selective inactivation of somatostatin-expressing CeA (CeA-SST) neurons during the priming phase prevented pain chronification. Further, electrophysiological recording revealed that CeA-SST neurons received increased excitatory transmission and showed enhanced excitability in chronic pain states. In line with the possible role of CeA-SST neurons in central sensitization, chemogenetic inactivation of CeA-SST neurons or pharmacological suppression of nociceptive afferents from the brainstem to CeA-SST neurons by pregabalin after the development of chronic muscle pain alleviated pain and negative emotions.

sentences: A signalling axis involving CNOT3, Aurora B and ERK promotes differentiation and survival of mesendodermal progenitor cells Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B, which requires sequences in the NOT box domain of CNOT3, and regulates MAPK/ERK signalling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse ES cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of ectoderm. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4 and Wnt3. The double mutation affects interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2, impacting on survival of the differentiated ME cells. Our results identify an adaptor function for CNOT3 that regulates a key pathway in embryogenesis and cancer.

sentences: The evolution of the cytochrome c6 family of photosynthetic electron transfer proteins During photosynthesis, electrons are transferred between the cytochrome b6f complex and photosystem I. This is carried out by the protein plastocyanin in plant chloroplasts. In contrast, electron transfer can be carried out by either plastocyanin or cytochrome c6 in many cyanobacteria and eukaryotic algal species. There are three further cytochrome c6 homologues: cytochrome c6A in plants and green algae, and cytochromes c6B and c6C in cyanobacteria. The function of these proteins is unknown. Here, we present a comprehensive analysis of the evolutionary relationship between the members of the cytochrome c6 family in photosynthetic organisms. Our phylogenetic analyses show that cytochrome c6B and cytochrome c6C are likely to be orthologues that arose from a duplication of cytochrome c6, but that there is no evidence for separate origins for cytochrome c6B and c6C. We therefore propose re-naming cytochrome c6C as cytochrome c6B. We show that cytochrome c6A is likely to have arisen from cytochrome c6B rather than by an independent duplication of cytochrome c6, and present evidence for an independent origin of a protein with some of the features of cytochrome c6A in peridinin dinoflagellates. We conclude with a new comprehensive model of the evolution of the cytochrome c6 family which is an integral part of understanding the function of the enigmatic cytochrome c6 homologues.

sentences: Implications of soil nitrogen enhancement on the yield performance of soybean (Glycine max) in cadmium-polluted soil The growth, development and yield of important crop plants like soybean (Glycine max) are constantly under threat by continuous inputs of cadmium in the biosphere as a result of various industrial activities. This study investigated the level to which, addition of nitrogen fertilization can enhance plant survival, growth and yield development in a cadmium-polluted. Three accessions of Glycine max (TGm - 1, TGm - 2 and TGm - 3) were sown in a 12 mg/kg-cadmium polluted, which was thereafter amended with urea (FU), ammonia (FA), and ammonium nitrate (FN) singly and in combinations of equal proportions. A non-fertilized Cd-polluted soil and a general control constituted the negative and positive controls. Results showed that although N application did not enhance yield dispositions of soybean in Cd-polluted soil, significant impact on vegetative development was noteworthy. Compared to yield of control plants, cadmium pollution imposed a 26.1 % reduction in per plant yield in TGm-1, compared 1.71 % in TGm-3. Generally, addition of nitrogenous fertilizer further suppressed crop yield by as much as 80 % in plants sown in cadmium-polluted soil. However, application of ammonia fertilizer to TGm-2 improved its yield performances in the cadmium-polluted soil.

sentences: Pre-exposure to mRNA-LNPs reprograms adaptive and innate immune responses in an inheritable fashion Billions of SARS-CoV-2 mRNA-LNP vaccine doses have already been administered to humans. However, we lack a comprehensive understanding of the immune effects of this platform. Here we bring experimental evidence that pre-exposure to mRNA-LNPs or its LNP component has effects on both innate and adaptive immune responses. Pre-exposure to mRNA-LNPs led to long-term, platform-specific inhibition of the adaptive immune responses. As such, mice responded with lower antibody responses when exposed the second time to the mRNA-LNP platform, but with undisturbed adaptive immune responses to protein-based Alum or AddaVax-adjuvanted vaccines. On the other hand, we report that after pre-exposure to mRNA-LNPs, resistance of mice to heterologous infections with influenza virus and Candida albicans is likely enhanced. Interestingly, mice pre-exposed to mRNA-LNPs can pass down the acquired immune traits to their offspring, providing better protection. In summary, the mRNA-LNP vaccine platform induces long-term immunological changes that can affect both humoral responses and heterologous protection against infections. More studies are needed to understand in-depth the mechanisms responsible for these effects.

sentences: Differential recognition of opioid analgesics by μ opioid receptors: Predicted interaction patterns correlate with ligand-specific voltage sensitivity The {micro} opioid receptor (MOR) is the key target for analgesia, but the application of opioids is accompanied by several issues. There is a wide range of opioid analgesics, differing in their chemical structure and their properties in receptor activation and subsequent effects. A better understanding of ligand-receptor interactions and resulting effects is important. Here, we calculated the respective binding modes for several opioids and analyzed fingerprints of ligand-receptor interactions. We further corroborated the binding modes experimentally by cellular assays. As ligand-induced modulation of activity due to changes in membrane potential was displayed by MOR, we further analyzed the effects of voltage sensitivity of this receptor. With a combined in silico and in vitro approach, we defined discriminating interaction patterns for the ligand-specific voltage sensitivity. With this, we present new insights for interactions likely in ligand recognition and their specific effects on activation of the MOR.

sentences: Negative regulation of IMD contributes to disease tolerance during systemic bacterial infection in Drosophila Disease tolerance is an infection phenotype where hosts show relatively high health despite harbouring elevated pathogen loads. Compared to the mechanisms of immune clearance our knowledge of the mechanisms underlying increased tolerance remains incomplete. Variation in the ability to reduce immunopathology may explain why some hosts can tolerate higher pathogen burdens with reduced pathology. Negative immune regulation would therefore appear to be a clear candidate for a mechanism underlying disease tolerance but this has not been tested directly for bacterial infections. Here, we examined how the negative regulation of the immune deficiency (IMD) pathway affects disease tolerance in Drosophila melanogaster when infected with the gram-negative bacterial pathogen Pseudomonas entomophila. We find that UASRNAi-mediated reduced expression of the negative regulators of IMD (pirk and caudal) severely reduced the ability to tolerate infection in both males and females across a wide range of infectious doses. While flies unable to regulate the IMD response exhibited higher expression of antimicrobial peptides and lower bacterial loads as expected, this was not accompanied by a proportional reduction in mortality. Instead, tolerance (measured as fly survival relative to its microbe load) was drastically reduced, likely due to the combination of increased immunopathology and cytotoxicity of elevated AMP expression. Our results therefore highlight that in addition to regulating an efficient pathogen clearance response, negative regulators of IMD also contribute to disease tolerance.

sentences: Plant-based expression of SARS-CoV-2 antigens for use in an oral vaccine Oral and intra-nasal vaccines represent a key means of inducing mucosal-based immunity against infection with SARS-CoV-2, yet such vaccines represent only a minority of candidates currently in development. In this brief communication, we assessed the expression of the SARS-CoV-2 Receptor Binding Domain (RBD) subunit of the surface-exposed Spike glycoprotein in the leaves of nine edible plant species (lettuce, spinach, collard greens, tomato, cucumber, radish, arugula, pepper, and Coho greens), with a goal of identifying a suitable candidate for the development of an oral vaccine against COVID-19. We report lettuce (Lactuca sativa L. cv. Hilde II Improved) to be a preferred host to support in planta expression of SARS-CoV-2 RBD, representing an important first step towards development of a plant-based oral vaccine.

sentences: Outsourced hearing in an orb-weaving spider that uses its web as an auditory sensor Hearing is a fundamental sense of many animals, including all mammals, birds, some reptiles, amphibians, fish, and arthropods. The auditory organs of these animals are extremely diverse in anatomy after hundreds of millions of years of evolution, yet all are made up of cellular tissues and are morphologically part of bodies of animals. Here we show hearing in the orb-weaving spider, Larinioides sclopetarius is not constrained by the organisms body but is extended through outsourcing hearing to its extended phenotype, the proteinaceous, self-manufactured orb-web. We find the wispy, wheel-shaped orb-web acts as a hyperacute acoustic "antenna" to capture the sound-induced air particle movements that approach the maximum physical efficiency, better than the acoustic responsivity of all previously known eardrums. By sensing the motion of web threads, the spider remotely detects and localizes the source of an incoming airborne acoustic wave such as those emitted by approaching prey or predators. By outsourcing its acoustic sensors to its web, the spider is released from body size constraints and permits the araneid spider to increase its sound-sensitive surface area enormously, up to 10,000 times greater than the spider itself. The spider also enables the flexibility to functionally adjust and regularly regenerate its "external eardrum" according to its needs. The "outsourcing" and "supersizing" of auditory function in spiders provides unique features for studying extended and regenerative sensing, and designing novel acoustic flow detectors for precise fluid dynamic measurement and manipulation.

sentences: Identification of an inhibitory pocket in falcilysin bound by chloroquine provides a new avenue for malaria drug development Despite their widespread use, our understanding of how malaria drugs work remains limited. This includes chloroquine (CQ), the most successful antimalarial ever deployed. Here, we used MS-CETSA and dose-response transcriptional profiling to elucidate protein targets and mechanism of action (MOA) of CQ, as well as MK-4815, a malaria drug candidate with a proposed MOA similar to CQ. We identified falcilysin (FLN) as the target of both compounds and found that hemoglobin digestion was the key biological pathway affected, with distinct MOA profiles between CQ-sensitive and CQ-resistant parasites. We showed that CQ and MK-4815 inhibit FLN proteolytic activity, and using X-ray crystallography, that they occupy a hydrophobic pocket situated within the large peptide substrate binding cavity of FLN. As a key protein in the MOA of CQ, FLN now constitute an interesting target for the development of novel anti-malarial drugs with improved resistance profiles.

sentences: Type II muscle fibre properties are not associated with balance recovery following large perturbations during walking in young and older adults Falls among older adults are often attributed to declining muscle strength with ageing. Associations between muscle strength and balance control have been reported, but the evidence for, and key mechanisms of resistance exercise in fall prevention are unclear. No studies have directly examined the relationship between muscle fibre characteristics and reactive balance control. Here, we address whether or not Type II muscle fibre characteristics associate with reactive balance during walking in young and older adults with varying muscle fibre type composition. We analyse muscle biopsy-derived fibre characteristics and stability during a treadmill-based walking perturbation (trip-like) task of healthy young adults, healthy, normally active older adults, trained older adults and physically impaired older adults. We find no significant associations between Type II muscle fibre properties and reactive balance during walking, indicating that practitioners and researchers should consider more than just the muscle tissue properties when assessing and intervening on fall risk.

sentences: A thermostable oral SARS-CoV-2 vaccine induces mucosal and protective immunity An ideal protective vaccine against SARS-CoV-2 should not only be effective in preventing disease, but also in preventing virus transmission. It should also be well accepted by the population and have a simple logistic chain. To fulfill these criteria, we developed a thermostable, orally administered vaccine that can induce a robust mucosal neutralizing immune response. We used our platform based on retrovirus-derived enveloped virus-like particles (e-VLPs) harnessed with variable surface proteins (VSPs) from the intestinal parasite Giardia lamblia, affording them resistance to degradation and the triggering of robust mucosal cellular and antibody immune responses after oral administration. We made e-VLPs expressing various forms of the SARS-CoV-2 Spike protein (S), with or without membrane protein (M) expression. We found that prime-boost administration of VSP-decorated e-VLPs expressing a pre-fusion stabilized form of S and M triggers robust mucosal responses against SARS-CoV-2 in mice and hamsters, which translate into complete protection from a viral challenge. Moreover, they dramatically boosted the IgA mucosal response of intramuscularly injected vaccines. We conclude that our thermostable orally administered e-VLP vaccine could be a valuable addition to the current arsenal against SARS-CoV-2, in a stand-alone prime-boost vaccination strategy or as a boost for existing vaccines.

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A single-cell clusters similarity measure for different batches, datasets, and samples SummarySince the inception of single-cell level measuring techniques, identification of distinct cell stages, phenotypes and populations has been a challenge. Cell clustering and dimensionality reduction methods are the most popular approaches to identify heterogeneity of single-cell data. But, as public repositories continue to grow in number, integrative analyses and merging of large pools of samples from different and heterogeneous datasets becomes a difficult challenge, which showcases the impossibility of scalability of some of the existing methods. Here we present ClusterFoldSimilarity, an R package that calculates a measure of similarity between clusters from different datasets/batches, without the need of correcting for batch effect or normalizing and merging the data, thus avoiding artifacts and the loss of information derived from these kinds of techniques. The similarity metric is based on the average vector module and sign of the product of logarithmic fold-changes. ClusterFoldSimilarity compares every single pair of clusters from any number of different samples/datasets, including different number of clusters for each sample. Additionally, the algorithm is able to select the top genes which contribute the most to the similarity of two specific clusters, serving also as a feature selection tool.

Availability and implementationThe algorithm is freely available as an R package at: https://github.com/OscarGVelasco/ClusterFoldSimilarity

Contactoscargvelasco@gmail.com

sentences: What is a supercoiling-sensitive gene? Insights from topoisomerase I inhibition in the Gram-negative bacterium Dickeya dadantii DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by gyrase inhibition were categorised as "supercoiling-sensitive". Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine. We showed that the drug is active against topoisomerase I from this species, and analysed the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in terms of expression level, spatial and orientational organisation along the chromosome. Altogether, these results highlight that the supercoiling-sensitivity is a complex feature, which depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after gyrase or topoisomerase I inhibition.

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Hot and cold spots of pest-induced US urban tree death, 2020-2050 O_LIUrban trees are important nature-based solutions for future wellbeing and livability but are at high risk of mortality from insect pests. In the United States (US), 82% of the population live in urban settings and this number is growing, making urban tree mortality a matter of concern for most of its population. Until now, the magnitudes and spatial distributions of risks were unknown.
C_LIO_LIHere, we combine new models of street tree populations in [~]30,000 US communities, species-specific spread predictions for 57 invasive insect species, and estimates of tree death due to insect exposure for 48 host tree genera.
C_LIO_LIWe estimate that 1.4 million street trees will be killed by invasive insects from 2020 through 2050, costing an annualized average of US$ 30M. However, these estimates hide substantial variation: 23% of urban centers will experience 95% of all insect-induced mortality. Further, 90% of all mortality will be due to emerald ash borer (Agrilus planipennis, EAB), which is expected to kill virtually all ash trees (Fraxinus spp.) in >6000 communities.
C_LIO_LIWe define an EAB high-impact zone spanning 902,500km2, largely within the southern and central US, within which we predict the death of 98.8% of all ash trees. "Mortality hotspot cities" include Milwaukee, WI; Chicago, IL; and New York, NY.
C_LIO_LIWe identify Asian wood borers of maple and oak trees as the highest risk future invaders, where a new establishment could cost US$ 4.9B over 30 years.
C_LIO_LIPolicy implications: To plan effective mitigation, managers must know which tree species in which communities will be at the greatest risk, as well as the highest-risk insects. We provide the first country-wide, spatial forecast of urban tree mortality due to invasive insect pests. This framework identifies dominant pest insects and spatial impact hotspots, which can provide the basis for spatial prioritization of spread control efforts such as quarantines and biological control release sites. Our results highlight the need for EAB early-detection efforts as far from current infestations as Seattle, WA. Further, these findings produce a list of biotic and spatiotemporal risk factors for future high-impact US urban forest insect pests.
C_LI

sentences: In silico design of bioactive chimeric peptide from archaeal antimicrobial peptides Novel peptide therapeutics have been the cardinal part of modern-day research. Such therapies are being incorporated to prevent the adverse effects of globally emerging multi-drug resistant bacteria and various chronic human diseases which pose a great risk to the present world. In this study, we have designed a novel peptide therapy involving archaeal antimicrobial peptides. In silico predictions assign the peptide construct to be antigenic, non-allergenic, non-toxic and having stable physicochemical properties. The secondary and tertiary structures of the construct were predicted. The tertiary structure was refined for improving the quality of the predicted model. Computational tools predicted intracellular receptors in Escherichia coli, Klebsiella pneumoniae and the human body to be possible binding targets of the construct. In silico docking of modelled peptide with predicted targets, showed prominent results against targets for complex human diseases and that of bacterial infections. The stability of those docked complexes was confirmed with computational studies of conformational dynamics. Certainly, the designed peptide could be a potent therapeutic against multi-drug resistant bacteria as well as several human diseases.

sentences: An Entropy-based Directed Random Walk for Pathway Activity Inference Using Topological Importance and Gene Interactions The integration of microarray technologies and machine learning methods has become popular in predicting pathological condition of diseases and discovering risk genes. The traditional microarray analysis considers pathways as simple gene sets, treating all genes in the pathway identically while ignoring the pathway networks structure information. This study, however, proposed an entropy-based directed random walk (e-DRW) method to infer pathway activity. This study aims (1) To enhance the gene-weighting method in Directed Random Walk (DRW) by incorporating t-test statistic scores and correlation coefficient values, (2) To implement entropy as a parameter variable for random walking in a biological network, and (3) To apply Entropy Weight Method (EWM) in DRW pathway activity inference. To test the objectives, the gene expression dataset was used as input datasets while the pathway dataset was used as reference datasets to build a directed graph. An equation was proposed to assess the connectivity of nodes in the directed graph via probability values calculated from the Shannon entropy formula. A direct proof of calculation based on the proposed mathematical formula was presented using e-DRW with gene expression data. Based on the results, there was an improvement in terms of sensitivity of prediction and accuracy of cancer classification between e-DRW and conventional DRW. The within-dataset experiments indicated that our novel method demonstrated robust and superior performance in terms of accuracy and number of predicted risk-active pathways compared to the other DRW methods. In conclusion, the results revealed that e-DRW not only improved prediction performance, but also effectively extracted topologically important pathways and genes that are specifically related to the corresponding cancer types.

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Disturbed retinoid metabolism upon loss of rlbp1a impairs cone function and leads to subretinal lipid deposits and photoreceptor degeneration in the zebrafish retina The RLBP1 gene encodes the 36 kDa cellular retinaldehyde binding protein, CRALBP, a soluble retinoid carrier, in the visual cycle of the eyes. Mutations in RLBP1 are associated with recessively inherited clinical phenotypes, including Bothnia dystrophy, retinitis pigmentosa, retinitis punctata albescens, fundus albipunctatus, and Newfoundland rod-cone dystrophy. However, the etiology of these retinal disorders is not well understood. Here, we generated homologous zebrafish models to bridge this knowledge gap. Duplication of the rlbp1 gene in zebrafish and cell-specific expression of the paralogs rlbp1a in the retinal pigment epithelium and rlbp1b in Muller glial cells allowed us to create intrinsically cell type-specific knockout fish lines. Using rlbp1a and rlbp1b single and double mutants, we investigated the pathological effects on visual function. Our analyses revealed that rlbp1a was essential for cone photoreceptor function and chromophore metabolism in the fish eyes. rlbp1a mutant fish displayed reduced chromophore levels and attenuated cone photoreceptor responses to light stimuli. They accumulated 11-cis and all-trans-retinyl esters which displayed as enlarged lipid droplets in the RPE reminiscent of the subretinal yellow-white lesions in patients with RLBP1 mutations. During aging, these fish developed retinal thinning and cone and rod photoreceptor dystrophy. In contrast, rlbp1b mutants did not display impaired vision. The double mutant essentially replicated the phenotype of the rlbp1a single mutant. Together, our study showed that the rlbp1a zebrafish mutant recapitulated many features of human blinding diseases caused by RLBP1 mutations and provided novel insights into the pathways for chromophore regeneration of cone photoreceptors.

Graphical Abstract

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sentences: Definition, modeling and detection of saccades in the face of post-saccadic oscillations When analyzing eye tracking data, one of the central tasks is the detection of saccades. Although many automatic saccade detection algorithms exist, the field still debates how to deal with brief periods of instability around saccade offset, so-called post-saccadic oscillations (PSOs), which are especially prominent in todays widely used video-based eye tracking techniques. There is good evidence that PSOs are caused by inertial forces that act on the elastic components of the eye, such as the iris or the lens. As this relative movement can greatly distort estimates of saccade metrics, especially saccade duration and peak velocity, video-based eye tracking has recurrently been considered unsuitable for measuring saccade kinematics. In this chapter, we review recent biophysical models that describe the relationship between pupil motion and eyeball motion. We found that these models were well capable of accurately reproducing saccade trajectories and implemented a framework for the simulation of saccades, PSOs, and fixations, which can be used - just like datasets hand-labelled by human experts - to evaluate detection algorithms and train statistical models. Moreover, as only pupil and corneal-reflection signals are observable in video-based eye tracking, one may also be able to use these models to predict the unobservable motion of the eyeball. Testing these predictions by analyzing saccade data that was registered with video-based and search-coil eye tracking techniques revealed strong relationships between the two types of measurements, especially when saccade offset is defined as the onset of the PSO. To enable eye tracking researchers to make use of this definition, we present and evaluate two novel algorithms - one based on eye-movement direction inversion, one based on linear classifiers previously trained on simulation data. These algorithms allow for the detection of PSO onset with high fidelity. Even though PSOs may still pose problems for a range of eye tracking applications, the techniques described here may help to alleviate these.

sentences: Comparative Analysis of Sorghum EMS Mutants and Natural Populations To build a large-scale genomic resource for functional validation of sorghum genes through EMS-mutagenized BTx623 seeds, we deep sequenced (30-60X) an additional 445 phenotyped EMS mutant lines. 4.2 million EMS mutations are called with nearly 36,800 mutations that could have a disruptive effect on functions of over 15,500 genes. Combining variants carried by both the natural population and previous EMS efforts, over 69% of sorghum coding genes (23644) are now presented with one or more mutations that are, or are predicted to be, disruptive to their functions. Our results show that the EMS population carries more significant mutations but less in each sample than the natural population, which makes it more powerful in elucidating sorghum gene functions on a large scale and requiring less work in validation of candidate causal genes. We have made the data available through two ways, one is the integration with the BSAseq workflow that supports retrieving independent EMS samples carrying the same genes with significant mutation for complementary testing, and the other is a web application for directly querying genes with significant mutations on SciApps.org.

sentences: Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin, to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a proteins binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

sentences: Don't think, feel: basal ganglia-cortical connectivity underlies self-regulation of brain oscillations in humans Brain-computer interfaces (BCIs) provide an artificial link by which the brain can directly interact with the environment. To achieve fine BCI control, participants must modulate the patterns of the cortical oscillations generated from the motor and somatosensory cortices. However, it remains unclear how humans regulate cortical oscillations, the controllability of which substantially varies across individuals. Here, we performed simultaneous electroencephalography (to assess BCI control) and functional magnetic resonance imaging (to measure brain activity) in healthy participants. Self-regulation of cortical oscillations induced activity in the basal ganglia-cortical network (BgCN) and the neurofeedback control network (NfCN). Successful self-regulation correlated with striatal activity in the BgCN, through which patterns of cortical oscillations were likely modulated. Moreover, BgCN and NfCN connectivity correlated with strong and weak self-regulation, respectively. The findings indicate that the BgCN is important for self-regulation, the understanding of which should help advance BCI technology.

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THC modifies the impact of heroin delivered by vapor inhalation in rats Opioids are effective medications, but they have several key limitations including the development of tolerance, establishment of dependence, diversion for non-medical use and the development of addiction. Therefore, any drugs which act in an additive or synergistic fashion with opioids to address medical applications have the potential to reduce opioid-related harms. This study was conducted to determine if heroin and {Delta}9-tetrahydrocannabinol (THC) interact in an additive or independent manner to alter nociception, body temperature and spontaneous locomotor activity when inhaled or injected.

Groups of male and female rats implanted with radiotelemetry transmitters were exposed to vapor for assessment of effects on temperature and activity. Heroin (50 mg/mL in the propylene glycol; PG) inhalation increased temperature and activity whereas THC (50 mg/mL) inhalation decreased temperature and activity. Effects of combined inhalation were in opposition, and additional experiments found the same outcome for the injection of heroin (0.5 mg/kg, s.c.) and THC (10 mg/kg, i.p.) alone and in combination. In contrast, the co-administration of Heroin and THC by either inhalation or injection produced additive effects on thermal nociception assessed with a warm water tail-withdrawal assay in male and female Sprague-Dawley and Wistar rats.

The conclusion of this study is that additive effects of THC with heroin on a medical endpoint such as analgesia may not generalize to other behavioral or physiological effects, which may be a positive outcome for unwanted side effects.

sentences: Radius-Optimized Efficient Template Matching for Lesion Detection from Brain Images Computer-aided detection of brain lesions from volumetric magnetic resonance imaging (MRI) is in demand for fast and automatic diagnosis of neural diseases. The template-matching technique can provide satisfactory outcome for automatic localization of brain lesions; however, finding the optimal template size that maximizes similarity of the template and the lesion remains challenging. This increases the complexity of the algorithm and the requirement for computational resources, while processing large MRI volumes with three-dimensional (3D) templates. Hence, reducing the computational complexity of template matching is needed. In this paper, we first propose a mathematical framework for computing the normalized cross-correlation coefficient (NCCC) as the similarity measure between the MRI volume and approximated 3D Gaussian template with linear time complexity, [Formula], as opposed to the conventional fast Fourier transform (FFT) based approach with the complexity [Formula], where N is the number of voxels in the image and amax is the number of tried template radii. We then propose a mathematical formulation to analytically estimate the optimal template radius for each voxel in the image and compute the NCCC with the location-dependent optimal radius, reducing the complexity to [Formula]. We test our methods on one synthetic and two real multiple-sclerosis databases, and compare their performances in lesion detection with FFT and a state-of-the-art lesion prediction algorithm. We demonstrate through our experiments the efficiency of the proposed methods for brain lesion detection and their comparable performance with existing techniques.

sentences: Large-scale genomic study reveals robust activation of the immune system following advanced Inner Engineering meditation retreat. The positive impact of meditation on human wellbeing is well documented, yet its molecular mechanisms are incompletely understood. We applied a comprehensive systems biology approach starting with whole blood gene expression profiling combined with multi-level bioinformatic analyses to characterize the co-expression, transcriptional, and protein-protein interaction networks to identify meditation-specific core network after an advanced 8-day Inner Engineering retreat program. We found the response to oxidative stress, detoxification, and cell cycle regulation pathways were downregulated after meditation. Strikingly, 220 genes directly associated with immune response, including 68 genes related to interferon (IFN) signaling were upregulated, with no significant expression changes in the inflammatory genes. This robust meditation-specific immune response network is significantly dysregulated in multiple sclerosis and severe COVID-19 patients. The work provides a foundation for understanding the effect of meditation and potential implications to voluntarily and non-pharmacologically improve the immune response before immunotherapy for many conditions, including multiple sclerosis and COVID-19 vaccination.

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On the application of BERT models for nanopore methylation detection MotivationDNA methylation is a common epigenetic modification, which is widely associated with various biological processes, such as gene expression, aging, and disease. Nanopore sequencing provides a promising methylation detection approach through monitoring abnormal signal shifts for detecting modified bases in target motif regions. Recently, model-based approaches, especially those with deep learning models, have achieved significant performance improvements on nanopore methylation detection. In this work, we explore using bidirectional encoder representations from transformers (BERT) for doing the task, which can provide non-recurrent neural structures for fast parallel computation.

ResultsWe find original BERT architecture does not work as well as the bidirectional recurrent neural network (biRNN) on the nanopore methylation prediction task. Through further analysis, we observe recurrent patterns of positional-signal-shift in the context window surrounding target 5-methylcytosine (5mC) and N6-methyladenine (6mA) motifs. We propose a refined BERT with relative position representation and center hidden units concatenation, which takes account of task-specific characters into modeling. We perform systematic evaluations in-sample and cross-sample. The experiment results show that the refined BERT model can achieve competitive or even better results than the state-of-the-art biRNN model, while the model inference speed is about 6x faster. Besides, on the cross-sample evaluation of datasets from the different research groups, BERT models demonstrate a good generalization performance.

AvailabilityThe source code and data are available at https://github.com/yaozhong/methBERT

Contactyaozhong@ims.u-tokyo.ac.jp

sentences: Screening of plant growth-promoting rhizobacterial strains for the degradation and utilization of exogenous pectin as a sole carbon source Universal primers for gyrB were used to screen 79 strains of Bacillus species. The genomic DNA from each strain was extracted, and the PCR product of gyrB was amplified and purified for gene sequencing. Gene sequences were edited, and aligned, and Bacillus amyloliquefaciens subsp. plantarum (Bap) strains identified based on the gyrB phylogenetic tree. Two primers (exuT and uxuB) were designed to detect pectin-associated transporter gene exuT and D-mannonate oxidoreductase gene uxuB. The Bap strains were then screened for the presence of the exuT and uxuB genes. These two pectin-utilizing genes were confirmed in 57 and 54 Bap strains by PCR amplification. Second, an in vitro tests were conducted to screen 59 Bap strains using pectin as a sole carbon source to confirm the pectate lyase and utilizing activity. Pectate Agar (PA) and Tris-Spizizen Salts (TSS) medium were used for the in vitro pectate lyase and degradation assays.

sentences: Convergence of MCR-8.2 and chromosome-mediated resistance to colistin and tigecycline in an NDM-5-producing ST656 Klebsiella pneumoniae from a lung transplant patient For infection caused by NDM-5-producing Klebsiella pneumoniae, tigecycline and colistin are the last treatment options. In this study, we characterized the first NDM-5 and MCR-8.2 co-harboring K. pneumoniae clinical isolate, combining with chromosomal gene-mediated resistance to colistin and tigecycline. The K. pneumoniae KP32558 was isolated from the bronchoalveolar lavage fluid from a lung transplant male patient. Whole genome sequencing was carried out using Illumina HiSeq sequencing platform as well as nanopore sequencing method. The K. pneumoniae KP32558 was identified as a pan-drug resistant bacteria, belonged to ST656, and harbored plasmid-encoded blaNDM-5 and mcr-8.2 genes. The blaNDM-5 gene was located on an IncX3 type plasmid, which was successfully transferred to Escherichia coli strain J53 without visible fitness cost. The mcr-8.2 gene was located on a conjugative plasmid pKP32558-2-mcr8. It had two replicons, IncFII(K) and IncQ1, harbored previously by another two mcr-8.2-carrying plasmids pMCR8_020135 and pMCR8_095845. These three plasmids were clustered into the same clade and derived from K. pneumoniae isolates of the same clonal complex, indicating that pKP32558-2-mcr8 may come from pMCR8_020135 and pMCR8_095845 related ancestor. The MIC of KP32558 for colistin was 256 mg/L, the 6 amino acid substitutions in the two-component system may involve in the high-level colistin resistance. The truncation in acrR gene, related to tigecycline resistance, was also identified. K. pneumoniae has evolved a variety of complex resistance mechanisms to the last-resort antimicrobials, close surveillance is urgently needed to monitor the prevalence of this clone.

sentences: Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory Amino Acid Transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl-) channels, but the physiological role of Cl- conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl- channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of five additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations with decreased hEAAT1 Cl- channel activity and functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl- homeostasis in glial cells for proper CNS function. We also identified a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

sentences: In vivo structure and dynamics of the RNA genome of SARS-Cov-2 The SARS-CoV-2 coronavirus, which causes the COVID-19 pandemic, is one of the largest positive strand RNA viruses. Here we developed a simplified SPLASH assay and comprehensively mapped the in vivo RNA-RNA interactome of SARS-CoV-2 RNA during the viral life cycle. We observed canonical and alternative structures including 3-UTR and 5-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in cells and in virions. We provide direct evidence of interactions between Transcription Regulating Sequences (TRS-L and TRS-Bs), which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE, forming a "high-order pseudoknot" embedding FSE, which might help ribosome stalling at frameshift sites. More importantly, we found that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome cyclization is weakened and genome domains remain stable. Our data provides a structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, describes dynamics of RNA structures during life cycle of SARS-CoV-2, and will help to develop antiviral strategies.

sentences: Evidence for a trap-and-flip mechanism in a proton-dependent lipid transporter Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters participate in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acids synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a  trap-and-flip mechanism that might be shared among MFS lipid transporters.

sentences: Hominoid-specific sulcal variability is related to face perception ability Human perception requires complex cortical networks that function at neuroanatomical scales of microns and temporal scales of milliseconds. Despite this complexity, what if just one morphological feature of the brain could predict perceptual ability? Here, we tested this hypothesis with pre-registered analyses of neuroanatomy and face perception in neurotypical controls (NTs) and individuals with developmental prosopagnosia (DPs). Results show that the length of the mid-fusiform sulcus (MFS), a hominoid-specific tertiary sulcus in ventral temporal cortex (VTC), was shorter in DPs than NTs. Furthermore, individual differences in MFS length in the right, but not left, hemisphere predicted individual differences in face perception. These results support theories linking brain structure and function to perception, as well as indicate that one feature - variability in MFS length - can predict face perception. Finally, these findings add to growing evidence supporting a role of morphological variability of late developing, tertiary sulci and individual differences in cognition.

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Intra and inter-annual climatic conditions have stronger effect than grazing intensity on root growth of permanent grasslands AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackground and AimsC_ST_ABSUnderstanding how direct and indirect changes in climatic conditions, management, and species composition affect root production and root traits is of prime importance for the delivery of carbon sequestration services of grasslands. This study considers the effects of climatic variability and gradients of herbage utilisation by grazing on root production over the course of two years. The root and leaf traits of the plant communities were determined to detect their capacity to predict above- and below-ground net primary production, ANPP and BNPP, respectively.

MethodsA long-term field experiment was used to compare the effects of abandonment and low (Ca-) and high (Ca+) grazing intensities (resulting in mean residual plant heights of 15.2 cm and 7.7 cm, respectively) induced by grazing rotations on upland fertile grasslands after 10 years of treatment application. Ingrowth cores and exclusion cages were used to measure, respectively, the root and shoot mass production several times each year and at an annual scale. The root and leaf traits of the communities were measured near the vegetations peak growing season.

ResultsWe observed strong seasonal root production across treatments in both a wet and a dry year, but the response to grazing intensity was hardly observable within growing seasons. In the abandonment treatment, the spring and autumn root growth peaks were delayed by approximately one month compared to the two cattle treatments, possibly due to a late plant canopy green-up induced by lower soil temperatures and an accumulation of litter. The BNPP was slightly lower in the abandonment treatment compared to the cattle treatments only during the dry year, whereas a decline of the ANPP in the abandonment treatment compared to the Ca+ treatment was observed during the wet year. In response to drought, which occurred during the second year, the root-to-shoot biomass ratio was stable in the cattle treatments but declined in the abandonment treatment. The higher allocation to root mass could benefit plant communities under drier conditions.

ConclusionsRotational grazing pressures and climatic condition variabilities had limited effects on root growth seasonality, although drought had stronger effects on the BNPP than on the ANPP. The stability of the root-to-shoot biomass ratio during the dry year evidenced a higher resistance to drought by grazed versus abandoned grassland communities.

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Treadmill exercise promotes retinal astrocyte plasticity and protects against retinal degeneration Exercise has been shown to be an effective neuroprotective intervention that preserves retinal function and structure in several animal models of retinal degeneration. However, retinal cell morphology and cell types governing exercise-induced retinal neuroprotection remain elusive. Previously, we found that the protective effects of exercise in animal models of retinal disease were accompanied by increased levels of circulating and retinal brain derived neurotrophic factor (BDNF) and required intact signal transduction with its high-affinity receptor, tropomyosin kinase B (TrkB). Studies of neurodegenerative diseases in the brain demonstrate that neurons and astrocytes express BDNF and TrkB. Additionally, astrocytes have been shown to alter their morphology in response to exercise. Here, we have investigated the role of retinal astrocytes as mediators of exercise-induced retinal neuroprotection in a light-induced retinal degeneration mouse model (LIRD). We found that treadmill exercise in both our dim (control maintenance light levels) and LIRD groups promote increased retinal astrocytic population, GFAP expression, branching and endpoints, dendritic complexity, and promotes BDNF-astrocyte interaction. In contrast, LIRD animals that were inactive had significant reductions in all measured parameters. Our findings indicate that exercise is sufficient to rescue retinal astrocyte morphology in a LIRD model maintaining branching and dendritic arborization similar to retinal astrocytes that are not undergoing degeneration. These studies provide essential information to current knowledge gaps in regards to exercise-induced neuroprotection and will additionally provide knowledge in exercise intervention optimization as a rehabilitative method.

Significance statementThis study represents an essential step in determining the cell-types governing and morphological alterations elicited from exercise which may provide neural repair and protection. Similar to astrocytes in the brain, retinal astrocytes alter their morphology in response to exercise. Our studies demonstrate exercise promotes increased interactions between retinal astrocytes and neural growth factors in healthy retinas as well as in retinas undergoing degeneration, which may ultimately protect dying retinal neurons. These studies provide insight into the potential neuroprotective role astrocytes play in neurodegenerative diseases.

sentences: Diaphragm muscle fibrosis involves changes in collagen organization with mechanical implications in Duchenne Muscular Dystrophy In Duchenne muscular dystrophy (DMD), diaphragm muscle dysfunction results in respiratory insufficiency, a leading cause of death in patients. Increased muscle stiffness occurs with buildup of fibrotic tissue, characterized by excessive accumulation of extracellular matrix (ECM) components such as collagen. However, changes in mechanical properties are not explained by collagen amount alone and we must consider the complex structure and mechanics of fibrotic tissue. The goals of our study were to (1) determine if and how collagen organization changes with the progression of DMD in diaphragm muscle tissue, and (2) predict how collagen organization influences the mechanical properties of ECM. We first visualized collagen structure with scanning electron microscopy (SEM) images and then developed an analysis framework to quantify collagen organization and generate image-based finite-element models. The image analysis revealed significant age- and disease-dependent increases in collagen fiber straightness and alignment, ranging from 4.7 to 13.4%, but collagen fibers retained a transverse orientation relative to muscle fibers. The mechanical models predicted significant age- and disease-dependent increases in transverse effective stiffness and average stress, ranging from 8.8 to 12.4%. Additionally, both healthy and diseased models revealed an increase in transverse stiffness relative to longitudinal stiffness, with significant age- and disease-dependent increases in the ratio of transverse to longitudinal stiffness, ranging from 19.7 to 24.5%. This study revealed changes in diaphragm ECM structure and mechanics during the progression of disease in the mdx muscular dystrophy mouse phenotype, highlighting the need to consider the role of collagen organization on diaphragm muscle function.

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Light Attention Predicts Protein Location from the Language of Life SummaryAlthough knowing where a protein functions in a cell is important to characterize biological processes, this information remains unavailable for most known proteins. Machine learning narrows the gap through predictions from expert-designed input features leveraging information from multiple sequence alignments (MSAs) that is resource expensive to generate. Here, we showcased using embeddings from protein language models (pLMs) for competitive localization prediction without MSAs. Our lightweight deep neural network architecture used a softmax weighted aggregation mechanism with linear complexity in sequence length referred to as light attention (LA). The method significantly outperformed the state-of-the-art (SOTA) for ten localization classes by about eight percentage points (Q10). So far, this might be the highest improvement of just embeddings over MSAs. Our new test set highlighted the limits of standard static data sets: while inviting new models, they might not suffice to claim improvements over the SOTA.

AvailabilityOnline predictions are available at http://embed.protein.properties. Predictions for the human proteome are available at https://zenodo.org/record/5047020. Code is provided at https://github.com/HannesStark/protein-localization.

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Adaptive reprogramming during early seed germination requires temporarily enhanced fermentation a critical role for alternative oxidase (AOX) regulation that concerns also microbiota effectiveness Plants respond to environmental cues via adaptive cell reprogramming that can affect whole plant and ecosystem functionality. Microbiota constitutes part of plants inner and outer environment. This Umwelt underlies steady dynamics, due to complex local and global biotic and abiotic changes. Hence, adaptive plant holobiont responses are crucial for continuous metabolic adjustment at systems levels. Plants require oxygen-dependent respiration for energy-dependent adaptive morphology, such as, germination, root and shoot growth, formation of adventitious, clonal and reproductive organs, fruits and seeds. Fermentative paths can help in acclimation and, to our view the role of alternative oxidase (AOX) in coordinating complex metabolic and physiologic adjustments is underestimated.

Cellular level of sucrose is an important sensor of environmental stress. We explored the role of exogenous sucrose and its interplay with AOX during early seed germination. We found that sucrose-dependent initiation of fermentation during the first 12 hours after imbibition (HAI) was beneficial to germination. However, parallel enhanced AOX expression was essential to control negative effects by prolonged sucrose treatment. Early down-regulated AOX activity until 12 HAI improved germination efficiency in the absence of sucrose, but suppressed early germination in its presence. Our results also suggest that seeds-inoculated arbuscular mycorrhizal fungi can buffer sucrose stress during germination to restore normal respiration more efficiently.

Following this approach, we propose a simple method to identify organic seeds and low-cost on-farm perspectives for early selection on disease tolerance, predicting plant holobiont behavior and improving germination. Furthermore, our research strengthens the view that AOX can serve as powerful functional marker source for seed hologenomes.

sentences: Targeting the MYC interaction network in B-cell lymphoma via histone deacetylase 6 inhibition Overexpression of oncogenic MYC is the hallmark of many lymphomas and is related to a poor prognosis. Although MYC is a potential cancer driver, therapeutic targeting is still challenging. Here, we report that histone deacetylase 6 (HDAC6) inhibition using the novel inhibitor Marbostat-100 (M-100) specifically alters protein-protein interactions in MYC-dependent cancer cells and targets MYC for proteasomal degradation. Subsequently, massive apoptosis is induced in MYC-overexpressing B-cell lymphoma cells after M-100 treatment. Besides, the application of M-100 prevents lymphoma formation in E-Myc transgenic mice and efficiently slows down tumor growth in already manifested lymphomas. Moreover, M-100 exclusively targets MYC-dependent tumor cells with little or no side effects on non-tumor cells and tissues. HDAC6 inhibition results in pleiotropic cellular effects, such as hyperacetylation of Tubulin. We propose a mechanism where the heat-shock protein DNAJA3 associates with acetylated Tubulin to control MYC turnover in malignant cells. Our data show a new mechanism how HDAC6 inhibition targets oncogenic MYC in lymphomas and demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.

sentences: A holistic insight of mycobacteriophage induced changes in mycobacterial cells Mycobacteriophages are phages that interact with mycobacteria resulting in their killing. Although lysis is the major mechanism by which mycobacteriophages cause cell death, other mechanisms may also be involved. The present study was initiated with the objective of investigating the changes that take place at the cellular level following the infection of mycobacterial cells by phage D29. To investigate this issue, we took recourse to performing immunofluorescence and electron microscopic studies. Transmission electron microscopic examination revealed the adsorption of phages on to the surface of mycobacteria, following which penetration of the tail through the thick mycoloic acid layer was seen. At later time points discrete populations of cells at different stages of lysis were observed, which comprised of completely lysed cells, in which the cells were fragmented and those at the early onset stage exhibited formation of membrane pores through which the phages and intracellular contents were released. SEM results also indicated that phages may come out through the entire surface of the cell, or alternatively through gaps in the surface. In some of the images we observed structures that apparently resembled membrane blebs which are normally encountered when cells undergo programmed cell death (PCD). In addition, we observed significant increase in DNA fragmentation as well as membrane depolarization, which are also indicative of occurrence of PCD. As several bacterial PCD pathways are mediated by the toxin-antitoxin (TA) modules, the expression profile of all the TA systems was examined before and after phage infection. Apart from specifically addressing the issue of PCD in mycobacteriophage infected cells, this investigation has led to the development of facile tools necessary for investigating mycobacteriophage-mycobacteria interactions by means of microscopic methods.

sentences: Unconstrained coevolution of bacterial size and the latent period of plastic phage Viruses play critical roles in the dynamics of microbial communities. Lytic viruses, for example, kill significant proportions of autotrophic and heterotrophic microbes. The dynamic interplay between viruses and microbes results from an overlap of physiological, ecological, and evolutionary responses: environmental changes trigger host physiological changes, affecting the ecological interactions of host and virus and, ultimately, the evolutionary pressures influencing the two populations. Recent theoretical work studied how the dependence of viral traits on host physiology (viral plasticity) affects the evolutionarily stable host cell size and viral infection time emerging from coevolution. Here, we broaden the scope of the framework to consider any coevolutionary outcome, including potential evolutionary collapses of the system. We used the case study of Escherichia coli and T-like viruses under chemo-stat conditions, but the framework can be adapted to any microbe-virus system. Oligotrophic conditions led to smaller, lower-quality but more abundant hosts, and infections that were longer but produced a reduced viral offspring. Conversely, eutrophic conditions resulted in fewer but larger higher-quality hosts, and shorter but more productive infections. The virus influenced host evolution decreasing host radius more noticeably for low than for high dilution rates, and for high than for low nutrient input concentration. For low dilution rates, the emergent infection time minimized host need/use, but higher dilution led to an opportunistic strategy that shortened the duration of infections. System collapses driven by evolution resulted from host failure to adapt quickly enough to the evolving virus. Our results contribute to understanding the eco-evolutionary dynamics of microbes and virus, and to improving the predictability of current models for host-virus interactions. The large quantitative and qualitative differences observed with respect to a classic description (in which viral traits are assumed to be constant) highlights the importance of including viral plasticity in theories describing short- and long-term host-virus dynamics.

sentences: ZIKV disrupts placental ultrastructure and drug transporter expression in mice Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome.

sentences: C. elegans enteric motor neurons fire synchronized action potentials underlying the defecation motor program The C. elegans nervous system was thought to be strictly analog, constituted solely by graded neurons. We recently discovered neuronal action potentials in the sensory neuron AWA; however, the extent to which the C. elegans nervous system relies on analog or digital neural signaling and coding is unclear. Here we report that the enteric motor neurons AVL and DVB fire all-or-none calcium-mediated action potentials that play essential roles in the rhythmic defecation behavior in C. elegans. Both AVL and DVB synchronously fire giant action potentials to faithfully execute all-or-none expulsion following the intestinal pacemaker. AVL fires unusual compound action potentials with each positive calcium-mediated spike followed by a potassium-mediated negative spike. The depolarizing calcium spikes in AVL are mediated by a CaV2 calcium channel UNC-2, while the negative potassium spikes are mediated by a repolarization-activated potassium channel EXP-2. Whole-body behavior tracking and simultaneous neural imaging in free-moving animals suggest that action potentials initiated in AVL in the head propagate along its axon to the tail and activate DVB through the INX-1 gap junction. Synchronized action potential spikes between AVL and DVB, as well as the negative spike and long-lasting afterhyperpolarization in AVL, play an important function in executing expulsion behavior. This work provides the first evidence that in addition to sensory coding, C. elegans motor neurons also use digital coding scheme to perform specific functions including long-distance communication and temporal synchronization, suggesting further, unforeseen electrophysiological diversity remains to be discovered in the C. elegans nervous system.

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Predicted structural mimicry of spike receptor-binding motifs from highly pathogenic human coronaviruses Viruses often encode proteins that mimic host proteins in order to facilitate infection. Little work has been done to understand the potential mimicry of the SARS-CoV-2, SARS-CoV, and MERS-CoV spike proteins, particularly the receptor-binding motifs, which could be important in determining tropism of the virus. Here, we use structural bioinformatics software to characterize potential mimicry of the three coronavirus spike protein receptor-binding motifs. We utilize sequence-independent alignment tools to compare structurally known or predicted three-dimensional protein models with the receptor-binding motifs and verify potential mimicry with protein docking simulations. Both human and non-human proteins were found to be similar to all three receptor-binding motifs. Similarity to human proteins may reveal which pathways the spike protein is co-opting, while analogous non-human proteins may indicate shared host interaction partners and overlapping antibody cross-reactivity. These findings can help guide experimental efforts to further understand potential interactions between human and coronavirus proteins.

HighlightsO_LIPotential coronavirus spike protein mimicry revealed by structural comparison
C_LIO_LIHuman and non-human protein potential interactions with virus identified
C_LIO_LIPredicted structural mimicry corroborated by protein-protein docking
C_LIO_LIEpitope-based alignments may help guide vaccine efforts
C_LI

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Dietary fat promotes antibiotic-induced Clostridioides difficile mortality in mice Clostridioides difficile infection (CDI), is the leading cause of hospital-acquired diarrhea and emerging evidence has linked dietary components with CDI pathogenesis, suggesting that dietary modulation may be an effective strategy for prevention. Here, we show that mice fed a high-fat/low-fiber "Western type" diet (WD) had dramatically increased mortality in a murine model of antibiotic-induced CDI compared to a low-fat/low-fiber (LF/LF) diet and standard mouse chow controls. We found that the WD had a pro- C. difficile bile acid composition that was driven in part by higher levels of primary bile acids that are produced to digest fat, and a lower level of secondary bile acids that are produced by the gut microbiome. This lack of secondary bile acids was associated with a greater disturbance to the gut microbiome with antibiotics in both the WD and LF/LF diet compared to mouse chow. Mice fed the WD also had the highest level of toxin TcdA just prior to the onset of mortality, but not of TcdB or increased inflammation. These findings indicate that dietary intervention to decrease fat may complement previously proposed dietary intervention strategies to prevent CDI in high-risk individuals.

One Sentence SummaryA high-fat/low-fiber Western type diet promoted mortality in a mouse model of antibiotic-induced C. difficile infection compared to a low-fat/low-fiber diet and chow diet, suggesting that lower dietary fat may be an effective strategy for preventing C. difficile pathology.

sentences: β-adrenergic stimulation synchronizes a broad spectrum of action potential firing rates of cardiac pacemaker cells towards a higher population average The heartbeat is initiated by pacemaker cells residing in the sinoatrial node (SAN). SAN cells generate spontaneous action potentials (APs), i.e. normal automaticity. The sympathetic nervous system increases heart rate commensurate with cardiac output demand via stimulation of SAN {beta}-adrenergic receptors ({beta}AR). While SAN cells reportedly represent a highly heterogeneous cell population, the current dogma is that in response to {beta}AR stimulation all cells increase their spontaneous AP firing rate in a similar fashion. The aim of the present study was to investigate cell-to-cell variability in the responses of a large population of SAN cells. We measured {beta}AR responses among 166 single SAN cells isolated from 33 guinea pig hearts. In contrast to the current dogma, the SAN cell responses to {beta}AR stimulation substantially varied. In each cell, changes in AP cycle length highly correlated (R2=0.97) with the AP cycle length before {beta}AR stimulation. While, as expected, on average the cells increased their pacemaker rate, greater responses were observed in cells with slower basal rates, and vice versa, cells with higher basal rates showed smaller responses, no responses, or even decreased their rate. Thus, {beta}AR stimulation synchronizes the operation of the SAN cell population towards a higher average rate, rather than uniformly shifting the rate in each cell, creating a new paradigm of {beta}AR-driven fight-or-flight response among individual pacemaker cells.

sentences: Environmental stressors induced strong small-scale phenotypic differentiation in a wide-dispersing marine snail Heterogeneous environments pose a particular challenge for organisms because the same phenotype is unlikely to perform best regardless of the variety of encountered stressors. To understand how species meet this challenge, we investigated the extent to which contrasting environmental pressures induced ecological and phenotypic responses in a natural population of a wide-dispersing marine snail at a small spatial scale. We analyzed several traits of Heleobia australis (Rissooidea: Cochliopidae) collected from heterogeneous, but highly connected, habitats from the intertidal area of the Bahia Blanca estuary, Argentina. We also conducted molecular analyses by amplifying the COI gene in individuals sampled from each habitat. We found that sympatric subpopulations of H. australis exhibited a strong phenotypic divergence in shell characters and body weight in response to thermal, saline, and dehydration stress, crab predation risk, and parasitic castrators. We proved that this differentiation occurred even early in life as most of the characters observed in juveniles mirrored those found in adults. We also found a divergence in penis size in snails collected from each habitat and raised in common garden laboratory conditions. The molecular analyses confirmed that the individuals studied constituted a single species despite the strong phenotypic differences among subpopulations. The small-scale phenotypic differentiation suggests that H. australis experienced a fine-grained environment where conditions imposed by different sources of stress favored the expression of beneficial traits. We discuss the role of plasticity in shaping adaptive phenotypic responses that increase the likelihood of persistence of subpopulations facing environmental stress conditions.

sentences: Defensive nymphs in the water-repellent gall of the social aphid Colophina monstrifica (Hemiptera: Aphididae: Eriosomatinae) The aphid Colophina monstrifica forms woolly colonies with sterile soldiers on the secondary host Clematis uncinata in Taiwan. However, the gall or primary-host generation of C. monstrifica has not been found to date. We successfully induced galls of the species on trees of Zelkova serrata through attaching its eggs onto the trees, and also found a few naturally formed galls on another Z. serrata tree. The identity of the aphids was confirmed by examining their morphology and mitochondrial DNA sequences. First- and second-instar nymphs in the galls exhibited attacking behavior toward artificially introduced moth larvae. Observations with a scanning electron microscope revealed that the gall inner surface was densely covered with minute trichomes. This indicates the water repellency of the inner surface, and strongly suggests that young nymphs of C. monstrifica dispose of honeydew globules outside the gall, as known in the congener C. clematis.

sentences: LBH is a cancer stem cell- and metastasis-driving oncogene essential for WNT stem cell function in breast cancer Cancer stem cells (CSCs) initiate tumors, resist treatment, and seed lethal metastases; yet CSC-specific treatments are lacking. Aggressive, treatment-resistant triple-negative breast cancers (TNBC) exhibit WNT pathway activation and are CSC enriched. Here, we show that Limb-Bud- and-Heart (LBH), a WNT/{beta}-catenin target required for normal mammary stem cell self-renewal, marks poor prognosis, stem-like TNBC, and is a key controller of breast cancer stemness. LBH is specifically expressed in tumor-initiating CD44+CD24-/low breast CSCs. LBH overexpression confers stem-like, metastatic traits on both TNBC and luminal origin, non-TNBC breast cancer cells by activating stem cell transcriptional programs. Importantly, silencing LBH potently suppresses tumor initiation and metastasis in vivo, and sensitizes TNBC cells to chemotherapy. LBH knockout in the MMTV-Wnt1 breast cancer mouse model, furthermore, revealed LBH is required for WNT-driven breast CSC expansion. Our findings identify LBH as an essential CSC driver downstream of WNT, and a new molecular target for anti-cancer stem cell therapy.

sentences: Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and twelve bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency rather than the antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. The anti-S2 antibody also neutralized SARS-CoV-1 and all four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

sentences: Influence of Fara-darmani Consciousness Field on Bacterial Population Growth The treatment of bacterial infections and the rising challenges of antibiotics resistance are global concerns and the primary topics in basic science and clinical microbiology. In the present study, the effectiveness of treatment of selected populations of bacteria using an immaterial and non-energetic method called Fara-darmani Consciousness Field treatment is investigated. Population growth was assessed by turbidimetry, colony counting and tetrazolium chloride reduction assays in non-treated control and Fara-darmani-treated groups. Our results suggest effectiveness of the Fara-darmani Consciousness Field on reducing various types of bacterial strain growth rates (up to 46%). In addition, along with a decrease in bacterial population, evidence of increased survival can be seen in the larger healthy population (up to about 60%). Thus, in this study, we confirm the effects of the Consciousness Field on bacterial population survival. This study also warrants additional research.

sentences: Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage ([≥]5 dpf). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA sequencing studies which identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells and interstitial stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, for select examples, we used gene knockout experiments to determine the role of newly identified genes. Our results reveal novel insights into ovarian development and function and the sequencing information will provide a valuable resource for future studies.

sentences: Annual dynamics of Zymoseptoria tritici populations in wheat cultivar mixtures: a compromise between the efficiency and durability of a recently broken-down resistance gene? Cultivar mixtures slow polycyclic epidemics but may also affect the evolution of pathogen populations by diversifying the selection pressures exerted by their plant hosts at field scale. We compared the dynamics of natural populations of the fungal pathogen Zymoseptoria tritici in pure stands and in three binary mixtures of wheat cultivars (one susceptible cultivar and one cultivar carrying the recently broken-down Stb16q gene) over two annual field epidemics. We combined analyses of population  size based on disease severity, and of population  composition based on changes in the frequency of virulence against Stb16q in seedling assays with more than 3000 strains. Disease reductions were observed in mixtures late in the epidemic, at the whole canopy scale and on both cultivars, suggesting the existence of a reciprocal  protective effect. The three cultivar proportions in the mixtures (0.25, 0.5 and 0.75) modulated the decrease in (i) the size of the pathogen population relative to the two pure stands, (ii) the size of the virulent subpopulation, and (iii) the frequency of virulence relative to the pure stand of the cultivar carrying Stb16q. Our findings suggest that optimal proportions may differ slightly between the three indicators considered. We argued potential trade-offs that should be taken into account when deploying a resistance gene in cultivar mixtures: between the dual objectives  efficacy and  durability, and between the  size and  frequency of the virulent subpopulation. Based on current knowledge, it remains unclear whether virulent subpopulation size or frequency has the largest influence on interepidemic virulence transmission.

sentences: Population-specific genome graphs improve high-throughput sequencing data analysis: A case study on the Pan-African genome Graph-based genome reference representations have seen significant development, motivated by the inadequacy of the current human genome reference to represent the diverse genetic information from different human populations and its inability to maintain the same level of accuracy for non-European ancestries. While there have been many efforts to develop computationally efficient graph-based toolkits for NGS read alignment and variant calling, methods to curate genomic variants and subsequently construct genome graphs remains an understudied problem that inevitably determines the effectiveness of the overall bioinformatics pipeline. In this study, we discuss obstacles encountered during graph construction and propose methods for sample selection based on population diversity, graph augmentation with structural variants and resolution of graph reference ambiguity caused by information overload. Moreover, we present the case for iteratively augmenting tailored genome graphs for targeted populations and demonstrate this approach on the whole-genome samples of African ancestry. Our results show that population-specific graphs, as more representative alternatives to linear or generic graph references, can achieve significantly lower read mapping errors and enhanced variant calling sensitivity, in addition to providing the improvements of joint variant calling without the need of computationally intensive post-processing steps.

sentences: Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival Zygotic Genome Activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance in shifting developmental control from primarily maternal stores in the oocyte to the embryo proper, we know little of the molecular events that initiate ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells (ESCs) have revealed Developmental Pluripotency Associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, while fertile, Dppa2/4 maternal knockout mice had reduced litter sizes, indicating decreased offspring survival. Immunofluorescence and transcriptome analyses of 2-cell embryos revealed while ZGA took place there were subtle defects in embryos lacking maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together our results show that while Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

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Ret loss-of-function decreases enteric neural crest progenitor proliferation and restricts developmental fate potential during enteric nervous system development The receptor tyrosine kinase gene RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. Pathogenic RET loss of function (LoF) alleles are associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. ENCDCs invade the developing GI tract, proliferate, migrate caudally, and differentiate into all of the major ENS cell types. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, its cell type and state-specific effects are unknown. Consequently, we performed single- cell RNA sequencing (scRNA-seq) on an enriched population of ENCDCs isolated from the developing GI tract of Ret null heterozygous and homozygous mouse embryos at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: (1) Ret-expressing ENCDCs are a heterogeneous population composed of ENS progenitors as well as glial and neuronal committed cells; (2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; (3) HSCR-associated and Ret gene regulatory network genes exhibit distinct expression patterns across Ret-expressing ENCDC with their expression impacted by Ret LoF; and (4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes associated with the proper development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment.

Summary StatementRet LoF affects proper development of the mouse ENS through multiple distinct cellular phenotypes including restriction of neuronal fate potential, disruption of ENCDC migration, and modulation of progenitor proliferation rate.

sentences: Elevated HLA-E and NKG2A as a consequence of chronic immune activation defines resistance to M. bovis BCG immunotherapy in Non-Muscle-Invasive Bladder Cancer Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the first-line treatment for non-muscle invasive bladder cancer (NMIBC), promotes the production of inflammatory cytokines, particularly interferon (IFN)-{gamma}. Prolonged inflammation and IFN-{gamma} exposure are known to cause an adaptive immune response, enabling immune escape and proliferation by tumor cells. We investigated HLA-E and NKG2A, a novel T and NK cell checkpoint pathway, as a driver of adaptive resistance in BCG unresponsive NMIBC. We observed ubiquitous inflammation in all patients after BCG immunotherapy, regardless of recurrence status. IFN-{gamma} was shown to drive tumor expression of HLA-E and PD-L1. Further, NKG2A-expressing NK and CD8 T cells were enriched in BCG unresponsive tumors and with enhanced capacity for cytolytic functions. Strikingly, in situ spatial analyses revealed that HLA-EHIGH tumors are activated to recruit NK and T cells via chemokine production, potentially sparing HLA-ELOW tumors that would otherwise be susceptible to lysis. Finally, blood-derived NK cells retained anti-tumor functions at the time of tumor recurrence. These data support combined NKG2A and PD-L1 blockade for BCG unresponsive disease.

sentences: Decline in constitutive proliferative activity in the zebrafish retina with ageing It is largely assumed that the teleost retina shows continuous and active proliferative and neurogenic activity throughout life. But when deepening in the teleost literature one finds that assumptions about a highly active and continuous proliferation in the adult retina are based on studies in which proliferation was not quantified in a comparative way at the different life stages or was mainly studied in juveniles/young adults. Here, we performed a systematic and comparative study of the constitutive proliferative activity of the retina from early developing (2 days post-fertilization) to aged (up to 3-4 years post-fertilization) zebrafish. Mitotic activity and cell cycle progression were analyzed by using immunofluorescence against pH3 and PCNA, respectively. We observed a decline in cell proliferation in the retina with ageing, even despite the occurrence of a wave of secondary proliferation during sexual maturation. During this wave of secondary proliferation the distribution of proliferating and mitotic cells changes from the inner to the outer nuclear layer in the central retina. Importantly, in aged zebrafish there is a virtual disappearance of mitotic activity. Our results showing a decline in proliferative activity of the zebrafish retina with ageing are of crucial importance since it is generally assumed that the fish retina has continuous proliferative activity throughout life.

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Paf1C denoises transcription and growth patterns to achieve organ shape reproducibility In multicellular systems, all cells exhibit transcriptional noise. However, its exact contribution to morphogenesis often remains unclear, especially in animals where cells can also move. Here we take advantage of walled plant cells, where transcriptional noise happens in tissues with a fixed topology. Using synchronously growing guard cells in stomata, we demonstrate that mutation in VIP3, a subunit of the conserved polymerase-associated factor 1 complex (Paf1C), increases transcriptional noise in Arabidopsis. This conclusion could be generalized to other group of cells at the shoot apex. Such noise translates into growth and shape defects. Indeed, in vip3 sepals, we measured higher growth heterogeneity between adjacent cells, with molecular evidence of increased local mechanical conflicts. This even culminated with the presence of negatively growing cells in specific growth conditions. Interestingly, such increased local noise makes the regional pattern of growth less clear-cut. Reduced regional conflicts lead to delay in organ growth arrest, ultimately making final organ shapes and sizes more variable. We propose that transcriptional noise is managed by Paf1C to ensure organ robustness by building up mechanical conflicts at the regional scale, instead of the local scale.

HIGHLIGHTSO_LIPaf1C controls transcriptional noise in Arabidopsis
C_LIO_LIPaf1C reduces growth heterogeneity and dampens local mechanical conflicts
C_LIO_LIImpairing both Paf1C and microtubules can trigger negative growth in Arabidopsis
C_LIO_LIScaling up local conflicts to regional ones with sharp boundaries contributes to organ shape robustness
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sentences: Multi-omics profiling of Earth's biomes reveals that microbial and metabolite composition are shaped by the environment As our understanding of the structure and diversity of the microbial world grows, interpreting how it functions is of critical interest for understanding and managing the many systems microbes influence. Despite advances in sequencing, lack of standardization challenges comparisons among studies that could provide global insight. Here, we present a multi-omics, meta-analysis of a novel, diverse set of microbial community samples collected for the Earth Microbiome Project. Standardized protocols and analytical methods for characterizing microbial communities, including assessment of function using untargeted metabolomics, facilitate identification of shared microbial and metabolite features, permitting us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of a community resource that will become more valuable with time. To provide an example of applying this database, we tested the hypothesis that metabolic potential is not limited or enriched by the environment. Our results show that the relative abundances of microbially-related metabolites vary across habitats, and highlight the power of certain chemistry - in particular alkaloids - in distinguishing Earths environments.

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DYT-TOR1A Subcellular Proteomics Reveals Selective Vulnerability of the Nuclear Proteome to Cell Stress TorsinA is a AAA+ ATPase that shuttles between the ER lumen and outer nuclear envelope in an ATP-dependent manner and is functionally implicated in nucleocytoplasmic transport. We hypothesized that the DYT-TOR1A dystonia disease-causing variant, {Delta}E TorsinA, may therefore disrupt the normal subcellular distribution of proteins between the nuclear and cytosolic compartments. To test this hypothesis, we performed proteomic analysis on nuclear and cytosolic subcellular fractions from DYT-TOR1A and wildtype mouse embryonic fibroblasts (MEFs). We further examined the compartmental proteomes following exposure to thapsigargin (Tg), an endoplasmic reticulum (ER) stressor, because DYT-TOR1A dystonia models have previously shown abnormalities in cellular stress responses. Across both subcellular compartments, proteomes of DYT-TOR1A cells showed basal state disruptions consistent with an activated stress response, and in response to thapsigargin, a blunted stress response. However, the DYT-TOR1A nuclear proteome under Tg cell stress showed the most pronounced and disproportionate degree of protein disruptions - 3-fold greater than all other conditions. The affected proteins extended beyond those typically associated with stress responses, including enrichments for processes critical for neuronal synaptic function. These findings highlight the advantage of subcellular proteomics to reveal events that localize to discrete subcellular compartments and refine thinking about the mechanisms and significance of cell stress in DYT-TOR1A pathogenesis.

HighlightsO_LIThe DYT-TOR1A nuclear proteome under cell stress showed 3-fold greater protein disruptions.
C_LIO_LIDYT-TOR1A MEFs show basal proteome alterations consistent with cell stress.
C_LIO_LIThapsigargin modulation of WT stress-responsive proteins is blunted in DYT-TOR1A MEFs.
C_LIO_LITorsinB was identified as part of the cell-stress responsive proteome in WT MEFs.
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Graphical Abstract

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sentences: m7G cap-eIF4E interaction stimulates polysome formation by enhancing first-round initiation kinetics Translation in eukaryotic cells occurs predominantly through a 7-methylguanosine (m7G) cap-dependent mechanism. m7G cap interactions with eukaryotic initiation factor 4E (eIF4E) facilitates 43S recruitment to the mRNA 5 end and enhances the translation efficiency of mRNA. However, it remains poorly understood how m7G cap-eIF4E interactions affect polysome formation kinetics. Here, we examine the role of the m7G cap in polysome formation by utilizing a single-molecule approach to track individual ribosomes during active translation. Translation was monitored in wheat germ extract with capped and uncapped synthetic mRNAs and in HeLa extract with purified human eIF4E titration. The presence of the m7G cap and the supplementation of eIF4E to eIF4E-deficient extract enhanced the kinetics of the first initiation event of polysomes. Subsequent to the first initiation event, efficient polysome-forming initiation events occurred independent of mRNA m7G capping status and eIF4E concentration. Our results indicate that m7G cap-eIF4E interactions in wheat germ and HeLa extracts promote polysome formation by enhancing first-round initiation kinetics. The dynamics of individual translation events on polysomal mRNAs suggest that first-round initiation events activate mRNAs for efficient subsequent rounds of polysome-forming initiation.

sentences: High-throughput analysis of B3GLCT regulation predicts phenotype of Peters' Plus Syndrome in line with the miRNA Proxy Hypothesis MicroRNAs (miRNAs, miRs) finely tune protein expression and target networks of 100s-1000s of genes that control specific biological processes. They are critical regulators of glycosylation, one of the most diverse and abundant posttranslational modifications. In recent work, miRs have been shown to predict the biological functions of glycosylation enzymes, leading to the "miRNA proxy hypothesis" which states, "if a miR drives a specific biological phenotype..., the targets of that miR will drive the same biological phenotype." Testing of this powerful hypothesis is hampered by our lack of knowledge about miR targets. Target prediction suffers from low accuracy and a high false prediction rate. Herein, we develop a high-throughput experimental platform to analyze miR:target interactions, miRFluR. We utilize this system to analyze the interactions of the entire human miRome with beta-3-glucosyltransferase (B3GLCT), a glycosylation enzyme whose loss underpins the congenital disorder Peters Plus Syndrome. Although this enzyme is predicted by multiple algorithms to be highly targeted by miRs, we identify only 27 miRs that downregulate B3GLCT, a >96% false positive rate for prediction. Functional enrichment analysis of these validated miRs predict phenotypes associated with Peters Plus Syndrome, although B3GLCT is not in their known target network. Thus, biological phenotypes driven by B3GLCT may be driven by the target networks of miRs that regulate this enzyme, providing additional evidence for the miRNA Proxy Hypothesis.

sentences: Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain Piezo2 mechanotransduction channel is a crucial mediator of sensory neurons for sensing and transducing touch, vibration, and proprioception. We here characterized Piezo2 expression and cell specificity in rat peripheral sensory pathway using a validated Piezo2 antibody. Immunohistochemistry using this antibody revealed Piezo2 expression in pan primary sensory neurons (PSNs) of dorsal rood ganglia (DRG) in naive rats, which was actively transported along afferent axons to both central presynaptic terminals innervating the spinal dorsal horn (DH) and peripheral afferent terminals in skin. Piezo2 immunoreactivity (IR) was also detected in the postsynaptic neurons of the DH and in the motor neurons of the ventral horn, but not in spinal GFAP- and Iba1-positive glia. Notably, Piezo2-IR was clearly identified in peripheral non-neuronal cells, including perineuronal glia, Schwann cells in the sciatic nerve and surrounding cutaneous afferent endings, as well as in skin epidermal Merkel cells and melanocytes. Immunoblots showed increased Piezo2 in DRG ipsilateral to plantar injection of complete Freunds adjuvant (CFA), and immunostaining revealed increased Piezo2-IR intensity in the DH ipsilateral to CFA injection. This elevation of DH Piezo2-IR was also evident in various neuropathic pain models and monosodium iodoacetate (MIA) knee osteoarthritis (OA) pain model, compared to controls. We conclude that 1) the pan neuronal profile of Piezo2 expression suggests that Piezo2 may function extend beyond simply touch/proprioception mediated by large-sized low-threshold mechanosensitive PSNs, 2) Piezo2 may have functional roles involving sensory processing in spinal cord, Schwann cells, and skin melanocytes, and 3) aberrant Piezo2 expression may contribute pain pathogenesis.

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POPEYE directly regulates bHLH Ib genes and its own expression Iron (Fe) is an essential trace element for plants. When suffering from Fe deficiency, plants modulate the expression of Fe deficiency responsive genes. POPEYE (PYE) is a key bHLH transcription factor involved in Fe homeostasis. However, the molecular mechanism of PYE regulating the Fe deficiency response remains elusive. We found that the over-expression of PYE attenuates the expression of Fe deficiency responsive genes. PYE directly represses the transcription of bHLH Ib genes (bHLH38, bHLH39, bHLH100, and bHLH101) by associating with their promoters. Although PYE contains an Ethylene response factor-associated Amphiphilic Repression (EAR) motif, it does not interact with the transcriptional corepressors TOPLESS/TOPLESS-RELATED (TPL/TPRs). Subcellular localization analysis indicated that PYE localizes in both the cytoplasm and nucleus. PYE contains a Nuclear Export Signal (NES) which is required for the cytoplasmic localization of PYE. The mutation of NES amplifies the repression function of PYE, resulting in downregulation of Fe deficiency responsive genes. Co-expression assays indicated that bHLH IVc members (bHLH104, bHLH105/ILR3, and bHLH115) facilitate the nuclear accumulation of PYE. Conversely, PYE indirectly represses transcription activation ability of bHLH IVc. Additionally, PYE directly negatively regulates its own transcription. This study provides insights into the complicated Fe deficiency response signaling pathway and enhances the understanding of PYE functions.

Short summaryPYE is a negative regulator of Fe homeostasis; however, it was still unclear how PYE integrates the Fe deficiency response signaling. Our study shows that conditional nuclear localization of PYE is crucial for Fe homeostasis. PYE not only negatively regulates FIT-dependent Fe uptake genes by directly targeting bHLH Ib genes, but also negatively regulates its own expression.

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Murine ex vivo cultured alveolar macrophages provide a novel tool to study tissue-resident macrophage behavior and function Tissue-resident macrophages are of vital importance as they preserve tissue homeostasis in all mammalian organs. Nevertheless, appropriate cell culture models are still limited. Here, we propose a novel culture model to study and expand murine primary alveolar macrophages (AMs), the tissue-resident macrophages of the lung, in vitro over several months. By providing a combination of GM-CSF, TGF{beta} and the PPAR{gamma} activator rosiglitazone, we maintain and expand mouse ex vivo cultured AMs, short mexAMs, over several months. MexAMs maintain typical morphologic features and stably express primary AM surface markers throughout in vitro culture. They respond to microbial ligands and exhibit an AM-like transcriptional profile, including the expression of AM specific transcription factors. Furthermore, when transferred into AM deficient mice, mexAMs efficiently engraft in the lung and fulfill key macrophage functions leading to a significantly reduced surfactant load in those mice. Altogether, mexAMs provide a novel, simple and versatile tool to study AM behavior in homeostasis and disease settings.

KEYPOINTSO_LIA novel method to culture and expand primary alveolar macrophages over several months ex vivo
C_LIO_LIMurine ex vivo cultured alveolar macrophages (mexAMs) restore lung function in a murine pulmonary alveolar proteinosis model
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sentences: Integrin β3 Targeting Biomaterial Preferentially Promotes Secretion of bFGF and Proliferation of iPSC-Derived Vascular Smooth Muscle Cells Human-induced pluripotent stem cell-derived-vascular smooth muscle cells (hiPSC-VSMC) have been shown to promote angiogenesis and wound healing. However, there is a paucity of research on how the extracellular matrix (ECM) microenvironment may impact the hiPSC-VSMCs function. In this study, our objective was to understand the effect of specific ECM ligand-integrin interaction on hiPSC- VSMCs paracrine secretion, cell proliferation, and morphology. We here showed a precise modulation of hiPSC-VSMC in a fibronectin functionalized fibrillar collagen scaffold by targeting their integrin {beta}3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin dependent via v{beta}3 integrin interactions. Also, our data indicate the possible role of a positive feedback loop between integrin {beta}3, bFGF, and matrix metalloproteinase-2 in regulating hiPSC- VSMCs morphology and cell proliferation. Finally, the secretome with improved proangiogenic activity shows potential for future regenerative applications.

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CulebrONT: a streamlined long reads multi-assembler pipeline for prokaryotic and eukaryotic genomes BackgroundUsing long reads provides higher contiguity and better genome assemblies. However, producing such high quality sequences from raw reads requires to chain a growing set of tools, and determining the best workflow is a complex task.

ResultsTo tackle this challenge, we developed CulebrONT, an open-source, scalable, modular and traceable Snakemake pipeline for assembling long reads data. CulebrONT enables to perform tests on multiple samples and multiple long reads assemblers in parallel, and can optionally perform, downstream circularization and polishing. It further provides a range of assembly quality metrics summarized in a final user-friendly report.

ConclusionsCulebrONT alleviates the difficulties of assembly pipelines development, and allow users to identify the best assembly options.

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The Voices of Medical Education Science: Describing the Published Landscape IntroductionMedical education has been described as a dynamic and growing field, driven in part by its unique body of scholarship. The voices of authors who publish medical education literature have a powerful impact on the discourses of the community. While there have been numerous studies looking at aspects of this literature, there has been no comprehensive view of recent publications.

MethodThe authors conducted a bibliometric analysis of all articles published in 24 medical education journals published between 2000-2020 to identify article characteristics, with an emphasis on author gender, geographic location, and institutional affiliation. This study replicates and greatly expands on two previous investigations by examining all articles published in these core medical education journals.

ResultsThe journals published 37,263 articles with the majority of articles published in 2020 (n=3,957, 10.7%) and the least in 2000 (n=711, 1.9%) representing a 456.5% increase. The articles were authored by 139,325 authors of which 62,708 were unique. Males were more prevalent across all authorship positions (n=62,828; 55.7%) than females (n=49,975; 44.3%). Authors listed 154 country affiliations with the United States (n=42,236, 40.4%), United Kingdom (n=12,967, 12.4%), and Canada (n=10,481, 10.0%) most represented. Ninety-three countries (60.4%) were low- or middle-income countries accounting for 9,684 (9.3%) author positions. Few articles were written by multinational teams (n=3,765; 16.2%). Authors listed affiliations with 4,372 unique institutions. Across all author positions, 48,189 authors (46.1%) were affiliated with a top 200 institution, as ranked by the Times Higher Education ranking.

DiscussionThere is a relative imbalance of author voices in medical education. If the field values a diversity of perspectives, there is considerable opportunity for improvement.

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How ancient forest fragmentation and riparian connectivity generate high levels of genetic diversity in a micro-endemic Malagasy tree AO_SCPLOWBSTRACTC_SCPLOWUnderstanding landscape changes is central to predicting evolutionary trajectories and defining conservation practices. While human-driven deforestation is intense throughout Madagascar, exception in areas like the Loky-Manambato region (North) raises questions. This region also harbors a rich and endemic flora, whose evolutionary origin remains poorly understood.

We assessed the genetic diversity of an endangered micro-endemic Malagasy olive species (Noronhia spinifolia) to better understand the vegetation dynamic in the Loky-Manambato region and its influence on past evolutionary processes. We characterized 72 individuals sampled across eight forests through nuclear and mitochondrial restriction associated sequencing data (RADseq) and chloroplast microsatellites (cpSSR). Extremely high genetic diversity was revealed in two genomic compartments (chloroplast h = 0.99, and mitochondrial h = 0.85). Combined population and landscape genetics analyses indicate that N. spinifolia diversity is best explained by the current forest cover (R2 = 0.90), highlighting a long-standing habitat mosaic in the region. Our results further suggest a predominant role of forest-dwelling organisms in mediating pollen and seed dispersals.

This sustains a major and long-term role of riparian corridors in maintaining connectivity across those antique mosaic-habitats, calling for the study of organismal interactions that promote gene flow.

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A genetic switch for male UV-iridescence in an incipient species pair of sulphur butterflies Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of UV iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern US with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knock-out, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation.

Significance statementIncipient species are at an intermediate stage of speciation where reproductive isolation is counteracted by the homogenizing effects of gene flow. Human activity sometimes leads such species to reunite, as seen in the Orange Sulphur butterfly, which forms large hybridizing populations with the Clouded Sulphur in alfalfa fields. Here we show that the sex chromosome maintains these species as distinct, while the rest of their genome is admixed. Sex chromosomes notably determine which males display to females a bright, iridescent ultraviolet signal on their wings. Genetic mapping, antibody stainings, and CRISPR knock-outs collectively indicate that the gene bric a brac controls whether UV-iridescent nanostructures develop in each species, elucidating how a master switch gene modulates a male courtship signal.

sentences: Uncontrolled CD21low age-associated and B1 B cell accumulation caused by failure of an EGR2/3 tolerance checkpoint CD21low age-associated or atypical memory B cells, enriched for autoantibodies and poised for plasma cell differentiation, accumulate in large numbers in chronic infections, autoimmune disease and immunodeficiency, posing the question of what checkpoints normally oppose their excessive accumulation. Here, we reveal a critical role for the calcium-NFAT-regulated transcription factors EGR2 and EGR3. In the absence of EGR2 and EGR3 within B cells, CD21low and B1 B cells accumulate and circulate in young mice in numbers 10-20 times greater than normal, over-express a large set of EGR2 ChIP-seq target genes including known drivers of plasma cell differentiation and under-express drivers of follicular germinal centers. Most follicular B cells constitutively express Egr2 proportionally to surface IgM down-regulation by self-antigens, and EGR2/3 deficiency abolishes this characteristic anergy response. These results define a key transcriptional checkpoint repressing CD21low B cell formation and inform how NFATC1 or EGR2 mutations promote B1 cell-derived chronic lymphocytic leukemias.

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SyNPL: Synthetic Notch pluripotent cell lines to monitor and manipulate cell interactions in vitro and in vivo. Cell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here we introduce SyNPL: clonal pluripotent stem cell lines which employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered "sender" and "receiver" cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new tool which could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and which can be adapted to generate synthetic patterning of cell fate decisions.

SUMMARY STATEMENTOptimised Synthetic Notch circuitry in mouse pluripotent stem cells provides a modular tool to monitor cell-cell interactions and program synthetic patterning of cell fates in culture and in embryos.

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Chaperones facilitate heterologous expression of naturally evolved putative de novo proteins Over the past decade, evidence has accumulated that new protein coding genes can emerge de novo from previously non-coding DNA. Most studies have focused on large scale computational predictions of de novo protein coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins is scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are predicted to be short and disordered. Here we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli.

We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30 % to 62 %. Our findings indicate that the best combination for expressing putative de novo proteins in E.coli is a GST-tag with T7 Express cells and co-expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express.

sentences: Molecular and Structural Basis of Olfactory Sensory Neuron Coalescence by Kirrel Receptors Projections from sensory neurons of olfactory systems coalesce into glomeruli in the brain. The Kirrel receptors are believed to homodimerize via their ectodomains and help separate sensory neuron axons into Kirrel2- or Kirrel3-expressing glomeruli. Here we present the crystal structures of homodimeric Kirrel receptors and show that the closely related Kirrel2 and Kirrel3 have evolved specific sets of polar and hydrophobic interactions, respectively, disallowing heterodimerization while preserving homodimerization, likely resulting in proper segregation and coalescence of Kirrel-expressing axons into glomeruli. We show that the dimerization interface at the N-terminal IG domains is necessary and sufficient to create homodimers, and fail to find evidence for a secondary interaction site in Kirrel ectodomains. Furthermore, we show that abolishing dimerization of Kirrel3 in vivo leads to improper formation of glomeruli in the mouse accessory olfactory bulb as observed in Kirrel3-/- animals. Our results provide strong evidence for Kirrel3 homodimerization controlling axonal coalescence.

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Protein interaction network analysis reveals growth conditions-specific crosstalk between chromosomal DNA replication and other cellular processes in E. coli E. coli and many other bacterial species can alter their cell cycle according to nutrient availability. Under optimal conditions bacteria grow and divide very fast but they slow down the cell cycle when conditions deteriorate. This adaptability is underlined by mechanisms coordinating cell growth with duplication of genetic material and cell division. Several mechanisms regulating DNA replication process in E. coli have been described with biochemical details so far. Nevertheless we still dont fully understand the source of remarkable precision that allows bacterial cells to coordinate their growth and chromosome replication. To shed light on regulation of E. coli DNA replication at systemic level, we used affinity purification coupled with mass spectrometry (AP-MS) to characterize protein-protein interactions (PPIs) formed by key E. coli replication proteins, under disparate bacterial growth conditions and phases. We present the resulting dynamic replication protein interaction network (PIN) and highlight links between DNA replication and several cellular processes, like outer membrane synthesis, RNA degradation and modification or starvation response.

ImportanceDNA replication is a vital process, ensuring propagation of genetic material to progeny cells. Despite decades of studies we still dont fully understand how bacteria coordinate chromosomal DNA duplication with cell growth and cell division under optimal and stressful conditions. At molecular level, regulation of processes, including DNA replication, is often executed through direct protein-protein interactions (PPIs). In this work we present PPIs formed by the key E. coli replication proteins under three different bacterial growth conditions. We show novel PPIs with confirmed impact on chromosomal DNA replication. Our results provide also alternative explanations of genetic interactions uncovered before by others for E.coli replication machinery.

sentences: Genome-scale modeling of Pseudomonas aeruginosa PA14 unveils its broad metabolic capabilities and role of metabolism in drug potentiation P. aeruginosa is an opportunistic human pathogen that is one of the leading causes of hospital-acquired infections. We have developed an updated genome-scale model (GEM) of Pseudomonas aeruginosa PA14 for systems-study of the pathogen. We used both automated and semi-manual approaches to reconstruct and curate the model. After an extensive literature research, we added organism-specific reactions (e.g., phenazine transport and redox metabolism, cofactor metabolism, carnitine metabolism, oxalate production, etc.) to the model. This effort led to a highly curated, three-compartment, and mass-and-charge balanced BiGG model of PA14 that contains 1509 genes, 1779 metabolic reactions and 1151 unique metabolites. The model (iSD1509) has the largest genome coverage of P. aeruginosa PA14 to date with 424 more genes than the previous model (iPau1129). It is also the most accurate with prediction accuracies as high as 92.4% (for gene essentiality) and 93.5% (for substrate utilization). The model simulates growth in both aerobic and anaerobic conditions. It predicts the biosynthesis of the virulence factor phenazine as a process for the pathogen to grow in low-oxygen environment. Further, a mechanism for the overproduction of a drug susceptibility biomarker (gluconate) can be elucidated by the principles of optimal growth. Finally, the model also simulates drug activity potentiation and protection by fumarate and glyoxylate, respectively, and provides mechanistic explanations for these processes. Overall, iSD1509 can be utilized to decipher the metabolic mechanisms associated with virulence and antibiotic susceptibility of P. aeruginosa PA14 to aid in the development of effective intervention strategies.

sentences: Effects of four antibiotics on the diversity of the intestinal microbiota Oral antibiotics remain the therapy of choice for severe bacterial infections; however, antibiotic use disrupts the intestinal microbiota, which increases the risk of colonization with intestinal pathogens. Currently, our understanding of antibiotic-mediated disturbances of the microbiota remains at the level of bacterial families or specific species, and little is known about the effect of antibiotics on potentially beneficial and potentially pathogenic bacteria under conditions of gut microbiota dysbiosis. Additionally, it is controversial whether the effects of antibiotics on the gut microbiota are temporary or permanent. In this study, we used 16S rRNA gene sequencing to evaluate the short-term and long-term effects of ampicillin, vancomycin, metronidazole, and neomycin on the murine intestinal microbiota by analyzing changes in the relative numbers of potentially beneficial and potentially pathogenic bacteria. We found that the changes in the intestinal microbiota reflected the antibiotics mechanisms of action and that dysbiosis of the intestinal microbiota led to competition between the different bacterial communities. Thus, destruction of bacteria with beneficial potential increased the abundance of bacteria with pathogenic potential. In addition, we found that these oral antibiotics had long-term negative effects on the intestinal microbiota and promoted the development of antibiotic-resistant bacterial strains. These results indicate that ampicillin, vancomycin, metronidazole, and neomycin have long-term negative effects and can cause irreversible changes in the diversity of the intestinal microbiota and the relative proportions of bacteria with beneficial potential and bacteria with pathogenic potential, thereby increasing the risk of host disease.

sentences: Evolution of reduced dormancy during range expansions There is increasing evidence that life-history traits can evolve rapidly during range expansion and that this evolution can impact the ecological dynamics of population spread. While dispersal evolution during range expansion has received substantial attention, dormancy (dispersal in time) has not. Here, we use an individual-based model to investigate the evolution of seed dormancy during range expansion. When a population is at spatial equilibrium our model produces results that are consistent with previous theoretical studies: seed dormancy evolves due to kin competition and the degree of dormancy increases as temporal environmental variation increases. During range expansions we consistently observe evolution towards reduced rates of dormancy at the front. Behind the front there is selection for higher rates of dormancy. Notably, the decreased dormancy towards the expanding margin reduces the regional resilience of recently expanded populations to a series of harsh years. We discuss how dormancy evolution during range expansion, and its consequences for spatial population dynamics, may impact other evolutionary responses to environmental change. We end with suggestions for future theoretical and empirical work.

sentences: A library for fMRI real-time processing systems in python (RTPSpy) with comprehensive online noise reduction, fast and accurate anatomical image processing, and online processing simulation Real-time fMRI (rtfMRI)has enormous potential for both mechanistic brain imaging studies or treatment-oriented neuromodulation. However, the adaption of rtfMRI has been limited due to technical difficulties implement an efficient computational framework. Here, we introduce a python library for real-time fMRI (rtfMRI) data processing systems, Real-Time Processing System in python (RTPSpy), to provide building blocks for a custom rtfMRI application with extensive and advanced functionalities. RTPSpy is a library package including 1) a fast, comprehensive, and flexible online fMRI image processing modules comparable to offline denoising, 2) utilities for fast and accurate anatomical image processing to define an anatomical target region, 3) a simulation system of online fMRI processing to optimize a pipeline and target signal calculation, 4) simple interface to an external application for feedback presentation, and 5) a boilerplate graphical user interface (GUI) integrating operations with RTPSpy library. The fast and accurate anatomical image processing utility wraps external tools, including FastSurfer, ANTs, and AFNI, to make tissue segmentation and region of interest masks. We confirmed that the quality of the output masks was comparable with FreeSurfer, and the anatomical image processing could complete in a few minutes. The modular nature of RTPSpy provides the ability to use it for a simulation analysis to optimize a processing pipeline and target signal calculation. We present a sample script for building a real-time processing pipeline and running a simulation using RTPSpy. The library also offers a simple signal exchange mechanism with an external application using a TCP/IP socket. While the main components of the RTPSpy are the library modules, we also provide a GUI class for easy access to the RTPSpy functions. The boilerplate GUI application provided with the package allows users to develop a customized rtfMRI application with minimum scripting labor. The limitations of the package as it relates to environment-specific implementations are discussed. These library components can be customized and can be used in parts. Taken together, RTPSpy is an efficient and adaptable option for developing rtfMRI applications. The package is available from GitHub (https://github.com/mamisaki/RTPSpy) with GPL3 license.

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Maximally predictive ensemble dynamics from data We leverage the interplay between microscopic variability and macroscopic order to connect physical descriptions across scales directly from data, without underlying equations. We reconstruct a state space by concatenating measurements in time, building a maximum entropy partition of the resulting sequences, and choosing the sequence length to maximize predictive information. Trading non-linear trajectories for linear, ensemble evolution, we analyze reconstructed dynamics through transfer operators. The evolution is parameterized by a transition time{tau} : capturing the source entropy rate at small{tau} and revealing timescale separation with collective, coherent states through the operator spectrum at larger{tau} . Applicable to both deterministic and stochastic systems, we illustrate our approach through the Langevin dynamics of a particle in a double-well potential and the Lorenz system. Applied to the behavior of the nematode worm C. elegans, we derive a "run-and-pirouette" navigation strategy directly from posture dynamics. We demonstrate how sequences simulated from the ensemble evolution capture both fine scale posture dynamics and large scale effective diffusion in the worms centroid trajectories and introduce a top-down, operator-based clustering which reveals subtle subdivisions of the "run" behavior.

POPULAR SUMMARYComplex structure is often composed from a limited set of relatively simple building blocks; such as novels from letters or proteins from amino acids. In musical composition, e.g., sounds and silences combine to form longer time scale structures; motifs form passages which in turn form movements. The challenge we address is how to identify collective variables which distinguish structures across such disparate time scales. We introduce a principled framework for learning effective descriptions directly from observations. Just as a musical piece transitions from one movement to the next, the collective dynamics we infer consists of transitions between macroscopic states, like jumps between metastable states in an effective potential landscape.

The statistics of these transitions are captured compactly by transfer operators. These operators play a central role, guiding the construction of maximally-predictive short-time states from incomplete measurements and identifying collective modes via eigenvalue decomposition. We demonstrate our analysis in both stochastic and deterministic systems, and with an application to the movement dynamics of an entire organism, unravelling new insight in long time scale behavioral states directly from measurements of posture dynamics. We can, in principle, also make connections to both longer or shorter timescales. Microscopically, postural dynamics result from the fine scale interactions of actin and myosin in the muscles, and from electrical impulses in the brain and nervous system. Macroscopically, behavioral dynamics may be extended to longer time scales, to moods or dispositions, including changes during aging, or over generations due to ecological or evolutionary adaptation. The generality of our approach provides opportunity for insights on long term dynamics within a wide variety of complex systems.

sentences: Redox potential linked to water loss from proteins in evolution and development Reactions involving water and oxygen are basic features of geological and biological processes. To understand how life interacts with its environment requires monitoring interactions with H2O and O2 at timescales relevant to not only organismal growth but also billions of years of geobiological evolution. Here, chemical transformations intrinsic to evolution and development were characterized by analyzing data from recent phylostratigraphic and proteomic studies. This two-stage analysis involves obtaining chemical metrics (carbon oxidation state and stoichiometric hydration state) from the elemental compositions of proteins followed by modeling the relative stabilities of target proteins against a proteomic background to infer thermodynamic parameters (oxygen fugacity, water activity, and virtual redox potential (Eh)). The main results of this study are a rise in carbon oxidation state of proteins spanning the time of the Great Oxidation Event, a rise in virtual redox potential that coincides with the likely emergence of aerobic metabolism, a rise in carbon oxidation state of proteins inferred from the transcriptome in late stages of Bacillus subtilis biofilm growth, and a drop in stoichiometric hydration state of the fruit fly developmental proteome at the same time as a drop in organismal water content. Stoichiometric hydration state also decreases for proteins with more recent gene ages and through stages of biofilm development, leading to predictions of higher hydration potentials at earlier time points. By building chemical representations of protein evolution and developmental proteomes, exciting new types of geobiochemical proxies can be developed.

sentences: The effects of chronic consumption of lipid-rich and delipidated bovine dairy milk on brown adipose tissue volume in wild-type mice Brown adipose tissue (BAT) activation is associated with increased energy expenditure by inducing non-shivering thermogenesis. Ingestion of a milk fat globule membrane (MFGM) supplement and a high calorie diet are reported gateways into BAT activation. However, little is known about the effect of MFGM and high calorie diets on BAT volume. To gain insight into this, mice were maintained on a high fat (HF) or low-fat (LF) diet in conjunction with either full-cream (FC) or skim bovine dairy milk (BDM). After being maintained on their respective diets for 13 weeks, body composition, including BAT volume, was measured using X-ray microtomography. A high calorie diet resulted in an increase in BAT volume and mice consuming a HF diet in conjunction with FC BDM had significantly greater BAT volume than all other groups. Conversely, mice consuming a HF diet in addition to skim milk had lower BAT volume compared to the HF control. The data presented suggests that consumption of a high calorie diet in conjunction with FC BDM increases BAT volume in wild-type mice. This study may provide valuable insight into future studies investigating BAT volume and BAT activity in relation to environmental factors including diet.

sentences: APOBEC1 mediated C-to-U RNA editing: target sequence and trans-acting factor contribution to 177 RNA editing events in 119 murine transcripts in-vivo Mammalian C-to-U RNA editing was described more than 30 years ago as a single nucleotide modification in APOB RNA in small intestine, later shown to be mediated by the RNA-specific cytidine deaminase APOBEC1. Reports of other examples of C-to-U RNA editing, coupled with the advent of genome-wide transcriptome sequencing, identified an expanded range of APOBEC1 targets. Here we analyze the cis-acting regulatory components of verified murine C-to-U RNA editing targets, including nearest neighbor as well as flanking sequence requirements and folding predictions. We summarize findings demonstrating the relative importance of trans-acting factors (A1CF, RBM47) acting in concert with APOBEC1. Using this information, we developed a multivariable linear regression model to predict APOBEC1 dependent C-to-U RNA editing efficiency, incorporating factors independently associated with editing frequencies based on 103 Sanger-confirmed editing sites, which accounted for 84% of the observed variance. Co-factor dominance was associated with editing frequency, with RNAs targeted by both RBM47 and A1CF observed to be edited at a lower frequency than RBM47 dominant targets. The model also predicted a composite score for available human C-to-U RNA targets, which again correlated with editing frequency.

sentences: The mirror neuron system compensates for amygdala dysfunction-associated social deficits in individuals with higher autistic traits The amygdala is a core node in the social brain which exhibits structural and functional abnormalities in Autism spectrum disorder and there is evidence that the mirror neuron system (MNS) can functionally compensate for impaired emotion processing following amygdala lesions. In the current study, we employed an fMRI paradigm in 241 subjects investigating MNS and amygdala responses to observation, imagination and imitation of dynamic facial expressions and whether these differed in individuals with higher as opposed to lower autistic traits. Results indicated that individuals with higher compared to lower autistic traits showed reduced left amygdala responses to imitation and enhanced responses in the left superior temporal sulcus (STS) of the MNS to observation, imagination and imitation. Additionally, functional connectivity between the left amygdala and the left STS as well as some other MNS regions was increased in individuals with higher autistic traits, especially during imitation of fearful expressions. The amygdala-MNS connectivity significantly moderated autistic group differences on recognition memory for fearful faces and real-life social network indices, indicating that increased amygdala-MNS connectivity could diminish the social behavioral differences between higher and lower autistic trait groups. Overall, findings demonstrate decreased imitation-related amygdala activity in individuals with higher autistic traits in the context of increased cortical MNS activity and amygdala-MNS connectivity which may functionally compensate for amygdala dysfunction and social deficits. Training targeting the MNS may capitalize on this compensatory mechanism for therapeutic benefits in Autism spectrum disorder.

sentences: Evolutionary Genetic Species Detected in Prokaryotes by Applying the K/θ Ratio to DNA Sequences Previous papers in this series described the "evolutionary genetic species concept" which is based on population genetic theory and should be applicable to any organism. Also described was a species criterion, the K/{theta} ratio, that delimits independently evolving evolutionary species based on single-gene sequences, and its application to sexual and asexual eukaryotes was illustrated. Here, we show how the evolutionary genetic species concept and the K/{theta} ratio can be applied to bacteria, using the sequences from some genes of the core genome that are rarely, if ever, transferred horizontally between different species. This completes the demonstration that K/{theta} is a general method for species delimitation, applicable to all organisms. Also, it adds to the evidence that bacteria have species in the most general sense, even though they have the ability to exchange genes across species boundaries. Finally, we show that a published critique of the use of K/{theta} [≥] 4 as a criterion for independently evolving species rests on two errors in the application of population/evolutionary genetic theory.

sentences: Molecular dissection of condensin II-mediated chromosome assembly using in vitro assays In vertebrates, condensin I and condensin II cooperate to assemble rod-shaped chromosomes during mitosis. Despite recent progress in our understanding of the mechanism of action of condensin I, how condensin II might work has remained largely unexplored. By introducing recombinant condensin II complexes into Xenopus egg extracts, we dissect the roles of its individual subunits in chromosome assembly. We show that two HEAT subunits, CAP-D3 and CAP-G2, play positive and negative roles, respectively, in condensin II-mediated chromosome assembly. The SMC ATPase and the basic amino acid clusters of the kleisin subunit are essential for this process. Deletion of the C-terminal tail of CAP-D3 increases the ability of condensin II to assemble chromosomes and further exposes a hidden function of CAP-G2 in the lateral compaction of chromosomes. Taken together, our results uncover a multilayered regulatory mechanism unique to condensin II, and provide profound implications in the evolution of condensin II.

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Intrinsic Mechanisms in the Gating of Resurgent Na+ Currents The resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps, which has been shown to contribute to regulating the firing properties of numerous neuronal cell types throughout the central and peripheral nervous systems. Although mediated by the same voltage-gated sodium (Nav) channels that underlie the transient and persistent Nav current components, the gating mechanisms that contribute to the generation of INaR remain unclear. Here, we characterized Nav currents in mouse cerebellar Purkinje neurons, and used tailored voltage-clamp protocols to define how the voltage and the duration of the initial membrane depolarization affect the amplitudes and kinetics of INaR. Using the acquired voltage-clamp data, we developed a novel Markov kinetic state model with parallel (fast and slow) inactivation pathways and, we show that this model reproduces the properties of the resurgent, as well as the transient and persistent, Nav currents recorded in (mouse) cerebellar Purkinje neurons. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state. Additional simulations reveal that extrinsic factors that affect the kinetics of fast or slow Nav channel inactivation and/or impact the relative distribution of Nav channels in the fast- and slow-inactivated states, such as the accessory Nav{beta}4 channel subunit, can modulate the amplitude of INaR.

SUMMARYThe resurgent component of the voltage-gated sodium current (INaR) is revealed on membrane hyperpolarizations following brief depolarizing voltage steps that activate the rapidly activating and inactivating, transient Nav current (INaT). To probe the mechanisms contributing to the generation and properties of INaR, we combined whole-cell voltage-clamp recordings from mouse cerebellar Purkinje neurons with computational modeling to develop a novel, blocking particle-independent, model for the gating of INaR that involves two parallel inactivation pathways, and we show that this model recapitulates the detailed biophysical properties of INaR measured in mouse cerebellar Purkinje neurons.

sentences: Milk-Way Algorithm applied in Imbalanced Dataset We wish to evaluate the algorithm Milk-Way, using a known dataset deposited in a public repository. The new algorithm, which converges various techniques from different areas of knowledge, can classify ligands and select potential new drugs. It was used a dataset of ligands, organized by 15 Bioassays and described by different fingerprints. Full details of the dataset architecture were already published in a public repository. Through the stratified feature selection, using the Milk-Way algorithm, the True Positive and False Positive Rates reached a higher performance compared to the published paper. Using all the features available for each Bioassay, we reached the lowest metrics in all of them. We demonstrated that adding more features have not made a significant impact on the performance. In all the Bioassays, the True Positives and False Positives reached 100% and 0%, respectively, only using 50% and 75% of the features available. The Milk-Way algorithm suggests a holistic approach, which will contribute to the machine-learning area, namely to classified ligands in the virtual screening.

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Off-target expression of Cre-dependent adeno-associated viruses in wild type C57BL/6J mice Adeno-associated viruses (AAVs) are a commonly used tool in neuroscience to efficiently label, trace, and/or manipulate neuronal populations. Highly specific targeting can be achieved through recombinase-dependent AAVs in combination with transgenic rodent lines that express Cre-recombinase in specific cell types. Visualization of viral expression is typically achieved through fluorescent reporter proteins (e.g., GFP or mCherry) packaged within the AAV genome. Although non-amplified fluorescence is usually sufficient to observe viral expression, immunohistochemical amplification of the fluorescent reporter is routinely used to improve viral visualization. In the present study, Cre-dependent AAVs were injected into the hippocampus and cortex of wild-type C57BL/6J mice. While we observed weak but consistent non-amplified off-target DIO expression in C57BL/6J mice, antibody amplification of the GFP or mCherry reporter revealed extensive Cre-independent viral expression. Off-target expression of DIO constructs in wild-type C57BL/6J mice occurred independent of vendor, AAV serotype or promoter. We also evaluated whether Cre-independent expression had functional effects via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). The DREADD agonist C21 had no effect on contextual fear conditioning or cFos expression in DIO-hM3Dq-mCherry+ cells of C57BL/6J mice. Taken together, our results indicate that DIO constructs have considerable off-target expression in wild type subjects. Our findings are particularly important for the design of experiments featuring sensitive systems and/or quantitative measurements that could be negatively impacted by off-target expression.

Significance StatementAdeno-associated viruses (AAV) are widely used in neuroscience because of their safety and ease of use. Combined with specific promoters, Cre/loxP, and stereotaxic injections, highly specific targeting of cells and circuits within the brain can be achieved. In the present study we injected Cre-dependent AAVs into wild-type C57BL/6J mice and found considerable Cre-independent viral expression of AAVs encoding mCherry, GFP, or hM3Dq following immunohistochemical amplification of the fluorescent reporter protein. Importantly, we observed no functional effects of the Cre-independent expression in the hippocampus, as C21 had no detectable effect on DIO-hM3Dq-mCherry infected neurons in C57BL/6J mice. Given the widespread use of DIO rAAVs by the neuroscience community, our data supports careful consideration when using DIO constructs in control animals.

sentences: Population structure of chum salmon and selection on the markers collected for stock identification Genetic stock identification (GSI) is a major management tool of Pacific salmon (Oncorhynchus Spp.) that has provided rich genetic baseline data of allozymes, microsatellites, and single nucleotide polymorphisms (SNPs) across the Pacific Rim. Here, we analyzed published data sets for adult chum salmon (Oncorhynchus keta), namely 10 microsatellites, 53 SNPs, and a mitochondrial DNA locus (mtDNA3, control region and NADH-3 combined) from 495 locations in the same distribution range (n = 61,813). TreeMix analysis of the microsatellite loci identified the highest level of genetic drift towards Japanese/Korean populations and suggested two admixture events from Japan/Korea to Russia and the Alaskan Peninsula. The SNPs had been purposively collected from rapidly evolving genes to increase the power of GSI. The highest expected heterozygosity was observed in Japanese/Korean populations for microsatellites, whereas it was highest in Western Alaskan populations for SNPs, reflecting the SNP discovery process. By regressing the SNP population structures on those of the microsatellites, we estimated the selection on the SNP loci according to deviations from the predicted structures. Specifically, we matched the sampling locations of the SNPs with those of the microsatellites according to geographical information and performed regression analyses of SNP allele frequencies on the two coordinates of multi-dimensional scaling (MDS) of matched locations obtained from microsatellite pairwise FST values. The MDS first axis indicated a latitudinal cline in American and Russian populations, whereas the second axis found a differentiation of Japanese/Korean populations. The top five outlier SNPs were mtDNA3 (combined locus of the control region and NADH-3), U502241 (unknown), GnRH373, ras1362, and TCP178, which were consistently identified by principal component analysis. We summarized the functions of the 53 nuclear SNPs and mtDNA3 locus by referring to a gene database system and discussed the functions of the outlier SNPs and fitness of chum salmon.

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Illusory tactile movement crosses arms and legs and is coded in external space Humans often misjudge where on the body a touch occurred. Theoretical accounts have ascribed such misperceptions to local interactions in peripheral and primary somatosensory neurons, positing that spatial-perceptual mechanisms adhere to limb boundaries and skin layout. Yet, perception often reflects integration of sensory signals with prior experience. On their trajectories, objects often touch multiple limbs; therefore, body-environment interactions should manifest in perceptual mechanisms that reflect external space.

Here, we demonstrate that humans perceived the cutaneous rabbit illusion - the percept of multiple identical stimuli as hopping across the skin - along the Euclidian trajectory between stimuli on two body parts and regularly mislocalized stimuli from one limb to the other. A Bayesian model based on Euclidian, as opposed to anatomical, distance faithfully reproduced key aspects of participants localization behavior.

Our results suggest that prior experience of touch in space critically shapes tactile spatial perception and illusions beyond anatomical organization.

sentences: Broad neutralization against SARS-CoV-2 variants induced by a modified B.1.351 protein-based COVID-19 vaccine candidate Beginning in late 2020, the emergence and spread of multiple variant SARS-CoV-2 strains harboring mutations which may enable immune escape necessitates the rapid evaluation of second generation COVID-19 vaccines, with the goal of inducing optimized immune responses that are broadly protective. Here we demonstrate in a mouse immunogenicity study that two doses of a modified B.1.351 spike (S)-Trimer vaccine (B.1.351 S-Trimer) candidate can induce strong humoral immune responses that can broadly neutralize both the original SARS-CoV-2 strain (Wuhan-Hu-1) and Variants of Concern (VOCs), including the UK variant (B.1.1.7), South African variant (B.1.351) and Brazil variant (P.1). Furthermore, while immunization with two doses (prime-boost) of Prototype S-Trimer vaccine (based on the original SARS-CoV-2 strain) induced lower levels of cross-reactive neutralization against the B.1.351 variant, a third dose (booster) administered with either Prototype S-Trimer or B.1.351 S-Trimer was able to increase neutralizing antibody titers against B.1.351 to levels comparable to neutralizing antibody titers against the original strain elicited by two doses of Prototype S-Trimer.

sentences: Attentional brain rhythms during prolonged cognitive activity As routine and lower demand cognitive tasks are taken over by automated assistive systems, human operators are increasingly required to sustain cognitive demand over long periods of time. This has been reported to have long term adverse effects on cardiovascular and mental health. However, it remains unclear whether prolonged cognitive activity results in a monotonic decrease in the efficiency of the recruited brain processes, or whether the brain is able to sustain functions over time spans of one hour and more. Here, we show that during working sessions of one hour or more, contrary to the prediction of a monotonic decline, behavioral performance in both humans and non-human primates consistently fluctuates between periods of optimal and suboptimal performance at a very slow rhythm of circa 5 cycles per hour. These fluctuations are observed in both high attentional (in non-human primates) and low attentional (in humans) demand conditions. They coincide with fluctuations in pupil diameter, indicating underlying changes in arousal and information-processing load. Accordingly, we show that these rhythmic behavioral fluctuations correlate, at the neurophysiological level, with fluctuations in the informational attention orientation and perception processing capacity of prefrontal neuronal populations. We further identify specific markers of these fluctuations in LFP power, LFP coherence and spike-field coherence, pointing towards long-range rhythmic modulatory inputs to the prefrontal cortex rather than a local prefrontal origin. These results shed light on the resilience of brain mechanisms to sustained effort and have direct implications on how to optimize high cognitive demand working and learning environments.

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Design considerations for workflow management systems use in production genomics research and the clinic BackgroundThe changing landscape of genomics research and clinical practice has created a need for computational pipelines capable of efficiently orchestrating complex analysis stages while handling large volumes of data across heterogeneous computational environments. Workflow Management Systems (WfMSs) are the software components employed to fill this gap.

ResultsThis work provides an approach and systematic evaluation of key features of popular bioinformatics WfMSs in use today: Nextflow, CWL, and WDL and some of their executors, along with Swift/T, a workflow manager commonly used in high-scale physics applications. We employed two use cases: a variant-calling genomic pipeline and a scalability-testing framework, where both were run locally, on an HPC cluster, and in the cloud. This allowed for evaluation of those four WfMSs in terms of language expressiveness, modularity, scalability, robustness, reproducibility, interoperability, ease of development, along with adoption and usage in research labs and healthcare settings. This article is trying to answer, "which WfMS should be chosen for a given bioinformatics application regardless of analysis type?".

ConclusionsThe choice of a given WfMS is a function of both its intrinsic language and engine features. Within bioinformatics, where analysts are a mix of dry and wet lab scientists, the choice is also governed by collaborations and adoption within large consortia and technical support provided by the WfMS team/community. As the community and its needs continue to evolve along with computational infrastructure, WfMSs will also evolve, especially those with permissive licenses that allow commercial use. In much the same way as the dataflow paradigm and containerization are now well understood to be very useful in bioinformatics applications, we will continue to see innovations of tools and utilities for other purposes, like big data technologies, interoperability, and provenance.

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The transposable element-rich genome of the cereal pest Sitophilus oryzae BackgroundThe rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions.

ResultsWe sequenced the Sitophilus oryzae genome using a combination of short and long reads to produce the best assembly for a Curculionidae species to date. We show that S. oryzae has undergone successive bursts of transposable element (TE) amplification, representing 72% of the genome. In addition, we show that many TE families are transcriptionally active, and changes in their expression are associated with insect endosymbiotic state. S. oryzae has undergone a high gene expansion rate, when compared to other beetles. Reconstruction of host-symbiont metabolic networks revealed that, despite its recent association with cereal weevils (30 Kyear), S. pierantonius relies on the host for several amino acids and nucleotides to survive and to produce vitamins and essential amino-acids required for insect development and cuticle biosynthesis.

ConclusionsHere we present the genome of an agricultural pest beetle, which may act as a foundation for pest control. In addition, S. oryzae may be a useful model for endosymbiosis, and studying TE evolution and regulation, along with the impact of TEs on eukaryotic genomes.

sentences: Over 1000 tools reveal trends in the single-cell RNA-seq analysis landscape Recent years have seen a revolution in single-cell technologies, particularly single-cell RNA-sequencing (scRNA-seq). As the number, size and complexity of scRNA-seq datasets continue to increase, so does the number of computational methods and software tools for extracting meaning from them. Since 2016 the scRNA-tools database has catalogued software tools for analysing scRNA-seq data. With the number of tools in the database passing 1000, we take this opportunity to provide an update on the state of the project and the field. Analysis of five years of analysis tool tracking data clearly shows the evolution of the field, and that the focus of developers has moved from ordering cells on continuous trajectories to integrating multiple samples and making use of reference datasets. We also find evidence that open science practices reward developers with increased recognition and help accelerate the field.

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Analyzing Brain Morphology in Alzheimer's Disease Using Discriminative and Generative Spiral Networks Several patterns of atrophy have been identified and strongly related to Alzheimers disease (AD) pathology and its progression. Morphological changes in brain shape have been identified up to ten years before clinical diagnoses of AD, making its early detection more relevant. We propose novel geometric deep learning frameworks for the analysis of brain shape in the context of neurodegeneration caused by AD. Our deep neural networks learn low-dimensional shape descriptors of multiple neuroanatomical structures, instead of handcrafted features for each structure. A discriminative network using spiral convolution on 3D meshes is constructed for the in-vivo binary classification of AD from healthy controls (HCs) using a fast and efficient "spiral" convolution operator on 3D triangular mesh surfaces of human brain subcortical structures extracted from T1-weighted magnetic resonance imaging (MRI). Our network architecture consists of modular learning blocks using residual connections to improve overall classifier performance.

In this work: (1) a discriminative network is used to analyze the efficacy of disease classification using input data from multiple brain structures and compared to using a single hemisphere or a single structure. It also outperforms prior work using spectral graph convolution on the same the same tasks, as well as alternative methods that operate on intermediate point cloud representations of 3D shapes. (2) Additionally, visual interpretations for regions on the surface of brain structures that are associated to true positive AD predictions are generated and fall in accordance with the current reports on the structural localization of pathological changes associated to AD. (3) A conditional generative network is also implemented to analyze the effects of phenotypic priors given to the model (i.e. AD diagnosis) in generating subcortical structures. The generated surface meshes by our model indicate learned morphological differences in the presence of AD that agrees with the current literature on patterns of atrophy associated to the disease. In particular, our inference results demonstrate an overall reduction in subcortical mesh volume and surface area in the presence of AD, especially in the hippocampus. The low-dimensional shape descriptors obtained by our generative model are also evaluated in our discriminative baseline comparisons versus our discriminative network and the alternative shape-based approaches.

sentences: Amyloid fibrils in frontotemporal lobar degeneration with TDP-43 inclusions are composed of TMEM106B, rather than TDP-43 FTLD is the third most common neurodegenerative condition, following only Alzheimers and Parkinsons diseases. FTLD typically presents in 45-64-year-olds with behavioral changes or progressive decline of language skills. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions detected by TDP-43 immunoreactivity. Here, we extracted amyloid fibrils from brains of four patients, representing four out of five FTLD-TDP subclasses and determined their near-atomic resolution structures by cryo-EM. Unexpectedly, all amyloid fibrils examined are composed of a 135-residue C-terminal fragment of TMEM106B, a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP. In addition to TMEM106B fibrils, abundant non-fibrillar aggregated TDP-43 is present, as revealed by immunogold labeling. Our observations confirm that FTLD-TDP is an amyloid-involved disease and suggest that amyloid involvement in FTLD-TDP is of protein TMEM106B, rather than of TDP-43.

sentences: Seasonal stability of SARS-CoV-2 in biological fluids Transmission of SARS-CoV-2 occurs by close contact with infected persons through droplets, the inhalation of infectious aerosols and the exposure to contaminated surface. Previously, we determined the virus stability on different types of surfaces under indoor and seasonal climatic conditions. SARS-CoV-2 survived the longest on surfaces under winter conditions, followed by spring/fall and summer conditions, suggesting the seasonal pattern of stability on surfaces. However, under natural conditions, the virus is secreted in various biological fluids from infected humans. In this respect, it remains unclear how long the virus survives in various types of biological fluids. This study explored the SARS-CoV-2 stability in human biological fluids under different environmental conditions and estimated the half-life. The virus was stable for up to 21 days in nasal mucus, sputum, saliva, tear, urine, blood, and semen; it remained infectious significantly longer under winter and spring/fall conditions than under summer conditions. In contrast, the virus was only stable up to 24 hours in feces and breast milk. These findings demonstrate the potential risk of infectious biological fluids in SARS-CoV-2 transmission and have implications for its seasonality.

sentences: General dimensions of human brain morphometry inferred from genome-wide association data Understanding the neurodegenerative mechanisms underlying cognitive declines in the general population may facilitate early detection of adverse health outcomes in late life. We develop Genomic Principal Components Analysis (genomic PCA) to elucidate the complex biological connections between macroscopic brain organisation, ageing, and cognitive ability. A genetic principal component (PC) representing dimensions of shared genetic architecture in brain morphometry across the whole brain accounted for 40% of the genetic variance across 83 individual brain regions. Genomic PCs corresponding to more specific canonical brain networks of interest accounted for 47-65% of the genetic variance in the corresponding brain regions. These genomic PCs were negatively associated with a measure of brain age (rg = -0.34) and the loadings of individual brain regions on the whole brain genomic PC corresponded to sensitivity of the corresponding region to age (r = -0.27). We identified positive genetic associations between the genomic PCs of brain morphometry and a general factor of cognitive ability (rg = 0.17-0.21 for different networks). Genomic PCA allowed us to model structural covariance within brain networks at the level of their underlying genetic architecture. We identified substantial genetic overlap between brain morphometry, ageing, and cognitive ability.

sentences: Examining the effect of chronic intranasal oxytocin administration on the neuroanatomy and behavior of three autism-related mouse models 1Although initially showing great potential, oxytocin treatment has encountered a translational hurdle in its promise of treating the social deficits of autism. Some debate surrounds the ability of oxytocin to successfully enter the brain, and therefore modify neuroanatomy. Moreover, given the heterogeneous nature of autism, treatment will only amerliorate symptoms in a subset of patients. Therefore, to determine whether oxytocin changes brain circuitry, and whether it does so variably, depending on genotype, we implemented a large randomized, blinded, placebo-controlled, preclinical study on chronic intranasal oxytocin treatment in three different mouse models related to autism with a focus on using neuroanatomical phenotypes to assess and subset treatment response. Intranasal oxytocin (0.6IU) was administered daily, for 28 days, starting at 5 weeks of age to the 16p11.2 deletion, Shank3 (exon 4-9) knockout, and Fmr1 knockout mouse models. Given the sensitivity of structural magnetic resonance imaging (MRI) to the neurological effects of interventions like drugs, along with many other advantages, the mice underwent in vivo longitudinal and high-resolution ex vivo imaging with MRI. The scans included three in vivo T1-weighted, 90um isotropic resolution scans and a T2-weighted, 3D fast spin echo with 40um isotropic resolution ex vivo scan to assess the changes in neuroanatomy using established automated image registration and deformation based morphometry approaches in response to oxytocin treatment. The behavior of the mice was assessed in multiple domains, including social behaviours and repetitive behaviours, among others. Treatment effect on the neuroanatomy did not reach significance, although the pattern of trending effects was promising. No significant effect of treatment was found on social behavior in any of the strains, although a significant effect of treatment was found in the Fmr1 mouse, with treatment normalizing a grooming deficit. No other treatment effect on behavior was observed that survived multiple comparisons correction. Overall, chronic treatment with oxytocin had limited effects on the three mouse models related to autism, and no promising pattern of response susceptibility emerged.

sentences: Linking CFTR modulators to opportunistic bacterial infections in cystic fibrosis Cystic fibrosis transmembrane conductance regulator (CFTR) modulators improve clinical outcomes with variable efficacy in patients with cystic fibrosis (CF). However, changes produced by bacterial persistence and adaptation in addition to antibiotic regimens could influence CFTR modulator efficacy and vice versa and hence clinical outcomes. We first evaluated the effects of ivacaftor (IVA), lumacaftor (LUM), tezacaftor, elexacaftor and elexacaftor/tezacaftor/ivacaftor (ETI), alone or combined with antibiotics, on sequential Staphylococcus aureus and Pseudomonas aeruginosa CF isolates. IVA and ETI showed the most potent direct antimicrobial activity against S. aureus, while P. aeruginosa was not affected. Additive effects or synergies were observed between the CFTR modulators and antibiotics against both S. aureus and P. aeruginosa, independently of the stage of colonization. IVA and LUM were the most effective in potentiating antibiotic activity against S. aureus, while IVA and ETI enhanced mainly polymyxins activity against P. aeruginosa. Next, we evaluated the effect of P. aeruginosa pneumonia on the pharmacokinetics of IVA in mice. The time-concentration curves of IVA and its metabolites in plasma, lung and epithelial lining fluid were influenced by P. aeruginosa infection. The area under the concentration-time curve showed that airway exposure to IVA was greater in infected than non-infected mice. These results suggest that CFTR modulators can have direct antimicrobial properties and/or enhance antibiotic activity against S. aureus and P. aeruginosa. Furthermore, bacterial infection impacts the IVA concentration and airway exposure, potentially affecting its efficacy. Our findings suggest optimizing host- and pathogen-directed drug regimens to improve efficacy for personalized treatment.

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Proteogenomics Reveals how Metastatic Melanoma Modulates the Immune System to Allow Immune Evasion Malignant melanoma (MM) develops from the melanocytes and in its advanced stage is the most aggressive type of skin cancer. Here we report a comprehensive analysis on a prospective cohort study, including non-tumor, primary and metastasis tissues (n=77) with the corresponding plasma samples (n=56) from patients with malignant melanoma. The tumors and surrounding tissues were characterized with a combination of high-throughput analyses including quantitative proteomics, phosphoproteomics, acetylomics, and whole exome sequencing (WES) combined with in-depth histopathology analysis. Melanoma cell proliferation highly correlates with dysregulation at the proteome, at the posttranslational- and at the transcriptome level. Some of the changes were also verified in the plasma proteome. The metabolic reprogramming in melanoma includes upregulation of the glycolysis and the oxidative phosphorylation, and an increase in glutamine consumption, while downregulated proteins involved in the degradation of amino acids, fatty acids, and the extracellular matrix (ECM) receptor interaction. The pathways most dysregulated in MM including the MAP kinases-, the PI3K-AKT signaling, and the calcium homeostasis, are among the most affected by mutations, thus, dysregulation in these pathways can be manifested as drivers in melanoma development and progression.

The phosphoproteome analysis combined with target-based prediction mapped 75% of the human kinome. Melanoma cell proliferation was driven by two key factors: i) metabolic reprogramming leading to upregulation of the glycolysis and oxidative phosphorylation, supported by HIF-1 signaling pathway and mitochondrial translation; and ii) a dysregulation of the immune system response, which was mirrored by immune system processes in the plasma proteome. Regulation of the melanoma acetylome and expression of deacetylase enzymes discriminated between groups based on tissue origin and proliferation, indicating a way to guide the successful use of HDAC inhibitors in melanoma. The disease progression toward metastasis is driven by the downregulation of the immune system response, including MHC class I and II, which allows tumors to evade immune surveillance. Altogether, new evidence is provided at different molecular levels to allow improved understanding of the melanoma progression, ultimately contributing to better treatment strategies.

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sentences: Impaired dynamics of brain precapillary sphincters and first order capillaries explains reduced neurovascular functions in aging The microvascular inflow tract (MIT), comprising the penetrating arterioles, precapillary sphincters, and first order capillaries, is the bottleneck for brain blood flow and energy supply. However, the exact structural and functional alterations of the MIT during aging remain elusive. In vivo 4-dimensional two-photon imaging showed an age-dependent decrease in vaso-responsivity, with reduced sensitivity of the MIT to pinacidil and papaverine, vasoconstrictor endothelin-1, and nitric oxide synthase inhibitor L-NAME. This was accompanied by an age-dependent decrease in capillary density close to the arterioles and loss of pericyte processes, though the number of pericyte somas and pericyte SMA density were preserved. The age-related reduction in vascular reactivity was most pronounced at precapillary sphincters, highlighting their crucial role in capillary blood flow regulation. Mathematical modeling revealed dysregulated but preserved pressure and flow in aged mice during vasoconstriction. Preventing reduced responsivity of the MIT may ameliorate the blood flow decrease associated with aging-related brain frailty.

sentences: Using Domain-Knowledge to Assist Lead Discovery in Early-Stage Drug Design We are interested in generating new small molecules which could act as inhibitors of a biological target, when there is limited prior information on target-specific inhibitors. This form of drug-design is assuming increasing importance with the advent of new disease threats for which known chemicals only provide limited information about target inhibition. In this paper, we propose the combined use of deep neural networks and Inductive Logic Programming (ILP) that allows the use of symbolic domain-knowledge (B) to explore the large space of possible molecules. Assuming molecules and their activities to be instances of random variables X and Y, the problem is to draw instances from the conditional distribution of X, given Y, B (DX|Y,B). We decompose this into the constituent parts of obtaining the distributions DX|B and DY|X,B, and describe the design and implementation of models to approximate the distributions. The design consists of generators (to approximate DX|B and DX|Y,B) and a discriminator (to approximate DY|X,B). We investigate our approach using the well-studied problem of inhibitors for the Janus kinase (JAK) class of proteins. We assume first that if no data on inhibitors are available for a target protein (JAK2), but a small numbers of inhibitors are known for homologous proteins (JAK1, JAK3 and TYK2). We show that the inclusion of relational domain-knowledge results in a potentially more effective generator of inhibitors than simple random sampling from the space of molecules or a generator without access to symbolic relations. The results suggest a way of combining symbolic domain-knowledge and deep generative models to constrain the exploration of the chemical space of molecules, when there is limited information on target-inhibitors. We also show how samples from the conditional generator can be used to identify potentially novel target inhibitors.

sentences: The histone chaperone FACT facilitates heterochromatin spreading through regulation of histone turnover and H3K9 methylation states Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT has been implicated in heterochromatin silencing, however, the mechanisms by which FACT engages in this process remain opaque. Here, we pin-pointed its function to the heterochromatin spreading process. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1 via nucleosome stabilization. Together, our study identifies FACT as a histone chaperone that specifically promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of epigenetic marks.

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NUCLEAR RETENTION OF LEPTIN/UPD2 REGULATES ORGANISMAL RESILIENCE TO NUTRIENT EXTREMES Adipokines released from the adipocytes function as a systemic adipometer; they impinge on neural circuits to signal nutrient status. On starvation, adipokines must be retained to signal energy deficit; else, it significantly reduces starvation survival. But how fat cells retain adipokines is unclear. Here, we demonstrate that Atg8, a cell-intrinsic autophagy factor, regulates the starvation-induced acute retention of the Leptin Drosophila ortholog Upd2. We show that on starvation, as a direct consequence of Atg8s lipidation, Upd2 accumulates in the nucleus. We illustrate that Upd2s nuclear retention is critical to fat mobilization and increased starvation resilience. Furthermore, nuclear Upd2 promotes the expression of a secreted innate immune gene signature. This hints at an unanticipated connection between adipokine nuclear retention and increased innate immunity. In conclusion, we propose that, during starvation, Atg8s role is not just limited to autophagy but is critical for withholding adipokines in the nucleus to promote starvation resilience.

GRAPHICAL ABSTRACT

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C_LIO_LIAtg8s lipidation on starvation results in Upd2s nuclear accumulation.
C_LIO_LIUpd2 nuclear retention on starvation increases fat mobilization and post-starvation hunger.
C_LIO_LIOn starvation Upd2 nuclear retention increases expression of a secreted innate immune signature.
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sentences: Generalization of Cortical Multivariate Genome-Wide Associations Within and Across Samples Genome-Wide Association studies have typically been limited to single phenotypes, given that high dimensional phenotypes incur a large multiple comparisons burden: ~1 million tests across the genome times the number of phenotypes. Recent work demonstrates that a Multivariate Omnibus Statistic Test (MOSTest) is well powered to discover genomic effects distributed across multiple phenotypes. Applied to cortical brain MRI morphology measures, MOSTest has resulted in a drastic improvement in power to discover loci when compared to established approaches (min-P). One question that arises is how well these discovered loci replicate in independent data. Here we perform 10 times cross validation within 35,644 individuals from UK Biobank for imaging measures of cortical area, thickness and sulcal depth (>1,000 dimensionality for each). By deploying a replication method that aggregates discovered effects distributed across multiple phenotypes, termed PolyVertex Score (PVS), we demonstrate a higher replication yield and comparable replication rate of discovered loci for MOSTest (# replicated loci: 348-845, replication rate: 94-95%) in independent data when compared with the established min-P approach (# replicated loci: 31-68, replication rate: 65-80%). An out-of-sample replication of discovered loci was conducted with a sample of 8,336 individuals from the Adolescent Brain Cognitive Development(R) (ABCD) study, who are on average 50 years younger than UK Biobank individuals. We observe a higher replication yield and comparable replication rate of MOSTest compared to min-P. This finding underscores the importance of using well-powered multivariate techniques for both discovery and replication of high dimensional phenotypes in Genome-Wide Association studies.

sentences: Pulvinar Slits: Cellulose-deficient and De-esterified Homogalacturonan-rich Structures in a Legume Motor Cell The cortical motor cells (CMCs) in a legume pulvinus execute the reversible deformation in leaf movement that is driven by changes in turgor pressure. In contrast to the underlying osmotic regulation, the cell wall structure of CMCs that contributes to the movement has yet to be characterized in detail. Here, we report that the cell wall of CMCs has tangential slits with low levels of cellulose deposition. This structure is unique and distinct from any other primary cell wall reported so far; thus, we named them " pulvinar slits." Notably, we predominantly detected de-methyl esterified homogalacturonan inside pulvinar slits, with a low deposition of highly methyl-esterified homogalacturonan inside the slits, as with cellulose. In addition, Fourier-transform infrared spectroscopy analysis indicated that the cell wall composition of pulvini is different from that of other axial organs, i.e. petioles or stems. Moreover, monosaccharide analysis showed that pulvini are pectin-rich organs like developing stems. Computer modeling suggested that pulvinar slits facilitate anisotropic extension in the direction perpendicular to the slits in the presence of turgor pressure. Mechanical tests revealed that the proximo-distal extensibility of pulvini is superior to that of the other axial organs. In this study, we thus established the distinctive cell wall structure of CMCs that contribute to plant movement, adding new knowledge to the structural diversity and function of the plant cell wall.

sentences: An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, peptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions - their membranes. However, suitable assays to quantify the efficacy of peptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure-activity relationships of two experimental series of peptide antibiotics. We reveal strong correlations between each peptides physicochemical activity at the membrane level and biological activity at the cellular level by assaying the membranolytic activities of the antibiotics on hundreds of individual giant lipid vesicles, and quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

sentences: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway Pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induces inflammatory cytokines and chemokines including IL-6, IL-1{beta}, TNFa, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and neucleocapsid (N) proteins. When stimulated with extracellular S protein, human lung epithelial cells A549 also produce inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly are non-inflammatory, but elicit an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-{kappa}B pathway in a MyD88-dependent manner. Further, such an activation of the NF-{kappa}B pathway is abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein induces IL-6, TNF-a, and IL-1 {beta} in wild-type, but not Tlr2-deficient mice. Together these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.

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Do males know? Evidence-driven rainmaking mating strategy by male bonobos to meet the fertile window of females Female bonobos exhibit prolonged receptivity, one suggested function of which is to lower male mating competition. However, it is questionable whether easier access to receptive females can reduce male-male competition, given the exclusive nature of male reproductive success. We tested whether males could determine the fertile phase of females. We found that ovulation probability predicted male mating effort. High-ranking males copulated with females with higher fertility, and male-male agonistic interactions increased when there were fertile females in the party. When there were multiple females with maximal swelling, males concentrated their mating effort on females with an older infant whose maximal swelling started earlier, and they continued mating efforts until detumescence (rainmaking). These findings suggest that male bonobos distinguish between fertile and non-fertile phases of females and that having more receptive females in the party does not reduce male-male competition for fertile females.

TeaserMales use the rainmakers rule to meet the periovulatory phase of the female bonobo for better reproductive success.

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Gli1 regulates the postnatal acquisition of peripheral nerve architecture Peripheral nerves are organized into discrete cellular compartments. Axons, Schwann cells (SCs), and endoneurial fibroblasts (EFs) reside within the endoneurium and are surrounded by the perineurium - a cellular sheath comprised of layers of perineurial glia (PNG). SC secretion of Desert Hedgehog (Dhh) regulates this organization. In Dhh nulls, the perineurium is deficient and the endoneurium is subdivided into small compartments termed minifascicles. Human Dhh mutations cause a peripheral neuropathy with similar defects. Here we examine the role of Gli1, a canonical transcriptional effector of hedgehog signaling, in regulating peripheral nerve organization. We identify PNG, EFs, and pericytes as Gli1-expressing cells by genetic fate mapping. Although expression of Dhh by SCs and Gli1 in target cells is coordinately regulated with myelination, Gli1 expression unexpectedly persists in Dhh null EFs. Thus, Gli1 is expressed in EFs non-canonically i.e., independent of hedgehog signaling. Gli1 and Dhh also have non-redundant activities. In contrast to Dhh nulls, Gli1 nulls have a normal perineurium. Like Dhh nulls, Gli1 nulls form minifascicles, which we show likely arise from EFs. Thus, Dhh and Gli1 are independent signals: Gli1 is dispensable for perineurial development but functions cooperatively with Dhh to drive normal endoneurial development. During development, Gli1 also regulates endoneurial extracellular matrix production, nerve vascular organization, and has modest, non-autonomous effects on SC sorting and myelination of axons. Finally, in adult nerves, induced deletion of Gli1 is sufficient to drive minifascicle formation. Thus, Gli1 regulates the development and is required to maintain the endoneurial architecture of peripheral nerves.

SIGNIFICANCE STATEMENTPeripheral nerves are organized into distinct cellular/ECM compartments: the epineurium, perineurium and endoneurium. This organization, with its associated cellular constituents, are critical for the structural and metabolic support of nerves and their response to injury. Here, we show Gli1 - a transcription factor normally expressed downstream of hedgehog signaling - is required for the proper organization of the endoneurium but not the perineurium. Unexpectedly, Gli1 expression by endoneurial cells is independent of, and functions non-redundantly with, Schwann Cell-derived Desert Hedgehog in regulating peripheral nerve architecture. These results further delineate how peripheral nerves acquire their distinctive organization during normal development and highlight mechanisms that may regulate their reorganization in pathologic settings including peripheral neuropathies and nerve injury.

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Phyllochron duration and changes through rice development shape the vertical leaf size profile O_LIWell-irrigated aerobic rice (Oryza sativa L.) plants are often shorter with smaller leaves, and the leaf appearance rate is slower in well-irrigated than in flooded environments. This observation questions the functional relationship between the leaf appearance rate, which is correlated with the apical cell production rate, and leaf length that is in turn correlated with the leaf division zone length.
C_LIO_LIMeristem size, blade size, and appearance were monitored for all leaves from leaf 6 on the main stem of rice plants in field experiments using two varieties, two watering systems, and three sowing dates.
C_LIO_LILeaf blade and division zone lengths were correlated in all leaves of the main stem of rice plants. New stable linear relationships were found between the leaf division zone growth duration and internal cell production rate, which changed after leaf 11. These stable relationships imply a stable link between the rates of cell production in the stem apex and leaf meristem, explaining the vertical leaf size profile.
C_LIO_LIOverall, faster leaf appearance leads to shorter leaves and organs in rice because of a functional link, which is counterbalanced by additional acclimation in transplanted plants.
C_LI

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TopoRoot: A method for computing hierarchy and fine-grained traits of maize roots from X-ray CT images Background3D imaging, such as X-ray CT and MRI, has been widely deployed to study plant root structures. Many computational tools exist to extract coarse-grained features from 3D root images, such as total volume, root number and total root length. However, methods that can accurately and efficiently compute fine-grained root traits, such as root number and geometry at each hierarchy level, are still lacking. These traits would allow biologists to gain deeper insights into the root system architecture (RSA).

ResultsWe present TopoRoot, a high-throughput computational method that computes fine-grained architectural traits from 3D X-ray CT images of field-excavated maize root crowns. These traits include the number, length, thickness, angle, tortuosity, and number of children for the roots at each level of the hierarchy. TopoRoot combines state-of-the-art algorithms in computer graphics, such as topological simplification and geometric skeletonization, with customized heuristics for robustly obtaining the branching structure and hierarchical information. TopoRoot is validated on both real and simulated root images, and in both cases it was shown to improve the accuracy of traits over existing methods. We also demonstrate TopoRoot in differentiating a maize root mutant from its wild type segregant using fine-grained traits. TopoRoot runs within a few minutes on a desktop workstation for volumes at the resolution range of 400^3, without need for human intervention.

ConclusionsTopoRoot improves the state-of-the-art methods in obtaining more accurate and comprehensive fine-grained traits of maize roots from 3D CT images. The automation and efficiency makes TopoRoot suitable for batch processing on a large number of root images. Our method is thus useful for phenomic studies aimed at finding the genetic basis behind root system architecture and the subsequent development of more productive crops.

sentences: Precise, Fast and Comprehensive Analysis of Intact Glycopeptides and Modified Saccharide Units with pGlyco3 We present a glycan-first glycopeptide search engine, pGlyco3, to comprehensively analyze intact N- and O-glycopeptides, including glycopeptides with modified saccharide units. A novel glycan ion-indexing algorithm developed in this work for glycan-first search makes pGlyco3 5-40 times faster than other glycoproteomic search engines without decreasing the accuracies and sensitivities. By combining electron-based dissociation spectra, pGlyco3 integrates a fast, dynamic programming-based algorithm termed pGlycoSite for site-specific glycan localization (SSGL). Our evaluation based on synthetic and natural glycopeptides showed that the SSGL probabilities estimated by pGlycoSite were proved to be appropriate to localize site-specific glycans. With pGlyco3, we found that N-glycopeptides and O-mannose glycopeptides in yeast samples were extensively modified by ammonia adducts on Hex (aH) and verified the aH-glycopeptide identifications based on released N-glycans and 15N/13C-labeled data. Thus pGlyco3, which is freely available on https://github.com/pFindStudio/pGlyco3/releases, is an accurate and flexible tool to identify glycopeptides and modified saccharide units.

sentences: Functional ultrasound imaging of the spreading activity following optogenetic stimulation of the rat visual cortex Optogenetic stimulation of the primary visual cortex (V1) is a promising therapy for sight restoration, but it remains unclear what total cerebral volume is activated after surface stimulation. In this study, we expressed the red-shifted opsin ChrimsonR in excitatory neurons within V1 in rats, and used the fine spatial resolution provided by functional ultrasound imaging (fUS) over the whole depth of the brain to investigate the brain response to focal surface stimulation. We observed optogenetic activation of a high proportion of the volume of V1. Extracellular recordings confirmed the neuronal origin of this activation. Moreover, neuronal responses were even located in deep layers under conditions of low irradiance, spreading to the LGN and V2, consistent with a normal visual information process. This study paves the way for the use of optogenetics for cortical therapies, and highlights the value of coupling fUS with optogenetics.

sentences: Simultaneous hybrid genome sequencing of Vermamoeba vermiformis and its Dependentiae endosymbiont Vermiphilus pyriformis A hybrid sequencing approach, using short and long reads sequencing, was employed for characterizing the genomes of the free-living amoeba host Vermamoeba vermiformis, along with its Dependentiae endosymbiont Vermiphilus pyriformis. The amoeba host reconstructed nuclear genome is 39.5 Mb, and its full mitochondrial genome is 61.7 kb. The closed, circular genome of the Dependentiae endosymbiont Vermiphilus pyriformis, naturally infecting V. vermiformis is 1.1 Mb.

sentences: Predicted epitope binding core motifs and common amino acid polymorphisms distinguish protective- from susceptibility-associated HLA alleles in leishmaniasis Susceptibility for leishmaniasis is largely dependent on genetic- and immune factors of the host. Despite the previously described association of human leukocyte antigen (HLA) gene cluster variants as genetic susceptibility factors, little is known on the mechanisms that mediate these associations. To characterize the functionality underpinning these associations between HLA and disease, we predicted the epitope binding repertoires for all known leishmaniasis-associated HLA variants collected in a thorough literature review. We identified several amino acid polymorphisms in the HLA sequences that distinguished protective-from risk-associated HLA-DRB1 alleles. Proteome-wide and multi-species T cell epitope binding predictions were carried out across these alleles, enabling us to map the effects on the epitope binding repertoires. The protective-associated HLA-DRB1 alleles were characterized by common binding core motifs, which map to the identified amino acid polymorphisms. These results strongly suggest that polymorphism in the HLA region, resulting in differential antigen presentation, affects the association between HLA and leishmaniasis disease development. Finally, we established a valuable open-access resource of putative epitopes, of which a set of 14 HLA-unrestricted strong-binding epitopes, conserved across species, were prioritized for further epitope discovery in the search for novel subunit-based vaccines.

sentences: Host-symbiont stress response to lack-of-sulfide in the giant ciliate mutualism The thiotrophic mutualism between the sulfur-oxidizing, chemoautotrophic (thiotrophic) bacterial ectosymbiont Candidatus Thiobius zoothamnicola and the giant ciliate Zoothamnium niveum thrives in a variety of shallow-water marine environments with highly fluctuating sulfide emission. To persist over time both partners must reproduce and ensure symbiont transmission prior cessation of sulfide, fueling the symbionts carbon fixation and host nourishment. We experimentally investigated the response of this mutualism to waning of sulfide. We found that colonies followed the r-strategy and released initially present but also newly produced macrozooids until death. A fraction of middle-sized longer-lived colonies were particularly proficient in producing and releasing swarmers. The symbionts on the colonies proliferated less and became larger and more rod-shaped under oxic conditions compared to symbionts from freshly collected colonies exposed to sulfide and oxygen. The symbiont monolayer was highly disturbed with epigrowth of other microbes and loss of symbionts that were subsequently found in the experimental seawater. We conclude that both partners response to cessation of sulfide emission was remarkably fast. The colony experienced death within two days but host reproduction through swarmers carrying the symbiont ensured the continuation of the association.

sentences: Insertions and deletions as phylogenetic signal in alignment-free sequence comparison Most methods for phylogenetic tree reconstruction are based on sequence alignments; they infer phylogenies from substitutions that may have occurred at the aligned sequence positions. Gaps in alignments are usually not employed as phylogenetic signal. In this paper, we explore an alignment-free approach that uses insertions and deletions (indels) as an additional source of information for phylogeny inference. For a set of four or more input sequences, we generate so-called quartet blocks of four putative homologous segments each. For pairs of such quartet blocks involving the same four sequences, we compare the distances between the two blocks in these sequences, to obtain hints about indels that may have happened between the blocks since the respective four sequences have evolved from their last common ancestor. A prototype implementation is presented to infer phylogenetic trees from these data, using a quartet-tree approach or, alternatively, under the maximum-parsimony paradigm. This approach should not be regarded as an alternative to established methods, but rather as a complementary source of phylogenetic information. Interestingly, however, our software is able to produce phylogenetic trees from putative indels alone that are comparable to trees obtained with existing alignment-free methods.

sentences: A novel, anatomy-similar in vitro model of 3D airway epithelial for anti-coronavirus drug discovery SARS-CoV-2 and its induced COVID-19 remains as a global health calamity. Severe symptoms and high mortality, caused by cytokine storm and acute respiratory distress syndrome in the lower respiratory airway, are always associated with elderly individuals and those with comorbidities; whereas mild or moderate COVID-19 patients have limited upper respiratory flu-like symptoms. There is an urgent need to investigate SARS-CoV-2 and other coronaviruses replication and immune responses in human respiratory systems. The human reconstituted airway epithelial air-liquid interface (ALI) models are the most physiologically relevant model for the investigation of coronavirus infection and virus-triggered innate immune signatures. We established ALI models representing both the upper and the lower respiratory airway to characterize the coronavirus infection kinetics, tissue pathophysiology, and innate immune signatures from upper and lower respiratory tract perspective. Our data suggested these in vitro ALI models maintain high physiological relevance with human airway tissues. The coronavirus induced immune response observed in these upper and lower respiratory airway models are similar to what has been reported in COVID-19 patients. The antiviral efficacy results of a few promising anti-coronavirus drugs in these models were consistent with previous reports and could be valuable for the human dose prediction. Taken together, our study demonstrates the importance of 3D airway epithelial ALI model for the understanding of coronavirus pathogenesis and the discovery and development of anti-coronavirus drugs.

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Rapid and Durable Protection Against Marburg Virus with a Single-Shot ChAd3-MARV GP Vaccine Marburg virus (MARV) causes a severe hemorrhagic fever disease in primates with mortality rates in humans up to 90%. Since 2018, MARV has been identified as a priority pathogen by the WHO, needing urgent research and development of countermeasures due to the high public health risk it poses. Recently, the first case of MARV in West Africa underscored the significant outbreak potential of this virus. The potential for cross border spread as had occurred during the Ebola 2014-2016 outbreak illustrates the critical need for Marburg vaccines. To support regulatory approval of the ChAd3-Marburg vaccine that has completed Phase I trials, we show that a non-replicating chimpanzee-derived adenovirus vector with a demonstrated safety profile in humans (ChAd3) protected against a uniformly lethal challenge with Marburg-Angola. Protective immunity was achieved within 7 days of vaccination and was maintained through one year post vaccination, antigen-specific antibodies were a significant immune correlate of protection in the acute challenge model (p=0.0003), and predictive for protection with an AUC = 0.88. These results demonstrate that a single-shot ChAd3 MARV vaccine generated a protective immune response that was both rapid and durable with a significant immune correlate of protection that will support advanced clinical development.

One Sentence SummaryA single-shot of non-replicating ChAd3-MARV vaccine demonstrated both rapid (within 1 week) and durable (12 months) protection against lethal Marburg virus infection in macaques.

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Co-evolutionary signals from Burkholderia pseudomallei genomics identify its survival strategies and highlight improving environmental health as prevention policy BackgroundThe soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis. It kills up to 40% of cases and contributes to human morbidity and mortality in many tropical and sub-tropical countries. As no vaccines are currently available, prevention is the key health policy and is achieved by avoiding direct contact with soil and standing water. The pathogen notoriously persists in ranges of environmental conditions which make disease prevention difficult. We aimed to scan B. pseudomallei genomes for signals of evolutionary adaptations that allow it to thrive across environmental conditions, which should ultimately inform prevention policy.

MethodsWe conducted three layers of analyses: a genome-wide epistasis and co-selection study (GWES) on 2,011 B. pseudomallei genomes to detect signals of co-selection; gene expression analyses across 82 diverse physical, chemical, biological and infectious conditions to identify specific conditions in which such selection might have acted; and gene knockout assays to confirm the function of the co-selection hotspot.

FindingsWe uncovered 13,061 mutation pairs in distinct genes and non-coding RNA that have been repeatedly co-selected through B. pseudomallei evolution. Genes under co-selection displayed marked expression correlation when B. pseudomallei was subjected to physical stress conditions including temperature stress, osmotic stress, UV radiation, and nutrient deprivation; highlighting these conditions as the major evolutionary driving forces for this bacterium. We identified a putative adhesin (BPSL1661) as a hub of co-selection signals, experimentally confirmed the role of BPSL1661 under nutrient deprivation, and explored the functional basis of the co-selection gene network surrounding BPSL1661 in facilitating bacterial survival under nutrient depletion.

InterpretationOur findings suggest that B. pseudomallei has a selective advantage to survive nutrient-limited conditions. Anthropogenic activities such as shifting cultivation systems with more frequent rotations of cropping and shortened fallow periods or continuous cultivation of cash crops could directly or indirectly contribute to loss of soil nutrient; these may lead to the preferential survival of B. pseudomallei and a subsequent rise of melioidosis. Successful disease control for melioidosis needs to consider improving environmental health in addition to current preventive efforts.

FundingWellcome Trust, European Research Council, UK Department of Health, Thailand Research Fund and Khon Kaen University

Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with terms (co-selection AND bacteria AND population) with no date or language restrictions from database inception until April 11, 2021. We identified 44 publications of which four were conducted at a genome-wide scale. These four studies were performed on human-restricted pathogens, detected co-selection of antibiotic resistance gene networks which highlight the use of antibiotics as major selection pressures and further inform treatment options. However, none of these studies were performed on Burkholderia pseudomallei or other opportunistic pathogens that have been adapted to both natural and host environments. The selection pressures exerted on these pathogens and the genetic determinants allowed for their adaptations remain unclear, which limit our understanding on the bacterial biology and the information used for disease control.

Added value of this studyBased on genomes of 2,011 B. pseudomallei collected from melioidosis endemic areas, we identified and confirmed genetic signals for co-selection. Using transcriptome profiling covering a broad spectrum of conditions and exposures, we showed that genes under co-selection displayed marked expression correlation under physical stress conditions with the gene at the co-selection hotspot conditionally expressed under nutrient starvation. Furthermore, we experimentally validated the function of the hotspot gene and demonstrated that unlike host-restricted pathogens, the B. pseudomallei co-selection network does not facilitate host infection but is focused on bacterial survival in a harsh environment, particularly under nutrient depletion. Aside from providing a data resource, the study also showcases the power of combined genetics, transcriptomics and functional analysis as a tool for biology discovery.

Implications of all available evidenceOur findings provide evolutionary and biological evidence for preferential survival of B. pseudomallei under nutrient starvation. Agricultural practice that induces soil loss, which is not uncommon in melioidosis endemic areas has been linked to soil nutrient depletion and may contribute to the prevalence of B. pseudomallei and a consequent rise of melioidosis in these regions. Successful melioidosis control has to consider environmental health in addition to existing prevention policy.

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The YAP/TAZ antagonist, PTPN14, stabilizes SMAD4 through direct interactions in endothelial cells: Implications for Hereditary Hemorrhagic Telangiectasia Hereditary Hemorrhagic Telangiectasia (HHT) is a vascular condition caused by germline heterozygous loss-of-function mutations of ENG, AVCRL1, and occasionally SMAD4, encoding components of TGF{beta}/BMP signaling. Telangiectases occur in most HHT patients, and pulmonary, visceral, or cerebral arteriovenous malformations (AVMs) occur in 20-50%, but our understanding of how HHT mutations disrupt down-stream signaling pathways causing clinical manifestations, and why some patients suffer more serious sequelae, is incomplete. We previously showed that genetic variation within PTPN14 at rs2936018 associates with presentation of PAVM in HHT patients. Here we show rs2936018 is a cis-eQTL for PTPN14, with lower expression of the HHT at-risk allele, and that in primary human endothelial cells, PTPN14 physically interacts with the transcription factor, SMAD4, protecting it from ubiquitylation to support higher SMAD4 expression levels.

In a panel of 69 lung samples, we find Ptpn14 RNA expression correlates with markers of angiogenesis, lymphangiogenesis, cell-cell interaction, BMP signaling and rho kinase signaling, indicating preferential expression in endothelial cells. RNAScope in situ hybridization analysis and use of transgenic Ptpn14-reporter mice (Ptpn14.tm1(KOMP) Vlcg) show that Ptpn14 is predominantly but not exclusively expressed in lymphatic and vascular ECs of lung, heart and skin. In lung, Ptpn14, Acvrl1, Eng and SMAD4 expression are tightly correlated as well as with Flt1, Ece1, Sash1, and Mapk3k. Additionally, Ptpn14, Acvrl1, Eng and SMAD4 show strong expression correlation with components of G protein coupled receptor (GPCR) signaling pathways impacting rho kinase, Cdc42, a regulator of cell migration. We report, for the first time, that in primary human endothelial cells PTPN14 binds SMAD4, as demonstrated by co-immunoprecipitation studies and confirmed by proximity ligation assay. In the nucleus, PTPN14 stabilizes SMAD4, protecting it from ubiquitylation and turnover and potentiating basal transcriptional activity from BMP and TGF{beta} responsive reporters, whereas in the cytoplasm PTPN14 sequesters phospho-TAZ (pTAZ) leading to TAZ turnover. PTPN14 therefore provides a physical link supporting BMP/ALK-1/SMAD4 signaling while inhibiting YAP/TAZ signaling in ECs to regulate a balance between these two pathways to maintain vascular stability. Polymorphisms in PTPN14 may alter tonic BMP/ALK-1/SMAD4 and TGF{beta}/TGF{beta}RI/SMAD4 signaling to magnify ligand-mediated responses. In this way, genetic variation within PTPN14 may influence clinical outcomes of HHT through its action on SMAD4.

sentences: Relative contributions of sex hormones, sex chromosomes, and gonads to sex differences in tissue gene regulation Sex differences in physiology and disease in mammals result from the effects of three classes of factors that are inherently unequal in males and females: reversible (activational) effects of gonadal hormones, permanent (organizational) effects of gonadal hormones, and cell-autonomous effects of sex chromosomes, as well as genes driven by these classes of factors. Often, these factors act together to cause sex differences in specific phenotypes, but the relative contribution of each and the interactions among them remain unclear. Here, we used the Four Core Genotypes (FCG) mouse model with or without hormone replacement to distinguish the effects of each class of sex-biasing factors on transcriptome regulation in liver and adipose tissues. We found that the activational hormone levels have the strongest influence on gene expression, followed by the organizational gonadal sex effect and, lastly, sex chromosomal effect, along with interactions among the three factors. Tissue specificity was prominent, with a major impact of estradiol on adipose tissue gene regulation, and of testosterone on the liver transcriptome. The networks affected by the three sex-biasing factors include development, immunity and metabolism, and tissue-specific regulators were identified for these networks. Furthermore, the genes affected by individual sex-biasing factors and interactions among factors are associated with human disease traits such as coronary artery disease, diabetes, and inflammatory bowel disease. Our study offers a tissue-specific account of the individual and interactive contributions of major sex-biasing factors to gene regulation that have broad impact on systemic metabolic, endocrine, and immune functions.

sentences: Linguistic laws are not the law in chimpanzee sexual solicitation gestures. Two language laws have been identified as manifestations of universal principles of animal behaviour, both acting on the organisation of numerous vocal and behavioural communicative systems. Zipfs law of brevity describes a negative relationship between behavioural length and frequency of behaviour. Menzeraths law defines a negative correlation between the number of behaviours in a sequence and average length of the behaviour composing it. Both laws have been linked with the information-theoretic principle of compression, which tends to minimise code length. We investigate the presence of these two laws in the repertoire of chimpanzee sexual solicitation gestures. We find that chimpanzee solicitation gestures do not follow either Zipfs law of brevity or Menzeraths law consistently. For the second time in ape gestural communication, evidence supporting Zipfs law of brevity was absent, and, here, the presence of Menzeraths law appears individually driven. Ape gesture does not appear to manifest a principle of compression or pressure for efficiency that has been previously proposed to be universal. Importantly, the same signals were shown to adhere to these laws when used in a different behavioural context; highlighting that signallers consider signalling efficiency broadly, and diverse factors play important roles in shaping investment in signal production.

sentences: Mechanistic Insights on ATP's role as Hydrotrope Hydrotropes are small amphiphilic molecules which help in solubilizing hydrophobic entities in aqueous medium. Recent experimental investigation has provided convincing evidences that, adenosine triphosphate (ATP), besides being an energy currency of cell, also can act as hydrotrope to inhibit the formation of protein condensates. In this work, we have designed computer simulations of prototypical macromolecules in aqueous ATP solution to dissect the molecular mechanism underlying ATPs newly discovered role as a hydrotrope. The simulation demonstrates that ATP can unfold a single-chain of hydrophobic macromolecule as well as can disrupt the aggregation process of a hydrophobic assembly. Moreover, the introduction of charges in the macromolecule is found to reinforce ATPs disaggregation effects in a synergistic fashion, a behaviour reminiscent of recent experimental observation of pronounced hydrotropic action of ATP in intrinsically disordered proteins. A molecular analysis indicates that this new-found ability of ATP are ingrained in its propensity of preferential binding to the polymer surface, which gets fortified in presence of charges. The investigation also renders evidence that the key to the ATPs superior hydrotropic role over chemical hydrotrope (Sodium xylene sulfonate, NaXS) may lie in its inherent self-aggregation propensity. Overall, via employing a bottom-up approach the current investigation provides fresh mechanistic insights into the dual solubilizing and denaturing abilities of ATP.

sentences: The ability of bumblebees Bombus terrestris (Hymenoptera: Apidae) to detect floral humidity is dependent upon environmental humidity Flowers produce local humidity that is often greater than that of the surrounding environment, and studies have shown that insect pollinators may be able to use this humidity difference to locate and identify suitable flowers. However, environmental humidity is highly heterogeneous, and is likely to affect the detectability of floral humidity, potentially constraining the contexts in which it can be used as a salient communication pathway between plants and their pollinators. In this study, we use differential conditioning techniques on bumblebees Bombus terrestris audax (Harris) to explore the detectability of an elevated floral humidity signal when presented against different levels of environmental noise. Artificial flowers were constructed that could be either dry or humid, and individual bumblebees were presented with consistent rewards in either the humid or dry flowers presented in an environment with four levels of constant humidity, ranging from low ([~]20% RH) to highly saturated ([~]95% RH). Ability to learn was dependent upon both the rewarding flower type and the environment: the bumblebees were able to learn rewarding dry flowers in all environments, but their ability to learn humid rewarding flowers was dependent on the environmental humidity, and they were unable to learn humid rewarding flowers when the environment was highly saturated. This suggests that floral humidity might be masked from bumblebees in humid environments, suggesting that it may be a more useful signal to insect pollinators in arid environments.

sentences: A Drosophila RNAi screen reveals conserved glioblastoma-related adhesion genes that regulate collective cell migration Migrating cell collectives are key to embryonic development but also contribute to invasion and metastasis of a variety of cancers. Cell collectives can invade deep into tissues, leading to tumor progression and resistance to therapies. Collective cell invasion is also observed in the lethal brain tumor glioblastoma, which infiltrates the surrounding brain parenchyma leading to tumor growth and poor patient outcomes. Drosophila border cells, which migrate as a small cell cluster in the developing ovary, are a well-studied and genetically accessible model used to identify general mechanisms that control collective cell migration within native tissue environments. Most cell collectives remain cohesive through a variety of cell-cell adhesion proteins during their migration through tissues and organs. In this study, we first identified cell adhesion, cell junction, and associated regulatory genes that are expressed in human brain tumors. We performed RNAi knockdown of the Drosophila orthologs in border cells to evaluate if migration and/or cohesion of the cluster was impaired. From this screen, we identified eight adhesion genes that disrupted border cell collective migration upon RNAi knockdown. Bioinformatics analyses further demonstrated that subsets of the orthologous genes were elevated in the margin and invasive edge of human glioblastoma patient tumors. These data together show that conserved cell adhesion and adhesion regulatory proteins with potential roles in tumor invasion also modulate collective cell migration. This dual screening approach for adhesion genes linked to glioblastoma and border cell migration thus may reveal conserved mechanisms that drive collective tumor cell invasion.

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Interaction between theta-phase and spike-timing dependent plasticity simulates theta induced memory effects Rodent studies suggest that spike timing relative to hippocampal theta activity determines whether potentiation or depression of synapses arise. Such changes also depend on spike timing between pre- and post-synaptic neurons, known as spike-timing-dependent plasticity (STDP). STDP, together with theta-phase-dependent learning, has inspired several computational models of learning and memory. However, evidence to elucidate how these mechanisms directly link to human episodic memory is lacking. In a computational model, we modulate long-term potentiation (LTP) and long-term depression (LTD) of STDP, by opposing phases of a simulated theta rhythm. We fit parameters to a hippocampal cell culture study in which LTP and LTD were observed to occur in opposing phases of a theta rhythm. Further, we modulated two inputs by cosine waves with synchronous and asynchronous phase offsets and replicate key findings in human episodic memory. Learning advantage was found for the synchronous condition, as compared to the asynchronous conditions, and was specific to theta modulated inputs. Importantly, simulations with and without each mechanism suggest that both STDP and theta-phase-dependent plasticity are necessary to replicate the findings. Together, the results indicate a role for circuit-level mechanisms, which bridges the gap between slice preparation studies and human memory.

Author SummaryLong-lasting changes in synaptic connectivity between neurons have been suggested to support learning and memory processes at the cellular level in the brain. Such synaptic modifications depend on synchronous activation of neurons, which leads to generate brain oscillations. Human memory studies focus on the relationships between brain oscillations and memory processes. Direct evidence on how the cellular mechanism links to human memory behaviour is lacking. To investigate the direct link between synaptic plasticity mechanisms and human memory formation, we built a computational neural network that implements two synaptic plasticity mechanisms, which are well-established in the rodents hippocampus. One mechanism shows that strengthening or weakening in synaptic connectivity depends on the phases of ongoing brain oscillation at theta frequency (4 - 8 Hz), which is a dominant signal in the hippocampus. The other mechanism suggests that synaptic modification depends on the precise timing of action potentials between two neurons. Our model successfully reproduces results from rodents, as well as several human episodic memory studies which demonstrated that human associative memory performance depends on phase synchronisation in theta frequency. These findings suggest a link between specific learning mechanisms at cellular level and human memory behaviour.

sentences: Viral origin of eukaryotic type IIA DNA topoisomerases Type II DNA topoisomerases of the family A (Topo IIA) are present in all bacteria (DNA gyrase) and eukaryotes. In eukaryotes, they play a major role in transcription, DNA replication, chromosome segregation and modulation of chromosome architecture. The origin of eukaryotic Topo IIA remains mysterious since they are very divergent from their bacterial homologues and have no orthologues in Archaea. Interestingly, eukaryotic Topo IIA have close homologues in viruses of the phylum Nucleocytoviricota, an expansive assemblage of large and giant viruses formerly known as the nucleocytoplasmic large DNA viruses (NCLDV). Topo IIA are also encoded by some bacterioviruses of the class Caudoviricetes (tailed bacteriophages). To elucidate the origin of the eukaryotic Topo IIA, we performed in-depth phylogenetic analyses combining viral and cellular Topo IIA homologs. Topo IIA encoded by bacteria and eukaryotes form two monophyletic groups nested within Topo IIA encoded by Caudoviricetes and Nucleocytoviricota, respectively. Importantly, Nucleocytoviricota remained well separated from eukaryotes after removing both bacteria and Caudoviricetes from the dataset, indicating that the separation of Nucleocytoviricota and eukaryotes is probably not due to long branch attraction artefact. The topology of our tree suggests that the eukaryotic Topo IIA was probably acquired from an ancestral member of the Nucleocytoviricota of the class Megaviricetes, before the emergence of the last eukaryotic common ancestor (LECA). This result further highlights a key role of these viruses in eukaryogenesis and suggests that early proto-eukaryotes used a Topo IIB instead of a Topo IIA for solving their DNA topological problems.

sentences: GAT-FD: An Integrated MATLAB Toolbox for Graph Theoretical Analysis of Task-Related Functional Dynamics Functional connectivity (FC) has been demonstrated to be varying over time during sensory and cognitive processes. Quantitative examinations of such variations can significantly advance our understanding on large-scale functional organizations and their topological dynamics that support normal brain functional connectome and can be altered in individuals with brain disorders. However, toolboxes that integrate the complete functions for analyzing task-related brain FC, functional network topological properties, and their dynamics, are still lacking. The current study has developed a MATLAB toolbox, the Graph Theoretical Analysis of Task-Related Functional Dynamics (GAT-FD), which consists of four modules for sliding-window analyses, temporal mask generation, estimations of network properties and dynamics, and result display, respectively. All the involved functions have been tested and validated using fMRI data collected from human subjects when performing a block-designed task. The results demonstrated that the GAT-FD allows for effective and quantitative evaluations of the functional network properties and their dynamics during the task period. As an open-source and user-friendly package, the GAT-FD and its detailed user manual are freely available at https://www.nitrc.org/projects/gat_fd and https://centers.njit.edu/cnnl/gat_fd/.

sentences: Metabolically-Incorporated Deuterium in Myelin Localized by Neutron Diffraction and Identified by Mass Spectrometry Myelin is a natural and dynamic multilamellar membrane structure that continues to be of significant biological and neurological interest, especially with respect to its biosynthesis and assembly during its normal formation, maintenance, and pathological breakdown. To explore the usefulness of neutron diffraction in the structural analysis of myelin, we investigated the use of in vivo labeling by metabolically incorporating non-toxic levels of deuterium (2H; D) via drinking water into a pregnant dam (D-dam) and her developing embryos. All of the mice were sacrificed when the pups (D-pups) were 55 days old. Myelinated sciatic nerves were dissected, fixed in glutaraldehyde and examined by neutron diffraction. Parallel samples that were unfixed (trigeminal nerves) were frozen for mass spectrometry (MS). The diffraction patterns of the nerves from deuterium-fed mice (D-mice) vs. the controls (H-mice) had major differences in the intensities of the Bragg peaks but no appreciable differences in myelin periodicity. Neutron scattering density profiles showed an appreciable increase in density at the center of the lipid-rich membrane bilayer. This increase was greater in D-pups than in D-dam, and its localization was consistent with deuteration of lipid hydrocarbon, which predominates over transmembrane protein in myelin. MS analysis of the lipids isolated from the trigeminal nerves demonstrated that in the pups the percentage of lipids that had one or more deuterium atoms was uniformly high across lipid species (97.6% {+/-} 2.0%), whereas in the mother the lipids were substantially less deuterated (60.6% {+/-} 26.4%) with levels varying among lipid species and subspecies. The mass distribution pattern of deuterium-containing isotopologues indicated the fraction (in %) of each lipid (sub-)species having one or more deuteriums incorporated: in the D-pups, the pattern was always bell-shaped, and the average number of D atoms ranged from a low of [~]4 in fatty acid to a high of [~]9 in cerebroside. By contrast, in D-dam most lipids had more complex, overlapping distributions that were weighted toward a lower average number of deuteriums, which ranged from a low of [~]3-4 in fatty acid and in one species of sulfatide to a high of 6-7 in cerebroside and sphingomyelin. The consistently high level of deuteration in D-pups can be attributed to their de novo lipogenesis during gestation and rapid, postnatal myelination. The widely varying levels of deuteration in D-dam, by contrast, likely depends on the relative metabolic stability of the particular lipid species during myelin maintenance. Our current findings demonstrate that stably-incorporated D label can be detected and localized using neutron diffraction in a complex tissue such as myelin; and moreover, that MS can be used to screen a broad range of deuterated lipid species to monitor differential rates of lipid turnover. In addition to helping to develop a comprehensive understanding of the de novo synthesis and turnover of specific lipids in normal and abnormal myelin, our results also suggest application to studies on myelin proteins (which constitute only 20-30% by dry mass of the myelin, vs. 70-80% for lipid), as well as more broadly to the molecular constituents of other biological tissues.

sentences: Cathelicidin-3 associated with serum extracellular vesicles enables early diagnosis of a transmissible cancer The identification of practical early diagnosis biomarkers is a cornerstone of improved prevention and treatment of cancers. Such a case is devil facial tumour disease (DFTD), a highly lethal transmissible cancer afflicting virtually an entire species, the Tasmanian devil (Sarcophilus harrisii). Despite a latent period that can exceed one year, to date DFTD diagnosis requires visual identification of tumour lesions. To enable earlier diagnosis, which is essential for the implementation of effective conservation strategies, we analysed the extracellular vesicle (EV) proteome of 87 Tasmanian devil serum samples. The antimicrobial peptide cathelicidin-3 (CATH3) was enriched in serum EVs of both devils with clinical DFTD (87.9% sensitivity and 94.1% specificity) and devils with latent infection (i.e., collected while overtly healthy, but 3-6 months before subsequent DFTD diagnosis; 93.8% sensitivity and 94.1% specificity). As antimicrobial peptides can play a variety of roles in the cancer process, our results suggest that the specific elevation of serum EV-associated CATH3 may be mechanistically involved in DFTD pathogenesis. This EV-based approach to biomarker discovery is directly applicable to improving understanding and diagnosis of a broad range of diseases in other species, and these findings directly enhance the capacity of conservation strategies to ensure the viability of the imperilled Tasmanian devil population.

sentences: Cross-covariance isolate detect: a new change-point method for estimating dynamic functional connectivity Evidence of the non stationary behavior of functional connectivity (FC) networks has been observed in task based functional magnetic resonance imaging (fMRI) experiments and even prominently in resting state fMRI data. This has led to the development of several new statistical methods for estimating this time-varying connectivity, with the majority of the methods utilizing a sliding window approach. While computationally feasible, the sliding window approach has several limitations. In this paper, we circumvent the sliding window, by introducing a statistical method that finds change-points in FC networks where the number and location of change-points are unknown a priori. The new method, called cross-covariance isolate detect (CCID), detects multiple change-points in the second-order (cross-covariance or network) structure of multivariate, possibly high-dimensional time series. CCID allows for change-point detection in the presence of frequent changes of possibly small magnitudes, can assign change-points to one or multiple brain regions, and is computationally fast. In addition, CCID is particularly suited to task based data, where the subject alternates between task and rest, as it firstly attempts isolation of each of the change-points within subintervals, and secondly their detection therein. Furthermore, we also propose a new information criterion for CCID to identify the change-points. We apply CCID to several simulated data sets and to task based and resting state fMRI data and compare it to recent change-point methods. CCID may also be applicable to electroencephalography (EEG), magentoencephalography (MEG) and electrocorticography (ECoG) data. Similar to other biological networks, understanding the complex network organization and functional dynamics of the brain can lead to profound clinical implications. Finally, the R package ccid implementing the method from the paper is available from CRAN.

sentences: Brazil Nut Effect Drives Pattern Formation in Early Mammalian Embryos The formation of three-dimensional ordered spatial patterns, which is essential for embryonic development, tissue regeneration, and cancer metastasis, is mainly guided by the chemical concentration gradient of morphogens. However, since no chemical concentration gradient has been observed in the early embryonic development (pre-implantation) of mammals, the pattern formation mechanism has been unsolved for a long time. During the second cell fate decision of mouse embryos, the inner cell mass (ICM) segregates into topographically regionalized epiblast (EPI) and primitive endoderm (PrE) layers. Here, we report that the segregation process of PrE/EPI precursors coincides with an emerged periodic expansion-contraction vibration of the blastocyst cavity, which induces phase transition in the ICM compartment to a higher fluidity state and generates directional tissue flows. By experiments and modeling, we demonstrate that the spatial segregation of PrE and EPI precursors is mediated by a "Brazil nut effect"-like viscous segregation mechanism in which PrE precursors with low affinity gradually migrate to the surface of ICM along with the tissue flow, while EPI precursors with high affinity remains inside ICM under cavity vibration. Artificially manipulation of the frequency and amplitude of cavity vibration could control the process of spatial separation as well as lineage specification of PrE/EPI. Furthermore, disruption of the cavity vibration in the initial stage after segregation could reverse the ICM cells back to a mixed state. Therefore, this study reveals a fundamental mechanism that guarantees the robustness of cell segregation and pattern formation without specific morphogens in early mammalian embryos. Our model also emphasizes a conserved function of cavity structure that widely exists in organisms as an energy reservoir and converter between different forms, such as chemical and mechanical energy.

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NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels Despite routine use of platinum-based chemotherapeutics in cancer treatment, there remains a need to improve efficacy and patient selection. Multi-omic assessment of human bladder cancer cell lines and their cisplatin resistant derivatives and whole-genome CRISPR screens identified NPEPPS, the puromycin-sensitive aminopeptidase as a novel driver of cisplatin resistance. NPEPPS depletion increased cisplatin import and sensitization of resistant cells in vitro and in vivo. Pharmacologic inhibition of NPEPPS in cells and chemoresistant, patient-derived tumor organoids improved response to cisplatin. NPEPPS was found in complex with volume regulated anion channel (VRAC) subunits LRRC8A and LRRC8D, whose loss is known to enhance resistance to cisplatin. Depletion of LRRC8A the only obligate subunit for normal VRAC function abrogated the effect of NPEPPS-mediated cisplatin import. Our findings describe the first mechanism by which VRACs can be targeted for therapeutic benefit.

HIGHLIGHTSO_LIMulti-omic screening found NPEPPS is a driver of cisplatin resistance in cancer
C_LIO_LINPEPPS is in protein complex with LRRC8A and D, volume regulated anion channel (VRAC) subunits
C_LIO_LILRRC8A and D loss increases cisplatin resistance and reduce its intracellular levels
C_LIO_LINPEPPS regulates cisplatin import and sensitivity through VRACs
C_LIO_LINPEPPS function is inhibited by small molecules offering potential for clinical translation
C_LI

sentences: Verbally mediated cooperation is consistently associated with inter-brain synchrony in frontal and temporoparietal areas: A mini-review and meta-analysis Single-brain neuroimaging studies have shown that human cooperation is associated with neural activity in frontal and temporoparietal regions. However, it remains unclear whether single-brain studies are informative about cooperation in real life, where people interact dynamically. Such dynamic interactions have become the focus of inter-brain studies. An advantageous technique in this regard is functional near-infrared spectroscopy (fNIRS) because it is less susceptible to movement artifacts than more conventional techniques like EEG or fMRI. We conducted a systematic review and the first quantitative meta-analysis of fNIRS hyperscanning of cooperation, based on thirteen studies with 890 participants. Overall, the meta-analysis revealed evidence of statistically significant inter-brain synchrony while people were cooperating, with large overall effect sizes in both frontal and temporoparietal areas. All thirteen studies observed significant inter-brain synchrony in the prefrontal cortex (PFC), suggesting that this region is particularly relevant for cooperative behavior. The consistency in these findings is unlikely to be due to task-related activations, given that the relevant studies used diverse cooperation tasks. Together, the present findings support the importance of inter-brain synchronization of frontal and temporoparietal regions in interpersonal cooperation. Moreover, the present article highlights the usefulness of meta-analyses as a tool for discerning patterns in inter-brain dynamics.

sentences: Nestmate recognition of early brood in ants Brood is critically important in social insect colonies. It carries the colony fitness through delivering future reproductive adults as well as workers that will increase the colonys workforce. Adoption of non-nestmate brood can be a mean to increase colonys workforce but entails the risk of rearing unrelated sexuals or social parasites. For early brood (eggs and L1 larvae), this balance is less positive as young brood need a substantial amount of resource before becoming workers. Thus, it appears beneficial for ant workers to discriminate between nestmate and alien brood using the chemical cues displayed at the broods surface. However, the chemical signature of ant early brood stages and its use by workers remains understudied. To fill this gap, we investigated the chemical basis of early brood nestmate and cross-species recognition in six Formicoid ants. We also tested the discrimination behaviour of workers in brood retrieval trials. We observed clear species-level cues and discrimination against heterospecific brood. We also found that eggs and most young larvae display a colony signature but that only some species discriminate against non-nestmate eggs and L1 larvae. Interestingly, these species appear to also be those belonging to genera subject to brood parasitism.

sentences: Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube In recent years, genome-editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need for Agrobacterium-mediated transfection and tissue culture, by using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable for driving expression of the delivered gene in pollen tubes. We also evaluated delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9 introduced pollen tubes but were not detected in the negative control. Bombarded pollen germinated pollen tubes on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.

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PCSK9 affects expression of key surface proteins in human pancreatic beta cells through intra- and extracellular regulatory circuits Aims/hypothesisProprotein convertase subtilisin/kexin 9 (PCSK9) is involved in the degradation of LDLR. However, PCSK9 can target other proteins in a cell-type specific manner. While PCSK9 has been detected in pancreatic islets, its expression in insulin-producing pancreatic beta cells is debated. Herein, we studied PCSK9 expression, regulation and function in the human pancreatic beta cell line EndoC-{beta}H1.

MethodsWe assessed PCSK9 expression in mouse and human pancreatic islets, and in the pancreatic beta cell line EndoC-{beta}H1. We also studied PCSK9 regulation by cholesterol, lipoproteins, Mevastatin, and by SREBPs transcription factors. To evaluate PCSK9 function in pancreatic beta cells, we performed PCSK9 gain-and loss-of-function experiments in EndoC-{beta}H1 using siPCSK9 or recombinant PCSK9 treatments, respectively.

ResultsWe demonstrate that PCSK9 is expressed and secreted by pancreatic beta cells. In EndoC-{beta}H1 cells, PCSK9 expression is regulated by cholesterol and by SREBPs transcription factors. Importantly, PCSK9 knockdown results in multiple transcriptome, proteome and secretome deregulations and impaired insulin secretion. By gain- and loss-of-function experiments, we observed that PCSK9 regulates the expression levels of LDLR and VLDLR through an extracellular mechanism while CD36, PD-L1 and HLA-ABC are regulated through an intracellular mechanism.

Conclusions/interpretationCollectively, these results highlight PCSK9 as an important regulator of CD36, PD-L1 and HLA-ABC cell surface expression in pancreatic beta cells.

Data availabilityRNA-seq data have been deposited to GEO database with accession number GSE182016. Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the following identifiers: PXD027921, PXD027911 and PXD027913.

sentences: A comparative life cycle analysis of electromicrobial production systems Electromicrobial production (EMP) processes represent an attractive strategy for the capture and conversion of CO2 into carbon-based products. We describe the development and application of comprehensive reactor, process, and life cycle impact models to analyze three major EMP systems relying on formate, H2, and acetate as intermediate molecules. Our results demonstrate that EMP systems can achieve a smaller carbon footprint than traditional bioprocessing strategies provided the electric grid is composed of >~90% renewable energy sources. For each of the three products we consider (biomass, enzymes, and lactic acid), the H2-mediated Knallgas bacteria system achieves the lowest overall global warming potential, indicating that this EMP strategy may be best-suited for industrial efforts based on current technology. We also identify environmental hotspots and process limitations that are key sites for future engineering and research efforts for each EMP system. Our analysis demonstrates the utility of an integrated bioelectrochemical model/life cycle assessment framework in both analyzing and aiding the ecodesign of electromicrobial processes and should help guide the design of working, scalable, and sustainable systems.

sentences: Identification of ACE2 mutations that modulate SARS-CoV-2 spike binding across multiple mammalian species Understanding how SARS-CoV-2 interacts with different mammalian angiotensin-converting enzyme II (ACE2) cell entry receptors elucidates determinants of virus transmission and facilitates development of vaccines for humans and animals. Yeast display-based directed evolution identified conserved ACE2 mutations that increase spike binding across multiple species. Gln42Leu increased ACE2-spike binding for human and four of four other mammalian ACE2s; Leu79Ile had a effect for human and three of three mammalian ACE2s. These residues are highly represented, 83% for Gln42 and 56% for Leu79, among mammalian ACE2s. The above findings can be important in protecting humans and animals from existing and future SARS-CoV-2 variants.

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TWIST1 interacts with adherens junction proteins during neural tube formation and regulates fate transition in cranial neural crest cells Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural fold borders into multiple cell lineages that differentiate into bone, cartilage, neurons, and glial cells. We previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicates that TWIST1 interacts with /{beta}/{gamma}-CATENINS during neural tube closure, and Irf6 is involved in the structural integrity of the neural tube. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects, and CNCC-derived structural abnormalities. Altogether, this study describes an uncharacterized function of Twist1 and Irf6 in the neural tube and CNCCs and provides new target genes of Twist1 involved in cytoskeletal remodeling. Furthermore, the association between DNA variations within TWIST1 putative enhancers and human facial morphology is also investigated.

SUMMARY STATEMENTThis study uncovers a new function of Twist1 in neural tube development and epithelial-to-mesenchymal transition in cranial neural crest cells. Data further shows that Twist1-interacting Irf6 is involved in regulating neural tube integrity.

sentences: Identification of highly cross-reactive mimotopes for a public T cell response in murine melanoma While immune checkpoint blockade results in durable responses for some patients, many others have not experienced such benefits. These treatments rely upon reinvigorating specific T cell-antigen interactions. However, it is often unknown what antigens are being recognized by T cells or how to potently induce antigen-specific responses in a broadly applicable manner. Here, we characterized the CD8+ T cell response to a murine model of melanoma following combination immunotherapy to determine the basis of tumor recognition. Sequencing of tumor-infiltrating T cells revealed a repertoire of highly homologous TCR sequences that were particularly expanded in treated mice and which recognized an antigen from an endogenous retrovirus. While vaccination against this peptide failed to raise a protective T cell response in vivo, engineered antigen mimotopes induced a significant expansion of CD8+ T cells cross-reactive to the original antigen. Vaccination with mimotopes resulted in killing of antigen-loaded cells in vivo yet showed modest survival benefit in a prophylactic vaccine paradigm. Together, this work demonstrates the identification of a dominant tumor-associated antigen and generation of mimotopes which can induce robust functional T cell responses that are cross-reactive to the endogenous antigen across multiple individuals.

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Temporal properties of painful contrast enhancement using repetitive stimulation Offset analgesia is characterized by a disproportionately large reduction in pain following a small decrease in a heat stimulus and is based on the phenomenon of temporal pain contrast enhancement (TPCE). The aim of this study is to investigate whether this phenomenon can also be induced by repetitive stimulation, i.e., by stimuli that are clearly separated in time. With this aim, the repetitive TPCE paradigm was induced in healthy, pain-free subjects (n=33) at the volar non-dominant forearm using heat stimuli. This paradigm was performed applying three different interstimulus intervals (ISIs): 5, 15, and 25 seconds. All paradigms were contrasted with a control paradigm without temperature change. Participants continuously rated the perceived pain intensity. In addition, electrodermal activity was recorded as a surrogate measure of autonomic arousal. Temporal pain contrast enhancement was confirmed for both ISI 5 seconds (p < 0.001) and ISI 15 seconds (p = 0.005), but not for ISI 25 seconds (p = 0.07), however the magnitude of TPCE did not differ between ISIs (p = 0.11). Electrodermal activity was consistent previous pain ratings, but showing significantly higher autonomic activity being measured. Thus, the phenomenon of temporal contrast enhancement of pain can also be induced by repetitive stimulation. Both the involvement of the autonomic nervous system and the involvement of habituation processes are conceivable, which consequently points to both central and peripheral mechanisms of TPCE.

SummaryThe temporal contrast enhancement of pain and electrodermal activity can be provoked by stimuli that are clearly separated in time.

sentences: New Segmentation and Feature Extraction Algorithm for Classification of White Blood Cells in Peripheral Smear Images This article addresses a new method for the classification of white blood cells (WBCs) using image processing techniques and machine learning methods. The proposed method consists of three steps: detecting the nucleus and cytoplasm, extracting features, and classification. At first, a new algorithm is designed to segment the nucleus. For the cytoplasm to be detected, only a part of it located inside the convex hull of the nucleus is involved in the process. This attitude helps us overcome the difficulties of segmenting the cytoplasm. In the second phase, three shapes and four novel color features are devised and extracted. Finally, by using an SVM model, the WBCs are classified. The segmentation algorithm can detect the nucleus with a dice similarity coefficient of 0.9675. The proposed method can categorize WBCs in Raabin-WBC, LISC, and BCCD datasets with accuracies of 94.65 %, 92.21 %, and 94.20 %, respectively. It is worth mentioning that the hyperparameters of the classifier are fixed only with the Raabin-WBC dataset, and these parameters are not readjusted for LISC and BCCD datasets. The obtained results demonstrate that the proposed method is robust, fast, and accurate.

sentences: Exclusion of the fittest predicts microbial community diversity in fluctuating environments Microorganisms live in environments that inevitably fluctuate between mild and harsh conditions. As harsh conditions may cause extinctions, the rate at which fluctuations occur can shape microbial communities and their diversity, but we still lack an intuition on how. Here, we build a mathematical model describing two microbial species living in an environment where substrate supplies randomly switch between abundant and scarce. We then vary the rate of switching as well as different properties of the interacting species, and measure the probability of the weaker species driving the stronger one extinct. We find that this probability increases with the strength of demographic noise under harsh conditions and peaks at either low, high, or intermediate switching rates depending on both species ability to withstand the harsh environment. This complex relationship shows why finding patterns between environmental fluctuations and diversity has historically been difficult. In parameter ranges where the fittest species was most likely to be excluded, however, the beta diversity in larger communities also peaked. In sum, how environmental fluctuations affect interactions between a few species pairs predicts their effect on the beta diversity of the whole community.

sentences: The pseudouridine synthase dyskerin binds to cytoplasmic H/ACA-box snoRNA retaining transcripts affecting nuclear hormone receptor dependence Dyskerin is a nuclear protein involved in H/ACA box snoRNA-guided uridine modification of RNA. Since its defective function induces specific alterations in gene expression, we sought to unbiasedly identify mRNAs regulated by dyskerin. We found that dyskerin depletion affects the expression or the association with polysomes of selected mRNA isoforms characterized by the retention of H/ACA box snoRNA-containing introns. These snoRNA retaining transcripts (snoRTs) are bound by dyskerin and can interact with cytoplasmic ribosomes. We then characterized the cytoplasmic dyskerin RNA interactome finding both H/ACA box snoRTs and protein-coding transcripts. Since a fraction of these latter transcripts is involved in the nuclear hormone receptor binding, we tested to see if this specific activity is affected by dyskerin. Results indicate that dyskerin dysregulation may alter the dependence on nuclear hormone receptor ligands in breast cancer. Our work suggests a cytoplasmic function for dyskerin which could affect mRNA post-transcriptional networks relevant for nuclear hormone receptor functions.

sentences: Reprogramming the neuroblastoma epigenome with a mitochondrial uncoupler Dysregulated DNA methylation is associated with poor prognosis in cancer patients, promoting tumorigenesis and therapeutic resistance1. DNA methyltransferase inhibitors (DNMTi) reduce DNA methylation and promote cancer cell differentiation, with two DNMTi already approved for cancer treatment2. However, these drugs rely on cell division to dilute existing methylation, thus the  demethylation effects are achieved in a passive manner, limiting their application in slow-proliferating tumor cells. In this study we use a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to actively achieve global DNA demethylation. NEN treatment promotes DNA demethylation by activating electron transport chain (ETC) to produce -ketoglutarate (-KG), a substrate for the DNA demethylase TET. In addition, NEN inhibits reductive carboxylation, a key metabolic pathway to support growth of cancer cells with defective mitochondria or under hypoxia. Importantly, NEN treatment reduces 2-hydroxyglutarate (2-HG) generation and blocks DNA hypermethylation under hypoxia. Together, these metabolic reprogramming effects of NEN actively alter the global DNA methylation landscape and promote neuroblastoma differentiation. These results not only support Warburgs original hypothesis that inhibition of ETC causes cell de-differentiation and tumorigenesis, but also suggest that mitochondrial uncoupling is an effective metabolic and epigenetic intervention that remodels the tumor epigenome for better prognosis.

sentences: IL-37 expression reduces acute and chronic neuroinflammation and rescues cognitive impairment in an Alzheimer s disease mouse model The anti-inflammatory cytokine interleukin-37 (IL-37) is a member of the IL-1 family but not expressed in mice. We used a human IL-37 (hIL-37tg) expressing mouse, which has been subjected to various models of local and systemic inflammation as well as immunological challenges. Those studies demonstrate an immune-modulatory role of IL-37 which can be characterized as an important suppressor of innate immunity. We investigated the functions of IL-37 in the CNS and explored the effects of IL-37 on neuronal architecture and function, microglia phenotype, cytokine production and behavior after inflammatory challenge by intraperitoneal LPS-injection. Reduced spine density, activated microglia phenotype and impaired long-term potentiation (LTP) were observed in wild-type mice after LPS injection, whereas hIL-37tg mice showed no impairment. In addition, we crossed the hIL-37tg mouse with an animal model of Alzheimers disease (APP/PS1) to investigate the anti-inflammatory properties of IL-37 under chronic neuroinflammatory conditions. Our results show that IL-37 is able to limit inflammation in the brain after acute inflammatory events and prevent the loss of cognitive abilities in a mouse model of AD.

sentences: Waves of sumoylation support transcription dynamics during adipocyte differentiation Tight control of gene expression networks required for adipose tissue formation and plasticity is essential for adaptation to energy needs and environmental cues. However, little is known about the mechanisms that orchestrate the dramatic transcriptional changes leading to adipocyte differentiation. We investigated the regulation of nascent transcription by the sumoylation pathway during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that the sumoylation pathway has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of specific transcription factors like Ppar{gamma}/RXR and their co-factors is associated with the transcription of adipogenic genes. Our data demonstrate that the sumoylation pathway coordinates the rewiring of transcriptional networks required for formation of functional adipocytes. This study also provides an in-depth resource of gene transcription dynamics, SUMO-regulated genes and sumoylation sites during adipogenesis.

sentences: New cellular models of undifferentiated pleomorphic sarcoma and malignant peripheral nerve sheath tumor Undifferentiated pleomorphic sarcoma (UPS) and malignant peripheral nerve sheath tumor (MPNST) are aggressive soft tissue sarcomas that do not respond well to current treatment modalities. The limited availability of UPS and MPNST cell lines makes it challenging to identify potential therapeutic targets in a laboratory setting. Understanding the urgent need for improved treatments for these tumors and the limited cellular models led us to generate additional cell lines to study these rare cancers further. Patient-derived tumors were used to establish 5 new UPS models, including one radiation-associated UPS--UPS060.1, UPS271.1, UPS511, UPS0103, and RIS620--and 3 new models of MPNST--MPNST007, MPNST3813E, and MPNST4970. This study examined the utility of the new cell lines as sarcoma models by assessing tumorigenic potential and mutation status for known sarcoma-related genes. All the cell lines formed colonies and migrated in vitro. The in vivo tumorigenic potential of each cell line was determined by either subcutaneous injection of cells or implantation of tumor tissue into immunocompromised mice. UPS060.1, UPS271.1, and UPS511 cells formed tumors in mice upon subcutaneous injection. UPS0103 and RIS620 tumor implants formed tumors in vivo, as did MPNST007 and MPNST3813E tumor implants. Mutation analysis of a panel of genes frequently mutated in sarcomas showed that two of the three MPNST cell lines had NF1 mutations. Two of the three MPNST cell lines had mutations in polycomb repressive complex 2 members. These new cellular models provide the scientific community with powerful tools for detailed studies of sarcomagenesis and investigate novel therapies for UPS and MPNST.

sentences: Electrophysiological evidence of synergistic auxin transport by interacting Arabidopsis ABCB4 and PIN2 proteins To understand why both ATP-binding cassette B (ABCB) and PIN-FORMED (PIN) proteins are required for polar auxin transport through tissues, even though only the latter is polarly localized, we biophysically studied their transport characteristics separately and together by whole-cell patch clamping. ABCB4 and PIN2 from Arabidopsis thaliana expressed in human embryonic kidney cells displayed electrogenic activity when CsCl-based electrolytes were used. Current-voltage (I-V) analysis of the activities and modeling the effects of adding the auxin anion (IAA-) as a potential substrate with the Goldman-Hodgkin-Katz equation, demonstrated that ABCB4 and PIN2 were 9-fold and 10-fold more selective for IAA- than Cl-, respectively. Thus, these proteins directly transport IAA-, which was not unequivocally established by previous auxin retention assays. Co-expression of ABCB4 and PIN2 produced an especially significant result. Co-expression synergistically doubled the selectivity for IAA-. An area of two-fold higher selectivity for IAA- that this result indicates will occur in cells with asymmetric PIN2 and symmetric ABCB4 matches what early models found to be necessary to create observed levels of polar auxin transport through tissues. Thus, the requirement for two different proteins appears to be explained by a synergistic effect on selectivity. More substrate details and important pharmacological results are reported.

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Biochemical analysis of deacetylase activity of rice sirtuin OsSRT1, a class IV member in plants The role of plant sirtuins is slowly unwinding. There are only reports of H3K9Ac deacetylation by OsSRT1. This belongs to class IV sirtuin family with a longer C-terminus. Here C-terminus is required for ligand binding and catalysis. OsSRT1 can deacetylate the lys residues at the N terminal tail of both H3 and H4. It can also target the non-histone target, OsPARP1 playing a role in DNA damage repair pathway. Changes in the extent of different histone deacetylation by OsSRT1 is also related with different abiotic stress conditions. NAM and ADP-ribose has negative effect on OsSRT1 deacetylation.

HighlightsO_LIOsSRT1 is capable of deacetylating various lysine residues of histone H3 and H4 as well as OsPARP1.
C_LIO_LIThe extra long C-terminus of OsSRT1 is required for its substrate binding and thus, its catalysis.
C_LIO_LIOn plants exposure to H2O2 and Arsenic toxicity, there is a relationship between increased expression of OsSRT1 and increased deacetylation of H3 and H4.
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Normative and mechanistic model of an adaptive circuit for efficient encoding and feature extraction One major question in neuroscience is how to relate connectomes to neural activity, circuit function, and learning. We offer an answer in the peripheral olfactory circuit of the Drosophila larva, composed of olfactory receptor neurons (ORNs) connected through feedback loops with interconnected inhibitory local neurons (LNs). We combine structural and activity data and, using a holistic normative framework based on similarity-matching, we propose a biologically plausible mechanistic model of the circuit. Our model predicts the ORN [->] LN synaptic weights found in the connectome and demonstrate that they reflect correlations in ORN activity patterns. Additionally, our model explains the relation between ORN [->] LN and LN - LN synaptic weight and the arising of different LN types. This global synaptic organization can autonomously arise through Hebbian plasticity, and thus allows the circuit to adapt to different environments in an unsupervised manner. Functionally, we propose LNs extract redundant input correlations and dampen them in ORNs, thus partially whitening and normalizing the stimulus representations in ORNs. Our work proposes a comprehensive framework to combine structure, activity, function, and learning, and uncovers a general and potent circuit motif that can learn and extract significant input features and render stimulus representations more efficient.

SignificanceThe brain represents information with patterns of neural activity. At the periphery, due to the properties of the external world and of encoding neurons, these patterns contain correlations, which are detrimental for stimulus discrimination. We study the peripheral olfactory neural circuit of the Drosophila larva, that preprocesses neural representations before relaying them to higher brain areas. A comprehensive understanding of this preprocessing is, however, lacking. Here, we propose a mechanistic and normative framework describing the function of the circuit and predict the circuits synaptic organization based on the circuits input neural activity. We show how the circuit can autonomously adapt to different environments, extracts stimulus features, and decorrelate and normalize input representations, which facilitates odor discrimination downstream.

sentences: Machine learning techniques to derive bioclimatic classifications for Colombia Bioclimatic classifications seek to divide a study region into geographic areas with similar bioclimatic characteristics. In this study we proposed two bioclimatic classifications for Colombia using machine learning techniques. We firstly characterized the precipitation space of Colombia using principal component analysis. Based on Lang classification, we then projected all background sites in the precipitation space with their corresponding categories. We sequentially fit logistic regression models to reclassify all background sites in the precipitation space with six redefined Lang categories. New categories were the used to define a new modified Lang and Caldas-Lang classifications.

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Silencing of an efflux pump coding gene decreases the efflux rate of pyrazinoic acid in Mycobacterium smegmatis BackgroundTuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). The recommended treatment for TB is based on the use of first-line drugs, including pyrazinamide (PZA). PZA is also a drug used in the treatment of multidrug-resistant TB (MDR-TB) because of its main effect against the latent stage. The main cause of resistance to PZA is mutations in the pncA gene, which compromise the activity of the encoded enzyme pyrazinamidase (PZAse), which hydrolyzes PZA into POA, the active antituberculosis molecule. The mechanism of action of PZA requires that POA is expelled from the bacterium by an efflux mechanism. After that, if the extracellular medium is sufficiently acidic, POA is protonated and returns to the cytosol, releasing the proton and repeating the cycle, resulting lethal to the bacteria. The efflux pump responsible for extruding the POA to the extracellular environment is not yet known. Mycobacterium smegmatis is naturally resistant to PZA and has a 900-fold faster POA efflux rate than MTB, and has the advantage to be a faster growing mycobacterium.

MethodsIn the present study we have silenced the transcription of several genes encoding efflux pumps in M. smegmatis by CRISPRi (CRISPR interference). These genes (MSMEG_0250, MSMEG_3815, MSMEG_0241, MSMEG_5046 and MSMEG_0410) were homologous to efflux pump genes in MTB. POA efflux rate was measured, and a quantitative Waynes test was performed after silencing each gene.

ResultsSilencing of MSMEG_0250, resulted in approximately 5-fold decrease in the POA efflux rate in M. smegmatis (P<0.0001). None of the other silenced genes showed a notable decrease in POA efflux rate.

sentences: Hippocampal sharp wave-ripples and the associatedsequence replay emerge from structured synapticinteractions in a network model of area CA3 Hippocampal place cells are activated sequentially as an animal explores its environment. These activity sequences are internally recreated ("replayed"), either in the same or reversed order, during bursts of activity (sharp wave-ripples; SWRs) that occur in sleep and awake rest. SWR-associated replay is thought to be critical for the creation and maintenance of long-term memory. In order to identify the cellular and network mechanisms of SWRs and replay, we constructed and simulated a data-driven model of area CA3 of the hippocampus. Our results show that the chain-like structure of recurrent excitatory interactions established during learning not only determines the content of replay, but is essential for the generation of the SWRs as well. We find that bidirectional replay requires the interplay of the experimentally confirmed, temporally symmetric plasticity rule, and cellular adaptation. Our model provides a unifying framework for diverse phenomena involving hippocampal plasticity, representations, and dynamics, and suggests that the structured neural codes induced by learning may have greater influence over cortical network states than previously appreciated.

sentences: Simultaneous functional MRI of socially interacting marmosets Social cognition is a dynamic process that requires the perception and integration of a complex set of idiosyncratic features between interacting conspecifics. Here we present a method for simultaneously measuring the whole-brain activation of two socially interacting marmoset monkeys using functional magnetic resonance imaging. MRI hardware (a radiofrequency coil and peripheral devices) and image-processing pipelines were developed to assess brain responses to socialization, both on an intra-brain and inter-brain level. Notably, brain-activation maps acquired during constant interaction demonstrated neuronal synchrony between marmosets in regions of the brain responsible for processing social interaction. This method enables a wide range of possibilities for studying social function and dysfunction in a non-human primate model, including using transgenic models of neuropsychiatric disorders.

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Bilaterally Reduced Rolandic Beta Band Activity in Minor Stroke Patients Stroke patients with hemiparesis display decreased beta band (13-25 Hz) rolandic activity, correlating to impaired motor function. However, patients without significant weakness, with small lesions far from sensorimotor cortex, nevertheless exhibit bilateral decreased motor dexterity and slowed reaction times. We investigate whether these minor stroke patients also display abnormal beta band activity.

Magnetoencephalographic (MEG) data were collected from nine minor stroke patients (NIHSS < 4) without significant hemiparesis, at ~1 and ~6 months postinfarct, and eight age-similar controls. Rolandic relative beta power during matching tasks and resting state, and Beta Event Related (De)Synchronization (ERD/ERS) during button press responses were analyzed.

Regardless of lesion location, patients had significantly reduced relative beta power and ERS compared to controls. Abnormalities persisted over visits, and were present in both ipsi- and contra-lesional hemispheres, consistent with bilateral impairments in motor dexterity and speed.

Minor stroke patients without severe weakness display reduced rolandic beta band activity in both hemispheres, which may be linked to bilaterally impaired dexterity and processing speed, implicating global connectivity dysfunction affecting sensorimotor cortex. Rolandic beta band activity may be a potential biomarker and treatment target, even for minor stroke patients with small lesions far from sensorimotor areas.

sentences: A tail of plumage colouration: disentangling geographic, seasonal, and dietary effects on plumage colour in a migratory songbird Plumage ornamentation in birds serves critical inter- and intra-sexual signaling functions. While carotenoid-based plumage colouration is often viewed as a classic condition-dependent sexually selected trait, plumage colouration can be influenced by a wide array of both intrinsic and extrinsic factors. Understanding the mechanisms underlying variation in colouration is especially important for species where the signaling function of ornamental traits is complex or the literature conflicting. Here, we examined variation in the yellow/orange tail feathers of American redstarts (Setophaga ruticilla) passing through two migratory stopover sites in eastern North America during both spring and fall migration to assess the role of geographic variation and seasonality in influencing feather colouration. In addition, we investigated whether diet during moult (inferred via stable isotope analysis of feather {delta}15N and {delta}13C) influenced plumage colouration. Our findings indicate that geographic variation, season, and diet all influence individual differences in American redstart colouration, represented by both traditional and tetrahedral colour variables. The extent to which these factors influence colour expression however is largely dependent on the colour metric under study, likely because different colour metrics reflect different attributes of the feather (e.g., structural components vs. pigment deposition). The effects of diet ({delta}15N) and season were pronounced for brightness, suggesting a strong effect of diet and feather wear/degradation on feather structure. Though hue, a metric that should strongly reflect pigment deposition, also changed from spring to fall, that effect was dependent on age, with only adults experiencing a reduction in ornamentation. Taken together, our results highlight the numerous sources of variation behind plumage coloration and underscores the difficulty of unraveling complex visual signaling systems, such as those in American redstarts.

sentences: Comparative transcriptome analysis of Cryptomeria japonica between solid and liquid culture. This manuscript has been withdrawn by the authors as it was submitted and made public without the appropriate authorizations. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

sentences: Role of Gasdermins in the Biogenesis of Apoptotic Cell-Derived Exosomes The gasdermins, GSDMA, GSDMB, GSDMC, GSDMD, DFNA5, and DFNB59, are a family of pore-forming proteins that has recently been suggested to play a central role in the pyroptosis and the release of inflammatory cytokines. Here, we describe the novel roles of gasdermins in the biogenesis of apoptotic cell-derived exosomes. In apoptotic cells, GADMA, GSDMC, GSDMD, and DFNA5 increased the release of ApoExos, and both their full-length and cleaved forms were localized in the exosomal membrane. GSDMB and DFNB59, on the other hand, negatively affected the release of ApoExos. The caspase-mediated cleavage of gasdermins, especially DFNA5, is suggested to enable cytosolic Ca2+ to flow through endosomal pores and thus increase the biogenesis of ApoExos. In addition, the DFNA5-meidiated biogenesis of ApoExos depended on the ESCRT-III complex and endosomal recruitment of Ca2+-dependent proteins: annexins A2 and A7, the PEF domain family proteins sorcin and grancalcin, and the Bro1 domain protein HD-PTP. Therefore, we propose that the biogenesis of ApoExos begins when gasdermin-mediated endosomal pores increase cytosolic Ca2+, continues through the recruitment of annexin-sorcin/grancalcin-HD-PTP, and is completed when the ESCRT-III complex synthesizes intraluminal vesicles in the multivesicular bodies of dying cells. Finally, we found that Dfna5-bearing tumors released ApoExos to induce inflammatory responses in the in vivo 4T1 orthotropic model of breast cancer. The data presented in this study indicate that the switch from apoptosis to pyroptosis could drive the transfer of mass signals to nearby or distant living cells and tissues by way of extracellular vesicles, and that gasdermins play critical roles in that process.

sentences: A Yap-dependent transcriptional program directs cell migration for embryo axis assembly The condensation of the embryo primary axis is a fundamental landmark in the establishment of the vertebrate body plan. Although the complex morphogenetic movements directing cell convergence towards the midline have been described extensively, little is known on how gastrulating cells interpret mechanical cues. Yap proteins are among the best characterized transcriptional mechanotransducers, yet their role in gastrulation has remained elusive. Here we show that the double knockout of yap and its paralog yap1b in medaka results in an axis assembly failure. Quantitative live imaging reveals that mutant cells display reduced displacement and migratory persistence. By characterizing the Yap-dependent transcriptional program, we identified genes involved in cytoskeletal organization and cell-ECM adhesion, rather than in germ layer specification, as direct Yap targets. Dynamic analysis of Tead sensors and downstream targets reveals Yap is acting in migratory cells, and not as a midline beacon, to direct gastrulating precursors trajectories by promoting cortical actin recruitment and focal adhesions assembly. We propose that Yap is engaged in a mechano-regulatory loop that is essential to maintain the directed cell migration sustaining embryo axis formation.

sentences: scCorr: A graph-based k-partitioning approach for single-cell gene-gene correlation analysis An important challenge in single-cell RNA-sequencing analysis is the abundance of zero values, which results in biased estimation of gene-gene correlations for downstream analyses. Here, we present a novel graph-based k-partitioning method by merging "homology" cells to reduce the number of zero values. Our method is robust and reliable for the detection of correlated gene pairs, which is fundamental to network construction, gene-gene interaction, and cellular -omic analyses.

sentences: Sex-specific regulation of metabolic health and vertebrate lifespan by AMP biosynthesis Energy homeostasis is disrupted with age, which then fuels multiple age-related pathologies. The AMP-activated protein kinase (AMPK) is the primary sensor of cellular energy in eukaryotes. However, the genetic regulation of vertebrate aging by AMPK remains poorly understood. Here, we manipulate energy levels in the turquoise killifish by mutating APRT, a key enzyme in AMP biosynthesis. These manipulations produced a male-specific lifespan extension and restored metabolic plasticity. Exploring the observed sex differences using an integrated omics approach implicated the mitochondria as an important player. Mechanistically, APRT regulated mitochondrial functions and AMPK activity, mimicking energy starvation in heterozygous cells. A fasting-like state was also detected, particularly in heterozygous males, which leads to resistance to high-fat diet. Finally, life-long intermittent fasting eliminated the male-specific longevity benefits mediated by the APRT mutation. These observations identify the AMP/AMPK axis as a sex-specific regulator of vertebrate longevity and metabolic health.

sentences: CloneSig can jointly infer intra-tumor heterogeneity and mutational signature activity in bulk tumor sequencing data Systematic DNA sequencing of cancer samples has highlighted the importance of two aspects of cancer genomics: intra-tumor heterogeneity (ITH) and mutational processes. These two aspects may not always be independent, as different mutational processes could be involved in different stages or regions of the tumor, but existing computational approaches to study them largely ignore this potential dependency. Here, we present CloneSig, a computational method to jointly infer ITH and mutational processes in a tumor from bulk-sequencing data. Extensive simulations show that CloneSig outperforms current methods for ITH inference and detection of mutational processes when the distribution of mutational signatures changes between clones. Applied to a large cohort of 8,951 tumors with whole-exome sequencing data from The Cancer Genome Atlas, and on a pan-cancer dataset of 2,632 whole-genome sequencing tumor samples from the Pan-Cancer Analysis of Whole Genomes initiative, CloneSig obtains results overall coherent with previous studies.

sentences: To denoise or to cluster? That is not the question. Optimizing pipelines for COI metabarcoding and metaphylogeography The recent blooming of metabarcoding applications to biodiversity studies comes with some relevant methodological debates. One such issue concerns the treatment of reads by denoising or by clustering methods, which have been wrongly presented as alternatives. It has also been suggested that denoised sequence variants should replace clusters as the basic unit of metabarcoding analyses, missing the fact that sequence clusters are a proxy for species-level entities, the basic unit in biodiversity studies. We argue here that methods developed and tested for ribosomal markers have been uncritically applied to highly variable markers such as cytochrome oxidase I (COI) without conceptual or operational (e.g., parameter setting) adjustment. COI has a naturally high intraspecies variability that should be assessed and reported, as it is a source of highly valuable information. We contend that denoising and clustering are not alternatives. Rather, they are complementary and both should be used together in COI metabarcoding pipelines. Using a typical dataset from benthic marine communities, we compared two denoising procedures (based on the UNOISE3 and the DADA2 algorithms), set suitable parameters for denoising and clustering COI datasets, and compared the outcome of applying these processes in different orders. Our results indicate that denoising based on the UNOISE3 algorithm preserves a higher intra-cluster variability. We suggest and test ways to improve this algorithm taking into account the natural variability of each codon position in coding genes. The order of the steps (denoising and clustering) has little influence on the final outcome. We recommend researchers to consider reporting their results in terms of both denoised sequences (a proxy for haplotypes) and clusters formed (a proxy for species), and to avoid collapsing the sequences of the latter into a single representative. This will allow studies at the cluster (ideally equating species-level diversity) and at the intra-cluster level, and will ease additivity and comparability between studies.

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MinION barcodes: biodiversity discovery and identification by everyone, for everyone BackgroundDNA barcodes are a useful tool for discovering, understanding, and monitoring biodiversity which are critical tasks at a time of rapid biodiversity loss. However, widespread adoption of barcodes requires cost-effective and simple barcoding methods. We here present a workflow that satisfies these conditions. It was developed via "innovation through subtraction" and thus requires minimal lab equipment, can be learned within days, reduces the barcode sequencing cost to <10 cents, and allows fast turnaround from specimen to sequence by using the portable, real-time sequencer MinION.

ResultsWe describe cost-effective and rapid procedures for barcoding individual specimens with MinION sequencing. We illustrate how tagged amplicons can be obtained and sequenced with the portable, real-time MinION sequencer in many settings (field stations, biodiversity labs, citizen science labs, schools). We also provide amplicon coverage recommendations that are based on several runs of the latest generation of MinION flow cells ("R10.3") which suggest that each run can generate barcodes for >10,000 specimens. Next, we present a novel software, ONTbarcoder, which overcomes the bioinformatics challenges posed by MinION reads. The software is compatible with Windows 10, Macintosh, and Linux, has a graphical user interface (GUI), and can generate thousands of barcodes on a standard laptop within hours based on only two input files (FASTQ, demultiplexing file). We document that MinION barcodes are virtually identical to Sanger and Illumina barcodes for the same specimens (>99.99%) and provide evidence that MinION flow cells and reads have improved rapidly since 2018.

ConclusionsWe propose that barcoding with MinION is the way forward for government agencies, universities, museums, and schools because it combines low consumable and capital cost with scalability. Small projects can use the flow cell dongle ("Flongle") while large projects can rely on MinION flow cells that can be stopped and re-used after collecting sufficient data for a given project.

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Omitting age-dependent mosquito mortality in malaria models underestimates the effectiveness of insecticide-treated nets Mathematical models of vector-borne infections, including malaria, often assume age-independent mortality rates of vectors, despite evidence that many insects senesce. In this study we present survival data on insecticide-resistant Anopheles gambiae s.l. from field experiments in Cote dIvoire. We fit a constant mortality function and two age-dependent functions (logistic and Gompertz) to the data from mosquitoes exposed (treated) and not exposed (control) to insecticidetreated nets (ITNs), to establish biologically realistic survival functions. This enables us to explore the effects of insecticide exposure on mosquito mortality rates, and the extent to which insecticide resistance might impact the effectiveness of ITNs. We investigate this by calculating the expected number of infectious bites a mosquito will take in its lifetime, and by extension the vectorial capacity. Our results show that the predicted vectorial capacity is substantially lower in mosquitoes exposed to ITNs, despite the mosquitoes in the experiment being highly insecticide-resistant. The more realistic age-dependent functions provide a better fit to the experimental data compared to a constant mortality function and, hence, influence the predicted impact of ITNs on malaria transmission potential. In models with age-independent mortality, there is a reduction of 56.52% ({+/-}14.66) for the vectorial capacity under exposure compared to no exposure. However, the two age-dependent functions predicted a larger reduction due to exposure: for the logistic function the reduction is 74.38% ({+/-}9.93) and for the Gompertz 74.35% ({+/-}7.11), highlighting the impact of incorporating age in the mortality rates. These results further show that multiple exposures to ITNs had a considerable effect on the vectorial capacity. Overall, the study highlights the importance of including age dependency in mathematical models of vector-borne disease transmission and in fully understanding the impact of interventions.

Author summaryInterventions against malaria are most commonly targeted on the adult mosquitoes, which transmit the infection from person to person. One of the most important interventions are bed-nets, treated with insecticides. Unfortunately, extensive exposure of mosquitoes to insecticide has led to widespread evolution of insecticide resistance, which might threaten control strategies. Piecing together the overall impact of resistance on the efficacy of insecticide-treated nets is complex, but can be informed by the use of mathematical models. However, there are some assumptions that the models frequently use which are not realistic in terms of the mosquito biology. In this paper, we formulate a model that includes age-dependent mortality rates, an important parameter in vector control since control strategies most commonly aim to reduce the lifespan of the mosquitoes. By using novel data collected using field-derived insecticide-resistant mosquitoes, we explore the effects that the presence of insecticides on nets have on the mortality rates, as well as the difference incorporating age dependency in the model has on the results. We find that including age-dependent mortality greatly alters the anticipated effects of insecticide-treated nets on mosquito transmission potential, and that ignoring this realism potentially overestimates the negative impact of insecticide resistance.

sentences: Cellular-level phenotyping of tumor-immune microenvironment (TiME) in patients in vivo reveals distinct inflammation and endothelial anergy signatures Immunotherapies have shown unprecedented clinical benefits in several malignancies1-3. However, clinical responses remain variable and unpredictable, indicating the need to develop predictive platforms that can improve patient stratification4. Phenotyping of tumors into hot, altered, or cold5 based on assessment of only T-cell infiltration in static tumor biopsies provides suboptimal prediction of immunotherapy response6,7. In vivo dynamic mechanisms within the tumor microenvironment such as tumor angiogenesis and leukocyte trafficking5,8,9 also play a central role in modulating anti-tumor immunity and therefore immunotherapy response. Here, we report novel tumor immune microenvironment (TiME) phenotyping in vivo in patients with non-invasive spatially-resolved cellular-level imaging based on endogenous contrast. Investigating skin cancers as a model, with reflectance confocal microscopy (RCM) imaging10, we determined four major phenotypes with variable prevalence of vasculature (Vasc) and inflammation (Inf) features: VaschiInfhi, VaschiInflo, VascloInfhi and Vascmed/hiInflo. The VaschiInfhi phenotype correlates with high immune activation, exhaustion, and vascular signatures while VaschiInflo with endothelial anergy and immune exclusion. Automated quantification of TiME features demonstrates moderate-high accuracy and correlation with corresponding gene expression. Prospectively analyzed response to topical immunotherapy show highest response in VascloInfhi, and reveals the added value of vascular features in predicting treatment response. Our novel in vivo cellular-level imaging and phenotyping approach can potentially advance our fundamental understanding of TiME, develop robust predictors for immunotherapy outcomes and identify novel targetable pathways in future.

sentences: High Content and High Throughout Phenotypic Assay for the Hourly Resolution of the Malaria Parasite Erythrocytic Cycle Over the last 20 years increased funding for malaria research has resulted in very significant technical advances to study the biology of Plasmodium species. High throughput phenotypic assays have been developed to screen millions of compounds and identify small molecules with antiparasitic activity. At the same time, advances in malaria genetic have greatly facilitated the generation of genetically modified parasites, and whole genome genetic screens are now feasible in Plasmodium species. Finally, there has been an increased interest to study malaria parasites at the population level, in particular in the area of drug resistance. Drug resistant field isolates have been collected around the world, and drug resistant strains are routinely generated in the lab to study the mechanisms of drug resistance. As a result, one of the current bottlenecks in malaria research is our ability to quickly characterize the phenotype associated with compound treatment or genetic modification, or to quickly compare differences in intracellular development between strains. Here, we present a high content/high throughput phenotypic assay that combines highly selective RNA, DNA, and RBC membrane dyes to provide hourly resolution of the full erythrocytic cycle for both P. falciparum and P. knowlesi. A flow cytometry assay allows the analysis of samples in a 384-well format and a quick way to determine the parasite developmental stage. On the other hand, the fluorescence microscopy format allows for a detailed visualization of parasite morphology. Finally, using open source software we have developed protocols for the automated cluster analysis of microscopy images. This assay can be applied to any Plasmodium species, requires very little amount of sample, is performed with fixed cells, and is easily scalable. Overall, we believe this assay will be a great tool for the malaria community to study Plasmodium species.

sentences: Cerebellar Dentate Connectivity Across Adulthood: A Large-Scale Resting State Functional Connectivity Investigation Cerebellar contributions to behavior in advanced age are of interest and importance, given its role in motor and cognitive performance. There are differences and declines in cerebellar structure in advanced age and cerebellar resting state connectivity is lower. However, the work on this area to date has focused on the cerebellar cortex. The deep cerebellar nuclei provide the primary cerebellar inputs and outputs to the cortex, as well as the spinal and vestibular systems. Dentate networks can be dissociated such that the dorsal region is associated with the motor cortex, while the ventral aspect is associated with the prefrontal cortex. However, whether dentato-thalamo-cortical networks differ across adulthood remains unknown. Here, using a large adult sample (n=590) from the Cambridge Center for Ageing and Neuroscience, we investigated dentate connectivity across adulthood. We replicated past work showing dissociable resting state networks in the dorsal and ventral aspects of the dentate. In both seeds, we demonstrated that connectivity is lower with advanced age, indicating that connectivity differences extend beyond the cerebellar cortex. Finally, we demonstrated sex differences in dentate connectivity. This expands our understanding of cerebellar circuitry in advanced age and underscores the potential importance of this structure in age-related performance differences.

sentences: Distinguishing and employing sister species of fish in assessment of stream quality Biological indicators (bioindicators) can be individual species or species groups used to assess habitat quality. Unfortunately, conservationists often lack information on species distribution, how to differentiate between similar species, and environmental conditions associated with the presence of a species. We addressed these problems using two "sister" species of fish, the Mottled Sculpin (Cottus bairdii) and the Slimy Sculpin (Cottus cognatus), as stream quality indicators in the Manistee River watershed in the Huron-Manistee National Forests in Michigan, USA. We determined the abundance and distribution of these species and related their presence to concurrent in-stream measurements of temperature, dissolved oxygen, pH, conductivity, turbidity, and stream quality score based on macroinvertebrate diversity. To be certain of identification, we sequenced the Cytochrome c Oxidase Subunit I (CO1) molecular marker for specimens and used it as a DNA barcode to determine a specimens species. Cladistic analyses of CO1 unambiguously supported recognition of Mottled Sculpin and Slimy Sculpin as distinct species, confirming initial 87.5% correct identification using morphological characteristics, with uncertainty limited to juvenile fish. Field determinations increased to 100% correct identification as investigators gained more experience. Both species were most abundant in headwater regions, decreased downstream, and were sympatric at several locations. Mottled Sculpin were more likely to be found at stream locations with lower conductivity, pH, and stream quality scores, whereas Slimy Sculpin presence was more strongly associated higher levels of DO and lower levels of turbidity. Such findings are important because Mottled Sculpin are a designated management indicator species of the US Forest Service in the Huron-Manistee National Forests, but may be ineffective as a habitat quality indicator when used alone. Concurrent use of Mottled Sculpin and Slimy Sculpin as a management indicator sister-species complex could allow sufficient landscape coverage to permit habitat assessment if species-specific differences in environmental tolerances are precisely determined.

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Cubam receptor-mediated endocytosis in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni Nutrient transfer from mother to the embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni belonging to the family Goodeidae, the intraovarian embryo intakes the maternal component secreted into the ovarian fluid via the trophotaenia. Our previous study reported that the epithelial layer cells of the trophotaenia incorporate a maternal protein via vesicle trafficking. However, the molecules responsible for the absorption were still elusive. Here, we focused on Cubam (Cubilin-Amnionless) as a receptor involved in the absorption, and cathepsin L as a functional protease in the vesicles. Our results indicated that the Cubam receptor is distributed in the apical surface of the trophotaenia epithelium and then is taken into the intracellular vesicles. The trophotaenia possesses acidic organelles in epithelial layer cells and cathepsin L-dependent proteolysis activity. This evidence does not conflict with our hypothesis that receptor-mediated endocytosis and proteolysis play roles in maternal macromolecule absorption via the trohotaenia in viviparous teleosts. Such nutrient absorption involving endocytosis is not a specific trait in viviparous fish. Similar processes have been reported in the larval stage of oviparous fish or the suckling stage of viviparous mammals. Our findings suggest that the viviparous teleost acquired trophotaenia-based viviparity from a modification of the intestinal absorption system common in vertebrates. This is a fundamental study to understand the strategic variation of the reproductive system in vertebrates.

Summary statementHere, we report that an endocytic pathway is a candidate for nutrient absorption in pseudoplacenta of a viviparous teleost. The trait may have developed from common intestinal mechanism among vertebrates.

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Accel-Align: A Fast Sequence Mapper and Aligner based on the Seed-Embed-Extend Method BackgroundImprovements in sequencing technology continue to drive sequencing cost towards $100 per genome. However, mapping sequenced data to a reference genome remains a computationally-intensive task due to the dependence on edit distance for dealing with indels and mismatches introduced by sequencing. All modern aligners use seed-filter-extend (SFE) methodology and rely on filtration heuristics to reduce the overhead of edit distance computation. However, filtering has inherent performance-accuracy trade-offs that limits its effectiveness.

ResultsMotivated by algorithmic advances in randomized low-distortion embedding, we introduce seed- embed-extend (SEE), a new methodology for developing sequence mappers and aligners. While SFE focuses on eliminating sub-optimal candidates, SEE focuses instead on identifying optimal candidates. To do so, SEE transforms the read and reference strings from edit distance regime to the Hamming regime by embedding them using a randomized algorithm, and uses Hamming distance over the embedded set to identify optimal candidates. To show that SEE performs well in practice, we present Accel-Align, an SEE-based short-read sequence mapper and aligner that is 3-12x faster than state-of-the-art aligners on commodity CPUs, without any special-purpose hardware, while providing comparable accuracy.

ConclusionsAs sequencing technologies continue to increase read length while improving throughput and accuracy, we believe that randomized embeddings open up new avenues for optimization that cannot be achieved by using edit distance. Thus, the techniques presented in this paper have a much broader scope as they can be used for other applications like graph alignment, multiple sequence alignment, and sequence assembly.

Availabilityhttps://github.com/raja-appuswamy/accel-align-release

sentences: From perception to action: Intracortical recordings reveal cortical gradients of human exogenous attention Exogenous attention, the process that makes external salient stimuli pop-out of a visual scene, is essential for survival. How attention-capturing events modulate processing dynamics in the human brain remains unclear. We obtained a comprehensive depiction of attentional cortical dynamics at high spatiotemporal resolution, by analyzing brain activity from 1,403 intracortical contacts implanted in 28 individuals, while they performed an exogenous attention task. The timing, location and task-relevance of attentional events defined a spatiotemporal gradient of three neural clusters, which mapped onto cortical core-periphery topography and presented a hierarchy of timescales, providing the scaffolding for attention temporal computations. Visual attributes modulated neural activity at one end of the gradient, while activity at the other end reflected the upcoming response timing, with attentional effects occurring at the intersection of visual and response signals. These findings reveal how the psychological construct of exogenous attention emerges over large-scale cortical gradients in the human brain.

sentences: NFkB-signaling suppresses Müller glia-mediated neuron regeneration in the mammalian retina Muller glia (MG) in mammalian retinas are incapable of regenerating neurons after damage, whereas the MG in lower vertebrates regenerate functional neurons. Identification networks that regulate MG-mediated regeneration is key to harnessing the regenerative potential of MG. Here we study how NFkB-signaling influences glial responses to damage and reprogramming of MG into neurons in the rodent retina. We find activation of NFkB and dynamic expression of NFkB-associated genes in MG after damage, however NFkB activity is inhibited by microglia ablation. Knockout of NFkB in MG suppressed the accumulation of immune cells after damage. Inhibition of NFkB following NMDA-damage significantly enhanced the reprogramming of Ascl1-overexpressing MG into neuron-like cells. scRNA-seq of retinal glia following inhibition of NFkB reveals coordination with signaling via TGF{beta}2 and suppression of NFI and Id transcription factors. Inhibition of Smad3 or Id transcription factors increased numbers of neuron-like cells produced by Ascl1-overexpressing MG. We conclude that NFkB is a key signaling hub that is activated in MG after damage, mediates the accumulation of immune cells, and suppresses the neurogenic potential of MG.

sentences: Integrative COVID-19 Biological Network Inference with Probabilistic Core Decomposition The SARS-CoV-2 coronavirus is responsible for millions of deaths around the world. To help contribute to the understanding of crucial knowledge and to further generate new hypotheses relevant to SARS-CoV-2 and human protein interactions, we make use of the information abundant Biomine probabilistic database and extend the experimentally identified SARS-CoV-2-human protein-protein interaction (PPI) network in silico. We generate an extended network by integrating information from the Biomine database, the PPI network, and other experimentally validated results. To generate novel hypotheses, we focus on the high-connectivity sub-communities that overlap most with the integrated experimentally validated results in the extended network. Therefore, we propose a new data analysis pipeline that can efficiently compute core decomposition on the extended network and identify dense subgraphs. We then evaluate the identified dense subgraph and the generated hypotheses in three contexts: literature validation for uncovered virus targeting genes and proteins, gene function enrichment analysis on subgraphs, and literature support on drug repurposing for identified tissues and diseases related to COVID-19. The majority types of the generated hypotheses are proteins with their encoding genes and we rank them by sorting their connections to the integrated experimentally validated nodes. In addition, we compile a comprehensive list of novel genes, and proteins potentially related to COVID-19, as well as novel diseases which might be comorbidities. Together with the generated hypotheses, our results provide novel knowledge relevant to COVID-19 for further validation.

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Processive translocation of cohesive and non-cohesive cohesin in vivo Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops. Purified cohesin translocates along DNA in a diffusive fashion that can be propelled by transcribing RNA polymerase. The complex also extrudes DNA loops in a process that consumes ATP. In this study we examine processive genomic translocation of cohesin in vivo. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by transcription in yeast. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore, cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers alter cohesin redistribution demonstrates that the complex translocates processively on chromatin in vivo. The approach provides a relative measure of the maximum size of the DNA binding chamber(s) of cohesin. Together, this study reveals unexplored limitations to cohesin movement on chromosomes.

Significance StatementCohesin is an architectural protein that brings distant chromosomal DNA sites together. The complex links sister chromatids cohesion but it also binds to single pieces of DNA in ways that do not generate cohesion. One class of non-cohesive complexes organizes chromosomal DNA into loops. All cohesin complexes move on DNA but the constraints on such movement are not fully explored. Here, we use size-calibrated obstacles in yeast to interrogate cohesin and the properties of its movement on DNA. We show that both cohesive and non-cohesive complexes translocate processively on chromosomes. In addition, we show that cohesive and non-cohesive complexes are blocked by obstacles of different size. Lastly, we show that stalled cohesive complexes block passage of non-cohesive complexes.

sentences: Adenovirus-α-defensin complexes induce NLRP3-associated maturation of human phagocytes via TLR4 engagement Intramuscular delivery of human adenovirus (HAdV)-based vaccines leads to rapid recruitment of neutrophils, which then release antimicrobial peptides/proteins (AMPs). How these AMPs influence vaccine efficacy over the subsequent 24 hr is poorly understood. In this study, we asked if human neutrophil protein 1 (HNP-1), an -defensin that influences the direct and indirect innate immune responses to a range of pathogens, impacts the innate response of human phagocytes to three HAdV species/types (HAdV-C5, -D26, -B35). We show that HNP-1 binds to the capsids, redirects HAdV-C5, -D26, -B35 to Toll-like receptor 4 (TLR4), which leads to internalization, an NLRP3-mediated inflammasome response, and IL-1{beta} release. Surprisingly, IL-1{beta} release was not associated with notable disruption of plasma membrane integrity. These data further our understanding of HAdV innate immunogenicity and may provide pathways to extend HAdV-based vaccines efficacy.

sentences: Optimal coordination and reorganization of photosynthetic properties in C4 grasses C3 and C4 are major functional types in terrestrial biosphere models, with photosynthesis traits as important input parameters. The evolution of C4 required reorganizations of Calvin-Benson-cycle and coordination of C4-cycle enzymes, resulting in divergences of physiological traits between C3 and C4. In addition, photosynthesis further optimized after the evolution of C4 causing diversification within C4 lineages due to different evolutionary histories. We combined optimality modeling, physiological measurements and phylogenetic analysis to examine how various aspects of C4 photosynthetic machinery were reorganized and coordinated within C4 lineages and as compared to closely-related C3 in grasses. Optimality models and measurements indicated a higher maximal electron transport to maximal Rubisco carboxylation ratio (Jmax/Vcmax) in C4 than C3, consistent with the optimal prediction to maximize photosynthesis. The coordination between Calvin-Benson and C4 cycles (Vpmax/Vcmax), however, is in line with the optimal modeling results under 200 ppm, as opposed to current CO2. Such inconsistencies can be explained by a slowly declining assimilation rate beyond optimal Vpmax/Vcmax. Although rapid coordination occurred early in C4 evolution, C4 is still under optimizing processes and photosynthetic measures have continued to increase across time. Lastly, better understandings of Jmax/Vcmax, Vpmax/Vcmax and fluorescence-based-electron-transport proffer enhanced approaches to parameterize terrestrial biosphere models.

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A multi-omics approach to visualize early neuronal differentiation in 4D Neuronal differentiation of pluripotent stem cells is an established method to study physiology, disease and medication safety. However, the sequence of events in human neuronal differentiation and the ability of in vitro models to recapitulate early brain development are poorly understood. We developed a protocol optimized for the study of early human brain development and neuropharmacological applications. We comprehensively characterized gene expression and epigenetic profiles at four timepoints, as the cells differentiate from embryonic stem cells towards a heterogenous population of progenitors, immature and mature neurons bearing telencephalic signatures. A multi-omics roadmap of neuronal differentiation, combined with searchable interactive gene analysis tools, allows for extensive exploration of early neuronal development and the effect of medications.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/478732v1_ufig1.gif" ALT="Figure 1">
View larger version (55K):
org.highwire.dtl.DTLVardef@163539forg.highwire.dtl.DTLVardef@72c09org.highwire.dtl.DTLVardef@dc04eforg.highwire.dtl.DTLVardef@c821d2_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIMulti-omics charting a new neuronal differentiation protocol for human ES cells
C_LIO_LISingle-cell analyses reveals marker genes during neuronal differentiation
C_LIO_LIIdentified transcriptional waves similar to early human brain development
C_LIO_LISearchable tools to visualize single-cell gene expression and chromatin state
C_LI

In BriefWe have developed a novel protocol for human embryonic stem cells to study neural induction and early neuronal differentiation. Multi-omics analyses uncovered cell populations, genes and transcriptional waves defining cell fate commitment. We comprehensively describe epigenetic landscapes and gene expression and provide searchable analysis tools for exploration of the data.

sentences: Structural insights into plasticity and discovery of remdesivir metabolite GS-441524 binding in SARS-CoV-2 macrodomain The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus, targeting this protein domain may offer a therapeutic strategy to combat the current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally-occurring nucleotides, small molecules as well as nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and it provides chemical starting points for future inhibitor development.

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Chronic stroke sensorimotor impairment is related to smaller hippocampal volumes: An ENIGMA analysis Persistent sensorimotor impairments after stroke can negatively impact quality of life. The hippocampus is involved in sensorimotor behavior but has not been widely studied within the context of post-stroke upper limb sensorimotor impairment. The hippocampus is vulnerable to secondary degeneration after stroke, and damage to this region could further weaken sensorimotor circuits, leading to greater chronic sensorimotor impairment. The purpose of this study was to investigate the cross-sectional association between non-lesioned hippocampal volume and upper limb sensorimotor impairment in people with chronic stroke. We hypothesized that smaller ipsilesional hippocampal volumes would be associated with worse upper-limb sensorimotor impairment.

Cross-sectional T1-weighted brain MRIs were pooled from 357 participants at the chronic stage after stroke (>180 days post-stroke) compiled from 18 research cohorts worldwide in the ENIGMA Stroke Recovery Working Group (age: median = 61 years, interquartile range = 18, range = 23-93; 135 women and 222 men). Sensorimotor impairment was estimated from the Fugl-Meyer Assessment of Upper Extremity scores. Robust mixed-effects linear models were used to test associations between post-stroke sensorimotor impairment and hippocampal volumes (ipsilesional and contralesional separately; Bonferroni-corrected, p-value < 0.025), controlling for age, sex, lesion volume, and lesioned hemisphere. We also performed an exploratory analysis to test whether sex differences influence the relationship between sensorimotor impairment and hippocampal volume.

Upper limb sensorimotor impairment was positively associated with ipsilesional (p = 0.005; d = 0.33) but not contralesional (p = 0.96; d = 0.01) hippocampal volume, such that impairment was worse for participants with smaller ipsilesional hippocampal volume. This association remained significant independent of lesion volume or other covariates (p = 0.001; d = 0.36). Evidence indicates an interaction between sensorimotor impairment and sex for both ipsilesional (p = 0.008; d = -0.29) and contralesional (p = 0.006; d = -0.30) hippocampal volumes, whereby women showed progressively worsening sensorimotor impairment with smaller hippocampal volumes compared to men.

The present study has identified a novel association between chronic post-stroke sensorimotor impairment and ipsilesional, but not contralesional, hippocampal volume. This finding was not due to lesion size and may be stronger in women. We also provide supporting evidence that smaller hippocampal volume post-stroke is likely a consequence of ipsilesional damage, which could provide a link between vascular disease and other disorders, such as dementia.

sentences: SplineDist: Automated Cell Segmentation with Spline Curves We present SplineDist, an instance segmentation convolutional neural network for bioimages extending the popular StarDist method. While StarDist describes objects as star-convex polygons, SplineDist uses a more flexible and general representation by modelling objects as planar parametric spline curves. Based on a new loss formulation that exploits the properties of spline constructions, we can incorporate our new object model in StarDists architecture with minimal changes. We demonstrate in synthetic and real images that SplineDist produces segmentation outlines of equal quality than StarDist with smaller network size and accurately captures non-star-convex objects that cannot be segmented with StarDist.

sentences: Nature-inspired Circular-economy Recycling (NaCRe) for Proteins: Proof of Concept The billion tons of synthetic polymer-based materials (i.e. plastics) produced every year are one of the greatest challenges that humanity has to face. Nature produces even more natural polymers, yet they are sustainable. For example, proteins are sequence-defined natural polymers, that are constantly recycled when living systems feed. Indeed, digestion is the protein depolymerization into amino acids (i.e. the monomers) followed by their re-assembly into new proteins of arbitrarily different sequence and function. This process breaks a common recycling paradigm where a material is recycled into itself. Organisms feed of random protein mixtures that are  recycled into new proteins whose identity depends on the cells needs at the time of protein synthesis. Currently, advanced materials are increasingly made of proteins, but the abovementioned ideal recyclability of such materials has yet to be recognized and established. In this study mixtures of several (up to >30) peptides and/or proteins were depolymerized into their amino acid constituents, and these amino acids were used to synthesize new fluorescent, and bio-active proteins extra-cellularly by using an amino acid-free cell-free transcription-translation system. Proteins with high relevance in materials engineering ({beta}-lactoglobulin films, used for water filtration, or silk fibroin solutions) were successfully recycled into biotechnologically relevant proteins (green, and red fluorescent proteins, catechol 2,3-dioxygenase). The potential long-term impact of this approach to recycling lies in its compatibility with circular-economy models where raw materials remain in use as long as possible, thus reducing the burden on the planet.

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Auxin-responsive (phospho)proteome analysis reveals regulation of cell cycle and ethylene signaling during rice crown root development The rice root system, which primarily consists of adventitious/crown roots (AR/CR) developed from the coleoptile base, is an excellent model system for studying shoot-to-root trans-differentiation process. We reveal global changes in protein and metabolite abundance, and protein phosphorylation in response to an auxin stimulus during CR development. Global proteome and metabolome analyses of developing crown root primordia (CRP) and emerged CRs uncovered that the biological processes associated with chromatin conformational change, gene expression, and cell cycle were translationally regulated by auxin signaling. Spatial gene expression pattern analysis of differentially abundant proteins disclosed their stage-specific dynamic expression pattern during CRP development. Further, our tempo-spatial gene expression and functional analyses revealed that auxin creates a regulatory feedback module during CRP development and activates ethylene biosynthesis exclusively during CRP initiation. Ethylene signaling promotes CR formation by repressing the cytokinin response regulator, OsRR2. Additionally, the (phospho)proteome analysis identified differential phosphorylation of the Cyclin-dependent kinase G-2 (OsCDKG;2), and cell wall proteins, in response to auxin signaling, suggesting that auxin-dependent phosphorylation may be required for cell cycle activation, and cell wall synthesis during root organogenesis. Thus, our study provides evidence for the translational and post-translational regulation during CRP trans-differentiation downstream of the auxin signaling pathway.

HighlightGlobal (phospho)proteome and metabolic profiling of rice CRP and CRs uncover differential proteins and metabolites associated with gene expression, cell cycle, ethylene signaling and cell wall synthesis during CR development.

sentences: Single cell profiling of functionally cured Chronic Hepatitis B patients reveals the emergence of activated innate and an altered adaptive immune response in the intra- hepatic envrionment Hepatitis B surface antigen (HBsAg) loss or functional cure (FC), is considered the desirable therapeutic outcome for chronic hepatitis B (CHB) patients. However, the immuno-pathological biomarkers and underlying mechanisms remain unclear. Here we present a comprehensive single cell-transcriptomic atlas together with immune-phenotyping of disease-associated cell states (DACS) isolated from intra-hepatic tissue and matched PBMCs of either CHB or FC patients. We find that the intra-hepatic environment displays specific cell identities and molecular signatures that are distinct from PBMCs. FC is associated with emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes (CD4-CTLs), and an activated innate response represented by liver-resident natural killer (LR-NK) cells. Overall, these findings provide novel insights into immuno-pathological cell states associated with FC that could serve as prognostic biomarkers.

sentences: Transmembrane signalling by a bionic receptor: biological input and output, chemical mechanism of signal transduction Signal transduction through sealed biological membranes is among the most important evolutionary achievements. Herein, we focus on the development of artificial signal transduction mechanisms and engineer a bionic receptor with capacity of transduction of biological signals across biological membranes using tools of chemistry. The bionic receptor described in this work exhibits similarity with the natural counterpart in the most essential characteristics: in having an exofacial ligand for signal capture, in being membrane anchored, and in featuring a releasable secondary messenger molecule, which performs enzyme activation in the endo volume. The main difference with the natural receptors is that signal transduction across the lipid bilayer was performed using the tools of organic chemistry, namely a self-immolative linker. The highest novelty of our work is that the artificial signalling cascade designed herein achieved transmembrane activation of enzymatic activity, as is the hallmark of activity by natural signalling receptors.

sentences: The N764K and N856K mutations in SARS-CoV-2 Omicron S protein generate potential cleavage sites for SKI-1/S1P protease Spike (S) protein is a key protein in coronaviruses life cycle. SARS-CoV-2 Omicron BA.1 variant of concern (VoC) presents an exceptionally high number of 30 substitutions, 6 deletions and 3 insertions in the S protein. Recent works revealed major changes in the SARS-CoV-2 Omicron biological properties compared to earlier variants of concern (VoCs). Here, these major changes could be explained, at least in part, by the mutations N764K and/or N856K in S2 subunit. These mutations were not previously detected in other VoCs. N764K and N856K generate two potential cleavage sites for SKI-1/S1P serine protease, known to cleave viral envelope glycoproteins. The new sites where SKI-1/S1P could cleave S protein might impede the exposition of the internal fusion peptide for membrane fusion and syncytia formation. Based on the human protein atlas, SKI-1/S1P protease is not found in lung tissues (alveolar cells type I/II and endothelial cells), but present in bronchus and nasopharynx. This may explain why Omicron has change of tissue tropism. Viruses have evolved to use several host proteases for cleavage/activation of envelope glycoproteins. Mutations that allow viruses to change of protease may have a strong impact in host range, cell and tissue tropism, and pathogenesis.

sentences: CULTURE AND MAINTENANCE OF URINE-DERIVED, 3-DIMENSIONAL CANINE TRANSITIONAL CELL CARCINOMA ORGANOIDS Bladder cancer is the ninth most common malignancy in the world. Transitional cell carcinoma (TCC), also referred to as urothelial carcinoma (UC) is the most common form of bladder cancer, occurring in 90% of cases. In this study, we explore urine-derived, 3-dimensional, canine TCC organoids as a possible model to study bladder TCC ex vivo. After establishing the cell lines, we subjected the 3D cells to RNA in situ hybridization (RNA-ISH) and cell viability assays. Overall, 3D cell culture from urine samples of TCC diagnosed canines expressed RNA biomarkers in a similar manner to parent tumors via RNA-ISH and showed more sensitivity to cisplatin treatment when compared to 2D human TCC cells. With further experimentation, canine TCC organoids could become an ideal model to study TCC ex vivo.

sentences: Representational dynamics of memories for real-life events The continuous flow of experience that characterizes real-life events is not recorded as such in episodic memory. The unfolding of events is represented as a succession of moments of prior experience (referred to as "experience units") that are separated by temporal discontinuities, such that events are temporally compressed when remembered. To unravel the neural basis of this temporal structure of episodic memories, we recorded a series of real-life events using wearable camera technology and then used fMRI to investigate brain activity during the temporal unfolding of these events in memory. We found that, compared to the representation of static scenes in memory, dynamically unfolding memory representations were associated with greater activation of posterior brain regions that support episodic recollection (i.e., the precuneus, posterior cingulate cortex, retrosplenial cortex, and angular gyrus). Strikingly, by analyzing the autocorrelation of brain activity patterns at successive time points throughout the retrieval period, we found that these brain regions showed higher temporal dynamics when remembering the unfolding of events compared to static scenes. Furthermore, these effects were more pronounced when recalling events that included a higher density of experience units (i.e., actions compared to spatial displacements). These results reveal the key role of the posterior medial network in representing the dynamic unfolding of event segments that constitute episodic memories.

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Accurate Protein Domain Structure Annotation with DomainMapper Automated domain annotation plays a number of important roles in structural informatics and typically involves searching query sequences against Hidden Markov Model (HMM) profiles. This process can be ambiguous or inaccurate when proteins contain domains with non-contiguous residue ranges, and especially when insertional domains are hosted within them. Here we present DomainMapper, an algorithm that accurately assigns a unique domain structure annotation to any query sequence, including those with complex topologies. We validate our domain assignments using the AlphaFold database and confirm that non-contiguity is pervasive (6.5% of all domains in yeast and 2.5% in human). Using this resource, we find that certain folds have strong propensities to be non-contiguous or insertional across the Tree of Life, likely underlying evolutionary preferences for domain topology. DomainMapper is freely available and can be run as a single command line function.

HIGHLIGHTSDomainMapper generates a unique domain structure annotation, including non-contiguous and insertional domains

Automated annotations of non-contiguous domains are validated against the AlphaFold database

DomainMapper can be easily installed and used by non-experts

Certain folds have strong preferences to be non-contiguous or insertional

GRAPHICAL ABSTRACT

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Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus.

ImportanceListeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacteriums major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

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Fast and Robust 2D Inverse Laplace Transformation of Single-Molecule Fluorescence Lifetime Data Fluorescence spectroscopy at the single-molecule scale has been indispensable for studying conformational dynamics and rare states of biological macromolecules. Single-molecule 2D-fluorescence lifetime correlation spectroscopy (sm-2D-FLCS) is an emerging technique that holds great promise for the study of protein and nucleic acid dynamics as it 1) resolves conformational dynamics using a single chromophore, 2) measures forward and reverse transitions independently, and 3) has a dynamic window ranging from microseconds to seconds. However, the calculation of a 2D fluorescence relaxation spectrum requires an inverse Laplace transition (ILT), which is an ill-conditioned inversion that must be estimated numerically through a regularized minimization. The current methods for performing ILTs of fluorescence relaxation can be computationally inefficient, sensitive to noise corruption, and difficult to implement. Here, we adopt an approach developed for NMR spectroscopy (T1-T2 relaxometry) to perform 1D and 2D-ILTs on single-molecule fluorescence spectroscopy data using singular-valued decomposition and Tikhonov regularization. This approach provides fast, robust, and easy to implement Laplace inversions of single-molecule fluorescence data.

Significance StatementInverse Laplace transformations are a powerful approach for analyzing relaxation data. The inversion computes a relaxation rate spectrum from experimentally measured temporal relaxation, circumventing the need to choose appropriate fitting functions. They are routinely performed in NMR spectroscopy and are becoming increasing used in single-molecule fluorescence experiments. However, as Laplace inversions are ill-conditioned transformations, they must be estimated from regularization algorithms that are often computationally costly and difficult to implement. In this work, we adopt an algorithm first developed for NMR relaxometry to provide fast, robust, and easy to implement 1D and 2D inverse Laplace transformations on single-molecule fluorescence data.

sentences: Copy number evolution in simple and complex tandem repeats across the C57BL/6 and C57BL/10 inbred mouse lines Simple sequence tandem repeats are among the most rapidly evolving compartments of the genome. Some repeat expansions are associated with mammalian disease or meiotic segregation distortion, yet the rates of copy number change across generations are not well known. Here, we use 14 distinct sub-lineages of the C57BL/6 and C57BL/10 inbred mouse strains, which have been evolving independently over about 300 generations, to estimate the rates of copy number changes in genome-wide tandem repeats. Rates of change varied across simple repeats and across lines. Notably, CAG, whose expansions in coding regions are associated with many neurological and other genetic disorders, was highly stable in copy number, likely indicating purifying selection. Rates of change were generally positively correlated with copy number, but the direction and magnitude of changes varied across lines. Some mouse lines experienced consistent losses or gains across most genome-wide simple repeats, but this did not correlate with copy number changes in complex repeats. Rates of copy number change were similar between simple repeats and the much more abundant complex repeats once they were normalized by copy number. Finally, the Y-specific centromeric repeat had a 4-fold higher rate of change than the homologous centromeric repeat on other chromosomes. Structural differences in satellite complexity, or restriction to the Y chromosome and the elevated mutation rate of the male germline, may explain the higher rate of change. Overall, our work underscores the mutational fluidity of long tandem arrays of repeats, and the correlations and constraints between genome-wide tandem repeats which suggest that turnover is not a completely neutral process.

sentences: Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an "aromatic cage" at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1-4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2-2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.

sentences: Automated Feeding and Fermentation Phase Transitions for the Production of Unspecific Peroxygenase by Pichia pastoris Fungal peroxygenases are promising biocatalysts for hydroxylation steps in various industry-relevant synthesis pathways. In this application note we describe a bioprocess for the production of unspecific peroxygenase (UPO) in Pichia pastoris. The process was divided in four phases, with different carbon requirements. Precise timing of culture feeding was crucial for optimal cell growth and protein expression. We demonstrate how the automation of culture feeding reduced manual work as well as the risk of process failure due to operator error.

sentences: Linearised loop kinematics to study pathways between conformations AO_SCPLOWBSTRACTC_SCPLOWConformational changes are central to the function of many proteins. Characterization of these changes using molecular simulation requires methods to effectively sample pathways between protein conformational states. In this paper we present an iterative algorithm that samples conformational transitions in protein loops, referred to as the Jacobian-based Loop Transition (JaLT) algorithm. The method uses internal coordinates to minimise the sampling space, while Cartesian coordinates are used to maintain loop closure. Information from the two representations is combined to push sampling towards a desired target conformation. The innovation that enables the simultaneous use of Cartesian coordinates and internal coordinate is the linearisation of the inverse kinematics of a protein backbone. The algorithm uses the Rosetta all-atom energy function to steer sampling through low-energy regions and uses Rosettas side-chain energy minimiser to update side-chain conformations along the way. Because the JaLT algorithm combines a detailed energy function with a low-dimensional conformational space, it is positioned in between molecular dynamics (MD) and elastic network model (ENM) methods. As a proof of principle, we apply the JaLT algorithm to study the conformational transition between the open and occluded state in the MET20 loop of the Escherichia coli dihydrofolate reductase enzyme. Our results show that the algorithm generates semi-continuous pathways between the two states with realistic energy profiles. These pathways can be used to identify energy barriers along the transition. The effect of a single point mutation of the MET20 loop was also investigated and the predicted increase in energy barrier is consistent with the experimentally observed reduction in catalytic rate of the enzyme. Additionally, it is demonstrated how the JaLT algorithm can be used to identify dominant degrees of freedom during a transition. This can be valuable input for a more extensive characterization of the free energy pathway along a transition using molecular dynamics, which is often performed with a reduced set of degrees of freedom. This study has thereby provided the first examples of how linearisation of inverse kinematics can be applied to the analysis of proteins.

sentences: Reinstatement of cortical outcome representations during higher-order learning Naturalistic learning scenarios are characterized by infrequent experience of external feedback to guide behavior. Higher-order learning mechanisms like second-order conditioning (SOC) may allow stimuli that were never experienced together with reinforcement to acquire motivational value. Despite its explanatory potential for real-world learning, surprisingly little is known about the neural mechanism underlying such associative transfer of value in SOC. Here, we used multivariate cross-session, cross-modality searchlight classification on functional magnetic resonance imaging data obtained from humans during SOC. We show that visual first-order conditioned stimuli (CS) reinstate cortical patterns representing previously paired gustatory outcomes in the lateral orbitofrontal cortex (OFC). During SOC, this OFC region showed increased functional covariation with amygdala, where neural pattern similarity between second-order CS and outcomes increased from early to late stages of SOC. Our data suggest a mechanism by which motivational value is conferred to stimuli that were never paired with reinforcement.

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TRIM67 drives tumorigenesis in oligodendrogliomas through Rho GTPase-dependent membrane blebbing Oligodendrogliomas are a subtype of isocitrate dehydrogenase (IDH) mutant gliomas defined by the co-deletion of chromosome arms 1p and 19q. Although the somatic genomic alterations of oligodendrogliomas have been well described, transcriptional changes unique to these tumors are not well studied. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas. We characterize the function of TRIM67 using high throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS) and co-immunoprecipitation (IP)-MS using human neural progenitor cells and patient-derived glioma tumorspheres constitutively overexpressing TRIM67. Our high throughput data suggest that TRIM67 overexpression alters the abundance of cytoskeletal proteins, which were validated by functional assays, including immunofluorescence (IF) staining, co-IP and western blotting (WB). Additionally, IF staining results indicate that TRIM67 ectopic expression induces formation of membrane blebs in glioma cells, which could be reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. GTP pulldown and WB assays further indicate that Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression. Phenotypically, TRIM67 expression resulted in higher cell motility in wound healing experiments, reduced cell adherence in adhesion assays, accelerated tumor growth and reduced survival in mouse orthotopic implantation models of an oligodendroglioma-derived patient tumorsphere line. Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis.

SignificanceWe identify TRIM67 as a novel oncogene in oligodendroglioma that leads to increased cell motility, tumor growth, reduced adhesion, and survival in mice. Our results also show that constitutive TRIM67 expression transforms cell morphology from an adherent to a rounded appearance with membrane blebs. Mechanistic alteration of actin cytoskeleton and Rho GTPase signaling upon TRIM67 upregulation underlies the rounded cell structure and the membrane blebbing phenotype. TRIM67-induced blebbing is specifically regulated by RHOA-RAC1-ROCK2 signaling axis. TRIM67 overexpression also alters pathways associated with cell migration and wound healing in various glioma cell lines and human neural progenitor cells, suggesting a general oncogenic mechanism in gliomas. Overall, our study highlights TRIM67 as a novel player orchestrating cytoskeleton, Rho GTPase signaling and bleb-based cell movement, ultimately causing tumorigenic outcomes in oligodendrogliomas.

sentences: Insights into the molecular mechanisms of cell fate decision making processes from chromosome structural dynamics Cell state transitions or cell fate decision making processes, such as cell development and cell pathological transformation, are believed to be determined by the regulatory network of genes, which intimately depend on the structures of chromosomes in the cell nucleus. The high temporal resolution picture of how chromosome reorganizes its 3D structure during the cell state transitions is the key to understanding the mechanisms of these fundamental cellular processes. However, this picture is still challenging to acquire at present. Here, we studied the chromosome structural dynamics during the cell state transitions among the pluripotent embryonic stem cell (ESC), the terminally differentiated normal cell and the cancer cell using landscape-switching model implemented in the molecular dynamics simulation. We considered up to 6 transitions, including differentiation, reprogramming, cancer formation and reversion. We found that the pathways can merge at certain stages during the transitions for the two processes having the same destination as the ESC or the normal cell. Before reaching the merging point, the two pathways are cell-type-specific. The chromosomes at the merging points show high structural similarity to the ones at the final cell states in terms of the contact maps, TADs and compartments. The post-merging processes correspond to the adaption of the chromosome global shape geometry through the chromosome compaction without significantly disrupting the contact formation. On the other hand, our detailed analysis showed no merging point for the two cancer formation processes initialized from the ESC and the normal cell, implying that cancer progression is a complex process and may be associated with multiple pathways. Our results draw a complete molecular picture of cell development and cancer at the dynamical chromosome structural level, and help our understanding of the molecular mechanisms of cell fate decision making processes.

sentences: Direct observation of the neural computations underlying a single decision Neurobiological investigations of perceptual decision-making have furnished the first glimpse of a flexible cognitive process at the level of single neurons1,2. Neurons in the parietal and prefrontal cortex3-6 are thought to represent the accumulation of noisy evidence, acquired over time, leading to a decision. Neural recordings averaged over many decisions have provided support for the deterministic rise in activity to a termination bound7. Critically, it is the unobserved stochastic component that is thought to confer variability in both choice and decision time8. Here, we elucidate this stochastic, diffusion-like signal on individual decisions by recording simultaneously from hundreds of neurons in the lateral intraparietal cortex (LIP). We show that a small subset of these neurons, previously studied singly, represent a combination of deterministic drift and stochastic diffusion--the integral of noisy evidence--during perceptual decision making, and we provide direct support for the hypothesis that this diffusion signal is the quantity responsible for the variability in choice and reaction times. Neuronal state space and decoding analyses, applied to the whole population, also identify the drift diffusion signal. However, we show that the signal relies on the subset of neurons with response fields that overlap the choice targets. This parsimonious observation would escape detection by these powerful methods, absent a clear hypothesis.

sentences: Activity-induced fluidization of arrested coalescence in fusion of cellular aggregates At long time scales, tissue spheroids may flow or appear solid depending on their capacity to reorganize their internal structure. Understanding the relationship between intrinsic mechanical properties at the single cell level, and the tissue spheroids dynamics at the long-time scale is key for artificial tissue constructs, which are assembled from multiple tissue spheroids that over time fuse to form coherent structures. The dynamics of this fusion process are frequently analyzed in the framework of liquid theory, wherein the time scale of coalescence of two droplets is governed by its radius, viscosity and surface tension. In this work, we extend this framework to glassy or jammed cell behavior which can be observed in spheroid fusion. Using simulations of an individual-cell based model, we demonstrate how the spheroid fusion process can be steered from liquid to arrested by varying active cell motility and repulsive energy as established by cortical tension. The divergence of visco-elastic relaxation times indicates glassy relaxation near the transition towards arrested coalescence. Finally, we investigate the role of cell growth in spheroid fusion dynamics. We show that the presence of cell division introduces plasticity in the material and thereby increases coalescence during fusion.

sentences: Dynamic neurogenomic responses to social interactions and dominance outcomes in female paper wasps Social interactions have large effects on individual physiology and fitness. In the immediate sense, social stimuli are often highly salient and engaging. Over longer time scales, competitive interactions often lead to distinct social ranks and differences in physiology and behavior. Understanding how initial responses lead to longer-term effects of social interactions requires examining the changes in responses over time. Here we examined the effects of social interactions on transcriptomic signatures at two points, at the end of a 45-minute interaction and 4 hours later, in female Polistes fuscatus paper wasp foundresses. Female P. fuscatus have variable facial patterns that are used for visual individual recognition, so we separately examined the transcriptional dynamics in the optic lobe and the central brain. Results demonstrate much stronger transcriptional responses to social interactions in the central brain compared to the optic lobe. Differentially regulated genes in response to social interactions are enriched for memory-related transcripts. Comparisons between winners and losers of the encounters revealed similar overall transcriptional profiles at the end of an interaction, which significantly diverged over the course of 4 hours, with losers showing changes in expression levels of genes associated with aggression and reproduction in paper wasps. On nests, subordinate foundresses are less aggressive, do more foraging and lay fewer eggs compared to dominant foundresses and we find losers shift expression of many genes, including vitellogenin, related to aggression, worker behavior, and reproduction within hours of losing an encounter. These results highlight the early neurogenomic changes that likely contribute to behavioral and physiological effects of social status changes in a social insect.

sentences: Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity The current COVID-19 pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III), in a wide range of human cell types. These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique conserved sequence motif (sense strand: 5-C, antisense strand: 3-GGG) that mediates end-to-end dimer self-assembly of these RNAs by Hoogsteen G-G base-pairing. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory siRNAs, their activity is independent of TLR7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad spectrum inhibition of infections by many respiratory viruses with pandemic potential, including SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza A, as well as the common cold virus HCoV-NL63 in both cell lines and human Lung Chips that mimic organ-level lung pathophysiology. These short dsRNAs can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics at low cost.

sentences: A 3D printable device allowing fast and reproducible longitudinal preparation of mouse intestines Accurate and reproducible analysis of mouse small and large intestinal lumen is key for research involving intestinal pathology in preclinical models. Currently, there is no easily accessible, standardized method that allows researchers of different skill levels to consistently dissect intestines in a time-efficient manner. Here, we describe the design and use of the 3D printed "Mouse Intestinal Slicing Tool" (MIST), which can be used to longitudinally prepare murine intestines for further analysis. We benchmarked the MIST against a commonly used procedure involving scissors to make a longitudinal cut along the intestines. Use of the MIST halved the time per mouse to prepare the intestines and outperformed alternative methods in smoothness of the cutting edge and general reproducibility. By sharing the plans for printing the MIST, we hope to contribute a uniformly applicable method for saving time and increasing consistency in studies of the mouse gastrointestinal tract.

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'Constituent length' effects in fMRI do not provide evidence for abstract syntactic processing How are syntactically and semantically connected word sequences, or constituents, represented in the human language system? An influential fMRI study, Pallier et al. (2011, PNAS), manipulated the length of constituents in sequences of words or pseudowords. They reported that some language regions (in the anterior temporal cortex and near the temporo-parietal junction) were sensitive to constituent length only for sequences of real words but not pseudowords. In contrast, language regions in the inferior frontal and posterior temporal cortex showed the same pattern of increased response to longer constituents--and similar overall response magnitudes-- for word and pseudoword sequences. Based on these results, Pallier et al. argued that the latter regions represent abstract sentence structure. Here we identify methodological and theoretical concerns with the Pallier et al. study and conduct a replication across two fMRI experiments. Our results do not support Pallier et al.s critical claim of distinct neural specialization for abstract syntactic representations. Instead, we find that all language regions show a similar profile of sensitivity to both constituent length and lexicality (stronger responses to real-word than pseudoword stimuli). In addition, we argue that the constituent length effect in these experiments i) is not readily grounded in established theories of sentence processing, and ii) may not actually derive from syntactic structure building, but may instead reflect the temporal receptive window of the human language system.

Significance StatementAn influential study (Pallier et al., 2011, PNAS) has been widely interpreted as evidence that inferior frontal and posterior temporal brain regions perform abstract syntactic processing during language comprehension, independent of lexical content. Here we identify theoretical, empirical, and methodological concerns with the original study and conduct a replication across two experiments. Contrary to Pallier et al. (2011), we find evidence that both syntactic and lexical processing are distributed throughout the human language network, consistent with prior arguments for a highly lexicalized and distributed human language processing system.

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THEWEIGHT: A simple and flexible algorithm for simulating non-ideal, age-structured populations O_LIThe Wright-Fisher model, which directs how matings occur and how genes are transmitted across generations, has long been a lynchpin of evolutionary biology. This model is elegantly simple, analytically tractable, and easy to implement, but it has one serious limitation: essentially no real species satisfies its many assumptions. With growing awareness of the importance of jointly considering both ecology and evolution in eco-evolutionary models, this limitation has become more apparent, causing many researchers to search for more realistic simulation models.
C_LIO_LIA recently described variation retains most of the Wright-Fisher simplicity but provides greater flexibility to accommodate departures from model assumptions. This generalized Wright-Fisher model relaxes the assumption that all individuals have identical expected reproductive success by introducing a vector of parental weights w that specifies relative probabilities different individuals have of producing offspring. With parental weights specified this way, expectations of key demographic parameters are simple functions of w. This allows researchers to quantitatively predict the consequences of non-Wright-Fisher features incorporated into their models.
C_LIO_LIAn important limitation of the Wright-Fisher model is that it assumes discrete generations, whereas most real species are age-structured. Here I show how an algorithm (TO_SCPLOWHEC_SCPLOWWO_SCPLOWEIGHTC_SCPLOW) that implements the generalized Wright-Fisher model can be used to model evolution in age-structured populations with overlapping generations. Worked examples illustrate simulation of seasonal and lifetime reproductive success and show how the user can pick vectors of weights expected to produce a desired level of reproductive skew or a desired Ne/N ratio. Alternatively, weights can be associated with heritable traits to provide a simple, quantitative way to model natural selection. Using TO_SCPLOWHEC_SCPLOWWO_SCPLOWEIGHTC_SCPLOW, it is easy to generate positive or negative correlations of individual reproductive success over time, thus allowing explicit modeling of common biological processes like skip breeding and persistent individual differences.
C_LIO_LIR code is provided to implement basic features of TO_SCPLOWHEC_SCPLOWWO_SCPLOWEIGHTC_SCPLOW and applications described here. However, required coding changes to the Wright-Fisher model are modest, so the real value of the new algorithm is to encourage users to adopt its features into their own or others models.
C_LI

sentences: Leafy and Weedy Seadragon Genomes Connect Genic and Repetitive DNA Features to the Extravagant Biology of Syngnathid Fishes Seadragons are a remarkable lineage of teleost fishes, and they are members of the family Syngnathidae renowned for having evolved male pregnancy. Comprising three known species, seadragons are widely recognized and admired for their fantastical body forms and coloration, and their specific habitat requirements have made them flagship representatives for marine conservation and natural history interests. Until recently, a gap has been the lack of significant genomic resources for seadragons. We have produced gene-annotated, chromosome-scale genome models for the leafy and weedy seadragon to advance investigations into evolutionary innovation and elaboration of morphological traits in seadragons as well as their pipefish and seahorse relatives. We identified several interesting features specific to seadragon genomes, including divergent non-coding regions near a developmental gene important for integumentary outgrowth, a high genome-wide density of repetitive DNA, and recent expansions of transposable elements and a vesicular trafficking gene family. Surprisingly, comparative analyses leveraging the seadragon genomes and additional syngnathid and outgroup genomes revealed striking, syngnathid-specific losses in the family of fibroblast growth factors (FGFs), which likely involve re-organization of highly conserved gene regulatory networks in ways that have not previously been documented in natural populations. The resources presented here serve as important tools for future evolutionary studies of developmental processes in syngnathids and will be a key resource for conservation studies of the extravagant seadragons and their relatives.

sentences: Increased excursions to functional networks in schizophrenia in the absence of task Brain activity during rest has been demonstrated to evolve through a repertoire of functional connectivity (FC) patterns, whose alterations may provide biomarkers of schizophrenia - a psychotic disorder characterized by dysfunctional brain connectivity. In this study, differences between the dynamic exploration of resting-state networks using functional magnetic resonance imaging (fMRI) data from 71 schizophrenia patients and 74 healthy controls were investigated using a method focusing on the dominant fMRI signal phase coherence pattern at each time point. Through the lens of dynamical systems theory, brain activity in the form of temporal FC state trajectories was examined for intergroup differences by calculating the fractional occupancy, dwell time, limiting probability of each state and the transition probabilities between states. Results showed reduced fractional occupancy of a globally synchronized state in schizophrenia. Conversely, FC states overlapping with canonical functional subsystems exhibited increased fractional occupancy and limiting probability in schizophrenia. Furthermore, state-to-state transition probabilities were altered in schizophrenia. This revealed a reduced probability of remaining in a global integrative state, increased probability of switching from this state to functionally meaningful networks and reduced probability of remaining in a state related to the Default Mode network. These results revealed medium to large effect sizes. Finally, this study showed that using K-medoids clustering did not influence the observed intergroup differences - highlighting the utility of dynamical systems theory to better understand brain activity. Combined, these findings expose pronounced differences between schizophrenia patients and healthy controls - supporting and extending current knowledge regarding disrupted brain dynamics in schizophrenia.

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Sod1 Integrates Oxygen Availability to Redox Regulate NADPH Production and the Thiol Redoxome Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2-) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major new aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH), which in turn re-routes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is exclusively dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2- to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this new mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2- toxicity, which only requires < 1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.

Significance StatementCu/Zn superoxide dismutase (Sod1) is a key antioxidant enzyme and its importance is underscored by the fact that its ablation in cell and animal models results in oxidative stress, metabolic defects, and reductions in cell proliferation, viability, and lifespan. Curiously, Sod1 detoxifies superoxide radicals (O2-) in a manner that produces an oxidant as a byproduct, hydrogen peroxide (H2O2). While much is known about the necessity of scavenging O2-, it is less clear what the physiological roles of Sod1-derived H2O2 are. Herein, we discovered that Sod1-derived H2O2 plays a very important role in antioxidant defense by stimulating the production of NADPH, a vital cellular reductant required for ROS scavenging enzymes, as well as redox regulating a large network of enzymes.

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Nanoscale Lubrication in Biosystems as Rationalized in Terms of Fractons and Spectral-Mechanical Properties of Networked Biopolymers in Ionic Solutions Articular cartilage is a natural tribochemical device just-designed by nature. Yet, a vivid debate goes on toward the mechanisms by which its nanoscopic viscoelastic properties facilitate lubrication in terms of ultralow static and kinetic friction coefficients. In this concisely conducted conceptual discussion, we wish to point out that a nanoscale tribomechanistic description based upon certain "viscoelastic quanta", called fractons, expressing spectral-mechanical properties of viscoelastic nets under the influence of force/pressure factor(s), may contribute substantially to the elucidation of ultralow coefficients of friction in the articular cartilage of predictable relaxational response. Our example unveils a part of a mechanically responsive viscoelastic network, such as a tied piece of hyaluronan molecule, fit in an Edwards type tube, in which upon water-mediated interaction of lipids with the hyaluronan when subjected to loading at the nanoscale, consecutive stress-field and ion diffusion actions occur simultaneously. It results in a natural-logarithmic formula that interrelates a number of hyaluronans interactive residues, N, with certain molecular-elastic (an exponent{gamma} ) and surface-to-volume (nano-colloid type) characteristics of around 2/3 to emerge near thermodynamic equilibrium, that is to say after a frictional loading action performed. It enables to relate uniquely a value of the exponent 0 <{gamma} < 1/2 with a virtual tribomicellization scenario of the nanoscale friction-lubrication event accompanied by inevitable tubular-milieu viscosity alterations at criticality when the quasi-static friction scenario shows up, preferably with{gamma} [->] 1/3 from above for large enough N -s. A periodic vibrational super-biopolymers mode is exploited, leading to a change in the nanoscale friction-lubrication period from which an opportunity to involve an essential contribution to the (nanoscale) coefficient of friction arises.

PACS numbers71.10.+x, 81.30.Fb, 05.70.Fh, 05.60.+w

sentences: Optimal strategies for learning multi-ancestry polygenic scores vary across traits Polygenic scores (PGSs) are individual-level measures that aggregate the genome-wide genetic predisposition to a given trait. As PGS have predominantly been developed using European-ancestry samples, trait prediction using such European ancestry-derived PGS is less accurate in non-European ancestry individuals. Although there has been recent progress in combining multiple PGS trained on distinct populations, the problem of how to maximize performance given a multiple-ancestry cohort is largely unexplored. Here, we investigate the effect of sample size and ancestry composition on PGS performance for fifteen traits in UK Biobank. For some traits, PGS estimated using a relatively small African-ancestry training set outperformed, on an African-ancestry test set, PGS estimated using a much larger European-ancestry only training set. We observe similar, but not identical, results when considering other minority-ancestry groups within UK Biobank. Our results emphasise the importance of targeted data collection from underrepresented groups in order to address existing disparities in PGS performance.

sentences: Mutational bias in spermatogonia impacts the anatomy of regulatory sites in the human genome Mutation in the germline is the ultimate source of genetic variation, but little is known about the influence of germline chromatin structure on mutational processes. Using ATAC-seq, we profile the open chromatin landscape of human spermatogonia, the most proliferative cell-type of the germline, identifying transcription factor binding sites (TFBSs) and PRDM9-binding sites, a subset of which will initiate meiotic recombination. We observe an increase in rare structural variant (SV) breakpoints at PRDM9-bound sites, implicating meiotic recombination in the generation of structural variation. Many germline TFBSs, such as NRF, are also associated with increased rates of SV breakpoints, apparently independent of recombination. Singleton short insertions (>=5 bp) are highly enriched at TFBSs, particularly at sites bound by testis active TFs, and their rates correlate with those of structural variant breakpoints. Short insertions often duplicate the TFBS motif, leading to clustering of motif sites near regulatory regions in this male-driven evolutionary process. Increased mutation loads at germline TFBSs disproportionately affect neural enhancers with activity in spermatogonia, potentially altering neurodevelopmental regulatory architecture. Local chromatin structure in spermatogonia is thus pervasive in shaping both evolution and disease.

sentences: A toolkit of highly selective and sensitive genetically encoded neuropeptide sensors Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes, including energy balance, sleep and circadian rhythms, stress, and social behaviors. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution; however, this has been hindered by the lack of direct, sensitive and non-invasive tools. Here, we developed a series of GRAB (G protein-coupled receptor activation-based) sensors for detecting somatostatin (SST), cholecystokinin (CCK), corticotropin-releasing factor (CRF), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors utilize the corresponding GPCRs as the neuropeptide-sensing module with the insertion of a circular-permutated GFP as the optical reporter. This design detects the binding of specific neuropeptides at nanomolar concentration with a robust increase in fluorescence. We used these GRAB neuropeptide sensors to measure the spatiotemporal dynamics of endogenous SST release in isolated pancreatic islets and to detect the release of both CCK and CRF in acute brain slices. Moreover, we detect endogenous CRF release induced by stressful experiences in vivo using fiber photometry and 2-photon imaging in mice. Together, these new sensors establish a robust toolkit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

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ElectroEncephaloGraphy robust statistical linear modelling using a single weight per trial Being able to remove or weigh down the influence of outlier data is desirable for any statistical models. While Magnetic and ElectroEncephaloGraphic (MEEG) data are often averaged across trials per condition, it is becoming common practice to use information from all trials to build statistical linear models. Individual trials can, however, have considerable weight and thus bias inferential results (effect sizes as well as thresholded t/F/p maps). Here, rather than looking for univariate outliers, defined independently at each measurement point, we apply the principal component projection (PCP) method at each channel, deriving a single weight per trial at each channel independently. Using both synthetic data and open EEG data, we show (1) that PCP is efficient at detecting a large variety of outlying trials; (2) how PCP-based weights can be implemented in the context of the general linear model (GLM) with accurate control of type 1 family-wise error rate; and (3) that our PCP-based Weighted Least Square (WLS) approach increases the statistical power of group analyses as well as a much slower Iterative Reweighted Least Squares (IRLS), although the weighting scheme is markedly different. Together, our results show that WLS based on PCP weights derived from whole trial profiles is an efficient method to weigh down the influence of outlier EEG data in linear models.

Data availabilityall data used are publicly available (CC0), all code (simulations and data analyzes) is also available online in the LIMO MEEG GitHub repository (MIT license).

sentences: Non-Newtonian Blood Rheology Impacts Left Atrial Stasis in Patient-Specific Simulations The lack of mechanically effective contraction of the left atrium (LA) during atrial fibrillation (AF) disturbs blood flow, increasing the risk of thrombosis and ischemic stroke. Thrombosis is most likely in the left atrial appendage (LAA), a small narrow sac where blood is prone to stagnate. Slow flow promotes the formation of erythrocyte aggregates in the LAA, also known as rouleaux, causing viscosity gradients that are usually disregarded in patient-specific simulations. To evaluate these non-Newtonian effects, we built atrial models derived from 4D computed tomography scans of patients and carried out computational fluid dynamics simulations using the Carreau-Yasuda constitutive relation. We examined six patients, three of whom had AF and LAA thrombosis or a history of transient ischemic attacks (TIAs). We modeled the effects of hematocrit and rouleaux formation kinetics by varying the parameterization of the Carreau-Yasuda relation and modulating non-Newtonian viscosity changes based on residence time. Comparing non-Newtonian and Newtonian simulations indicates that slow flow in the LAA increases blood viscosity, altering secondary swirling flows and intensifying blood stasis. While some of these effects can be subtle when examined using instantaneous metrics like shear rate or kinetic energy, they are manifested in the blood residence time, which accumulates over multiple heartbeats. Our data also reveal that LAA blood stasis worsens when hematocrit increases, offering a potential new mechanism for the clinically reported correlation between hematocrit and stroke incidence. In summary, we submit that hematocrit-dependent non-Newtonian blood rheology should be considered in calculating patient-specific blood stasis indices by computational fluid dynamics.

sentences: Competition contributes to both warm and cool range edges Competition plays an important role in shaping species spatial distributions. However, it remains unclear where and how competition regulates species range limits. In a field experiment with plants originating from low and high elevations and conducted across an elevation gradient in the Swiss Alps, we find that both lowland and highland species can better persist in the presence of competition within than beyond their elevation ranges, suggesting that competition helps set both lower and upper elevation range limits of these species. Furthermore, the reduced ability of pairs of lowland or highland species to coexist beyond their range edges is mainly driven by diminishing niche differences; changes in both niche differences and relative fitness differences drive weakening competitive dominance of lowland over highland species with increasing elevation. These results highlight the need to account for competitive interactions and investigate underlying coexistence mechanisms to understand current and future species distributions.

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Genomic epidemiology of Mycobacterium bovis infection in sympatric badger and cattle populations in Northern Ireland. BackgroundBovine tuberculosis (bTB) is a costly, epidemiologically complex, multi-host, endemic disease. Pathogen whole genome sequencing can improve the resolution of epidemiological tracing. We genome sequenced an exceptional data set of 619 Mycobacterium bovis isolates from badgers and cattle in a 100km2 bTB  hotspot. Historical molecular subtyping data permitted the targeting of an endemic pathogen lineage, whose long-term persistence provided an opportunity to study genome epidemiology in detail. To assess whether badger population genetic structure was associated with the spatial distribution of pathogen genetic diversity, we microsatellite genotyped hair samples from 769 badgers trapped in this area.

ResultsEight lineages of M. bovis were circulating in the study area, seven of which were likely non-endemic, and imported by animal movement. The endemic lineage exhibited low genetic diversity with an average inter-isolate genetic distance of 7.6 SNPs (s.d. {+/-} 4.0), consistent with contemporary transmission. Bayesian phylogenetic methods determined an evolutionary rate of 0.30 substitutions per genome per year for this lineage, estimating its emergence 40-50 years before present, while Bayesian Skyline analysis identified significant population expansion of the endemic lineage in the 1990s and again in 2011-2012. The phylogeny revealed distinct sub-lineages, all of which contained isolates from both cattle and badger hosts, indicative of the sharing of closely related strains and inter-species transmission. However, the presence of significant badger population genetic structure was not associated with the spatial distribution of M. bovis genetic diversity.

ConclusionsOur data provided unparalleled detail on the evolutionary history of an endemic M. bovis lineage. Findings are consistent with ongoing interspecies transmission in the study area but suggest that badger intra-species transmission may not be a major driver of persistence in this area. In addition, the data collected permitted the tracking of incursions of novel pathogen lineages into the study area and means to determine if they were involved in disease transmission.

sentences: Endosomal cAMP production broadly impacts the cellular phosphoproteome Endosomal signaling downstream of G protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. Yet, our knowledge of the functional consequences of intracellular signaling is incomplete. To begin to address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger generated by active GPCRs, cyclic AMP (cAMP), with unbiased mass spectrometry-based analysis of phosphoproteomic effects. We identified 218 unique, highconfidence sites whose phosphorylation is either increased or decreased in response to cAMP production. We next determined that the same amount of cAMP produced from endosomes led to more robust changes in phosphorylation than the plasma membrane. Remarkably, this was true for the entire repertoire of identified targets, and irrespective of their annotated sub-cellular localization. Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A-B56{delta} as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.

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Maternal cradling bias drives early handedness in infant monkeys: A longitudinal study of grasping lateralization in baboons The most emblematic behavioral manifestation of human brain asymmetries is handedness. While the precise mechanisms behind the development of handedness are still widely debated, empirical evidences highlight that besides genetic factors, environmental factors may play a crucial role. As one of these factors, maternal cradling behavior may play a key role in the emergence of early handedness in the offspring. In the present study we followed 41 olive baboon (Papio anubis) infants living in different social groups with their mother for which direction (e.g., left- or right-arm) and degree of maternal cradling-side bias were available from our previous published study. We assessed hand preferences for an unimanual grasping task at 3 developmental stages: (1) 0-4, (2) 4-6 and (3) 9-10 months of age. We found that individual hand preferences for grasping exist as soon as the first months of age, with a population-level left-handedness predominance, being stable until 6 months; to wit the period during which juveniles are mainly carried by their mothers. More importantly, this early postnatal handedness is positively correlated with maternal cradling lateralization. Interestingly, hand preferences assessed later in the development, once juveniles are no longer carried (i.e., from 9 to 10 months of age), are less consistent with the earlier developmental stages and no longer dependent from the maternal cradling bias. Our findings suggest that the ontogenetic dynamics of the infants hand preference and its changes might ultimately rely on the degree of infant dependence from the mother across development.

Research HighlightsO_LIEarly postnatal individual hand preferences are detected for unimanual food grasping within the first four months of age.
C_LIO_LIEarliest measures of infant hand preference are positively correlated with measures of maternal cradling lateralization.
C_LIO_LIHand preferences assessed later in the development, from 9 to 10 months of age are less consistent with the earlier developmental stages and independent from maternal cradling bias.
C_LI

sentences: GRAMMAR-Lambda: An Extreme Simplification for Genome-wide Mixed Model Association Analysis A highly efficient genome-wide association method, GRAMMAR-Lambda is proposed to make simple genomic control for the test statistics deflated by GRAMMAR, producing statistical power as high as exact mixed model association method. Using the simulated and real phenotypes, we show that at a moderate or above genomic heritability, polygenic effects can be estimated using a small number of randomly selected markers, which extremely simplify genome-wide association analysis with an approximate computational complexity to naive method in large-scale complex population. Upon a test at once, joint association analysis offers significant increase in statistical power over existing methods.

sentences: Mosaic cis-regulatory evolution drives transcriptional partitioning of HERVH endogenous retrovirus in the human embryo The human endogenous retrovirus type-H (HERVH) family is expressed in the preimplantation embryo. A subset of these elements are specifically transcribed in pluripotent stem cells where they appear to exert regulatory activities promoting self-renewal and pluripotency. How HERVH elements achieve such transcriptional specificity remains poorly understood. To uncover the sequence features underlying HERVH transcriptional activity, we performed a phyloregulatory analysis of the long terminal repeats (LTR7) of the HERVH family, which harbor its promoter, using a wealth of regulatory genomics data. We found that the family includes at least 8 previously unrecognized subfamilies that have been active at different timepoints in primate evolution and display distinct expression patterns during human embryonic development. Notably, nearly all HERVH elements transcribed in ESCs belong to one of the youngest subfamilies we dubbed LTR7up. LTR7 sequence evolution was driven by complex mutational processes, including multiple recombination events between subfamilies, that led to transcription factor binding motif modules characteristic of each subfamily. Using a reporter assay, we show that one such motif, a predicted SOX2/3 binding site unique to LTR7up, is essential for robust promoter activity in induced pluripotent stem cells. Together these findings illuminate the mechanisms by which HERVH diversified its expression pattern during evolution to colonize distinct cellular niches within the human embryo.

sentences: Robust regulatory architecture of pan-neuronal gene expression Pan-neuronally expressed genes, such as genes involved in the synaptic vesicle release cycle or in neuropeptide maturation, are critical for proper function of all neurons, but the transcriptional control mechanisms that direct such genes to all neurons of a nervous system remain poorly understood. We show here that six members of the CUT family of homeobox genes control pan-neuronal identity specification in C. elegans. Single CUT mutants show barely any effects on pan-neuronal gene expression or global nervous system function, but such effects become apparent and progressively worsen upon removal of additional CUT family members, indicating a critical role of gene dosage. Overexpression of each individual CUT gene rescued the phenotype of compound mutants, corroborating that gene dosage, rather than the activity of specific members of the gene family, is critical for CUT gene function. Genome-wide binding profiles as well as mutation of CUT binding sites by CRISPR/Cas9 genome engineering show that CUT genes directly control the expression of pan-neuronal features. Moreover, CUT genes act in conjunction with neuron-type specific transcription factors to control pan-neuronal gene expression. Our study, therefore, provides a previously missing key insight into how neuronal gene expression programs are specified and reveals a highly buffered and robust mechanism that controls the most critical functional features of all neuronal cell types.

sentences: A Chromodomain Mutation Identifies Separable Roles for C. elegans MRG-1 in Germline and Somatic Development The packaging of DNA into chromatin strongly influences gene regulation. Post-translational modifications of histones, and the proteins that bind to them, alter the accessibility of chromatin and contribute to the activation and repression of genes. The human MRG15 (MORF4- Related Gene on chromosome 15) protein is a conserved chromodomain-containing protein that binds to methylated lysine 36 on histone H3 (H3K36me) and plays important roles in development, genome integrity, and gene regulation. MRG15 affects transcriptional regulation through its interactions with both histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes. MRG-1, its C. elegans homolog, has similarly been shown to have important roles in genomic integrity and development, and has also been shown to co- purify with HDAC complexes. Like MRG15, MRG-1 is predicted to bind to H3K36me through its chromodomain, yet despite mrg-1 mutants displaying developmental and germline phenotypes that overlap with H3K36 methyltransferase mutants, the role of the MRG-1 chromodomain has never been characterized. In this study, we examined meiotic cells lacking H3K36me3 to compare to mrg-1 mutant germ cell phenotypes, and mutated key residues in the MRG-1 chromodomain (CD) to assess its function. The CD mutations cause embryonic lethality but few post-embryonic germline defects, in contrast to mrg-1 deletion mutants which are viable but sterile. The CD mutations therefore disrupt somatic development despite the apparent absence of a requirement for MRG-1 protein in embryogenesis. Furthermore, the CD mutants exhibit a dominant RNAi resistance phenotype that is not seen in other mrg-1 mutant alleles. This suggests that the function of MRG-1, and the chromatin modifying complexes with which it interacts, includes tissue-specific interactions involving different requirements for a functional chromodomain. We propose that the CD mutation disrupts proper guidance of complexes within which it acts, and this guidance defect results in improper HDAC and/or HAT regulation causing an indirect defect in RNAi machinery expression or targeting.

sentences: Artificial Hsp100-mediated systems for re-localizing protein aggregates Spatial Protein Quality Control (sPQC) sequesters misfolded proteins into specific, organelle-associated inclusions within the cell to harness their toxicity. To approach the role of sPQC in cellular fitness, neurodegenerative diseases and aging, we report on the construction of Hsp100-based systems in yeast cells, which can artificially target protein aggregates to non-canonical locations. We demonstrated that aggregates of mutant Huntingtin (mHtt), the disease-causing agent of Huntingtons disease can be artificially targeted to daughter cells as well as to eisosomes and endosomes with this approach. Removing aggregates from mother cells did not significantly affect their lifespan and targeting mHtt to multiple smaller aggregates rather than one large inclusion did not alter its toxicity. We demonstrated that this approach is able to manipulate mHtt inclusion formation also in human cells and has the potential to be a useful complementation to present therapeutic approaches aimed at alleviating age-related neurodegenerative diseases.

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Immunomodulatory actions of a kynurenine-derived endogenous electrophile. The inflammatory upregulation of kynurenine metabolism induces immunomodulatory responses via incompletely understood mechanisms. We report that increases in cellular and systemic kynurenine levels yield the electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA), as evidenced by the accumulation of thiol-conjugates and saturated metabolites. Under physiological conditions, Kyn-CKA induces Nrf2-regulated genes and inhibits NF-{kappa}B and NLRP3-dependent pro-inflammatory signaling. Sickle Cell Disease (SCD) is a hereditary hemolytic condition characterized by basal inflammation and recurrent vaso-occlusive crises. Both a transgenic SCD murine model and SCD patients exhibit increased kynurenine synthesis and elevated Kyn-CKA metabolite levels. Plasma hemin and kynurenine concentrations are positively correlated, indicating that Kyn-CKA synthesis in SCD is upregulated during pathogenic vascular stress. Remarkably, exogenous administration of Kyn-CKA abrogated pulmonary microvasculature occlusion in SCD mice, an important factor in the development of lung injury. These findings demonstrate that the upregulation of kynurenine synthesis and its metabolism to Kyn-CKA is an adaptive response that attenuates inflammation and protects tissues.

One-Sentence SummaryKyn-CKA is a kynurenine-derived signaling mediator that transduces its immunomodulatory protective actions and attenuates vaso-occlusion in sickle cell disease.

sentences: The use of barcoded Asaia bacteria in mosquito in vivo screens for identification of systemic insecticides and inhibitors of malaria transmission This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the bloodmeals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonized Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects, and identifies novel candidate small molecules for disease control.

sentences: Maximizing GWAS discovery and genomic prediction accuracy in Biobank data Genetically informed and deep-phenotyped biobanks are an important research resource. The cost of phenotyping far outstrips that of genotyping, and therefore it is imperative that the most powerful, versatile and efficient analysis approaches are used. Here, we apply our recently developed Bayesian grouped mixture of regressions model (GMRM) in the UK and Estonian Biobanks and obtain the highest genomic prediction accuracy reported to date across 21 heritable traits. On average, GMRM accuracies were 15% (SE 7%) greater than prediction models run in the LDAK software with SNP annotation marker groups, 18% (SE 3%) greater than a baseline BayesR model without SNP markers grouped into MAF-LD-annotation categories, and 106% (SE 9%) greater than polygenic risk scores calculated from mixed-linear model association (MLMA) estimates. For height, the prediction accuracy R2 was 47% in a UK Biobank hold-out sample, which was 76% of the estimated [Formula]. We then extend our GMRM prediction model to provide MLMA SNP marker estimates for GWAS discovery, which increased the independent loci detected to 7,910 in unrelated UK Biobank individuals, as compared to 5,521 from BoltLMM and 5,727 from Regenie, a 43% and 38% increase respectively. The average{chi} 2 value of the leading markers was 34% (SE 5.11) higher for GMRM as compared to Regenie, and increased by 17% for every 1% increase in prediction accuracy gained over a baseline BayesR model across the traits. Thus, we show that modelling genetic associations accounting for MAF and LD differences among SNP markers, and incorporating prior knowledge of genomic function, is important for both genomic prediction and for discovery in large-scale individual-level biobank-scale studies.

sentences: Cortical wiring by synapse-specific control of local protein synthesis Neurons use local protein synthesis as a mechanism to support their morphological complexity, which requires independent control across multiple subcellular compartments including individual synapses. However, to what extent local translation is differentially regulated at the level of specific synaptic connections remains largely unknown. Here, we identify a signaling pathway that regulates the local synthesis of proteins required for the formation of excitatory synapses on parvalbumin-expressing (PV+) interneurons in the mouse cerebral cortex. This process involves the regulation of the mTORC1 inhibitor Tsc2 by the receptor tyrosine kinase ErbB4, which enables the local control of mRNA translation in a cell type-specific and synapse-specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins that regulates the formation of excitatory inputs on PV+ interneurons downstream of ErbB4 signaling. Our work demonstrates that local protein translation is regulated at the level of specific connections to control synapse formation in the nervous system.

sentences: Development of early postnatal inhibitory function in the mouse medial prefrontal and primary somatosensory cortex The prefrontal cortex (PFC) is characterized by protracted maturation. The cellular mechanisms controlling the early development of prefrontal circuits are still largely unknown. Our study delineates the developmental cellular processes in the mouse medial PFC (mPFC) during the second and third postnatal weeks and characterizes their contribution to the changes in network activity. We show that spontaneous inhibitory postsynaptic currents (sIPSC) are increased while spontaneous excitatory postsynaptic currents (sEPSC) are reduced from the second to the third postnatal week. Drug application suggested that the increased sEPSC frequency in mPFC at P10 is due to depolarizing GABAA receptor (GABAAR) function. To further validate this, perforated patch-clamp recordings were obtained and the expression levels of K-Cl co-transporter 2 (KCC2) protein were examined. The reversal potential of IPSCs in response to current stimulation was significantly more depolarized at P10 compared to P20 while KCC2 expression is decreased. Moreover, the number of parvalbumin-expressing GABAergic interneurons increase from P10 to P20 in the mPFC and their intrinsic electrophysiological properties significantly mature. Using computational modeling, we show that the developmental changes in synaptic and intrinsic properties of mPFC neurons contribute to the enhanced network activity in the juvenile compared to neonatal mPFC.

sentences: Distinctiveness and continuity in transcriptome and connectivity in the anterior-posterior axis of the paraventricular nucleus of thalamus The paraventricular nucleus of the thalamus (PVT) projects axons to multiple areas and mediates a wide range of behaviors. Heterogeneity of functions and axonal projections in PVT have been reported, but what cell types exist in PVT and how different they are have not been addressed. We applied single-cell RNA sequencing to depict transcriptomic characteristics of mouse PVT neurons. The transcriptome of PVT neurons had a continuous distribution with the largest variance corresponding to the anterior-posterior axis. Although the single-cell transcriptome classified PVT neurons into four types, transcriptomic and histological analyses showed their continuity. Similarly, anterior and posterior subpopulations had nearly non-overlapping axon projection patterns, while another population showed intermediate patterns. In addition, they responded differently to appetite-related neuropeptides, and their chemogenetic activation showed opposing effects in food consumption. Our studies showed contrasts and continuity of PVT neurons underlying their function as a behavior-modulating hub.

sentences: Chi3l1 is a modulator of glioma stem cell states and a therapeutic vulnerability for glioblastoma Chi3l1 (Chitinase 3-like 1) is a secreted protein highly expressed in glioblastoma. Here, we show that exposure of glioma stem cells (GSCs) to Chi3l1 reduces the CD133+/SOX2+ cells and increases the CD44+/Chi3l1+ cells. Chi3l1 binds to CD44 and induces phosphorylation and nuclear translocation of beta-catenin, Akt and STAT3. Single cell RNA-seq and RNA velocity following incubation of GSCs with Chi3l1 show significant changes in GSC state dynamics driving GSCs towards a mesenchymal expression profile and reducing transition probabilities towards terminal cellular states. ATAC-seq reveals that Chi3l1 increases accessibility of promoters containing MAZ transcription factor footprint. Inhibition of MAZ directly regulates genes with highest expression in cellular clusters exhibiting significant cell state transitions. Finally, targeting Chi3l1 in vivo with a blocking antibody, resets the transcriptomic profile of glioblastoma and inhibits tumor growth. Our work implicates Chi3l1 as modulator of GSC cellular states and demonstrates pre-clinical efficacy of anti-Chi3l1 antibody treatment.

sentences: Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics and live-cell microscopy, we determined the contribution of the pilotin to assembly, function and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In the absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. In line, the majority of export apparatus subcomplexes is mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, the export of late T3SS substrates including the virulence effectors is hardly affected in these bacteria, whereas early T3SS substrates, such as the needle subunits, are only exported to a very low degree. We found that pilotins transiently interact with the secretin and the large export apparatus component SctV, but mostly localize throughout the bacterial membrane, where they form transient mobile clusters, which do not colocalize with assembled injectisomes. In addition, pilotins interact with non-T3SS components, including sugar transporters. Pilotins therefore have additional functions downstream injectisome assembly, which include the regulation of type III secretion and a potential new link to the cellular energy metabolism.

sentences: Long-Short Term Memory Networks for Electric Source Imaging with Distributed Dipole Models 1Magneto- and electroencephalography (M/EEG) are widespread techniques to measure neural activity in vivo at a high temporal resolution but relatively low spatial resolution. Locating the sources underlying the M/EEG poses an inverse problem, which is itself ill-posed. In recent years, a new class of source imaging methods was developed based on artificial neural networks. We present a long-short term memory (LSTM) network to solve the M/EEG inverse problem. It integrates several aspects essential for qualitative inverse solutions: Low computational cost, exploitation of both spatial and temporal information, input flexibility and robustness to noise. Using simulation data, the LSTM shows higher accuracy on multiple metrics and for varying numbers of neural sources, compared to classical inverse solutions but also compared to our alternative architectures without integration of temporal information. It successfully integrates the temporal context given by sequential data, which is particularly useful with noisy data. Real data of a median nerve stimulation paradigm was used to show that the LSTM predicts plausible sources that are in concordance with classical inverse solutions. The performance of the LSTM regarding its robustness to noise renders it a promising and easy-to-apply inverse solution to be considered in future source localization studies and for clinical applications.

sentences: FIT-GWA: A new method for the genetic analysis of small gene effects, high precision in phenotype measurements and small sample sizes. Small gene effects involved in complex traits remains difficult to analyse using current genome-wide association methods (GWAS) due to the number of individuals required to return meaningful association(s), a.k.a. study power. Inspired by physics fields theory we provide a different method called Fields Informational Theory for Genome-Wide Associations (FIT-GWA). Contrary to GWAS, FIT-GWA that the phenotype is measured precisely enough and/or the number of individuals in the population is too small, to permit categories. To extract information FIT-GWA use the difference in the cumulated sum of gene microstates between two configurations: (i) when the individuals are taken at random without information on phenotype values and, (ii) when individuals are ranked as a function of their phenotype value. Such difference can be accounted through the emergence of  phenotypic fields. We demonstrate that FIT-GWA recovers GWAS, i.e., Fishers theory, when the phenotypic fields are linear. However, unlike GWAS, FIT-GWA permits to demonstrate how the variance of microstate distribution density functions are also involved in genotype-phenotype associations. Using genotype-phenotype simulations based on Fishers theory we illustrate the application and power of the method with a small sample size of 1000 individuals.

sentences: Critical contribution of 3' non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA Base-pairing of the seed region (g2-g8) is essential for microRNA targeting, however, the in vivo function of the 3 non-seed region (g9-g22) are less well understood. Here we report the first systematic investigation of the in vivo roles of 3 non-seed nucleotides in microRNA let-7a, whose entire g9-g22 region is conserved among bilaterians. We found that the 3 non-seed sequence functionally distinguishes let-7a from its family paralogs. The complete pairing of g11-g16 is essential for let-7a to fully repress multiple key targets, including evolutionarily conserved lin-41, daf-12 and hbl-1. Nucleotides at g17-g22 are less critical but may compensate for mismatches in the g11-g16 region. Interestingly, the 3 non-seed pairing of let-7a can be critically required with certain minimal complementarity for sites with perfect seed pairing. These results provide evidence that the specific configurations of both seed and 3 non-seed base-pairing can critically influence microRNA-mediated gene regulation in vivo.

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Integrative experimental/computational approach establishes active cellular protrusion as the primary driving force of phagocytic spreading by immune cells The dynamic interplay between cell adhesion and protrusion is a critical determinant of many forms of cell motility. When modeling cell spreading on adhesive surfaces, traditional mathematical treatments often consider passive cell adhesion as the primary, if not exclusive, mechanistic driving force of this cellular motion. To better assess the contribution of active cytoskeletal protrusion to immune-cell spreading during phagocytosis, we here develop a computational framework that allows us to optionally investigate both purely adhesive spreading ("Brownian zipper hypothesis") as well as protrusion-dominated spreading ("protrusive zipper hypothesis"). We model the cell as an axisymmetric body of highly viscous fluid surrounded by a cortex with uniform surface tension and incorporate as potential driving forces of cell spreading an attractive stress due to receptor-ligand binding and an outward normal stress representing cytoskeletal protrusion, both acting on the cell boundary. We leverage various model predictions against the results of a directly related experimental companion study of human neutrophil phagocytic spreading on substrates coated with different densities of antibodies. We find that the concept of adhesion-driven spreading is incompatible with experimental results such as the independence of the cell-spreading speed on the density of immobilized antibodies. In contrast, the protrusive zipper model agrees well with experimental findings and, when adapted to simulate cell spreading on discrete adhesion sites, it also reproduces the observed positive correlation between antibody density and maximum cell-substrate contact area. Together, our integrative experimental/computational approach shows that phagocytic spreading is driven by cellular protrusion, and that the extent of spreading is limited by the density of adhesion sites.

Author SummaryTo accomplish many routine biological tasks, cells must rapidly spread over different types of surfaces. Here, we examine the biophysical underpinnings of immune cell spreading during phagocytosis, the process by which white blood cells such as neutrophils engulf pathogens or other foreign objects. Our computational framework models the case in which a human neutrophil spreads over a flat surface coated with antibodies, which we also test experimentally in a companion paper. Our primary purpose is to assess whether phagocytic spreading is actively driven by protrusive forces exerted by the cell, or passively by adhesive forces acting between receptors in the cell membrane and antibodies on the surface. By directly comparing our model predictions to experimental results, we demonstrate that phagocytic spreading is primarily driven by protrusion, but the extent of spreading is still limited by the availability of binding sites. Our findings improve the fundamental understanding of phagocytosis and may also pave the way for future investigations of the balance between adhesion and protrusion in other forms of cell spreading, such as wound healing or cancer cell migration.

sentences: Accurate and automated high-coverage identification of chemically cross-linked peptides with MaxLynx Cross-linking combined with mass spectrometry (XL-MS) provides a wealth of information about the 3D structure of proteins and their interactions. We introduce MaxLynx, a novel computational proteomics workflow for XL-MS integrated into the MaxQuant environment. It is applicable to non-cleavable and MS-cleavable cross linkers. For both we have generalized the Andromeda peptide database search engine to efficiently identify cross-linked peptides. For non-cleavable peptides, we implemented a novel di-peptide Andromeda score, which is the basis for a computationally efficient N-squared search engine. Additionally, partial scores summarize the evidence for the two constituents of the di-peptide individually. A posterior error probability based on total and partial scores is used to control false discovery rates. For MS-cleavable cross linkers a scoring of signature peaks is combined with the conventional Andromeda score on the cleavage products. The MaxQuant 3D-peak detection was improved to ensure more accurate determination of the monoisotopic peak of isotope patterns for heavy molecules, which cross-linked peptides typically are. A wide selection of filtering parameters can replace manual filtering of identifications, which is often necessary when using other pipelines. On benchmark datasets of synthetic peptides, MaxLynx outperforms all other tested software on data for both types of cross linkers as well as on a proteome-wide dataset of cross-linked D. melanogaster cell lysate. The workflow also supports ion-mobility enhanced MS data. MaxLynx runs on Windows and Linux, contains an interactive viewer for displaying annotated cross-linked spectra and is freely available at https://www.maxquant.org/.

sentences: Visual association cortex links cues with conjunctions of reward and locomotor contexts Postrhinal cortex (POR) and neighboring lateral visual association areas are necessary for identifying objects and interpreting them in specific contexts, but how POR neurons encode the same object across contexts remains unclear. Here, we imaged excitatory neurons in mouse POR across tens of days throughout initial cue-reward learning and reversal learning. As such, neurons were tracked across sessions/trials where the same cue was rewarded or unrewarded, during both locomotor and stationary contexts. Surprisingly, a large class of POR neurons were minimally cue-driven prior to learning. After learning, distinct clusters within this class responded selectively to a given cue when presented in a specific conjunction of reward and locomotion contexts. In addition, another class involved clusters of neurons whose cue responses were more transient, insensitive to reward learning, and adapted over thousands of presentations. These two classes of POR neurons may support context-dependent interpretation and context-independent identification of sensory cues.

sentences: Transoceanic voyages of drywood termites (Isoptera: Kalotermitidae) inferred from extant and extinct species Termites are major decomposers of organic matter in terrestrial ecosystems and the second most diverse lineage of social insects. The Kalotermitidae, the second-largest termite family, are widely distributed across tropical and subtropical ecosystems, where they typically live in small colonies confined to single wood items inhabited by individuals with no foraging abilities. How the Kalotermitidae have acquired their global distribution patterns remains unresolved. Similarly, it is unclear whether foraging is ancestral to Kalotermitidae or was secondarily acquired in a few species. These questions can be addressed in a phylogenetic framework. We inferred time-calibrated phylogenetic trees of Kalotermitidae using mitochondrial genomes and nuclear ribosomal RNA genes of [~]120 species, about 27% of kalotermitid diversity, including representatives of 22 of the 23 kalotermitid genera. We found that extant kalotermitids shared a common ancestor 81 Mya (72-91 Mya 95% HPD), indicating that a few disjunctions among early-diverging kalotermitid lineages may predate Gondwana breakup. However, most of the [~]40 disjunctions among biogeographic realms were dated at less than 50 Mya, indicating that transoceanic dispersals, and more recently human-mediated dispersals, have been the major drivers of the global distribution of Kalotermitidae. Our phylogeny also revealed that the capacity to forage is often found in early-diverging kalotermitid lineages, implying that the ancestors of Kalotermitidae were able to forage among multiple wood pieces. Our phylogenetic estimates provide a platform for a critical taxonomic revision of the family and for future comparative analyses of Kalotermitidae.

sentences: Classification and clustering of RNA crosslink-ligation data reveal complex structures and homodimers The recent development and application of methods based on the general principle of "crosslinking and proximity ligation" (crosslink-ligation) are revolutionizing RNA structure studies in living cells. However, extracting structure information from such data presents unique challenges. Here we introduce a set of computational tools for the systematic analysis of data from a wide variety of cross-link-ligation methods, specifically focusing on read mapping, alignment classification and clustering. We design a new strategy to map short reads with irregular gaps at high sensitivity and specificity. Analysis of previously published data reveals distinct properties and bias caused by the crosslinking reactions. We perform rigorous and exhaustive classification of alignments and discover 8 types of arrangements that provide distinct information on RNA structures and interactions. To deconvolve the dense and inter-twined gapped alignments, we develop a network/graph-based tool CRSSANT (Crosslinked RNA Secondary Structure Analysis using Network Techniques), which enables clustering of gapped alignments and discovery of new alternative and dynamic conformations. We discover that multiple crosslinking and ligation events can occur on the same RNA, generating multi-segment alignments to report complex high level RNA structures and multi-RNA interactions. We find that alignments with overlapped segments are produced from potential homodimers and develop a new method for their de novo identification. Analysis of overlapping alignments revealed potential new homodimers in cellular noncoding RNAs and RNA virus genomes in the Picornaviridae family. Together, this suite of computational tools enables rapid and efficient analysis of RNA structure and interaction data in living cells.

sentences: Stem cell-laden hydrogel bioink for generation of high resolution and fidelity engineered tissues with complex geometries Recently, 3D bioprinting has been explored as a promising technology for biomedical applications with the potential to create complex structures with precise features. Cell encapsulated hydrogels composed of materials such as gelatin, collagen, hyaluronic acid, alginate and polyethylene glycol have been widely used as bioinks for 3D bioprinting. However, since most hydrogel-based bioinks may not allow rapid stabilization immediately after 3D bioprinting, achieving high resolution and fidelity to the intended architecture is a common challenge in 3D bioprinting of hydrogels. In this study, we have utilized shear-thinning and self-healing ionically crosslinked oxidized and methacrylated alginates (OMAs) as a bioink, which can be rapidly gelled by its self-healing property after bioprinting and further stabilized via secondary crosslinking. It was successfully demonstrated that stem cell-laden calcium-crosslinked OMA hydrogels can be bioprinted into complicated 3D tissue structures with both high resolution and fidelity. Additional photocrosslinking enables long-term culture of 3D bioprinted constructs for formation of functional tissue by differentiation of encapsulated human mesenchymal stem cells.

sentences: FAMOSS, a conserved 41-aa peptide involved in plant tip growth regulation Recent evidence shows that small open reading frame (smORF; <100 codons)-encoded peptides (SEPs) containing transmembrane domains are preadapted to be progenitors of novel functional genes. A dozen of such SEPs translated from long non-coding RNAs (lncRNAs) are already functionally characterised in animals. However, functional plant lncRNA-smORF-coded peptides are not yet described. Here, we report detailed functional characterization of a 41-aa peptide encoded by lncRNA-smORFs in the moss Physcomitrium patens, which was named "FAst-growing MOSS" (FAMOSS). We found that the FAMOSS interacts with the Rab-type small GTPase proteins and its overexpression leads to faster moss growth rate and more intensive vesicular transport in apical cells, while its knockout results in the opposite effect. The FAMOSS contains a predicted transmembrane domain and possible orthologs from streptophyta algae to flowering plants have a very conserved structure. Thus, the FAMOSS peptide is a previously unknown conserved player of Rab-mediated processes in plants. Our findings are in line with functional studies of transmembrane SEPs in animals and prove the principles of SEPs evolution. This study provides new insights into functions of plant lncRNA-smORFs.

sentences: Non-coding function for mRNAs in Focal Adhesion Architecture and Mechanotransduction Messenger RNA (mRNA) compartmentalization within the cytosol is well-recognized as a key mechanism of local translation-mediated regulation of protein levels, but whether such localization could be a means of exercising non-coding mRNA function is unknown. Here, we explore non-coding functions for mRNAs associated with focal adhesions (FAs), cellular structures responsible for mediating cell adhesion and response to changes in the extracellular matrix (ECM). Using high-throughput single molecule imaging and genomic profiling approaches, we find that mRNAs with distinct sequence characteristics localize to FAs in different human cell types. Notably, [~]85% of FA-mRNAs are not translationally active at steady state or under conditions of FA dissolution or activation. Untranslated mRNA sequences are anchored to FA based on their functional states by the RNA binding protein, G3BP1, forming biomolecular granules. Removing RNA or G3BP1, but not blocking new polypeptide synthesis, dramatically changes FA protein composition and organization, resulting in loss of cell contractility and cellular ability to adapt to changing ECM. We have therefor uncovered a novel, non-coding role for mRNAs as scaffolds to maintain FA structure and function, broadening our understating of noncanonical mRNA functions.

sentences: Process engineering of biopharmaceutical production in moss bioreactors via model-based description and evaluation of phytohormone impact The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 {micro}M) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-Kynurenine (100 {micro}M) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 hours after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first two hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/ g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.

sentences: Loss of Nuclear DNA Ligase III Reverts PARP Inhibitor Resistance in BRCA1/53BP1 Double-deficient Cells by Exposing ssDNA Gaps Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

sentences: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells Cells can help each other replicate by communicating with diffusible molecules. In cell cultures, molecules may diffuse within a cell colony or between adjacent or distant colonies. Determining which cell helps which cells replication is challenging. We developed a systematic approach, integrating modeling and experiments, for determining the length-scales of cell-cell communication (from microns to centimeters). With this approach, we discovered that differentiating murine ES cells, scattered across centimeters on a dish, communicate over millimeters to form one macroscopic entity that survives if and only if its centimeter-scale population-density is above a threshold value. Single-cell-level measurements, transcriptomics, and modeling revealed that this "macroscopic quorum sensing" arises from differentiating ES cells secreting and sensing survival-promoting FGF4 that diffuses over millimeters and activates YAP1-induced survival mechanisms. Through the same mechanism, a lone macroscopic, but not microscopic, colony survives differentiation. Our work rigorously establishes that in vitro ES-cell differentiation relies on macroscopic cooperation.

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A billion synthetic 3D-antibody-antigen complexes enable unconstrained machine-learning formalized investigation of antibody specificity prediction Machine learning (ML) is a key technology to enable accurate prediction of antibody-antigen binding, a prerequisite for in silico vaccine and antibody design. Two orthogonal problems hinder the current application of ML to antibody-specificity prediction and the benchmarking thereof: (i) The lack of a unified formalized mapping of immunological antibody specificity prediction problems into ML notation and (ii) the unavailability of large-scale training datasets. Here, we developed the Absolut! software suite that allows the parameter-based unconstrained generation of synthetic lattice-based 3D-antibody-antigen binding structures with ground-truth access to conformational paratope, epitope, and affinity. We show that Absolut!-generated datasets recapitulate critical biological sequence and structural features that render antibody-antigen binding prediction challenging. To demonstrate the immediate, high-throughput, and large-scale applicability of Absolut!, we have created an online database of 1 billion antibody-antigen structures, the extension of which is only constrained by moderate computational resources. We translated immunological antibody specificity prediction problems into ML tasks and used our database to investigate paratope-epitope binding prediction accuracy as a function of structural information encoding, dataset size, and ML method, which is unfeasible with existing experimental data. Furthermore, we found that in silico investigated conditions, predicted to increase antibody specificity prediction accuracy, align with and extend conclusions drawn from experimental antibody-antigen structural data. In summary, the Absolut! framework enables the development and benchmarking of ML strategies for biotherapeutics discovery and design.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/451258v2_ufig1.gif" ALT="Figure 1">
View larger version (81K):
org.highwire.dtl.DTLVardef@14295d2org.highwire.dtl.DTLVardef@7a12bdorg.highwire.dtl.DTLVardef@647102org.highwire.dtl.DTLVardef@375112_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical abstractC_FLOATNO The software framework Absolut! enables (A,B) the generation of virtually arbitrarily large numbers of in silico 3D-antibody-antigen structures, (C,D) the formalization of antibody specificity as machine learning (ML) tasks as well as the exploration of ML strategies for paratope-epitope prediction.

C_FIG Highlights- Software framework Absolut! to generate an arbitrarily large number of in silico 3D-antibody-antigen structures
- Generation of one billion in silico antigen-antibody structures reflecting biological layers of complexity that make ML predictions challenging
- Immunological antibody specificity prediction problems formalized as machine learning tasks for which the in silico complexes are immediately usable as benchmark.
- Exploration of machine learning architectures for paratope-epitope interaction prediction accuracy as a function of neural network depth, dataset size, and sequence-structure encoding

sentences: Kdm6b confers Tfdp1 with the competence to activate p53 signalling in regulating palatogenesis Epigenetic regulation plays extensive roles in diseases and development. Disruption of epigenetic regulation not only increases the risk of cancer, but can also cause various developmental defects. However, it is still unclear how epigenetic regulators coordinate with tissue-specific regulatory factors during morphogenesis of specific organs. Using palatogenesis as a model, we reveal the functional significance of Kdm6b, a H3K27me3 demethylase, in regulating embryonic development. Our study shows that Kdm6b plays an essential role in neural crest development, and loss of Kdm6b disturbs p53 pathway-mediated activity, leading to complete cleft palate along with cell proliferation and differentiation defects. Furthermore, activity of H3K27me3 on the promoter of p53 is precisely controlled by Kdm6b, and Ezh2 in regulating p53 expression in cranial neural crest cells. More importantly, Kdm6b renders chromatin accessible to the transcription factor Tfdp1, which binds to the promoter of p53 along with Kdm6b to specifically activate p53 expression during palatogenesis. Collectively our results highlight the important role of the epigenetic regulator Kdm6b and how it cooperates with Tfdp1 to achieve its functional specificity in regulating p53 expression, and further provide mechanistic insights into the epigenetic regulatory network during organogenesis.

sentences: Acidophilic Micrarchaeon Seems to Maintain a Slightly Alkaline Cytosolic pH Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota remains largely enigmatic. "Candidatus Micrarchaeum harzensis A_DKE", for example, highly expresses numerous genes encoding hypothetical proteins and their function is difficult to elucidate due to a lacking genetic system. Still, not even the intracellular pH value of A_DKE is known, and heterologous production attempts are generally missing so far. Hence, A_DKEs isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified for bio-chemical characterisation. MhIDH appeared to be specific for NADP+, yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed KM-values of 53.03{+/-}5.63 {micro}M and 1.94{+/-}0.12 mM and kcat-values of 38.48{+/-}1.62 s-1 and 43.99{+/-}1.46 s-1 for DL-isocitrate and NADP+, respectively. MhIDHs exceptionally low affinity for NADP+, potentially limiting its reaction rate, can be likely attributed to the presence of a proline residue in the NADP+ binding-pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure. Furthermore, a pH optimum of 7.89 implies, that A_DKE applies potent mechanisms of proton homoeostasis, to maintain a slightly alkaline cytosolic milieu in a highly acidic environment.

sentences: Gut Microbiota predicts Healthy Late-life Aging in Male Mice Calorie restriction (CR) extends lifespan and retards age-related chronic diseases in most species. There is growing evidence that the gut microbiota has a pivotal role in host health and age-related pathological conditions. Yet, it is still unclear how CR and the gut microbiota are related to healthy aging. Here we report findings from a small longitudinal study of male C57BL/6 mice maintained on either ad libitum or mild (15%) CR diets from 21 months of age and tracked until natural death. We demonstrate that CR results in a significant reduction in frailty index (FI), a well-established indicator of aging. We observed significant alterations in bacterial load, diversity, and compositional patterns of the mouse gut microbiota during the aging process. Interrogating the FI-related microbial features using machine learning techniques, we show that gut microbial signatures from 21-month-old mice can predict the healthy aging of 30-month-old mice with reasonable accuracy. This study deepens our understanding of the links between CR, gut microbiota, and frailty in the aging process of mice.

sentences: Bioinformatics analysis of potential key genes and mechanisms in type 2 diabetes mellitus Type 2 diabetes mellitus (T2DM) is etiologically related to metabolic disorder. The aim of our study was to screen out candidate genes of T2DM and to elucidate the underlying molecular mechanisms by bioinformatics methods. Expression profiling by high throughput sequencing data of GSE154126 was downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) between T2DM and normal control were identified. And then, functional enrichment analyses of gene ontology (GO) and REACTOME pathway analysis was performed. Protein-protein interaction (PPI) network and module analyses were performed based on the DEGs. Additionally, potential miRNAs of hub genes were predicted by miRNet database. Transcription factors (TFs) of hub genes were detected by NetworkAnalyst database. Further, validations were performed by receiver operating characteristic curve (ROC) analysis and real-time polymerase chain reaction (RT-PCR). In total, 925 DEGs were identified in T2DM, including 447 up regulated genes and 478 down-regulated genes. Functional enrichment analysis results showed that up regulated DEGs were significantly enriched in defense response, neutrophil degranulation, cell adhesion and extracellular matrix organization. The top 10 hub genes, JUN, VCAM1, RELA, U2AF2, ADRB2, FN1, CDK1, TK1, A2M and ACTA2 were identified from the PPI network, modules, miRNA-hub gene regulatory network and TF-hub gene regulatory network. Furthermore, ROC analysis and RT-PCR revealed that JUN, VCAM1, RELA, U2AF2, ADRB2, FN1, CDK1, TK1, A2M and ACTA2 might serve as biomarkers in T2DM. Bioinformatics analysis is a useful tool to explore the molecular mechanism and pathogenesis of T2DM. The identified hub genes may participate in the onset and advancement of T2DM and serve as therapeutic targets.

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A comparison of hard and soft direct methods for DNA extraction from soil Nucleic acid extraction is the first step in molecular biology studies of soil bacterial communities. The most common used soil DNA extraction method is the direct, hard extraction Mobio method, which uses bead beating to lyse bacteria. In this study we compared the Mobio method with a soft, enzymatic lysis extraction method. Next generation sequencing (Illumina and Pyrosequencing) of amplicons generated from four 16S primer pairs and DNA from 12 soils and 3 composts was used to compare the two extraction methods.

Four bacterial orders, the delta proteobacterial Desulfuromonadales and gamma proteobacterial Pseudomonadales, Enterobacteriales, and Alteromonadales were more common in amplicons from soft extracted DNA, sometimes by two orders of magnitude. These groups can be a significant fraction of the bacterial population. For example the Pseudomonadales made up to 16 % and Enterobacteriales 10% of amplicons from Soft extracted DNA. The JG30-KF-CM45 order was under extracted by the enzymatic lysis extraction method. Results differed more by primer choice than extraction method and the phylogenetic resolution of differences between extraction methods changed with primer choice.

Given how often Mobio extraction is used, these proteobacterial orders are probably under-represented in the studies of soil bacteria that use nucleic acid methods. Further improvements in soil DNA extraction are needed. Amplicons sequencing studies should use a range of different primers to confirm the phylogenetic resolution of their results.

ImportanceSeveral large scale studies of soil bacteria that compare thousands of soil samples across continents have used the Mobio method for DNA extraction. Large scale studies like these are increasing with the recent establishment of the Global Soil Biodiversity Observation Network (Soil BON), which also uses the Mobio method. The results of this work will be used to make policy decisions about how to manage the soil and may be a guide for bioprospectors. As the Mobio method is so widely used, it is important to know its limitations. Studies that use the Mobio method underestimate the fraction of several proteobacterial groups. Most notably the Enterobacteria and Pseudomonas can be under extracted by 10-100 fold. The degree of under extraction varies with different soils.

sentences: Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation Nonstructural protein 1 (Nsp1) is a major pathogenicity factor of SARS-CoV-2. It inhibits host-cell translation, primarily through a direct interaction between its C-terminal domain and the mRNA entry channel of the 40S small ribosomal subunit, with an N-terminal {beta}-barrel domain fine-tuning the inhibition and promoting selective translation of viral mRNA. SARS-CoV-2 nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. To provide the biochemical basis for this, it is essential to characterize the efficiency of translational inhibition by the said protein variants. Here, we use an in vitro translation system to investigate the translation inhibition capacity of a series of clinically observed Nsp1 deletion variants. We find that a frequently observed deletion of residues 79-89 destabilized the N-terminal domain (NTD) and severely reduced the capacity of Nsp1 to inhibit translation. Interestingly, shorter deletions in the same region have been reported to effect the type I interferon response but did not affect translation inhibition, indicating a possible translation-independent role of the Nsp1 NTD in interferon response modulation. Taken together, our data provide a mechanistic basis for understanding how deletions in Nsp1 influence SARS-CoV-2 induction of interferon response and COVID-19 progression.

sentences: Loneliness and the social brain: how perceived social isolation impairs human interactions Loneliness is a painful condition associated with increased risk for premature mortality. The formation of new, positive social relationships can alleviate feelings of loneliness, but requires rapid trustworthiness decisions during initial encounters and it is still unclear how loneliness hinders interpersonal trust. Here, we use a multimodal approach including behavioral, psychophysiological, hormonal, and neuroimaging measurements to probe a trust-based mechanism underlying impaired social interactions in loneliness. Pre-stratified healthy individuals with high loneliness scores (n = 42 out of a screened sample of 3678 adults) show reduced oxytocinergic and affective responsiveness to a positive conversation, report less interpersonal trust, and prefer larger social distances compared to controls (n = 40). Moreover, lonely individuals are rated as less trustworthy compared to controls and identified by the blinded confederate better than chance. During initial trust decisions, lonely individuals exhibit attenuated limbic and striatal activation and blunted functional connectivity between the anterior insula and occipitoparietal regions, which correlates with the diminished affective responsiveness to the positive social interaction. This neural response pattern is not mediated by loneliness-associated psychological symptoms. Thus, our results indicate compromised integration of trust-related information as a shared neurobiological component in loneliness, yielding a reciprocally reinforced trust bias in social dyads.

sentences: A Coupled Flow-Thermoregulation Lumped Model to Investigate Cardiac Function AO_SCPLOWBSTRACTC_SCPLOWLumped (zero-dimensional) technique is a robust and widely used approach to mathematically model and explore bulk behaviour of different physical phenomena in a lower expense. In modelling of cardio/cerebrovascular fluid dynamics, this technique facilitates the assessment of relevant metrics such as flow, pressure, and temperature at different locations over a large network/domain. Furthermore, they can be employed as boundary conditions in multiscale modelling of physiological flows. In this methodology paper, a lumped model for the cardiovascular flow simulation along with a two-node thermoregulation model are employed. The lumped models are built upon previous studies and are amended appropriately to focus on cardiac function. The output of the coupled model can either be used for assessing the cardiac function in different physiological conditions or it can provide the input data for other investigations. Noteworthy to mention that, the present model has been specifically developed for investigation on the effects of atrial fibrillation on cardiac performance.

sentences: The role of syndapin-2 mediated transcytosis across the blood-brain barrier   on amyloid-beta accumulation in the brain A deficient transport of amyloid-(A{beta}) across the blood-brain barrier (BBB), and its diminished clearance from the brain, contributes to neurodegenerative and vascular pathologies, including Alzheimers (AD) and cerebral angiopathy, respectively. At the BBB, A{beta} efflux transport is associated with the low-density receptor-related protein 1 (LRP1). However, the precise mechanisms governing A{beta} transport across the BBB, in health and disease, remain to be fully understood. Recent evidence indicates that the LRP1 transcytosis occurs through a tubulation-mediated mechanism stabilised by syndapin-2. Here, we show that syndapin-2 is associated with A{beta} clearance via LRP1 across the BBB. We further demonstrate that risk factors for AD, A{beta} expression and ageing, are associated with a decline in the native expression of syndapin-2 within brain endothelium. Our data reveal that the syndapin-2-mediated pathway, and its balance with the endosomal sorting, are important for A{beta} clearance proposing a measure to evaluate AD and ageing, as well as a target for counteracting A{beta} build-up. Moreover, we provide evidence for the impact of the avidity of A{beta} assemblies in their trafficking across the brain endothelium and in LRP1 expression levels, which may affect the overall clearance of A{beta} across the BBB.

sentences: A Novel Local Explainability Approach for Spectral Insight into Raw EEG-Based Deep Learning Classifiers The frequency domain of electroencephalography (EEG) data has developed as a particularly important area of EEG analysis. EEG spectra have been analyzed with explainable machine learning and deep learning methods. However, as deep learning has developed, most studies use raw EEG data, which is not well-suited for traditional explainability methods. Several studies have introduced methods for spectral insight into classifiers trained on raw EEG data. These studies have provided global insight into the frequency bands that are generally important to a classifier but do not provide local insight into the frequency bands important for the classification of individual samples. This local explainability could be particularly helpful for EEG analysis domains like sleep stage classification that feature multiple evolving states. We present a novel local spectral explainability approach and use it to explain a convolutional neural network trained for automated sleep stage classification. We use our approach to show how the relative importance of different frequency bands varies over time and even within the same sleep stages. Furthermore, to better understand how our approach compares to existing methods, we compare a global estimate of spectral importance generated from our local results with an existing global spectral importance approach. We find that the {delta} band is most important for most sleep stages, though {beta} is most important for the non-rapid eye movement 2 (NREM2) sleep stage. Additionally, {theta} is particularly important for identifying Awake and NREM1 samples. Our study represents the first approach developed for local spectral insight into deep learning classifiers trained on raw EEG time series.

sentences: Generation and analysis of context-specific genome-scale metabolic models derived from single-cell RNA-Seq data Single-cell RNA sequencing has the potential to unravel the differences in metabolism across cell types and cell states in both the healthy and diseased human body. The use of existing knowledge in the form of genome-scale metabolic models (GEMs) holds promise to strengthen such analyses, but the combined use of these two methods requires new computational methods. Here, we present a method for generating cell-type-specific genome-scale models from clusters of single-cell RNA-Seq profiles. Specifically, we developed a method to estimate the number of cells required to pool to obtain stable models, a bootstrapping strategy for estimating statistical inference, and a faster version of the tINIT algorithm for generating context-specific GEMs. In addition, we evaluated the effect of different RNA-Seq normalization methods on model topology and differences in models generated from single-cell and bulk RNA-Seq data. We applied our methods on data from mouse cortex neurons and cells from the tumor microenvironment of lung cancer and in both cases found that almost every cell subtype had a unique metabolic profile, emphasizing the need to study them separately rather than to build models from bulk RNA-Seq data. In addition, our approach was able to detect cancer-associated metabolic differences between cancer cells and healthy cells, showcasing its utility. With the ever-increasing availability of single-cell RNA-Seq datasets and continuously improved GEMs, their combination holds promise to become an important approach in the study of human metabolism.

sentences: E2F4 as a single multifactorial target against Alzheimer's disease Alzheimers disease (AD) has a multifactorial etiology, which requires a single multi-target approach for an efficient treatment. We have focused on E2F4, a transcription factor that regulates cell quiescence and tissue homeostasis, controls gene networks affected in AD, and is upregulated in the brain of Alzheimers patients and of APPswe/PS1dE9 and 5xFAD transgenic mice. E2F4 contains an evolutionarily-conserved Thr-motif that, when phosphorylated, modulates its activity, thus constituting a potential target for intervention. Here we show that neuronal expression in 5xFAD mice of a dominant negative form of E2F4 lacking this Thr-motif (E2F4DN) potentiates a transcriptional program consistent with the attenuation of the immune response and global brain homeostasis. This correlates with reduced microgliosis and astrogliosis, modulation of A{beta} proteostasis, and blockade of neuronal tetraploidization. Moreover, E2F4DN prevents cognitive impairment and body weight loss, a known somatic alteration associated with AD. Our finding is relevant for AD, since E2F4 is expressed in cortical neurons from Alzheimer patients in association with Thr-specific phosphorylation, as evidenced by an anti-E2F4/anti-phosphoThr proximity ligation assay. We propose E2F4DN-based gene therapy as a promising multifactorial approach against AD.

sentences: Blocking cell fusion inhibits age-induced polyploidy and maintains epithelial organization in Drosophila A characteristic of normal aging and age-related diseases is the remodeling of a tissues cellular organization through polyploid cell growth. Polyploidy arises from an increase in nuclear ploidy or the number of nuclei per cell. However, it is not known whether age-induced polyploidy is an adaption to stressors or a precursor to degeneration. Here, we find that the adult fruit flys abdominal epithelium becomes polyploid with age through generation of large multinucleated cells that make up more than 40% of the tissue area. The syncytia arise by cell fusion, not endomitosis. Epithelial multinucleation is also a characteristic of macular degeneration, including Ctnna1tvrm5, a mouse model for pattern dystrophy. Similarly, we find that the knockdown of alpha-catenin enhances multinucleation in the fly epithelium. We further show that age-induced polyploidy can be suppressed by inhibiting cell fusion revealing a means to maintain tissue organization in older animals.

sentences: Evolutionary rates of testes-expressed genes differ in monogamous and promiscuous Peromyscus species Reproductive proteins, including those expressed in the testes, are among the fastest evolving proteins across the tree of life. Sexual selection on traits involved in sperm competition is thought to be a primary driver of testes gene evolution and is expected to differ between promiscuous and monogamous species due to intense competition between males to fertilize females in promiscuous lineages and lack thereof in monogamous ones. Here, we employ the rodent genus Peromyscus as a model to explore differences in evolutionary rates between testes-expressed genes in monogamous and promiscuous species. We find candidate genes that may be associated with increased sperm production in promiscuous species and gene ontology categories that show patterns of molecular convergence associated with phenotypic convergence in independently evolved monogamous species. Overall, our results highlight the possible molecular consequences of differences in mating system, likely due to differences in selective pressures.

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Preparatory attention incorporates contextual expectations Humans are remarkably proficient at finding objects within a complex visual world. Current theories of attentional selection propose that this ability is mediated by target-specific preparatory activity in visual cortex, biasing visual processing in favor of the target object. In real-world situations, however, the retinal image that any object will produce is unknown in advance; its size, for instance, varies dramatically with the objects distance from the observer. Using fMRI, we show that preparatory activity is systematically modulated by expectations derived from scene context. Human participants searched for objects at different distances in scenes. Activity patterns in object-selective cortex during search preparation (while no objects were presented), resembled activity patterns evoked by viewing targets object in isolation. Crucially, this preparatory activity was modulated by distance, reflecting the predicted retinal image of the object at each distance. These findings reconcile current theories of attentional selection with the challenges of real-world vision.

HighlightsO_LIVisual cortex contains object-specific representations during search preparation.
C_LIO_LIWe demonstrate this for the first time during concurrent visual scene processing.
C_LIO_LIPreparatory object representations are scaled to account for viewing distance.
C_LIO_LIPreparatory biases reflect the predicted retinal image inferred from scene context.
C_LI

eTOC blurbAttentional selection is thought to be mediated by target-specific preparatory activity in visual cortex. Gayet and Peelen provide evidence that such preparatory biases incorporate contextual expectations about object appearance, reconciling attention theories with the challenges of naturalistic vision.

sentences: Host preferences of coexisting Perkinsea parasitoids during coastal dinoflagellate blooms Parasites in aquatic systems are highly diverse and ubiquitous. In marine environments, parasite-host interactions contribute substantially to shaping microbial communities, but their nature and complexity remain poorly understood. In this study, we examined the relationship between Perkinsea parasitoids and bloom-forming dinoflagellate species. Our aim was to determine whether parasite-host species interactions are specific and whether the diversity and distribution of parasitoids are shaped by their dinoflagellate hosts. Several locations along the Catalan coast (NW Mediterranean Sea) were sampled during the blooms of five dinoflagellate species and the diversity of Perkinsea was determined by combining cultivation-based methods with metabarcoding of the V4 region of 18S rDNA. Most known species of Parviluciferaceae, and others not yet described, were detected, some of them coexisting in the same coastal location, and with a wide distribution. The specific parasite-host interactions determined for each of the studied blooms demonstrated the host preferences exhibited by parasitoids in nature. The dominance of a species within the parasitoid community is driven by the presence and abundances of its preferred host(s).The absence of parasitoid species, often associated with a low abundance of their preferred hosts, suggested that high infection rates are reached only under conditions that favour parasitoid propagation, especially dinoflagellate blooms.

sentences: Principles of sensor-effector organization in six-transmembrane ion channels Receptor proteins sense stimuli and generate downstream signals via sensor and effector domains. Presently, the structural constraints on sensor-effector organization across receptor protein superfamilies are not clear. Here, we perform statistical coupling analysis (SCA) on the transient receptor potential (TRP) and voltage-gated potassium (Kv) ion channel superfamilies to characterize the networks of coevolving residues, or protein sectors, that mediate their receptor functions. Comparisons to structural and functional studies reveal a conserved "core" sector that extends from the pore and mediates effector functions, including pore gating and sensor-pore coupling, while sensors correspond to family-specific "accessory" sectors and localize according to three principles: Sensors (1) may emerge in any region with access to the core, (2) must maintain contact with the core, and (3) must preserve the integrity of the core. This sensor-core architecture may represent a conserved and generalizable paradigm for the structure-function relationships underlying the evolution of receptor proteins.

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Multi-regional characterisation of renal cell carcinoma and microenvironment at single cell resolution Tumour behaviour is dependent on the oncogenic properties of cancer cells and their multicellular interactions. These dependencies were examined through 270,000 single cell transcriptomes and 100 micro-dissected whole exomes obtained from 12 patients with kidney tumours. Tissue was sampled from multiple regions of tumour core, tumour-normal interface, normal surrounding tissues, and peripheral blood. We found the principal spatial location of CD8+ T cell clonotypes largely defined exhaustion state, with clonotypic heterogeneity not explained by somatic intra-tumoural heterogeneity. De novo mutation calling from single cell RNA sequencing data allows us to lineagetrace and infer clonality of cells. We discovered six meta-programmes that distinguish tumour cell function. An epithelial-mesenchymal transition meta-programme, enriched at the tumour-normal interface appears modulated through macrophage expressed IL1B, potentially forming a therapeutic target.

Single sentence summaryKidney cancer evolution, prognosis and therapy are revealed by a single cell multi-regional study of the microenvironment.

sentences: Individual-Specific Areal-Level Parcellations Improve Functional Connectivity Prediction of Behavior Resting-state functional MRI (rs-fMRI) allows estimation of individual-specific cortical parcellations. We have previously developed a multi-session hierarchical Bayesian model (MS-HBM) for estimating high-quality individual-specific network-level parcellations. Here, we extend the model to estimate individual-specific areal-level parcellations. While network-level parcellations comprise spatially distributed networks spanning the cortex, the consensus is that areal-level parcels should be spatially localized, i.e., should not span multiple lobes. There is disagreement about whether areal-level parcels should be strictly contiguous or comprise multiple non-contiguous components, therefore we considered three areal-level MS-HBM variants spanning these range of possibilities. Individual-specific MS-HBM parcellations estimated using 10min of data generalized better than other approaches using 150min of data to out-of-sample rs-fMRI and task-fMRI from the same individuals. Resting-state functional connectivity (RSFC) derived from MS-HBM parcellations also achieved the best behavioral prediction performance. Among the three MS-HBM variants, the strictly contiguous MS-HBM (cMS-HBM) exhibited the best resting-state homogeneity and most uniform within-parcel task activation. In terms of behavioral prediction, the gradient-infused MS-HBM (gMS-HBM) was numerically the best, but differences among MS-HBM variants were not statistically significant. Overall, these results suggest that areal-level MS-HBMs can capture behaviorally meaningful individual-specific parcellation features beyond group-level parcellations. Multi-resolution trained models and parcellations are publicly available (https://github.com/ThomasYeoLab/CBIG/tree/master/stable_projects/brain_parcellation/Kong2022_ArealMSHBM).

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Identification and comparative analysis of long non-coding RNAs in the brain of fire ant queens in two different reproductive states BackgroundMany long non-coding RNAs (lncRNAs) have been extensively identified in many higher eukaryotic species. The function of lncRNAs has been reported to play important roles in diverse biological processes, including developmental regulation and behavioral plasticity. However, there are no reports of systematic characterization of long non-coding RNAs in the fire ant Solenopsis invicta.

ResultsIn this study, we performed a genome-wide analysis of lncRNAs in the brains of S. invicta from RNA-seq. In total, 1,393 novel lncRNA transcripts were identified in the fire ant. In contrast to the annotated lncRNA transcripts having at least two exons, novel lncRNAs are monoexonic transcripts with a shorter length. Besides, the transcriptome from virgin alate and dealate mated queens were analyzed and compared. The results showed 295 differentially expressed mRNA genes (DEGs) and 65 differentially expressed lncRNA genes (DELs) between virgin and mated queens, of which 17 lncRNAs were highly expressed in the virgin alates and 47 lncRNAs were highly expressed in the mated dealates. By identifying the DEL:DEG pairs with high association in their expression (Spearmans |rho| > 0.8 and p-value < 0.01), many DELs were co-regulated with DEGs after mating. Furthermore, several remarkable lncRNAs (MSTRG.6523, MSTRG.588, and nc909) that were found to associate with particular coding genes may play important roles in the regulation of brain gene expression in reproductive transition in fire ants.

ConclusionThis study provides the first genome-wide identification of S. invicta lncRNAs in the brains in different reproductive states and will contribute to a fuller understanding of the transcriptional regulation underpinning reproductive changes.

sentences: Two pathways drive meiotic chromosome axis assembly in Saccharomyces cerevisiae Successful meiotic recombination, and thus fertility, depends on conserved axis proteins that organize chromosomes into arrays of anchored chromatin loops and provide a protected environment for DNA exchange. Here, we show that the stereotypic chromosomal distribution of axis proteins in S. cerevisiae is the additive result of two independent pathways: a cohesin-dependent pathway, which was previously identified and mediates focal enrichment of axis proteins at gene ends, and a parallel cohesin-independent pathway that recruits axis proteins to broad genomic islands with high gene density. These islands exhibit elevated markers of crossover recombination as well as increased nucleosome density, which we show is a direct consequence of the underlying DNA sequence. A predicted PHD domain in the center of the axis factor Hop1 specifically mediates cohesin-independent axis recruitment. Intriguingly, other chromosome organizers, including cohesin, condensin, and topoisomerases, are differentially depleted from the same regions even in non-meiotic cells, indicating that these DNA sequence-defined chromatin islands exert a general influence on the patterning of chromosome structure.

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Using cGMP analogues to modulate photoreceptor light sensitivity: Perspectives for the treatment of retinal degeneration. Cyclic nucleotide-gated (CNG) channels play an essential role within the phototransduction cascade in vertebrates. Although rod and cone light responses are mediated through similar pathways, each photoreceptor type relies on a specific CNG-channel isoform. In many forms of retinal degeneration, increased cGMP levels initiate a pathophysiological rollercoaster, which starts with an enhanced CNG-channel activation, often in rod photoreceptors. This causes cell death of both rods and cones, and eventually leads to complete blindness. Unfortunately, the targeting of the desired channel isoform still constitutes the bottleneck for many therapeutic schemes.

Here, we present a novel strategy, based on cGMP analogues with opposite types of action, which allows for the selective modulation of either rod or cone photoreceptors. A combined treatment with a weak rod-selective CNG-channel inhibitor (Rp-8-Br-PET-cGMPS) and a cone-selective CNG-channel activator (8-pCPT-cGMP) preserved the normal CNG-channel function at physiological and pathological cGMP levels. The effectiveness of this approach was tested and confirmed in explanted mouse retina. Under physiological conditions, the inhibitor silenced the rods selectively and decreased the dependency of cone responses on light intensity. Remarkably, the activator, when applied together with the inhibitor, reinstated only the light responsiveness of cones. Yet, when applied alone, the activator dampened rod responses more strongly than those of cones.

Hence, combinations of cGMP analogues with desired properties may elegantly address the isoform-specificity problem in future pharmacological therapies. Beyond therapies for retinal degeneration diseases, treatments based on this strategy may allow modulation of visual performance in certain light environments or disease conditions.

One Sentence SummaryA combination of cGMP analogues with opposite types of action can selectively modulate either rod or cone photoreceptor function.

sentences: Translational control of lipogenesis links protein synthesis and phosphoinositide signaling with nuclear division Continuously dividing cells coordinate their growth and division. How fast cells grow in mass determines how fast they will multiply. Yet, there are few, if any, examples of a metabolic pathway that actively drives a cell cycle event instead of just being required for it. Here, we show that translational upregulation of lipogenic enzymes in yeast increased the abundance of lipids and accelerated nuclear elongation and division. De-repressing translation of acetyl CoA carboxylase and fatty acid synthase also suppressed cell cycle-related phenotypes, including delayed nuclear division, associated with Sec14p phosphatidylinositol transfer protein deficiencies, and the irregular nuclear morphologies of mutants defective in phosphatidylinositol 4-OH kinase activities. Our results show that increased lipogenesis drives a critical cell cycle landmark and report a phosphoinositide signaling axis in control of nuclear division. The broad conservation of these lipid metabolic and signaling pathways raises the possibility these activities similarly govern nuclear division in mammals.

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Prevalence of heterotrophic methylmercury detoxifying bacteria across oceanic regions Microbial reduction of inorganic divalent mercury (Hg2+) and methylmercury (MeHg) demethylation is performed by the mer operon, specifically by merA and merB genes respectively, but little is known about the mercury tolerance capacity of marine microorganisms and its prevalence in the global ocean. Here, we explored the distribution of these genes in 290 marine heterotrophic bacteria (Alteromonas and Marinobacter spp.) isolated from different oceanographic regions and depths, and assessed their tolerance to diverse concentrations of Hg2+ and MeHg. About 25% of the isolates presented merA and only 8.9% presented both merAB genes, including the strain ISS312 that exhibited the highest tolerance capacity and a degradation efficiency of 98.2% in 24 h. Fragment recruitment analyses of ISS312 genome against microbial metagenomes indicated an extensive distribution across the global bathypelagic ocean. Our findings highlighted that mercury resistance genes are widely distributed in a non-highly polluted environment such as the pelagic marine environment, and that degradation of the neurotoxic MeHg can be performed through the ocean water column by some heterotrophic bacteria at high efficiency with important implications in the biogeochemical cycle of mercury and potentially for the environment and human health.

TeaserActive mercury resistance genes detected in marine cultured bacteria are widely distributed in the ocean including the bathypelagic zone.

sentences: Quantifying the potential for red blood cell β-adrenergic sodium-proton exchangers to protect oxygen transport in hypoxic and hypercapnic white seabass White seabass (Atractoscion nobilis) are increasingly experiencing periods of low oxygen (O2; hypoxia) and high carbon dioxide (CO2, hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O2 carrier in the blood and in many teleost fishes Hb-O2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically-stimulated sodium-proton-exchangers ({beta}-NHE). We hypothesized that RBC {beta}-NHEs in white seabass are an important mechanism that can protect the blood O2-carrying capacity during hypoxia and hypercapnia. We determined the O2-binding characteristics of white seabass blood, the response of RBCs to adrenergic stimulation, and quantified the protective effect of {beta}-NHE activity on Hb-O2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O2 affinity (PO2 at half-saturation; P50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient -0.92; Root effect 52%). The presence of RBC {beta}-NHEs was confirmed by functional, molecular and bioinformatic data and super-resolution imaging revealed, for the first time, the subcellular location of {beta}-NHE protein in vesicle-like structures and on the RBC membrane, and its translocation after adrenergic stimulation. The activation of RBC {beta}-NHEs increased Hb-O2 saturation by [~]8% in normoxia at 1 kPa PCO2, and by up to 20% in hypoxia. Our results confirm that RBC {beta}-NHE activity in white seabass has great potential to protect arterial O2 transport in environmentally relevant conditions of hypoxia and hypercapnia, but also reveal a potential vulnerability of fish to combinations of these stressors.

sentences: The Endoplasmic Reticulum pool of Bcl-xL dampens the Unfolded Protein Response through IP3R-dependent Calcium Release Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localization and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localization is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we showed that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs demonstrated a reduced expression of the Unfolded Protein Response (UPR) markers upon ER stress and displayed reduced inositol trisphosphate receptor (IP3R)-mediated ER calcium release. Collectively, our data show that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the ER, apart from its cliche regulation of apoptosis.

sentences: A sex-specific evolutionary interaction between ADCY9 and CETP Pharmacogenomic studies have revealed associations between rs1967309 in the adenylyl cyclase type 9 (ADCY9) gene and clinical responses to the cholesteryl ester transfer protein (CETP) modulator dalcetrapib, however, the mechanism behind this interaction is still unknown. Here, we characterized selective signals at the locus associated with the pharmacogenomic response in human populations and we show that rs1967309 region exhibits signatures of positive selection in several human populations. Furthermore, we identified a variant in CETP, rs158477, which is in long-range linkage disequilibrium with rs1967309 in the Peruvian population. The signal is mainly seen in males, a sex-specific result that is replicated in the LIMAA cohort of over 3,400 Peruvians. Analyses of RNA-seq data further suggest an epistatic interaction on CETP expression levels between the two SNPs in multiple tissues, which also differs between males and females. We also detected interaction effects of the two SNPs with sex on cardiovascular phenotypes in the UK Biobank, in line with the sex-specific genotype associations found in Peruvians at these loci. We propose that ADCY9 and CETP coevolved during recent human evolution due to sex-specific selection, which points towards a biological link between dalcetrapibs pharmacogene ADCY9 and its therapeutic target CETP.

sentences: Transient demographic approaches can drastically expand the toolbox of coral reef science Coral communities are threatened by an increasing plethora of abiotic and biotic disturbances. Preventing the ensuing loss of coral coverage and diversity calls for a mechanistic understanding of resilience across coral species and populations that is currently lacking in coral reef science. Assessments into the dynamics of coral populations typically focus on their long-term (i.e. asymptotic) characteristics, tacitly assuming stable environments in which populations can attain their long-term characteristics. Instead, we argue that greater focus is needed on investigating the transient (i.e. short-term) dynamics of coral populations to describe and predict their characteristics and trajectories within unstable environments. Applying transient demographic approaches to the evaluation and forecasting of the responses of coral populations to disturbance holds promise for expediting our capacity to predict and manage the resilience of coral populations, species, and communities.

sentences: Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

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vcf2gwas - Python API for comprehensive GWAS analysis using GEMMA We present a new software package vcf2gwas to perform reproducible genome-wide association studies (GWAS). vcf2gwas is a Python API for bcftools, PLINK and GEMMA. Before running the analysis a traditional GWAS workflow requires the user to edit and format the genotype information from commonly used Variant Call Format (VCF) file and phenotype information. Post-processing steps involve summarizing and visualizing the analysis results. This workflow requires a user to utilize the command-line, manual text-editing and knowledge of one or more programming/scripting languages which can be time-consuming especially when analyzing multiple phenotypes. Our package provides a convenient pipeline performing all of these steps, reducing the GWAS workflow to a single command-line input without the need to edit or format the VCF file beforehand or to install any additional software. In addition, features like reducing the dimensionality of the phenotypic space and performing analyses on the reduced dimensions or comparing the significant variants from the results to specific genes/regions of interest are implemented. By integrating different tools to perform GWAS under one workflow, the package ensures reproducible GWAS while reducing the user efforts significantly.

Availability and implementationThe source code of vcf2gwas is available under the GNU General Public License. The package can be easily installed using conda. Installation instructions and a manual including tutorials can be accessed on the package website at https://github.com/frankvogt/vcf2gwas

sentences: A Genome-Scale Metabolic Model of Marine Heterotroph Vibrio splendidus sp. 1A01 While the Vibrio splendidus species is best known as an opportunistic pathogen in oysters, the Vibrio splendidus sp. 1A01 strain was first identified as an early colonizer of synthetic chitin particles incubated in seawater. To gain a better understanding of its metabolism, a genome-scale metabolic model (GSMM) of V. splendidus sp. 1A01 was reconstructed. GSMMs enable us to simulate all metabolic reactions in a bacterial cell using Flux Balance Analysis. A draft model was built using an automated pipeline from BioCyc. Manual curation was then performed based on experimental data, in part by gap-filling metabolic pathways and tailoring the models biomass reaction to V. splendidus sp. 1A01. The challenges of building a metabolic model for a marine microorganism like V. splendidus sp. 1A01 are described.

sentences: Genomic features of Mycobacterium avium subsp. hominissuis isolated from pigs in Japan. Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in humans and pigs. Genome analysis on MAH of human isolates has been proceeding, however, those of pigs are limited despite its potential source of infection to human. In the current study, we obtained 30 draft genome sequences of MAH of pigs reared in Japan. The 30 draft genomes consisted of 4,848,678 - 5,620,788 bp length, 4,652 - 5,388 coding genes and 46 - 75 (Med: 47) tRNAs. All isolates had restriction modification associated genes and 185 - 222 predicted virulence genes. Two isolates had tRNA arrays and one isolate had a clustered regularly interspaced short palindromic repeat (CRISPR) region. Our results will be useful for evaluation of the ecology of MAH by providing a foundation for genome-based epidemiological studies.

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Histone neutralization in a rat model of acute lung injury induced by double-hit lipopolysaccharide BackgroundAcute respiratory distress syndrome (ARDS) remains a challenge because of its high morbidity and mortality. Circulation histones levels in ARDS patients were correlated to disease severity and mortality. This study examined the impact of histone neutralization in a rat model of acute lung injury (ALI) induced by a lipopolysaccharide (LPS) double-hit.

MethodsSixty-eight male Sprague-Dawley rats were randomized to sham (N=8, received saline only) or LPS (N=60). The LPS double-hit consisted of a 0.8 mg/kg intraperitoneal injection followed after 16 hours by 5 mg/kg intra-tracheal nebulized LPS. The LPS group was then randomized into five groups: LPS only (N=12); LPS + 5, 25, or 100 mg/kg intravenous STC3141 every 8 hours (LPS+L, LPS+M, LPS+H, respectively, each N=12); or LPS + intraperitoneal dexamethasone 2.5 mg/kg every 24 hours for 56 hours (LPS+D, N=12) The animals were observed for 72 hours.

ResultsLPS animals developed ALI as suggested by lower oxygenation, lung edema formation, and histological changes compared to the sham animals. Compared to the LPS group, LPS+H and +D animals had significantly lower circulating histone levels; only the LPS+D group had significantly lower bronchoalveolar lavage fluid (BALF) histone concentrations. The LPS+L, +M, +H and +D groups had improved oxygenation compared to the LPS group and the LPS+H and +D groups had a lower lung wet-to-dry ratio. All animals survived.

ConclusionNeutralization of histone using STC3141, especially at high dose, had similar therapeutic effects to dexamethasone in this LPS double-hit rat ALI model, with significantly decreased circulating histone concentration, improved oxygenation, and decreased lung edema formation.

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Clock time: a foreign measure to brain dynamics Human thought is highly flexible and dynamic, achieved by evolving patterns of brain activity across groups of cells. Neuroscience aims to understand cognition in the brain by analysing these intricate patterns. Here, we argue that this goal is impeded by the time format of our data - clock time. The brain is a system with its own dynamics and regime of time, with no intrinsic concern for the human-invented second. A more appropriate time format is cycles of brain oscillations, which coordinate neural firing and are widely implicated in cognition. These brain dynamics do not obey clock time - they start out of tune with clock time and drift apart even further as oscillations unpredictably slow down, speed up, and undergo abrupt changes. Since oscillations clock cognition, their dynamics should critically inform our analysis. We describe brain time warping as a new method to transform data in accordance with brain dynamics, which sets the time axis to cycles of clocking oscillations (a native unit) rather than milliseconds (a foreign unit). We also introduce the Brain Time Toolbox, a software library that implements brain time warping for electrophysiology data and tests whether it reveals information patterns in line with how the brain uses them.

Glossary

O_TBL View this table:
org.highwire.dtl.DTLVardef@d81c2org.highwire.dtl.DTLVardef@c1ff2corg.highwire.dtl.DTLVardef@57dfa4org.highwire.dtl.DTLVardef@11b8c43org.highwire.dtl.DTLVardef@195a419_HPS_FORMAT_FIGEXP  M_TBL C_TBL

sentences: Acute vagus nerve stimulation does not affect liking or wanting ratings of food in healthy participants The vagus nerve plays a vital role in the regulation of food intake and vagal afferent signals may help regulate food cue reactivity by providing negative homeostatic feedback. Despite strong evidence from preclinical studies on vagal afferent "satiety" signals in guiding food intake, evidence from human studies is largely inconclusive to date. Here, we investigated the acute effects of left or right transcutaneous auricular vagus nerve stimulation (taVNS) on subjective ratings of wanting and liking of various food and non-food items in 82 healthy participants (46 women, MBMI = 23.1 kg/m2). In contrast to previous reports in patients with depression, we found moderate to anecdotal evidence supporting the absence of taVNS-induced changes in food ratings. To test whether the absence of taVNS effects on food ratings is due to heterogeneity in the sample, we conducted post hoc subgroup analyses by splitting the data according to stimulation side and sex (between-subject factors) as well as caloric density, perceived healthiness, and flavor (sweet vs. savory) of the food (within-subject factors). This multiverse analysis largely supported the absence of taVNS-induced changes since the strongest subgroup effects provided only anecdotal evidence in favor of taVNS-induced changes. We conclude that acute taVNS only has a marginal effect on subjective ratings of food, suggesting that it is an unlikely mechanism for the reported long-term effects of VNS on body weight. In light of an absence of acute taVNS effects on food craving, our results call for future research on the correspondence between acute and chronic effects of vagal afferent stimulation.

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Hyperdirect connectivity of opercular speech network to the subthalamic nucleus The importance of the basal ganglia in modulating cognitive and motor behaviors is well known, yet how the basal ganglia participate in the uniquely human behavior of speech is poorly understood. The subthalamic nucleus (STN) is well positioned to facilitate two basal ganglia functions critical for speech: motor learning and gain modulation. Using a novel paradigm to study cortical-subcortical interactions during speech in patients undergoing awake DBS surgery, we found evidence for a left opercular hyperdirect pathway in humans by stimulating in the STN and examining antidromic evoked activity in the left temporal, parietal and frontal opercular cortex. These high resolution cortical and subcortical mapping data provided evidence for hyperdirect connectivity between Brocas area (typically corresponding to pars triangularis and pars opercularis of the inferior frontal gyrus) and the STN. In addition, we observed evoked potentials consistent with the presence of monosynaptic projections from areas of opercular speech cortex that are primarily sensory, including auditory cortex, to the STN. These connections may be unique to humans, evolving alongside the ability for speech.

SIGNIFICANCEUsing intracranial recordings from the basal ganglia and cortex in subjects undergoing deep brain stimulation, this study provides evidence for monosynaptic cortical inputs from motor planning, motor sensory, and auditory sensory cortices to the subthalamic nucleus. These observations suggest that in humans, the cortical-basal ganglia hyperdirect pathway is uniquely positioned to participate in speech production. Moreover, the existence of a monosynaptic connection between human sensory cortical areas and the subthalamic nucleus indicates a need to update traditional models of information transfer within cortical-basal ganglia-thalamocortical circuitry, which has significant implications for understanding other human cognitive behavior or dysfunction.

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Enhancing Quantitative and Data Science Education for Graduate Students in Biomedical Science Substantial guidance is available on undergraduate quantitative training for biologists, including reports focused on biomedical science, but far less attention has been paid to the graduate curriculum. In this setting, we propose an innovative approach to quantitative education that goes beyond recommendations of a course or set of courses or activities. Due to the diversity of quantitative methods, it is infeasible to expect that biomedical PhD students can be exposed to more than a minority of the quantitative concepts and techniques employed in modern biology. We developed a novel prioritization approach in which we mined and analyzed quantitative concepts and skills from publications that faculty in relevant units deemed central to the scientific comprehension of their field. The analysis provides a prioritization of quantitative skills and concepts and could represent an effective method to drive curricular focus based upon program-specific faculty input for biological science programs of all types. Our results highlight the disconnect between typical undergraduate quantitative education for life science students, focused on continuous mathematics, and the concepts and skills in graphics, statistics, and discrete mathematics that arise from priorities established by biomedical science faculty.

One Sentence SummaryWe developed a novel approach to prioritize quantitative concepts and methods for inclusion in a graduate biomedical science curriculum based upon approaches included in faculty-identified key publications.

sentences: Stress Evaluation of Various Breeding Environments in Mouse Laboratory animals are raised in a fixed space during the study period and are environmentally bound. Laboratory animal may be under stress on the constrained environment, which changes physiological indicators, affecting the reproducibility and accuracy of animal study. Therefore, reducing animal stress by providing proper breeding environment and environmental enrichment can be the basis for animal study. In this study, the stress level was assessed according to the mouse breeding environment. According to the results of the experiment, it was determined that the individual ventilation cage had less cortisol concentration in serum and body weight increased in the individual ventilation cage than individual isolated cage, when providing environmental enrichment rather than group breeding or not providing environmental enrichment. The results will provide appropriate guidelines for laboratory animal welfare.

sentences: Large-scale geographic survey provides insights into the colonization history of a major aphid pest on its cultivated apple host in Europe, North America and North Africa AO_SCPLOWBSTRACTC_SCPLOWWith frequent host shifts involving the colonization of new hosts across large geographical ranges, crop pests are good models for examining the mechanisms of rapid colonization. The microbial partners of pest insects may also be involved in or affected by colonization processes, which has been little studied so far. We investigated the demographic history of the rosy apple aphid, Dysaphis plantaginea, a major pest of the cultivated apple (Malus domestica) in Europe, North Africa and North America, as well as the diversity of its microbiota. We genotyped a comprehensive sample of 714 colonies from Europe, Morocco and the US using mitochondrial (CytB and CO1), bacterial (16s rRNA and TrnpB), and 30 microsatellite markers. We detected five populations spread across the US, Morocco, Western and Eastern Europe and Spain. Populations showed weak genetic differentiation and high genetic diversity, except the ones from Morocco and North America that are likely the result of recent colonization events. Coalescent-based inferences revealed high levels of gene flow among populations during the colonization but did not allow determining the sequence of colonization of Europe, North America and Morroco by D. plantaginea, likely because of the weak genetic differentiation and the occurrence of gene flow among populations. We found that D. plantaginea rarely hosts other endosymbiotic bacteria than its obligate nutritional symbiont Buchnera aphidicola. This suggests that secondary endosymbionts did not play an important role in the rapid spread of the rosy apple aphid. These findings have fundamental importance for understanding pest colonization processes and implications for sustainable pest control programs.

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Biochemical and subcellular characterization of a squid hnRNPA/B-like protein in osmotic stress activated cells reflects molecular properties conserved in this protein family In previous works, we characterized a novel, strongly basic, squid hnRNPA/B-like Protein 2 in presynaptic terminals of squid neurons. Here, we show that squid hnRNPA/B-like Protein 2 are exclusively nuclear localization and relocated to cytoplasmic granules containing hnRNPA1 and Poly-A binding protein-1 (PABP-1) when the cells are treated with sorbitol. Also, we show an interaction of hnRNPA/B like Protein 2 with squid RNA, its interfered with dynamic of formation of hnRNPA/B like Protein 2 dimers, whereas possibly involved disulfide bounds and postranslations modification in a distinct stage of dimers formation. An understanding of the molecular and biochemical mechanisms involved in the stability of the dimeric form, and the regulation of the transition between monomeric and dimeric forms may bring insights into evolution of several neurodegenerative diseases.

HighlightsO_LIWe identified monomeric (p37) and dimeric (p65) forms of squid hnRNPA/B-like Protein 2 in squid optic lobes
C_LIO_LIOur data indicate a conserved structure and cellular properties of squid hnRNPA/B-like Protein 2 and human hnRNPA1 protein colocalizing with PABP into stress granules (SGs)
C_LIO_LIThe stability of hnRNPA/B-like dimers involved the squid RNAs and disulfide bonds to promote higher SDS-stable dimers formation
C_LIO_LIAn understanding of the transition between monomeric and dimeric forms of squid hnRNPA/B-like Protein 2 may give clues to misfolding processes in neuropathologies
C_LI

sentences: Naturally enhanced neutralizing breadth to SARS-CoV-2 after one year Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naive individuals2,5-8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

sentences: Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype. There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations upregulated several anterior HOX genes and downregulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment upregulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

sentences: Woody species do not differ in dormancy progression: differences in time to budbreak due to forcing and cold hardiness Budbreak is one of the most observed and studied phenological phases in perennial plants. Two dimensions of exposure to temperature are generally used to model budbreak: accumulation of time spent at low temperatures (chilling); and accumulation of heat units (forcing). These two effects have a well-established negative correlation: the more chilling, the less forcing required for budbreak. Furthermore, temperate plant species are assumed to vary in amount of chilling required to complete endodormancy and begin the transition to breaking bud. Still, prediction of budbreak remains a challenge. The present work demonstrates across a wide range of species how bud cold hardiness must be accounted for to study dormancy and accurately predict time to budbreak. Cold hardiness defines the path length to budbreak, meaning the difference between the cold hardiness buds attain during the winter, and the cold hardiness at which deacclimated buds are predicted to open. This distance varies among species and throughout winter within a species. Increases in rate of cold hardiness loss (deacclimation) measured throughout winter show that chilling controls deacclimation potential - the proportion of the maximum rate response attained at high chill accumulation - which has a sigmoid relationship to chilling accumulation. For forcing, rates of deacclimation increase non-linearly in response to temperature. Comparisons of deacclimation potential show a dormancy progresses similarly for all species. This observation suggests that comparisons of physiologic and genetic control of dormancy requires an understanding of cold hardiness dynamics and the necessity for an update of the framework for studying dormancy and its effects on spring phenology.

sentences: Coherent mapping of position and head direction across auditory and visual cortex Neurons in primary visual cortex (V1) may not only signal current visual input but also relevant contextual information such as reward expectancy and the subjects spatial position. Such location-specific representations need not be restricted to V1 but could participate in a coherent mapping throughout sensory cortices. Here we show that spiking activity in primary auditory cortex (A1) and lateral, secondary visual cortex (V2L) of freely moving rats coherently represents a location-specific mapping in a sensory detection task performed on a figure-8 maze. Single-unit activity of both areas showed extensive similarities in terms of spatial distribution, reliability and position coding. Importantly, reconstructions of subject position on the basis of spiking activity displayed decoding errors that were correlated between areas in magnitude and direction. In addition to position, we found that head direction, but not locomotor speed or head angular velocity, was an important determinant of activity in A1 and V2L. Finally, pairs of units within and across areas showed significant correlations in instantaneous variability of firing rates (noise correlations). These were dependent on the spatial tuning of cells as well as the spatial position of the animal. We conclude that sensory cortices participate in coherent, multimodal representations of the subjects sensory-specific location. These may provide a common reference frame for distributed cortical sensory and motor processes and may support crossmodal predictive processing.

sentences: Embryonic valproate exposure alters mesencephalic dopaminergic neurons distribution and septal dopaminergic gene expression in domestic chicks In recent years, the role of the dopaminergic system in the regulation of social behavior is being progressively outlined, and dysfunctions of the dopaminergic system are increasingly associated with neurodevelopmental disorders, including ASD. To further elucidate the role of the dopaminergic system in ASD, we investigated the effects of embryonic exposure to valproic acid (VPA) on the postnatal development of the mesencephalic DA system in the domestic chick. We found that VPA affected the rostro-caudal distribution of DA neurons, without changing the expression levels of several dopaminergic markers in the mesencephalon. We also investigated a potential consequence of this altered DA neuronal distribution in the septum, a social brain area previously associated to social behaviour in several vertebrate species, describing alterations in the expression of genes linked to DA neurotransmission. These findings support the emerging hypothesis of a role of DA dysfunction in ASD pathogenesis. Together with previous studies showing impairments of early social orienting behaviour, these data also support the use of the domestic chick model to investigate the neurobiological mechanisms involved in early ASD symptoms.

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Measuring arterial pulsatility with Dynamic Inflow MAgnitude Contrast (DIMAC) The pulsatility of blood flow through cerebral arteries is clinically important, as it is intrinsically associated with cerebrovascular health. In this study we outline a new MRI approach to measuring the real-time pulsatile flow in cerebral arteries, which is based on the inflow phenomenon associated with fast gradient-recalled-echo acquisitions. Unlike traditional phase-contrast techniques, this new method, which we dub Dynamic Inflow Magnitude Contrast (DIMAC), does not require velocity-encoding gradients as sensitivity to flow velocity is derived purely from the inflow effect. We achieved this using a highly accelerated single slice EPI acquisition with a very short TR (15 ms) and a 90{degrees} flip angle, thus maximizing inflow contrast. We simulate the spoiled GRE signal in the presence of large arteries and perform a sensitivity analysis to demonstrate that in the regime of high inflow contrast it shows much greater sensitivity to flow velocity over blood volume changes. We support this theoretical prediction with in-vivo data collected in two separate experiments designed to demonstrate the utility of the DIMAC signal contrast. We perform a hypercapnia challenge experiment in order to experimentally modulate arterial tone within subjects, and thus modulate the arterial pulsatile flow waveform. We also perform a thigh-cuff release challenge, designed to induce a transient drop in blood pressure, and demonstrate that the continuous DIMAC signal captures the complex transient change in the pulsatile and non-pulsatile components of flow. In summary, this study proposes a new role for a well-established source of MR image contrast and demonstrates its potential for measuring both steady-state and dynamic changes in arterial tone.

HighlightsO_LIWe present a novel method for measuring pulsatility of cerebral arteries.
C_LIO_LIThe inflow effect on fast GRE imaging can be exploited to yield a flow velocity dependent signal.
C_LIO_LIWe measure pulsatile flow through cerebral arteries dynamically on a beat-to-beat basis.
C_LIO_LIWe use physiological challenges to demonstrate sensitivity to dynamic and steady-state changes in vascular tone.
C_LI

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Regulation of invasion-associated actin dynamics by the Chlamydia trachomatis effectors TarP and TmeA The obligate intracellular pathogen Chlamydia trachomatis manipulates the host actin cytoskeleton to assemble actin-rich structures that drive pathogen entry. The recent discovery of TmeA, which like TarP is an invasion-associated type III effector implicated in actin remodeling, raised questions regarding the nature of their functional interaction. Quantitative live-cell imaging of actin remodeling at invasion sites revealed differences in recruitment and turnover kinetics associated with TarP and TmeA pathways, with the former accounting for most of the robust actin dynamics at invasion sites. TarP-mediated recruitment of the actin nucleators formin and the Arp2/3 complex were crucial for rapid actin kinetics, generating a collaborative positive feedback loop that enhanced their respective actin-nucleating activities within invasion sites. In contrast, Fmn1 is neither recruited to invasion sites nor collaborates with Arp2/3 within the context of TmeA-associated actin recruitment. While the TarP-Fmn1-Arp2/3 signaling axis is responsible for the majority of actin dynamics, its inhibition had similar effects as deletion of TmeA on invasion efficiency, consistent with the proposed model that TarP and TmeA acting on different stages of the same invasion pathway.

Summary StatementKinetic analysis of actin recruitment during C. trachomatis invasion reveals TarP as the major contributor relative to TmeA, via its ability to facilitate collaboration between actin nucleators Formin 1 and Arp2/3.

sentences: Wnt-dependent activation of ERK mediates repression of chondrocyte fate during calvarial development. Wnt signaling regulates cell fate decisions in diverse contexts during development, and loss of Wnt signaling in the cranial mesenchyme results in a robust and binary cell fate switch from cranial bone to ectopic cartilage. The Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and Wnt signaling pathways are activated during calvarial osteoblast cell fate selection. Here, we test the hypothesis that ERK signaling is a mediator of Wnt-dependent cell fate decisions in the cranial mesenchyme. First, we show that loss of Erk1/2 in the cranial mesenchyme results in a diminished domain of osteoblast marker expression and increased expression of cartilage fate markers and ectopic cartilage formation in the frontal bone primordia. Second, we show that mesenchyme Wnt/{beta}-catenin signaling and Wntless are required for ERK activation in calvarial osteoblasts. Third, we demonstrate that Wnt and ERK signaling pathways function together to repress Sox9 expression in mouse cranial mesenchyme. Our results demonstrate a link between the Wnt and ERK signaling pathways in regulating lineage selection in a subset of calvarial cells and provide new insights into Wnt-dependent cell fate decisions.

sentences: RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity The RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA, RecBCD a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks and also facilitating decoration of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA and the RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA crosslinker mitomycin C, we induce DNA damage and quantify the resulting changes in stoichiometry, copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA filamental stoichiometries equivalent to several hundred molecules that act largely in RecA tetramers before DNA damage, but switch to approximately hexameric subunits when mature filaments are formed. Unexpectedly, we find that the physiologically predominant form of RecB is a dimer.

sentences: REVERSE: A user-friendly web server for analyzing next-generation sequencing data from in vitro selection/evolution experiments Next-generation sequencing (NGS) enables the identification of functional nucleic acid sequences from in vitro selection/evolution experiments and illuminates the evolutionary process at single nucleotide resolution. However, analyzing the vast output from NGS can be daunting, especially with limited programming skills. No single platform exists that performs all the steps necessary to generate publishable results starting with raw sequence data. We developed REVERSE (Rapid EValuation of Experimental RNA Selection/Evolution) (https://www.reverseserver.org/), a web server that incorporates an integrated computational pipeline through a graphical user interface, which performs both pre-processing and sequence level analyses within minutes. FASTQ files from multiple rounds are quality filtered, dereplicated, and trimmed before being analyzed by two pipelines. The first pipeline counts, sorts, and tracks enrichment of unique sequences and tracks the enrichment of sequence motifs. It also identifies mutational intermediates present in the sequence data that connect two input sequences. The second pipeline sorts similar sequences into clusters and tracks enrichment of peak sequences. It also performs nucleotide conservation analysis on the cluster of choice. Both pipelines generate downloadable high-resolution figures. Collectively, REVERSE is a one stop-solution for the rapid analysis of NGS data obtained from in vitro selection/evolution experiments that obviates the need for computational expertise.

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Conformational entropy limits the transition from nucleation to elongation in amyloid aggregation The formation of {beta}-sheet rich amyloid fibrils in Alzheimers disease and other neurodegenerative disorders is limited by a slow nucleation event. To understand the initial formation of {beta}-sheets from disordered peptides, we used all-atom simulations to parameterize a lattice model that treats each amino acid as a binary variable with {beta} and non-{beta} states. We show that translational and conformational entropy give the nascent {beta}-sheet an anisotropic surface tension which can be used to describe the nucleus with two-dimensional Classical Nucleation Theory. Since translational entropy depends on concentration, the aspect ratio of the critical {beta}-sheet changes with protein concentration. Our model explains the transition from the nucleation phase to elongation as the point where the {beta}-sheet core becomes large enough to overcome the conformational entropy cost to straighten the terminal molecule. At this point the {beta}-strands in the nucleus spontaneously elongate, which results in a larger binding surface to capture new molecules. These results suggest that nucleation is relatively insensitive to sequence differences in co-aggregation experiments because the nucleus only involves a small portion of the peptide.

SIGNIFICANCEThe conversion of soluble proteins to amyloid aggregates is associated with many neurodegenerative diseases. Experiments have shown that this conversion occurs by a slow nucleation step followed by rapid growth. This work identifies the principle contributions to the free energy barrier that separates these two stages. It also shows how factors like protein concentration, sidechain interactions, and interactions with the environment can modify the barrier and affect nucleation times.

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Disorder in Ca2+ Release Unit Locations Confers Robustness but Cuts Flexibility of Heart Pacemaking Excitation-contraction coupling kinetics are dictated by the rate and rhythm of the excitations generated by sinoatrial-nodal cells. These cells generate local Ca releases (LCRs) that activate Na/Ca exchanger current, which accelerates diastolic depolarization and determines the rate and rhythm of the excitations. The LCRs are generated by clusters of ryanodine receptors, Ca release units (CRUs), residing in the sarcoplasmic reticulum. While the spatial CRU distribution in pacemaker cells exhibits substantial heterogeneity, it remains unknown if it has any functional importance. Using numerical modeling, here we showed that with a square lattice distribution of CRUs, Ca-induced-Ca-release propagation during diastolic depolarization is insufficient for pacemaking within a broad lower range of realistic ICaL densities. Allowing each CRU to deviate from its original lattice position fundamentally changes the model behavior: during diastolic depolarization sparks propagate, forming LCRs observed experimentally. As disorder in the CRU positions increases, the CRU distribution exhibits larger empty spaces but simultaneously CRU clusters, as in Poisson clumping. Propagating within the clusters, Ca release becomes synchronized, increasing AP firing rate and reviving pacemaker function within lower ICaL densities at which cells with lattice CRU distribution were dormant/non-firing. However, cells with fully disordered CRU positions cannot reach low firing rates and their {beta}-adrenergic receptor stimulation effect was substantially decreased. Thus, order/disorder in CRU locations regulates Ca release propagation and could be harnessed by pacemaker cells to regulate their function. Excessive disorder is expected to limit heart rate range that may contribute to heart rate range decline with age and in disease.

SummaryThe present numerical modeling study shows that disorder in locations of Ca release units in cardiac pacemaker cells has substantial functional impact by creating release clusters, similar to Poisson clumping, and opportunity of Ca release to propagate within the clusters.

sentences: Establishing regulated unconventional secretion for production of heterologous proteins Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream pro-cessing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. Here, we are first to develop regulatory systems for unconventional protein secretion. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

sentences: In silico analysis for potential proteins and microRNAs in Glioblastoma and Parkinsonism. In todays world, neurodegenerative diseases such as Alzheimers disease, Parkinsons Disease, Huntingtons Disease as well as brain cancers such as astrocytomas, ependymomas, glioblastomas have become a great threat to us. In this study, we are trying to find a probable molecular connection associated with two very much different diseases, Glioblastoma, also known as Glioblastoma Multiforme (cancers of microglial cells of our brain) and Parkinsons disease. We at first downloaded the microarray datasets of these two diseases from Gene Expression Omnibus (GEO) and then analyzed them by the GEO2R tool. After analysis, we found 249 common upregulated differential expressed genes and 135 common downregulated differential expressed genes of these two diseases. Therefore the common differentially expressed genes, both upregulated and downregulated, were imported into STRING online tool to find out the protein-protein interactions. Now, this whole network was subjected to Cytoscape and the top ten hub genes were found by Cyto-Hubba plug-in. The top then hub genes are EGFR, CCNB1, CDK1, CCNA2, CHEK1, RAD51, MAD2L1, KIF20A, BUB1, and CCNB2. These all genes are upregulated in both diseases. To find out the biological processes, molecular functions, cellular components, and pathways associated with these hub genes Enrichr online software was used. We used miRNet software to determine the interactions of hub genes with microRNAs. This study will be useful in the future for drug targets discovery for these diseases.

sentences: A combinatorial anticancer drug screen identifies off-target effects of epigenetic chemical probes Anticancer drug response is determined by genetic and epigenetic mechanisms. To identify the epigenetic regulators of anticancer drug response, we conducted a chemical epigenetics screen using chemical probes that target different epigenetic modulators. In this screen, we tested 31 epigenetic probes in combination with 14 mechanistically diverse anticancer agents and identified 8 epigenetic probes that significantly potentiate the cytotoxicity of TAK-243, a first-in-class ubiquitin-activating enzyme (UBA1) inhibitor evaluated in several solid and hematologic malignancies. These probes are TP-472, GSK-864, A-196, UNC1999, SGC-CBP30 and PFI-4 (and its related analogs GSK6853 and GSK5959), and they target BRD9/7, mutant IDH1, SUV420H1/2, EZH2/1, p300/CBP and BRPF1B, respectively. In contrast to epigenetic probes, negative control compounds did not have a significant impact on TAK-243 cytotoxicity. Potentiation of TAK-243 cytotoxicity was associated with reduced ubiquitylation and induction of apoptosis. Mechanistically, epigenetic probes exerted their potentiation by inhibiting the efflux transporter ABCG2 without inducing significant changes in the ubiquitylation pathways or ABCG2 expression levels. The identified probes shared chemical scaffold similarities with TAK-243 and could potentially interact with ABCG2 as assessed by docking analysis. Based on these data, we have developed a cell-based assay that can quantitatively evaluate ABCG2 inhibition by drug candidates. In conclusion, our study identifies epigenetic chemical probes that profoundly potentiate TAK-243 cytotoxicity through off-target ABCG2 inhibition. We also provide experimental evidence that several negative control compounds cannot exclude a subset of off-target effects of chemical probes. Finally, potentiation of TAK-243 cytotoxicity can serve as a quantitative measure of ABCG2-inhibitory activity.

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RABL2 Regulates Ciliation via Controlling IFT-B1 Basal Body Recruitment and ARL3-mediated BBSome Ciliary Retrieval Highly conserved intraflagellar transport (IFT) trains and certain small GTPases coordinate to direct ciliation and to maintain the ciliary dynamics of signaling molecules via the IFT cargo adaptor BBSome. Unlike murine Rab-like 2 (RABL2) GTPase that enters cilia to drive outward transition zone (TZ) passage of the BBSome, human orthologue fails to enter cilia but resides at the ciliary base, indispensable for ciliation. However, mechanisms underlying how RABL2 regulates ciliation and BBSome barrier passage remain elusive. Here, we show that Chlamydomonas RABL2 regulates basal body targeting of the IFT-B1 subcomplex component of IFT trains as a RABL2-specific effector, mediating ciliation via controlling IFT-B1 basal body amount available for assembling anterograde IFT trains. RABL2GTP binds IFT-B1 to perform IFT; sheds from retrograde IFT trains at the proximal ciliary region right above the TZ; and converts to RABL2GDP rapidly. Next, RABL2GDP activates the ciliary membrane anchored Arf-like 3 (ARL3) GTPase (ARL3GDP) as a ARL3-specific guanine nucleotide exchange factor. Upon detaching from the ciliary membrane, the active ARL3GTP recruits its BBSome effector, autonomous of retrograde IFT train association, to move cross the TZ for ciliary retrieval. This ensures proper BBSome ciliary turnover for maintaining phototactic response of Chlamydomonas cells. For finishing RABL2 ciliary cycle, RABL2GDP passes the TZ for ciliary retrieval by loading onto the ARL3GTP/BBSome as a BBSome cargo. Our data thus propose that RABL2 mediates ciliation and BBSome ciliary retrieval simultaneously but via distinct molecular pathways.

Significance statementIntraflagellar transport (IFT) and its cargo adaptor BBSome are indispensable for ciliation and ciliary singling. Rab-like 2 (RABL2) GTPase mediates ciliation and outward transition zone (TZ) passage of BBSomes with mechanisms yet to be determined. Here, we report that RABL2 decides ciliation by controlling the basal body amount of its effector IFT-B1 available for the assembly of anterograde IFT trains. RABL2GTP cycles through cilia as an IFT-B1 cargo; sheds from IFT at the ciliary base; and undergoes nucleotide exchange for activating ARL3 as an ARL3-specfic guanine nucleotide exchange factor. ARLGTP recruits IFT-shed BBSomes to pass the TZ for ciliary retrieval. RABL2GDP exists cilia via ARL3GTP/BBSome as a BBSome cargo. Therefore, RABL2 functions both outside and inside cilia for initiating IFT and BBSome ciliary retrieval, respectively.

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Changes in antennal gene expression underlying sensory system maturation in Rhodnius prolixus BackgroundTriatomine bugs are the blood feeding insect vectors transmitting Chagas disease to humans, a neglected tropical disease that affects over 8 million people, mainly in Latin America. The behavioral responses to host cues and bug signals in Rhodnius prolixus are state dependent, i.e., they vary as a function of post-ecdysis age. At the molecular level, these changes in behavior are probably due to a modulation of peripheral and central processes. In the present study, we report a significant modulation of the expression of a large set of sensory-related genes. Results were generated by means of antennal transcriptomes of 5th instar larvae along the first week (days 0, 2, 4, 6 and 8) after ecdysis sequenced using the Illumina platform.

ResultsAge induced significant changes in transcript abundance were established in more than 6,120 genes (54,7 % of 11,186 genes expressed) in the R. prolixus antenna. This was especially true between the first two days after ecdysis when more than 2,500 genes had their expression significantly altered. In contrast, expression profiles were almost identical between day 6 and 8, with only a few genes showing significant modulation of their expression. A total of 86 sensory receptors, odorant carriers and odorant degrading enzymes were significantly modulated across age points and clustered into three distinct expression profiles.

ConclusionsThe set of sensory genes whose expression increased with age (profile 3) may include candidates underlying the increased responsiveness to host cues shown by R. prolixus during the first days after molting. For the first time, we describe the maturation process undergone at the molecular level by the peripheral sensory system is described in an hemimetabolous insect.

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Neural correlates of individual differences in speech-in-noise performance in a large cohort of cochlear implant users ObjectivesUnderstanding speech in noise (SiN) is a complex task that recruits multiple cortical subsystems. Individuals vary in their ability to understand SiN. This cannot be explained by simple peripheral hearing profiles, but recent work by our group (Kim et al., 2021, Neuroimage) highlighted central neural factors underlying the variance in SiN ability in normal hearing (NH) subjects. The current study examined neural predictors of speech-in-noise ability in a large cohort of cochlear-implant (CI) users, with the long-term goal of developing a simple electrophysiological correlate that could be implemented in clinics.

DesignWe recorded electroencephalography (EEG) in 114 post-lingually deafened CI users while they completed the California Consonant Test (CCT): a word-in-noise task. In many subjects, data were also collected on two other commonly used clinical measures of speech perception: a word-in-quiet task (Consonant-Nucleus-Consonant [CNC]) word and a sentence-in-noise task (AzBio sentences). Neural activity was assessed at a single vertex electrode (Cz), to maximize generalizability to clinical situations. The N1-P2 complex of event-related potentials (ERPs) at this location were included in multiple linear regression analyses, along with several other demographic and hearing factors as predictors of speech in noise performance.

ResultsIn general, there was a good agreement between the scores on the three speech perception tasks. ERP amplitudes did not predict AzBio performance which was predicted by the duration of device use, low-frequency hearing thresholds, and age. However, ERP amplitudes were strong predictors for performance for both word recognition tasks: the CCT (which was conducted simultaneously with EEG recording), and the CNC (conducted offline). These correlations held even after accounting for known predictors of performance including residual low-frequency hearing thresholds. In CI-users, better performance was predicted by an increased cortical response to the target word, in contrast to previous reports in normal-hearing subjects in whom speech perception ability was accounted for by the ability to suppress noise.

ConclusionsThese data indicate a neurophysiological correlate of speech-in-noise performance that can be relatively easily captured within the clinic, thereby revealing a richer profile of an individuals hearing performance than shown by psychoacoustic measures alone. These results also highlight important differences between sentence and word recognition measures of performance and suggest that individual differences in these measures may be underwritten by different mechanisms. Finally, the contrast with prior reports of NH listeners in the same task suggests CI-users performance may be explained by a different weighting of neural processes than NH listeners.

sentences: Single-cell motion of magnetotactic bacteria in microfluidic confinement: interplay between surface interaction and magnetic torque Swimming microorganisms often experience complex environments in their natural habitat. The same is true for microswimmers in envisioned biomedical applications. The simple aqueous conditions typically studied in the lab differ strongly from those found in these environments and often exclude the effects of small volume confinement or the influence that external fields have on their motion. In this work, we investigate magnetically steerable microswimmers, specifically magnetotactic bacteria, in strong spatial confinement and under the influence of an external magnetic field. We trap single cells in micrometer-sized microfluidic chambers and track and analyze their motion, which shows a variety of different trajectories, depending on the chamber size and the strength of the magnetic field. Combining these experimental observations with simulations using a variant of an active Brownian particle model, we explain the variety of trajectories by the interplay between the wall interactions and the magnetic torque. We also analyze the pronounced cell-to-cell heterogeneity, which makes single-cell tracking essential for an understanding of the motility patterns. In this way, our work establishes a basis for the analysis and prediction of microswimmer motility in more complex environments.

sentences: Integrative analysis of the plasma proteome and polygenic risk of cardiometabolic diseases Summary ParagraphCommon human diseases are frequently polygenic in architecture, comprising a large number of risk alleles with small effects spread across the genome1-3. Polygenic scores (PGSs) aggregate these alleles into a metric which represents an individuals genetic predisposition to a specific disease. PGSs have shown promise for early risk prediction4-7, and there is potential to use PGSs to understand disease biology in parallel8. Here, we investigate the role plasma protein levels play in cardiometabolic disease risk in a cohort of 3,087 healthy individuals using PGSs. We found PGSs for coronary artery disease (CAD), type 2 diabetes (T2D), chronic kidney disease (CKD), and ischaemic stroke (IS) were associated with levels of 49 plasma proteins. These associations were polygenic in architecture, largely independent of cis protein QTLs, and robust to environmental variation. Over a median 7.7 years follow-up, 28 of these plasma proteins were associated with future myocardial infarction (MI) or T2D events, 16 of which were causal mediators between polygenic risk and incident disease. These protein mediators of polygenic disease risk included targets of approved therapies which may have repurposing potential. Our results demonstrate that PGSs can identify proteins with causal roles in disease, and may have utility in drug development.

sentences: Alternative Splicing Regulates the Physiological Adaptation of the Mouse Hind Limb Postural and Phasic Muscles to Microgravity Muscle atrophy and fiber type alterations are well-characterized physiological adaptations to microgravity with both understood to be primarily regulated by differential gene expression (DGE). While microgravity-induced DGE has been extensively investigated, adaptations to microgravity due to alternative splicing (AS) have not been studied in a mammalian model. We sought to comprehensively elucidate the transcriptomic underpinnings of microgravity-induced muscle phenotypes in mice by evaluating both DGE and changes in AS due to extended spaceflight. Tissue sections and total RNA were isolated from the gastrocnemius and quadriceps, postural and phasic muscles of the hind limb, respectively, of 32-week-old female BALB/c mice exposed to microgravity or ground control conditions for nine weeks. Immunohistochemistry disclosed muscle type-specific physiological adaptations to microgravity that included i) a pronounced reduction in muscle fiber cross-sectional area in both muscles and ii) a prominent slow-to-fast fiber type transition in the gastrocnemius. RNA sequencing revealed that DGE and AS varied across postural and phasic muscle types with preferential employment of DGE in the gastrocnemius and AS in the quadriceps. Gene ontology analysis indicated that DGE and AS regulate distinct molecular processes. Various non-differentially expressed transcripts encoding musculoskeletal proteins (Tnnt3, Tnnt1, Neb, Ryr1, and Ttn) and muscle-specific RNA binding splicing regulators (Mbnl1 and Rbfox1) were found to have significant changes in AS that altered critical functional domains of their protein products. In striking contrast, microgravity-induced differentially expressed genes were associated with lipid metabolism and mitochondrial function. Our work serves as the first comprehensive investigation of coordinate changes in DGE and AS in large limb muscles across spaceflight. We propose that substantial remodeling of pre-mRNA by AS is a major component of transcriptomic adaptation of skeletal muscle to microgravity. The alternatively spliced genes identified here could be targeted by small molecule splicing regulator therapies to address microgravity-induced changes in muscle during spaceflight.

sentences: DRP1-mediated mitochondrial fission is essential to maintain cristae morphology and bioenergetics Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission is known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin-related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF1). EMPF1 is a devastating neurodevelopmental disease with no effective treatment. To interrogate the mechanisms by which DRP1 mutations cause cellular dysfunction, we utilized human-derived fibroblasts from patients with mutations in DRP1 who present with EMPF1. As expected, patient cells display elongated mitochondrial morphology and lack of fission. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and upregulation of glycolysis. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in aberrant cristae structure and hyperpolarized mitochondrial membrane potential, both of which are tightly linked to the changes in metabolism. Peroxisome structure is also severely elongated in patient cells and results in a potential functional compensation of fatty acid oxidation. Understanding the mechanism by which DRP1 mutations cause these metabolic changes will give insight into the role of mitochondrial dynamics in cristae maintenance and the metabolic capacity of the cell, as well as the disease mechanism underlying EMPF1.

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HuR-dependent expression of Wisp1 is necessary for TGF????-induced cardiac myofibroblast activity Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGF{beta}-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGF{beta}-induced myofibroblast activation upon HuR inhibition. To delineate HuR-dependent mechanisms, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify eleven mRNA transcripts that showed enriched HuR binding following TGF{beta} treatment as well as significant co-expression correlation with HuR, SMA, and periostin using single-cell RNA-sequencing from ischemic-zone isolated fibroblasts. Of these, Wnt1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 is able to partially rescue myofibroblast contractile function following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGF{beta}. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGF{beta}, in part through post-transcriptional regulation of Wisp1.

HIGHLIGHTSO_LIThe RNA binding protein HuR is highly expressed in cardiac fibroblasts and its expression strongly correlates with markers of active myofibroblasts
C_LIO_LIHuR inhibition reduces migration, contraction, and ECM production activity of cardiac fibroblasts
C_LIO_LIExpression of the secreted matricellular protein, Wisp1, is increased in a HuR-dependent manner following TGF{beta} treatment
C_LIO_LIRecombinant Wisp1 rescues myofibroblast contractile function following HuR inhibition
C_LIO_LIHuR-dependent expression of Wisp1 is necessary for myofibroblast activation
C_LI

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High contrast, moving targets in an emerging target paradigm promote fast visuomotor responses during visually guided reaching Humans have a remarkable capacity to rapidly interact with the surrounding environment, often by transforming visual input into motor output on a moment-to-moment basis. But what visual features promote rapid reaching? High contrast, fast-moving targets elicit strong responses in the superior colliculus (SC), a structure associated with express saccades and implicated in rapid electromyographic (EMG) responses on upper limb muscles. To test the influence of stimulus properties on rapid reaches, we had human subjects perform visually guided reaches to moving targets varied by speed (experiment 1) or speed and contrast (experiment 2), in an emerging target paradigm which has recently been shown to robustly elicit fast visuomotor responses. Our analysis focused on stimulus-locked responses (SLRs) on upper limb muscles. SLRs appear within <100 ms of target presentation, and as the first wave of muscle recruitment, they have been hypothesized to arise from the SC. Across 32 subjects studied in both experiments, 97% expressed SLRs in the emerging target paradigm, whereas only 69% expressed SLRs in an immediate response paradigm towards static targets. Faster moving targets (experiment 1) evoked large magnitude SLRs, while high contrast fast moving targets (experiment 2) evoked short latency, large magnitude SLRs. In some instances, SLR magnitude exceeded the magnitude of movement aligned activity. Both large magnitude and short latency SLRs were correlated with short latency reach reaction times. Our results support the hypothesis that, in scenarios requiring expedited responses, a subcortical pathway originating in the SC elicits the earliest wave of muscle recruitment, expediting reaction times.

New & NoteworthyHow does the brain rapidly transform vision into action? Here, by recording upper limb muscle activity, we find that high contrast and fast moving targets are highly effective at evoking rapid visually guided reaches. We surmise that a brainstem circuit originating in the superior colliculus contributes to the most rapid reaching responses. When time is of the essence, cortical areas may serve to prime this circuit, and elaborate subsequent phases of recruitment.

sentences: Precise estimation of genome size from NGS data Precise estimates of genome sizes are important parameters for both theoretical and practical biodiversity genomics. We present here a fast, easy-to-implement and precise method to estimate genome size from the number of bases sequenced and the mean sequence coverage. To estimate the latter, we take advantage of the fact that a precise estimation of the Poisson distribution parameter lambda is possible from truncated data, restricted to the part of the coverage distribution representing the true underlying distribution. With simulations we could show that reasonable genome size estimates can be gained even from low-coverage (10X), highly discontinuous genome drafts. Comparison of estimates from a wide range of taxa and sequencing strategies with flow-cytometry estimates of the same individuals showed a very good fit and suggested that both methods yield comparable, interchangeable results.

sentences: Pin Electrode Reactor: A novel cold atmospheric plasma device and its potential in glioblastoma treatment Glioblastoma multiforme (GBM) is the most common and biologically aggressive brain tumour. The current standard therapy for GBM consists in surgical resection, followed by radiotherapy and chemotherapy. Yet, the treatment is limited due to the area for the surgical resection and for the inability of some drugs to cross the brain blood barrier, leading to a general prognostic of no more than a year. Cold atmospheric plasma (CAP) is a new approach in the treatment of this challenging disease. CAP interaction with cells is dependent on physical and chemical factors, with different plasma discharges, cell type, and culture conditions leading to different CAP activity. Considering the plasma self-adaptation that different plasma discharge modes can undergo, which leads to different interaction plasma/cells, the characterization of a new device is essential. In this study we analysed the effect of a novel large pin-to-plate non-thermal atmospheric plasma on U-251 MG cells under different conditions. The analysis of reactive oxygen and nitrogen species (RONS) on plasma, media and cells were also assessed. We were able to demonstrate that the pin-to-plate device is cytotoxic to GBM cells in a dose, time and ROS dependent manner. The measurements of RONS on plasma/media also give us an insight on the chemical effect of this novelty device, and the possibility to better understand the use of this device as a promising GBM therapy.

sentences: Social deficits induced by pervasive environmental stressors are prevented by microbial or dopaminergic modulation Environmental toxicant exposure, including air pollution, is increasing worldwide. However, toxicant exposures are not equitably distributed. Rather, low-income and minority communities bear the greatest burden, along with higher levels of psychosocial stress. Both air pollution and maternal stress during pregnancy have been linked to neurodevelopmental disorders such as autism, but biological mechanisms and targets for therapeutic intervention remain poorly understood. We demonstrate that combined prenatal exposure to air pollution (diesel exhaust particles, DEP) and maternal stress (MS) in mice induces social behavior deficits only in male offspring, in line with the male bias in autism. These behavioral deficits are accompanied by changes in microglia and dopaminergic circuits in the brain, along with changes in the structure of the gut epithelium and microbiome. Importantly, DEP/MS-induced social deficits in males are prevented by shifting the gut microbiome by cross-fostering at birth and reversed by chemogenetic activation of the dopamine system.

sentences: Porous yet Dense matrices: using ice to shape collagen 3D cell culture systems with increased physiological relevance Standard in vitro cell culture is one of the pillars of biomedical science. However, there is increasing evidence that 2D systems provide biological responses that are often in disagreement with in vivo observations, partially due to limitations in reproducing the native cellular microenvironment. 3D materials that are able to mimic the native cellular microenvironment to a greater extent tackle these limitations. Here, we report Porous yet Dense (PyD) type I collagen materials obtained by ice-templating followed by topotactic fibrillogenesis. These materials combine extensive macroporosity, favouring the cell migration and nutrients exchange, as well as dense collagen walls, which mimic locally the Extracellular Matrix. When seeded with Normal Human Dermal Fibroblasts (NHDFs), PyD matrices allow for a faster and more extensive colonisation when compared with equivalent Non-Porous matrices. The textural properties of the PyD materials also impact cytoskeletal and nuclear 3D morphometric parameters. Due to the effectiveness in creating a biomimetic 3D environment for NHDFs and the ability to promote cell culture for more than 28 days without subculture, we anticipate that PyD materials could configure an important step towards in vitro systems applicable to other cell types and with higher physiological relevance.

sentences: Single cell variations in expression of codominant alleles A and B on RBC of AB blood group individuals. One of the key questions in biology is whether all cells of a "cell type" have more or less the same phenotype, especially with relation to non-imprinted autosomal loci. Recent studies point to differential allelic expression of autosomal genes being a prevalent phenomenon responsible to confer phenotypic variability at individual cell level. However, most studies have been carried out in actively transcribing cells. Here we display cellular mosaicism arising from differential allelic expression for the cell surface glycoprotein in the enucleated RBCs. We studied the expression of the A and B histo-blood group antigens encoded by the co-dominant alleles in individual RBCs using immunofluorescence. We assessed the relative levels of the co-dominant alleles IA and IB in 2512 RBC from 24 individuals with AB blood group using Cy3- and FITC- tagged antibodies. Quantification of individual fluorescence intensities from each cell and test of their normal distribution revealed that contrary to the general belief that all RBC in AB individuals express both antigens in comparable amounts, they segregated into 4 groups: showing normal distribution for both antigens, either antigen, and neither antigen; the deviation from normal distribution could not be correlated to maternal/paternal origin, thus appear to be stochastic. Surprisingly, very few people showed any correlation between the amounts of these two antigens on RBC. In fact, the ratio of antigen A to B in the entire set of samples spanned over 5 orders of magnitude. This variability in amount of the antigens A and/or B, combined with a lack of correlation between the amounts of these two antigens resulted in unique staining patterns for RBC, generating widespread mosaicism in the RBC population of AB blood group individuals.

sentences: The stem cell-specific protein TRIM71 inhibits maturation and activity of the pro-differentiation miRNA let-7 via two independent molecular mechanisms The stem cell-specific RNA-binding protein TRIM71/LIN-41 was the first identified target of the pro-differentiation and tumor suppressor miRNA let-7. TRIM71 has essential functions in embryonic development and a proposed oncogenic role in several cancer types, such as hepatocellular carcinoma. Here, we show that TRIM71 regulates let-7 expression and activity via two independent mechanisms. On the one hand, TRIM71 enhances pre-let-7 degradation through its direct interaction with LIN28 and TUT4, thereby inhibiting let-7 maturation and indirectly promoting the stabilization of let-7 targets. On the other hand, TRIM71 represses the activity of mature let-7 via its RNA-dependent interaction with the RNA-Induced Silencing Complex (RISC) effector protein AGO2. We found that TRIM71 directly binds and stabilizes let-7 targets, suggesting that let-7 activity inhibition occurs on active RISCs. MiRNA enrichment analysis of several transcriptomic datasets from mouse embryonic stem cells and human hepatocellular carcinoma cells suggests that these let-7 regulatory mechanisms shape transcriptomic changes during developmental and oncogenic processes. Altogether, our work reveals a novel role for TRIM71 as a miRNA repressor and sheds light on the precise mechanistic dual regulation of let-7.

sentences: Antiviral and Neuroprotective Abilities of Influenza Virus Infection in Tractable Brain Organoids Human pluripotent stem cell (hPSC)-derived brain organoids offer an unprecedented opportunity for various applications as an in vitro model, such as modeling virus infection and drug screening. In this study, we present an experimental brain organoid platform for modeling infection with multiple viruses (e.g., influenza virus or enterovirus). Brain organoids challenged by influenza viruses (H1N1-WSN and H3N2-HKT68) had decreased overall organoid size, similar to ZIKA virus infection, while enteroviruses (EV68 and EV71) infected brain organoids displayed the opposite result. Then, we studied the molecular events in WSN-infected organoids, and we found that WSN could widely infect multiple cell types, and preferentially infected MAP2+ neurons compared to SOX2+ neural stem cells (NSCs) and GFAP+ astrocytes in brain organoids, and induced apoptosis of NSCs and neurons, but not astrocytes. The inflammatory responses in organoids observed to occur (Tumor necrosis factor alpha, interferon gamma, and interleukin 6) after WSN infection may further facilitate brain damage. Furthermore, transcriptional profiling revealed several upregulated genes (CSAG3 and OAS2) and downregulated genes (CDC20B, KCNJ13, OTX2-AS1, CROCC2, and F5) after WSN infection for 24 hpi and 96 hpi, implicating antiviral drugs development responses to WSN. Finally, we explored neurotrophic factors (e.g., BDNF, GDNF, and NT3) and PYC-12 as antiviral and neuroprotective reagents, which could significantly suppress virus infection, apoptosis, and inflammatory responses. Collectively, we established a tractable experimental model system to investigate the impact and mechanism of virus infection on human brain development, and provide a platform for rapidly screening therapeutic compounds, advancing the development of antiviral strategies.

sentences: An insight into SARS-CoV-2 Membrane protein interaction with Spike, Envelope, and Nucleocapsid proteins Intraviral protein-protein interactions are crucial for replication, pathogenicity, and viral assembly. Among these, virus assembly is a critical step as it regulates the arrangements of viral structural proteins and helps in the encapsulation of genomic material. SARS-CoV-2 structural proteins play an essential role in the self-rearrangement, RNA encapsulation, and mature virus particle formation. In SARS-CoV, the membrane protein interacts with the envelope and spike protein in Endoplasmic Reticulum Golgi Intermediate Complex (ERGIC) to form an assembly in the lipid bilayer, followed by membrane-ribonucleoprotein (nucleocapsid) interaction. In this study, we tried to understand the interaction of membrane proteins interaction with envelope, spike, and nucleocapsid proteins using protein-protein docking. Further, simulation studies performed up to 100 ns to examine the stability of protein-protein complexes of Membrane-Envelope, Membrane-Spike, and Membrane-Nucleocapsid. Prime MM-GBSA showed high binding energy calculations than the docked complex. The interactions identified in our study will be of great importance, as it provides valuable insight into the protein-protein complex, which could be the potential drug targets for future studies.

sentences: Dorsal raphe nucleus to anterior cingulate cortex 5-HTergic neural circuit modulates consolation and sociability Consolation is a common response to the distress of others in humans and some social animals, but the neural mechanisms underlying this behavior are not well characterized. By using socially monogamous mandarin voles, we found that optogenetic or chemogenetic inhibition of 5-HTergic neurons in the dorsal raphe nucleus (DR) or optogenetic inhibition of 5-HT terminals in the anterior cingulate cortex (ACC) significantly decreased allogrooming time in the consolation test and reduced sociability in the three-chamber test. The release of 5-HT within the ACC and the activity of DR neurons were significantly increased during allogrooming, sniffing and social approaching. Finally, we found that the activation of 5-HT1A receptors in the ACC was sufficient to reverse consolation and sociability deficits induced by the chemogenetic inhibition of 5-HTergic neurons in the DR. Our study provided first direct evidence that DR-ACC 5-HTergic neural circuit is implicated in consolation-like behaviors and sociability.

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Mechanical control of morphogenetic robustness in an inherently challenging environment Epithelial sheets undergo highly reproducible remodeling to shape organs. This stereotyped morphogenesis depends on a well-defined sequence of events leading to the regionalized expression of developmental patterning genes that finally triggers downstream mechanical forces to drive tissue remodeling at a pre-defined position. However, how tissue mechanics controls morphogenetic robustness when challenged by intrinsic perturbations in close proximity has never been addressed.

Here, we show that a bias in force propagation ensures stereotyped morphogenesis despite the presence of mechanical noise in the environment. We found that knockdown of the Arp2/3 complex member Arpc5 specifically affects fold directionality without altering neither the developmental nor the force generation patterns. By combining in silico modeling, biophysical and ad hoc genetic tools, our data reveal that junctional Myosin II planar polarity favors long-range force channeling and ensures folding robustness, avoiding force scattering and thus isolating the fold domain from surrounding mechanical perturbations.

sentences: The zebrafish presomitic mesoderm elongates through compression-extension In vertebrate embryos the presomitic mesoderm become progressively segmented into somites at the anterior end while extending along the anterior-posterior axis. A commonly adopted model to explain how this tissue elongates is that of posterior growth, driven in part by the addition of new cells from uncommitted progenitor populations in the tailbud. However, in zebrafish, much of somitogenesis is associated with an absence of overall volume increase and posterior progenitors do not contribute new cells until the final stages of somitogenesis. Here, we perform a comprehensive 3D morphometric analysis of the paraxial mesoderm and reveal that extension is linked to a volumetric decrease, compression in both dorsal-ventral and medio-lateral axes, and an increase in cell density. We also find that individual cells decrease in their cell volume over successive somite stages. Live cell tracking confirms that much of this tissue deformation occurs within the presomitic mesoderm progenitor zone and is associated with non-directional rearrangement. Furthermore, unlike the trunk somites that are laid down during gastrulation, tail somites develop from a tissue that can continue to elongate in the absence of functional PCP signalling. Taken together, we propose a compression-extension mechanism of tissue elongation that highlights the need to better understand the role of tissue intrinsic and extrinsic forces play in regulating morphogenesis.

sentences: Cell type-specific inference of differential expression in spatial transcriptomics Spatial transcriptomics enables spatially resolved gene expression measurements at near single-cell resolution. There is a pressing need for computational tools to enable the detection of genes that are differentially expressed (DE) within specific cell types across tissue context. We show that current approaches cannot learn cell type-specific DE due to changes in cell type composition across space and the fact that measurement units often detect transcripts from more than one cell type. Here, we introduce a statistical method, Cell type-Specific Inference of Differential Expression (C-SIDE), that identifies cell type-specific patterns of differential gene expression while accounting for localization of other cell types. We model spatial transcriptomics gene expression as an additive mixture across cell types of general log-linear cell type-specific expression functions. This approach provides a unified framework for defining and identifying gene expression changes in a wide-range of relevant contexts: changes due to pathology, anatomical regions, physical proximity to specific cell types, and cellular microenvironment. Furthermore, our approach enables statistical inference across multiple samples and replicates when such data is available. We demonstrate, through simulations and validation experiments on Slide-seq and MER-FISH datasets, that our approach accurately identifies cell type-specific differential gene expression and provides valid uncertainty quantification. Lastly, we apply our method to characterize spatially-localized tissue changes in the context of disease. In an Alzheimers mouse model Slide-seq dataset, we identify plaque-dependent patterns of cellular immune activity. We also find a putative interaction between tumor cells and myeloid immune cells in a Slide-seq tumor dataset. We make our C-SIDE method publicly available as part of the open source R package https://github.com/dmcable/spacexr.

sentences: Significant organic carbon acquisition by Prochlorococcus in the oceans Marine phytoplankton are responsible for about half of the photosynthesis on Earth. Many are mixotrophs, combining photosynthesis with heterotrophic assimilation of organic carbon but the relative contribution of these two carbon sources is not well quantified. Here, single-cell measurements reveal that Prochlorococcus at the base of the photic zone in the Eastern Mediterranean Sea are obtaining only ~20% of carbon required for growth by photosynthesis. Consistently, laboratory-calibrated evaluations of Prochlorococcus photosynthesis indicate that carbon fixation is systematically too low to support published in situ growth rates in the deep photic layer of the Pacific Ocean. Furthermore, agent-based model simulations show that mixotrophic cells maintain realistic growth rates and populations 10s of meters deeper than obligate photo-autotrophs, deepening the nutricline and Deep Chlorophyll Maximum by ~20 m. Time-series of Prochlorococcus ecotype-abundance from the subtropical North Atlantic and North Pacific suggest that up to 30% of the Prochlorococcus cells live where light intensity is not enough to sustain obligate photo-autotrophic populations during warm, stratified periods. Together, these data and models suggest that mixotrophy underpins the ecological success of a large fraction of the global Prochlorococcus population and its collective genetic diversity.

sentences: Newly recruited intraepithelial Ly6A+CCR9+CD4+ T cells protect against enteric viral infection The intestinal epithelium comprises the bodys largest surface exposed to viruses. However, a role for intraepithelial T lymphocytes in resistance against viral infections remain elusive. By fate-mapping T cells recruited to the murine intestinal epithelium, we observed accumulation of CD4+ T cells after infection with murine norovirus (MNV) or mouse adenovirus type-2 (AdV), but not after reovirus infection. Intraepithelial CD4+ T cells recruited after MNV or AdV infection co-express Ly6A and CCR9, and exhibit T helper 1 and cytotoxic profiles. Although these cells display a diverse TCR repertoire, they conferred protection against AdV and MNV both in vivo and in an organoid co-culture model in an IFN-{gamma}-dependent manner. Ablation of the T cell receptor (TCR) or the transcription factor ThPOK in CD4+ T cells prior to infection prevented viral control, while TCR ablation during infection did not impact viral clearance. These results uncover a protective role for intraepithelial Ly6A+CCR9+CD4+ T cells against enteric viruses.

sentences: Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice Since the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

sentences: Robust statistical evaluation of tumor incidences in long-term rodent carcinogenicity studies: the reformulated poly-k trend test Mortality-adjusted tumor rates in long-term carcinogenicity rodent bioassays are commonly evaluated by means of the poly-k=3 Armitage trend test. However, this assumes exactly a linear dose-response curve and the Weibull parameter of k=3 for all tumor sites. These unrealistic assumptions can be circumvented by multiple testing across multiple possible dose-response shapes, multiple Weibull parameters, multiple effect sizes, multiple correlated tumors as well as pairwise and trend tests using the multiple marginal models approach. Based on data examples, different multiple tests are demonstrated using the CRAN R packages multcomp, tukeytrend, coin, MCPAN and multfisher.

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Epigenetic Memory of COVID-19 in Innate Immune Cells and Their Progenitors Severe coronavirus disease 2019 (COVID-19) is characterized by systemic inflammation and can result in protracted symptoms. Robust systemic inflammation may trigger persistent changes in hematopoietic cells and innate immune memory through epigenetic mechanisms. We reveal that rare circulating hematopoietic stem and progenitor cells (HSPC), enriched from human blood, match the diversity of HSPC in bone marrow, enabling investigation of hematopoiesis and HSPC epigenomics. Following COVID-19, HSPC retain epigenomic alterations that are conveyed, through differentiation, to progeny innate immune cells. Epigenomic changes vary with disease severity, persist for months to a year, and are associated with increased myeloid cell differentiation and inflammatory or antiviral programs. Epigenetic reprogramming of HSPC may underly altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

One Sentence SummaryTranscriptomic and epigenomic analysis of blood reveal sustained changes in hematopoiesis and innate immunity after COVID-19.

Graphical Abstract

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sentences: Structural dynamics of cytochrome P450 3A4 in the presence of substrates and cytochrome P450 reductase Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds, as well as by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects however is poorly understood. We have used hydrogen-deuterium exchange mass spectroscopy (HDX-MS) to investigate the impact of CPR and three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced form of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from a suppression of deuterium exchange in several regions of CYP3A4, including those known to be involved in protein-protein interactions (C-helix) as well as substrate binding and specificity (B-, E-helices and K/{beta}1-loop). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted by CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of allostery, ligand binding, and catalysis in CYP enzymes.

sentences: Whole plastid genome-based phylogenomics supports an inner placement of the O. insectifera group rather than a basal position in the rapidly diversifying Ophrys genus (Orchidaceae) Some lineages of the Orchid genus Ophrys exhibit among the highest diversification rates reported so far. As a consequence of a such intense and rapid evolution, the systematics and the taxonomy of this genus remains unclear. A hybrid assembly approach based-on long- and short-read genomic data allowed us to outperform classical methods to successfully assemble whole plastid genomes for two new Ophrys species: O. aymoninii and O. lutea. Along with three other previously Ophrys plastid genome sequences, we then reconstructed the first whole plastome-based molecular phylogeny including representatives of the three mains recognized Ophrys lineages. Our results support the placement of the O. insectifera clade as sister group of  non-basal Ophrys rather than a basal position. Our findings corroborate recent results obtained from genomic data (RAD-seq and transcriptomes) but contrast with previous ones. These results therefore confirm that molecular phylogenetic hypotheses based on a limited number of loci (e.g. nrITS, matK, rbcL) may have provided a biased picture of phylogenetic relationships within Ophrys and possibly other plant taxa.

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Unique Features of Different Classes of G-Protein-Coupled Receptors Revealed from Sequence Coevolutionary and Structural Analysis G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and represent the primary targets of about one third of currently marketed drugs. Despite the critical importance, experimental structures have been determined for only a limited portion of GPCRs and functional mechanisms of GPCRs remain poorly understood. Here, we have constructed novel sequence coevolutionary models of the A and B classes of GPCRs and compared them with residue contact frequency maps generated with available experimental structures. Significant portions of structural residue contacts were successfully detected in the sequence-based covariational models. "Exception" residue contacts predicted from sequence coevolutionary models but not available structures added missing links that were important for GPCR activation and allosteric modulation. Moreover, we identified distinct residue contacts involving different sets of functional motifs for GPCR activation, such as the Na+ pocket, CWxP, DRY, PIF and NPxxY motifs in the class A and the HETx and PxxG motifs in the class B. Finally, we systematically uncovered critical residue contacts tuned by allosteric modulation in the two classes of GPCRs, including those from the activation motifs and particularly the extracellular and intracellular loops in class A GPCRs. These findings provide a promising framework for rational design of ligands to regulate GPCR activation and allosteric modulation.

SignificanceG-protein-coupled receptors (GPCRs) play key roles in cellular signaling and serve as the primary targets of [~]1/3 of currently marketed drugs. In this work, we have presented the first analysis of both residue sequence coevolution and structural contact maps in different classes of GPCRs. We have inferred pathways for GPCR signal transduction that could not be determined from structural analysis alone. Distinct residue contacts have been identified in the signaling pathways of class A and B GPCRs. Our combined sequence coevolutionary and structural contact analysis has thus revealed important insights into the mechanism of GPCR signal transduction, which is expected to facilitate rational drug design of the GPCRs.

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CARP2 regulates the Golgi dynamics upon EGF stimulation Golgi apparatus regulate diverse cellular functions like protein sorting, vesicular trafficking, secretion, protein modifications like glycosylation etc. In mammalian cells though, Golgi apparatus appear as ribbon architecture, individual stacks laterally linked to each other by tubular structure, its architecture changes dynamically to cater to the needs of the cell under physiological and stress conditions. Loss of Golgi integrity is reported to be associated with pathological conditions like cancer and neurodegeneration. Very little is known of molecular regulators of Golgi dynamics. Here, we demonstrate that CARP2 (Caspase -8 and -10 associated RING containing protein 2), an endosomal ubiquitin ligase and a known regulator of cell migration, modulates the Golgi structure. Stimulation with EGF (Epidermal growth factor) modestly increased CARP2 protein levels. CARP2 exogenous expression or EGF treatment resulted in dispersal of the Golgi apparatus. Conversely, CARP2 deletion suppressed EGF induced Golgi dispersal. CARP2 variants that are defective in their endosomal-association or E3 ligase activity were unable to exhibit Golgi dispersal, indicating importance of both the endosomal localization and the E3 activity for this function. Importantly, we provide evidence that in EGF stimulated cells CARP2 mechanistically functions by targeting one of the Golgi structural proteins, Golgin45 for ubiquitination and degradation. Taken together, our findings unravel the existence of crosstalk between endosomal ubiquitin signaling and the Golgi dynamics.

SignificanceThe Golgi is an organelle that exists in mammals in ribbon form - individual stacks laterally linked with each other - is central to protein and lipid modifications, trafficking and secretion. The Golgi architecture is changed dynamically to cater to the physiological needs of the cells (eg: cell division, migration). Dysfunctional or altered Golgi is reported under pathological conditions like cancer, neurodegenerative diseases etc. This study unravels a complex signaling between endosomal ubiquitin ligase, CARP2 and one of the Golgi structural proteins, Golgin45. Here, we delineate CARP2-Golgin45 signaling as a fundamental mechanism that regulates Golgi dynamics underlying in EGF-stimulated cell migration.

sentences: Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms to control cell motility and neurite outgrowth Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent, nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knock-down phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.

sentences: Transcriptional states of retroelement-inserted regions and KRAB zinc finger protein association regulate DNA methylation of retroelements in human male germ cells DNA methylation, repressive histone modifications, and PIWI-interacting RNAs are essential for controlling retroelement silencing in mammalian germ lines. Dysregulation of retroelement silencing is associated with male sterility. Although retroelement silencing mechanisms have been extensively studied in mouse germ cells, little progress has been made in humans. Here, we show that the Kruppel-associated box domain zinc finger proteins (KRAB-ZFPs) are associated with DNA methylation of retroelements in human primordial germ cells (hPGCs), and hominoid-specific retroelement SINE-VNTR-Alus (SVA) is subjected to transcription-directed de novo DNA methylation during human spermatogenesis. Furthermore, we show that the degree of de novo DNA methylation in SVAs varies among human individuals, which confers a significant inter-individual epigenetic variation in sperm. Collectively, our results provide potential molecular mechanisms for the regulation of retroelements in human male germ cells.

sentences: Suberin plasticity to developmental and exogenous cues is regulated by a set of MYB transcription factors Suberin is a hydrophobic biopolymer that can be deposited at the periphery of cells, forming protective barriers against biotic and abiotic stress. In roots, suberin forms lamellae at the periphery of endodermal cells where it plays crucial roles in the control of water and mineral transport. Suberin formation is highly regulated by developmental and environmental cues. However, the mechanisms controlling its spatiotemporal regulation are poorly understood. Here, we show that endodermal suberin is regulated independently by developmental and exogenous signals to fine tune suberin deposition in roots. We found a set of four MYB transcription factors (MYB41, MYB53, MYB92 and MYB93), that are regulated by these two signals, and are sufficient to promote endodermal suberin. Mutation of these four transcription factors simultaneously through genome editing, lead to a dramatic reduction of suberin formation in response to both developmental and environmental signals. Most suberin mutants analyzed at physiological levels are also affected in another endodermal barrier made of lignin (Casparian strips), through a compensatory mechanism. Through the functional analysis of these four MYBs we generated plants allowing unbiased investigations of endodermal suberin function without accounting for confounding effects due to Casparian strip defects, and could unravel specific roles of suberin in nutrient homeostasis.

sentences: Mapping microglia and astrocytes activation in vivo using diffusion MRI Glia, and particularly microglia, are increasingly implicated in the pathophysiology of psychiatric and neurodegenerative disorders. However, to date the only methods for imaging these cells in vivo involve either invasive procedures (e.g. multi-photon imaging in rodents) or TSPO-PET radiotracers, which afford low resolution and specificity, since TSPO expresses across multiple cell types. Here, we present a non-invasive diffusion-weighted MRI method to image changes in glia morphometry in vivo. Using two rat models of neuroinflammation, with and without neurodegeneration, we demonstrate that diffusion-weighted MRI carries the fingerprint of microglia and astrocytes activation, and that specific signatures from each population can be quantified non-invasively. We demonstrate that the method can further detect glia proliferation, and provide a quantitative account of neuroinflammation regardless of the existence of a concomitant neuronal loss. We prove the translational value of the approach showing significant correlations between MRI and histological microglia markers measured across different brain regions in humans. This framework holds the potential to transform basic and clinical research by providing a tool to clarify the role of inflammation in health and disease across the lifespan.

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Aberrant Temporal-spatial Patterns to Sad expressions in Major Depressive Disorders via Hidden Markov Model BackgroundThe pathological mechanisms of Major depressive disorders (MDD) is associated with over-expressing of negative emotions, and the overall temporal-spatial patterns underlying over-representation in depression still remained to be revealed to date. We hypothesized that the aberrant spatio-temporal attributes of the process of sad expressions relate to MDD and help to detect depression severity.

MethodsWe enrolled a total of 96 subjects including 47 MDDs and 49 healthy controls (HCs), and recorded their Magnetoencephalography data under a sad expressions recognition task. A hidden Markov model (HMM) was applied to separate the whole neural activity into several brain states, then to characterize the dynamics. To find the disrupted spatial-temporal features, power estimations and fractional occupancy of each state were contrasted between MDDs and HCs.

ResultsThree states were found over the period of emotional stimuli processing procedure. The visual state was mainly distributed in early stage (0 - 270ms) and the limbic state in middle and later stage (270ms - 600ms) of the task, while the fronto-parietal state remained a steady proportion across the whole period. MDDs activated statistically more in limbic system during limbic state (p = 0.0045) and less in frontoparietal control network during fronto-parietal state (p = 5.38*10-5) relative to HCs. Hamilton-Depression-Rating Scale scores was significantly correlated with the predicted severity value using the state descriptors (p = 0.0062, r = 0.3933).

DiscussionAs human brain exhibited varied activation patterns under the negative stimuli, MDDs expressed disrupted temporal-spatial activated patterns across varied stages involving the primary visual perception and emotional contents processing compared to HCs, indicting disordered regulation of brain functions. Furthermore, descriptors built by HMM could be potential biomarkers for identifying the severity of depression disorders.

sentences: The value of genotype-specific reference for transcriptome analyses High quality transcriptome assembly using short reads from RNA-seq data still heavily relies upon reference-based approaches, of which the primary step is to align RNA-seq reads to a single reference genome of haploid sequence. However, it is increasingly apparent that while different genotypes within a species share "core" genes, they also contain variable numbers of "specific" genes that are only present a subset of individuals. Using a common reference may thus lead to a loss of genotype-specific information in the assembled transcript dataset and the generation of erroneous, incomplete or misleading transcriptomics analysis results. With the recent development of pan-genome information in many species, it is important that we understand the limitations of single genotype references for transcriptomics analysis. In this study, we quantitively evaluated the advantages of using genotype-specific reference genomes for transcriptome assembly and analysis using cultivated barley as a model. We mapped barley cultivar Barke RNA-seq reads to the Barke genome and to the cultivar Morex genome (common barley genome reference) to construct a genotype specific Reference Transcript Dataset (sRTD) and a common Reference Transcript Datasets (cRTD), respectively. We compared the two RTDs according to their transcript diversity, transcript sequence and structure similarity and the accuracy they provided for transcript quantification and differential expression analysis. Our evaluation shows that the sRTD has a significantly higher diversity of transcripts and alternative splicing events. Despite using a high-quality reference genome for assembly of the cRTD, we miss ca. 40% transcripts present in the sRTD and cRTD only has ca. 70% true assemblies. We found that the sRTD is more accurate for transcript quantification as well as differential expression and differential alternative splicing analysis. However, gene level quantification and comparative expression analysis are less affected by the source RTD, which indicates that analysing transcriptomic data at the gene level may be a reasonable compromise when a high-quality genotype-specific reference is not available.

sentences: Effects of mucin and DNA concentrations in airway mucus on Pseudomonas aeruginosa biofilm recalcitrance The pathological properties of airway mucus in cystic fibrosis (CF) are dictated by mucus concentration and composition, with mucins and DNA being responsible for mucus viscoelastic properties. As CF pulmonary disease progresses, the concentrations of mucins and DNA increase and are associated with increased mucus viscoelasticity and decreased transport. Similarly, the biophysical properties of bacterial biofilms are heavily influenced by the composition of their extracellular polymeric substances (EPS). While the roles of polymer concentration and composition in mucus and biofilm mechanical properties have been evaluated independently, the relationship between mucus concentration and composition and the biophysical properties of biofilms grown therein remains unknown. Pseudomonas aeruginosa biofilms were grown in airway mucus as a function of overall concentration and DNA concentration to mimic healthy and CF physiology and biophysical properties were evaluated with macro- and microrheology. Biofilms were also characterized after exposure to DNase or DTT to examine the effects of DNA and mucin degradation, respectively. Identifying critical targets in biofilms for disrupting mechanical stability in highly concentrated mucus may lead to the development of efficacious biofilm therapies and ultimately improve CF patient outcomes. Overall mucus concentration was the predominant contributor to biofilm viscoelasticity and both DNA degradation and mucin reduction resulted in compromised biofilm mechanical strength.

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Pparα and fatty acid oxidation coordinate hepatic transcriptional architecture Fasting requires tight coordination between the metabolism and transcriptional output of hepatocytes to maintain systemic glucose and lipid homeostasis. Genetically-defined deficits in hepatic fatty acid oxidation result in dramatic fasting-induced hepatocyte lipid accumulation and induction of genes for oxidative metabolism, thereby providing a mouse model to interrogate the mechanisms by which the liver senses and transcriptionally responds to fluctuations in lipid levels. While fatty acid oxidation is required for a rise in acetyl-CoA and subsequent lysine acetylation following a fast, changes in histone acetylation (total, H3K9ac, and H3K27ac) associated with transcription do not require fatty acid oxidation. Instead, excess fatty acids prompt induction of lipid catabolic genes largely via ligand-activated Ppar. We observe that active enhancers in fasting mice are enriched for Ppar binding motifs, and that inhibition of hepatic fatty acid oxidation results in elevated enhancer priming and acetylation proximal to Ppar binding sites within regulatory elements largely associated with genes in lipid metabolism. Also, a greater number of Ppar-associated H3K27ac signal changes occur at active enhancers compared to promoters, suggesting a genomic mechanism for Ppar to tune target gene expression levels. Overall, these data demonstrate the requirement for Ppar activation in maintaining transcriptionally permissive hepatic genomic architecture particularly when fatty acid oxidation is limiting.

HIGHLIGHTSO_LIFasting-induced transcription and histone acetylation are largely independent of acetyl-CoA concentration.
C_LIO_LIDeficits in fatty acid oxidation prompt epigenetic changes and Ppar-sensitive transcription.
C_LIO_LIFasting prompts enhancer priming and acetylation proximal to Ppar binding sites independent of Ppar.
C_LIO_LIPatterns of Ppar target genes can be distinguished by epigenetic marks at promoters and enhancers.
C_LI

sentences: A tissue-aware machine learning framework enhances the mechanistic understanding and genetic diagnosis of Mendelian and rare diseases Genetic studies of Mendelian and rare diseases face the critical challenges of identifying pathogenic gene variants and their modes-of-action. Previous efforts rarely utilized the tissue-selective manifestation of these diseases for their elucidation. Here we introduce an interpretable machine learning (ML) platform that utilizes heterogeneous and large-scale tissue-aware datasets of human genes, and rigorously, concurrently and quantitatively assesses hundreds of candidate mechanisms per disease. The resulting tissue-aware ML platform is applicable in gene-specific, tissue-specific, or patient-specific modes. Application of the platform to selected Mendelian disease genes pinpointed mechanisms that lead to tissue-specific disease manifestation. When applied jointly to diseases that manifest in the same tissue, the models revealed common known and previously underappreciated factors that underlie tissue-selective disease manifestation. Lastly, we harnessed our ML platform toward genetic diagnosis of tissue-selective rare diseases. Patient-specific models of candidate disease-causing genes from 50 patients successfully prioritized the pathogenic gene in 86% of the cases, implying that the tissue-selectivity of rare diseases aids in filtering out unlikely candidate genes. Thus, interpretable tissue-aware ML models can boost mechanistic understanding and genetic diagnosis of tissue-selective heritable diseases. A webserver supporting gene prioritization is available at https://netbio.bgu.ac.il/trace/.

sentences: Machine Learning for the Identification of Viral Attachment Machinery from Respiratory Virus Sequences At the outset of an emergent viral respiratory pandemic, sequence data is among the first molecular information available. As viral attachment machinery is a key target for therapeutic and prophylactic interventions, rapid identification of viral "spike" proteins from sequence can significantly accelerate the development of medical countermeasures. For five families of respiratory viruses, covering the vast majority of airborne and droplet-transmitted diseases, host cell entry is mediated by the binding of viral surface glycoproteins that interact with a host cell receptor. In this report it is shown that sequence data for an unknown virus belonging to one of the five families above provides sufficient information to identify the protein(s) responsible for viral attachment and to permit an assignment of viral family. Random forest models that take as input a set of respiratory viral sequences can classify the protein as "spike" vs. non-spike based on predicted secondary structure elements alone (with 97.8 % correctly classified) or in combination with N-glycosylation related features (with 98.1 % correctly classified). In addition, a Random Forest model developed using the same dataset and only secondary structural elements was able to predict the respiratory virus family of each protein sequence correctly 89.0 % of the time. Models were validated through 10-fold cross-validation as well as bootstrapping. Surprisingly, we showed that secondary structural element and N-glycosylation features were sufficient for model generation. The ability to rapidly identify viral attachment machinery directly from sequence data holds the potential to accelerate the design of medical countermeasures for future pandemics.

sentences: Achieving natural behavior in a robot using neurally inspired hierarchical control Terrestrial locomotion presents tremendous computational challenges on account of the enormous degrees of freedom in legged animals, and the complex and unpredictable properties of the natural environment and the effectors. Yet the nervous system can achieve locomotion with ease. Here we introduce a quadrupedal robot capable of goal-directed posture control and locomotion over rough terrain. The underlying control architecture is a hierarchical network of simple negative feedback control systems inspired by the organization of the vertebrate nervous system. Without using an internal model or feedforward planning, and without any training, our robot shows robust posture control and locomotor behavior in novel environments with unpredictable disturbances.

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Naturally occurring combinations of receptors from single cell transcriptomics in endothelial cells VEGF inhibitor drugs have been successful, especially in ophthalmology, but not all patients respond to them. Combinations of drugs are likely to be needed for more effective therapies of angiogenesis-related diseases. In this paper we describe naturally occurring combinations of receptors in endothelial cells that might help to understand how cells communicate and to identify targets for drug combinations. We also develop and share a new software tool called DECNEO to identify them.

Single-cell gene expression data are used to identify a set of co-expressed endothelial cell receptors, conserved among species (mice and humans) and enriched, within a network, of connections to up-regulated genes. This set includes several receptors previously shown to play a role in angiogenesis. Multiple statistical tests from large datasets, including an independent validation set, support the reproducibility, evolutionary conservation and role in angiogenesis of these naturally occurring combinations of receptors. We also show tissue-specific combinations and, in the case of choroid endothelial cells, consistency with both well-established and recent experimental findings, presented in a separate paper.

The results and methods presented here advance the understanding of signaling to endothelial cells. The methods are generally applicable to the decoding of intercellular combinations of signals.

sentences: Insulin-dependent and -independent dynamics of insulin receptor trafficking in muscle cells Insulin resistance contributes to type 2 diabetes and can be driven by hyperinsulinemia. Insulin receptor (INSR) internalization and cell-surface dynamics at rest and during insulin exposure are incompletely understood in muscle cells. Using surfacing labeling and live-cell imaging, we observed robust basal internalization of INSR in C2C12 myoblasts, without a robust effect of added insulin. Mass-spectrometry analysis of INSR-binding proteins identified potential molecular mechanisms associated with internalization. We confirmed known interactors, including IGF1R, but also identified underappreciated INSR-binding factors such as ANXA2. Protein-protein interaction network mapping suggested links between INSR and caveolin-mediated endocytosis. INSR interacted with both caveolin and clathrin heavy chain (CLTC) in mouse skeletal muscle and C2C12 myoblasts. Whole cell 2D super-resolution imaging revealed that high levels of insulin (20 nM) increased INSR colocalization with CAV1 but decreased its colocalization with CLTC. Single particle tracking confirmed the colocalization of cell-surface INSR with both over-expressed CAV1-mRFP and CLTC-mRFP. INSR tracks that colocalized with CAV1 exhibited longer radii and lifetimes, regardless of insulin exposure, compared to non-colocalized tracks, whereas insulin further increased the lifetime of INSR/CLTC colocalized tracks. Overall, these data suggest that muscle cells utilize both CAV1 and CLTC-dependent pathways for INSR dynamics and internalization.

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gcFront: a tool for determining a Pareto front of growth-coupled cell factory designs MotivationA widely applicable strategy to create cell factories is to knock out (KO) genes or reactions to redirect cell metabolism so that chemical synthesis is made obligatory when the cell grows at its maximum rate. Synthesis is thus growth-coupled, and the stronger the coupling the more deleterious any impediments in synthesis are to cell growth, making high producer phenotypes evolutionarily robust. Additionally, we desire that these strains grow and synthesise at high rates. Genome-scale metabolic models can be used to explore and identify KOs that growth-couple synthesis, but these are rare in an immense design space, making the search difficult and slow.

ResultsTo address this multi-objective optimization problem, we developed a software tool named gcFront - using a genetic algorithm it explores KOs that maximise cell growth, product synthesis, and coupling strength. Moreover, our measure of coupling strength facilitates the search so that gcFront not only finds a growth coupled design in minutes but also outputs many alternative Pareto optimal designs from a single run - granting users flexibility in selecting designs to take to the lab.

Availability and ImplementationgcFront, with documentation and a workable tutorial, is freely available at GitHub: https://github.com/lLegon/gcFront, the repository of which is archived at Zenodo, DOI: 10.5281/zenodo.6338595 (Legon et al., 2022).

Supplementary InformationSupplementary notes and data files are available at Bioinformatics online.

sentences: Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of extracellular vesicle inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed and high-throughput assays. We captured 4 subpopulations of EVs from colorectal cancer cell lines HT29 and SW403 based on EpCAM, CD9, CD63 and CD81 expression, and quantified the co-expression of 8 outer (integrins and tetraspanins) and 4 inner (heat shock, endosomal and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

sentences: A large-sample crisis? Exaggerated false positives by popular differential expression methods We report a surprising phenomenon about identifying differentially expressed genes (DEGs) from population-level RNA-seq data: two popular bioinformatics methods, DESeq2 and edgeR, have unexpectedly high false discovery rates (FDRs). Via permutation analysis on an immunotherapy RNA-seq dataset, we observed that DESeq2 and edgeR identified even more DEGs after samples condition labels were randomly permuted. Motivated by this, we evaluated six DEG identification methods (DESeq2, edgeR, limma-voom, NOISeq, dearseq, and the Wilcoxon rank-sum test) on population-level RNA-seq datasets. We found that the FDR control was often failed by the three popular parametric methods--DESeq2, edgeR, and limma-voom-- and the new non-parametric method dearseq. In particular, the actual FDRs of DESeq2 and edgeR sometimes exceeded 20% when the target FDR threshold was only 5%. Although NOISeq, a non-parametric method used by GTEx, controlled the FDR better than the other four methods did, its power was much lower than that of the Wilcoxon rank-sum test, a classic nonparametric test that consistently controlled the FDR and achieved good power in our evaluation. Based on these results, for population-level RNA-seq studies, we recommend the Wilcoxon rank-sum test.

sentences: An abundant myeloid progenitor shapes neonatal hematopoiesis of naked mole-rats The naked mole-rat became an attractive model for biomedical research due to its exceptional longevity and resistance to disease, yet the developmental properties of naked mole-rat stem and progenitor compartments are poorly understood. Here we report a high frequency bone marrow progenitor, which we termed TC90, exclusively present in neonatal animals. TC90s were marked by an immunophenotype resembling adult naked mole-rat common myeloid progenitors (CMP) with mature neutrophilic Thy1.1-antigen levels and showed a clear differentiation bias and transcriptome signature towards myelopoiesis. TC90s overexpressed both CXCL12 and its alternative receptor CXCR7, thereby abrogating their homing capacity and produced multiple secreted matrix components related to neonatal bone marrow niche formation. We hypothesize that the TC90 cell population protects newborns during the critical transition at birth and shortly beyond by boosting a supply of innate immune cells. In summary, our results identify a transient cell stage during postnatal hematopoiesis unique to naked mole-rats.

sentences: SARS-CoV-2 Genome Sequencing Methods Differ In Their Ability To Detect Variants From Low Viral Load Samples SARS-CoV-2 genomic surveillance has been vital in understanding the spread of COVID-19, the emergence of viral escape mutants and variants of concern. However, low viral loads in clinical specimens affect variant calling for phylogenetic analyses and detection of low frequency variants, important in uncovering infection transmission chains. We systematically evaluated three widely adopted SARS-CoV-2 whole genome sequencing methods for their sensitivity, specificity, and ability to reliably detect low frequency variants. Our analyses highlight that the ARTIC v3 protocol consistently displays high sensitivity for generating complete genomes at low viral loads compared with the probe-based Illumina respiratory viral oligo panel, and a pooled long-amplicon method. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high quality sequence data from low viral load samples for public health and genomic surveillance purposes.

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A COMPARATIVE ANALAYSIS OF INTER-SITE GENE EXPRESSION HETEROGENICITY OF NORMAL HUMAN BUCCAL MUCOSA WITH NORMAL GINGIVAL MUCOSA BACKGROUNDDescription of heterogeneity of gene expression of various human intraoral sites are not adequate. The aim of this study was to explore the difference of gene expression profiles of whole tissue obtained from apparently normal human gingiva and buccal mucosa (HGM, HBM).

MATERIALS AND METHODSGene sets fulfilling inclusion and exclusion criteria of HGM and HBM in gene Expression Omnibus(GEO) database were identified, segregated, filtered and analysed using the ExAtlas online web tool using pre-determined cut-off. The differentially expressed genes were studied for epithelial keratinization related, housekeeping(HKG), extracellular matrix related(ECMRG) and epithelial-mesenchymal transition related genes(EMTRGs).

RESULTSIn all 40 HBM and 64 HGM formed the study group. In all there were 18012 significantly expressed genes. Of this, 1814 were over-expressed and 1862 under-expressed HBM genes as compared to HGM. One in five of all studied genes significantly differed between HBM and HGM. For the keratinization genes, 1 in 6 differed. One of every 5 HKG-proteomics genes differed between HBM and HGM, while this ratio was 1-in 4 for all ECMRGs and EMTRGs.

DISCUSSIONThis difference in the gene expression between the HBM and HGM could possibly influence a multitude of biological pathways. This result could explain partly the difference in clinicopathological features of oral lesions occurring in HBM and HGM. The innate genotypic difference between the two intra-oral niches could serve as confounding factor in genotypic studies. Hence studies that compare the HBM and HGM should factor-in these findings while evaluating their results.

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Hunger improves reinforcement-driven but not planned action Human decisions can be reflexive or planned, being governed respectively by model-free and model-based learning systems. These two systems might differ in their responsiveness to our needs. Hunger drives us to specifically seek food rewards, but here we ask whether it might have more general effects on these two decision systems. On one hand, the model-based system is often considered flexible and context-sensitive, and might therefore be modulated by metabolic needs. On the other hand, the model-free systems primitive reinforcement mechanisms may have closer ties to biological drives. Here, we tested participants on a well-established two-stage sequential decision-making task that dissociates the contribution of model-based and model-free control. Hunger enhanced overall performance by increasing model-free control, without affecting model-based control. These results demonstrate a generalised effect of hunger on decision-making that enhances reliance on primitive reinforcement learning, which in some situations translates into adaptive benefits.

Significance statementThe prevalence of obesity and eating disorder is steadily increasing. To counteract problems related to eating, people need to make rational decisions. However, appetite may switch us to a different decision mode, making it harder to achieve long-term goals. Here we show that planned and reinforcement-driven actions are differentially sensitive to hunger. Hunger specifically affected reinforcement-driven actions, and did not affect the planning of actions. Our data shows that people behave differently when they are hungry. We also provide a computational model of how the behavioural changes might arise.

sentences: Hippocampal and thalamic afferents form distinct synaptic microcircuits in the mouse frontal cortex Neural circuits within the frontal cortex support the flexible selection of goal-directed behaviors by integrating input from brain regions associated with sensory, emotional, episodic, and semantic memory functions. From a connectomics perspective, determining how these disparate afferent inputs target their synapses to specific cell types in the frontal cortex may prove crucial in understanding circuit-level information processing. Here, we used monosynaptic retrograde rabies mapping to examine the distribution of afferent neurons targeting four distinct classes of local inhibitory interneurons and four distinct classes of excitatory projection neurons in mouse infralimbic cortex. Interneurons expressing parvalbumin, somatostatin, or vasoactive intestinal peptide received a large proportion of inputs from hippocampal regions, while interneurons expressing neuron-derived neurotrophic factor received a large proportion of inputs from thalamic regions. A more moderate hippocampal-thalamic dichotomy was found among the inputs targeting excitatory neurons that project to the basolateral amygdala, lateral entorhinal cortex, nucleus reuniens of the thalamus, and the periaqueductal gray. Together, these results show a prominent bias among hippocampal and thalamic afferent systems in their targeting to genetically or anatomically defined sets of frontal cortical neurons. Moreover, they suggest the presence of two distinct local microcircuits that control how different inputs govern frontal cortical information processing.

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Distinct and common features of numerical and structural chromosomal instability across different cancer types A large proportion of tumours is characterised by numerical or structural chromosomal instability (CIN), defined as an increased rate of gaining or losing whole chromosomes (W-CIN) or of accumulating structural aberrations (S-CIN). Both W-CIN and S-CIN are associated with tumourigenesis, cancer progression, treatment resistance and clinical outcome. Although W-CIN and S-CIN can co-occur, they are initiated by different molecular events. By analysing tumour genomic data from 33 cancer types, we show that the majority of tumours with high levels of W-CIN underwent whole genome doubling, whereas S-CIN levels are strongly associated with homologous recombination deficiency. Both CIN phenotypes are prognostic in several cancer types. Most drugs are less efficient in high-CIN cell lines, but we also report compounds and drugs which should be investigated as targets for W-CIN or S-CIN. By analysing associations between CIN and bio-molecular entities with pathway and gene expression levels, we complement gene signatures of CIN and report that the drug resistance gene CKS1B is strongly associated with S-CIN. Finally, we propose a potential copy number-dependent mechanism to activate the PI3K pathway in high-S-CIN tumours.

1. Simple summaryMany cancer cells are chromosomally unstable, a phenotype describing a tendency for accumulating chromosomal aberrations. Entire chromosomes tend to be gained or lost, which is called whole chromosome instability (W-CIN). Structural chromosomal instability (S-CIN) describes an increased rate of gaining, losing or translocating smaller parts of chromosomes. Here, we analyse data from 33 cancer types to find differences and commonalities between W-CIN and S-CIN. We find that W-CIN is strongly linked to whole genome doubling (WGD), whereas S-CIN is associated with a specific DNA damage repair pathway. Both W-CIN and S-CIN are difficult to target using currently available compounds and have distinct prognostic values. The activity of the drug resistance gene CKS1B is associated with S-CIN, which merits further investigation. In addition, we identify a potential copy number-based mechanism promoting signalling of the important PI3K cancer pathway in high-S-CIN tumours.

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Convergent evolution of SARS-CoV-2 spike mutations, L452R, E484Q and P681R, in the second wave of COVID-19 in Maharashtra, India As the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expands, genomic epidemiology and whole genome sequencing are being constantly used to investigate its transmissions and evolution. In the backdrop of the global emergence of "variants of concern" (VOCs) during December 2020 and an upsurge in a state in the western part of India since January 2021, whole genome sequencing and analysis of spike protein mutations using sequence and structural approaches was undertaken to identify possible new variants and gauge the fitness of current circulating strains.

Phylogenetic analysis revealed that the predominant clade in circulation was a distinct newly identified lineage B.1.617 possessing common signature mutations D111D, G142D, L452R, E484Q, D614G and P681R, in the spike protein including within the receptor binding domain (RBD). Of these, the mutations at residue positions 452, 484 and 681 have been reported in other globally circulating lineages. The structural analysis of RBD mutations L452R and E484Q along with P681R in the furin cleavage site, revealed that these may possibly result in increased ACE2 binding and rate of S1-S2 cleavage resulting in better transmissibility. The same two RBD mutations indicated decreased binding to select monoclonal antibodies (mAbs) and may affect their neutralization potential. Experimental validation against a wider panel of mAbs, sera from vaccinees and those that recovered from natural infection needs to be studied.

The emergence of such local variants through the accumulation of convergent mutations during the COVID-19 second wave needs to be further investigated for their public health impact in the rest of the country and its possibility of becoming a VOC.

sentences: Quantitative Profiling of Adaptation to Cyclin E Overproduction Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Cyclin E overproduction is toxic to mammalian cells, although the gene encoding cyclin E (CCNE1) is overexpressed in some cancers. To gain insight into how cancer cells tolerate high cyclin E, we extensively characterized non-transformed epithelial cells throughout a time course of chronic cyclin E overproduction. Cells overproducing human cyclin E, but not cyclin D or cyclin A, briefly experienced truncated G1 phases, then endured a transient period of DNA replication origin underlicensing, replication stress, and severely impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite maintaining high cyclin E-associated activity. Transcriptome analysis revealed that adapted cells downregulated a cohort of G1-regulated genes. Withdrawing cyclin E induction only partially reversed the intermediate licensing phenotype of adapted cells indicating that adaptation is at least partly independent of mutations. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing by an indirect mechanism through premature S phase onset and provides further insight into the relationship between CDK activity and licensing in mammals. It serves as an example of specific oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

sentences: Human iPSC-derived cerebral organoids model features of Leigh Syndrome and reveal abnormal corticogenesis Leigh syndrome (LS) is a rare, inherited neurometabolic disorder that presents with bilateral brain lesions, caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded proteins. We generated iPSCs from three patient-derived LS fibroblast lines and identified, by whole-exome and mitochondrial sequencing, unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). LS-derived iPSC lines were viable and generally capable of differentiating into key progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. LS-derived cerebral organoids showed defects in neural epithelial bud generation, size, and cortical architecture at 100 days. The double mutant MT-ATP6/PDH line produced organoid neural progenitor cells with abnormal mitochondrial morphology characterized by fragmentation and disorganization and showed an increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that can be used as LS model systems.

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Caveolin-1 temporal modulation enhances trastuzumab and trastuzumab-drug conjugate efficacy in heterogeneous gastric cancer Resistance mechanisms and heterogeneity in HER2-positive gastric cancers (GC) limit trastuzumab benefit in 32% of patients, and other targeted therapies have failed in clinical trials. Using genomic data from patient tissue, patient-derived xenografts (PDXs), partially humanized biological models, and HER2-targeted imaging we identified caveolin-1 (CAV1) as a complementary biomarker in GC selection for trastuzumab therapy. In retrospective analyses of samples from patients enrolled on trastuzumab trials, the CAV1-high profile was associated with low membrane HER2 density and reduced patient survival. We found a negative correlation between CAV1 tumoral protein levels - a major protein of cholesterol-rich membrane domains - and trastuzumab-drug conjugate TDM1 tumor uptake. Finally, CAV1 depletion using knockdown or pharmacologic approaches was shown to increase HER2-directed immunoPET uptake and TDM1 efficacy in GC with incomplete HER2 membranous reactivity. In support of these findings, background statin use in patients is associated with enhanced antibody efficacy. Together, this work provides mechanistic justification and clinical evidence that require prospective investigation of HER2-targeted therapies combined with statins to delay drug resistance in GC.

SIGNIFICANCEThis study identifies how CAV1 protein expression and statin use relate to GC response in HER2-targeted imaging and therapeutic approaches. In addition, to support the synergy of CAV1 depletion with TDM1 observed in mouse models, we demonstrate that statin users had better clinical responses to antibody-based therapies in HER2+ GC.

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Probiotic consortia improve anti-viral immunity to SARS-CoV-2 in ferrets Probiotics have been suggested as one solution to counter detrimental health effects by SARS-CoV-2, however, data so far is scarce. We tested the effect of two probiotic consortia, OL-1 and OL-2, against SARS-CoV-2 in ferrets and assessed their effect on cytokine production and transcriptome in a human monocyte-derived macrophage (Mf) and dendritic cell (DC) model.

The results showed that the consortia significantly reduced the viral load, modulated immune response, and regulated viral receptor expression in ferrets compared to placebo. In human Mf and DC model, OL-1 and OL-2 induced cytokine production and genes related to SARS-CoV-2 anti-viral immunity.

The study results indicate that probiotic stimulation of the ferret immune system leads to improved anti-viral immunity against SARS-COV-2 and that critical genes and cytokines for anti-SARS-CoV-2 immunity are stimulated in human immune cells in vitro. The effect of the consortia against SARS-CoV-2 warrants further investigations in human clinical trials.

sentences: Astrocyte-derived thrombospondin induces cortical synaptogenesis in a sex-specific manner. The regulation of synaptic connectivity in the brain is vital to proper functioning and development of the central nervous system (CNS). Formation of neural networks in the CNS has been shown to be heavily influenced by astrocytes, which secrete factors, including thrombospondin (TSP) family proteins, that promote synaptogenesis. However, whether this process is different between males and females has not been thoroughly investigated. In this study, we found that cortical neurons purified from newborn male rats showed a significantly more robust synaptogenic response compared to female-derived cells when exposed to factors secreted from astrocytes. This difference was driven largely by the neuronal response to TSP2, which increased synapses in male neurons while showing no effect on female neurons. Blockade of endogenous 17{beta}-estradiol production with letrozole normalized the TSP response between male and female cells, indicating a level of regulation by estrogen signaling. Our results suggest that TSP-induced synaptogenesis is critical for the development of male but not female cortical synapses, contributing to sex differences in astrocyte-mediated synaptic connectivity.

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A locus conferring tolerance to Theileria infection in African cattle East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Boran cattle demonstrating heritable tolerance to infection by T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on Bos taurus chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained polymorphism in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africas most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.

Author SummaryMore than a million cattle die of East Coast fever in Africa each year, the impact of which disproportionately falls onto low-income, smallholder farmers. The lack of a widely accessible vaccine, heavy reliance on chemicals to control the tick vector and inadequate drug treatments means that new approaches for controlling the disease are urgently required. Through a genetic study of an extended pedigree of Boran cattle that are more than three times less likely to succumb to the disease than matched controls, we identify a region on chromosome 15 of the cattle genome associated with a high level of tolerance to the disease. We show that a variant in this region is also associated with survival in an independent cohort, and is linked to rates of cell expansion during infection. This genetic variant can therefore support marker-assisted selection, allowing farmers to breed tolerant cattle and offers a route to introduce this beneficial DNA to non-native breeds, enabling reduced disease incidence and increased productivity, which would be of benefit to millions of rural smallholder farmers across Africa.

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IPMK modulates insulin-mediated suppression of hepatic glucose production Hepatic glucose production is crucial for the maintenance of normal glucose homeostasis. Although hepatic insulin resistance contributes to excessive glucose production, its mechanism is not well understood. Here, we show that inositol polyphosphate multikinase (IPMK), a key enzyme in inositol polyphosphate biosynthesis, plays a role in regulating hepatic insulin signaling and gluconeogenesis both in vitro and in vivo.IPMK-deficient hepatocytes exhibit decreased insulin-induced activation of Akt-FoxO1 signaling. The expression of mRNA levels of phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose 6-phosphatase (G6pc), key enzymes mediating gluconeogenesis, are increased in IPMK-deficient hepatocytes compared to wild type (WT) hepatocytes. Importantly, re-expressing IPMK restores insulin sensitivity and alleviates glucose production in IPMK-deficient hepatocytes. Moreover, hepatocyte-specific IPMK deletion exacerbates hyperglycemia and insulin sensitivity in mice fed a high-fat diet (HFD), accompanied by an increase in hepatic glucose production during pyruvate tolerance test and reduction in Akt phosphorylation in IPMK deficient liver. Our results demonstrate that IPMK mediates insulin signaling and gluconeogenesis and may be potentially targeted for treatment of diabetes.

HighlightsIPMK expression is reduced in livers of HFD-fed mice.

Hepatocyte-specific deletion of IPMK in mice aggravated HFD-induced insulin resistance.

Loss of IPMK decreased insulin-induced activation of Akt-FoxO1 signaling, leading to the increase of glucose production in hepatocytes.

sentences: Aβ/APP-induced hyperexcitability and dysregulation of homeostatic synaptic plasticity in models of Alzheimer's disease The proper function of the nervous system is dependent on the appropriate timing of neuronal firing. Synapses continually undergo rapid activity-dependent modifications that require feedback mechanisms to maintain network activity within a window in which communication is energy efficient and meaningful. Homeostatic synaptic plasticity (HSP) and homeostatic intrinsic plasticity (HIP) are such negative feedback mechanisms. Accumulating evidence implicates that Alzheimers disease (AD)-related amyloid precursor protein (APP) and its cleavage product amyloid-beta (A{beta}) play a role in the regulation of neuronal network activity, and in particular HSP. AD features impaired neuronal activity with regional early hyper-activity and A{beta}-dependent hyperexcitability has also been demonstrated in AD transgenic mice. We demonstrate similar hyper-activity in AD transgenic neurons in culture that have elevated levels of both human APP and A{beta}. To examine the individual roles of APP and A{beta} in promoting hyperexcitability we used an APP construct that does not generate A{beta}, or elevated A{beta} levels independently of APP. Increasing either APP or A{beta} in wild type (WT) neurons leads to increased frequency and amplitude of calcium transients. Since HSP/HIP mechanisms normally maintain a setpoint of activity, we examined whether homeostatic synaptic/intrinsic plasticity was altered in AD transgenic neurons. Using methods known to induce HSP/HIP, we demonstrate that APP protein levels are regulated by chronic modulation of activity and show that AD transgenic neurons have an impaired response to global changes in activity. Further, AD transgenic compared to WT neurons failed to adjust the length of their axon initial segments (AIS), an adaptation known to alter excitability. Thus, we present evidence that both APP and A{beta} influence neuronal activity and that mechanisms of HSP/HIP are disrupted in neuronal models of AD.

sentences: MESOMELIC DYSPLASIAS ASSOCIATED WITH THE HOXD LOCUS ARE CAUSED BY REGULATORY REALLOCATIONS Some human families display severe shortening and bending of the radius and ulna, a condition referred to as mesomelic dysplasia. Many of these families contain chromosomal rearrangements at 2q31, where the human HOXD locus maps. In mice, the dominant X-ray-induced Ulnaless inversion of the HoxD gene cluster produces a similar phenotype suggesting that the same mechanism is responsible for this pathology in humans and mice. Amongst the proposed explanations, the various alterations to the genomic structure of HOXD could expose Hoxd13 to proximal limb enhancers, leading to its deleterious gain-of-expression in the embryonic forelimb. To assess this hypothesis, we used an engineered 1Mb large inversion including the HoxD gene cluster, in order to position Hoxd13 within a chromatin domain rich in proximal limb enhancers. We show that these enhancers contact and activate Hoxd13 in proximal cells, concomitant to the formation of a mesomelic dysplasia phenotype. A secondary mutation in the coding frame of the HOXD13 protein in-cis with the inversion completely rescued the limb alterations, demonstrating that ectopic HOXD13 is indeed the unique cause of this bone anomaly. Single cell expression analysis and evaluation of HOXD13 binding sites in cells from this ectopic expression domain suggests that the phenotype arises primarily by acting through genes normally controlled by HOXD13 in distal limb cells. Altogether, these results provide a conceptual and mechanistic framework to understand and unify the molecular origins of human mesomelic dysplasia associated with 2q31.

sentences: A Microengineered Brain-Chip to Model Neuroinflammation in Humans Species differences in the brain and the blood-brain barrier (BBB) biology hamper the translation from animal models to humans and impede the development of specific therapeutics for brain diseases. Here we present a human Brain-Chip engineered to recapitulate critical aspects of the complex brain cell-cell interactions that mediate neuroinflammation development. Our human organotypic microphysiological system (MPS) includes endothelial-like cells, pericytes, glia, and cortical neurons and maintains BBB permeability at in vivo relevant levels, providing a significant improvement in complexity and clinical mimicry compared to previous MPS models. This is the first report of a Brain-Chip with an RNA expression profile close to that of the adult human cortex and that demonstrates advantages over Transwell culture. Through perfusion of TNF-, we recreated key inflammatory features, such as glia activation, the release of proinflammatory cytokines, and increased barrier permeability. Our model may provide a reliable tool for mechanistic studies in neuron-glial interactions and dysregulation of BBB function during neuroinflammation.

sentences: Clonal evolution in serially passaged Cryptococcus neoformans x deneoformans hybrids reveals a heterogenous landscape of genomic change Cryptococcus neoformans x deneoformans hybrids (also known as serotype AD hybrids) are basidiomycete yeasts that are common in a clinical setting. Like many hybrids, the AD hybrids are largely locked at the F1 stage and are mostly unable to undergo normal meiotic reproduction. However, these F1 hybrids, which display a high ([~]10%) sequence divergence are known to genetically diversify through mitotic recombination and aneuploidy, and this diversification may be adaptive. In this study, we evolved a single AD hybrid genotype in six diverse environments by serial passaging and then used genome resequencing of evolved clones to determine evolutionary mechanisms of adaptation. The evolved clones generally increased fitness after passaging, accompanied by an average of 3.3 point mutations, 2.9 loss of heterozygosity (LOH) events, and 0.7 trisomic chromosomes per clone. LOH occurred through nondisjunction of chromosomes, crossing over consistent with break-induced replication, and gene conversion, in that order of prevalence. The breakpoints of these recombination events were significantly associated with regions of the genome with lower sequence divergence between the parents and clustered in subtelomeric regions, notably in regions that had undergone introgression between the two parental species. Parallel evolution was observed, particularly through repeated homozygosity via nondisjunction, yet there was little evidence of environment-specific parallel change for either LOH, aneuploidy, or mutations. These data show that AD hybrids have both a remarkable genomic plasticity and yet are challenged in the ability to recombine through sequence divergence and chromosomal rearrangements, a scenario likely limiting the precision of adaptive evolution to novel environments.

sentences: Group size planning of breedings of gene-modified animals Animal breeding is time-consuming, costly, and affected by stochastic events related to Mendelian genetics, fertility, and litter size. Careful planning is mandatory to ensure a successful outcome using the least number of animals, hence adhering to the 3Rs of animal welfare. We have developed an R package, accessible also through an interactive website, that optimizes breeding design and provides a comprehensive report suitable for any breeder of genetically defined traits.

sentences: Molecular architecture of SARS-CoV-2 envelope by integrative modeling Despite tremendous efforts by research community during the COVID-19 pandemic, the exact structure of SARS-CoV-2 and related betacoronaviruses remains elusive. Here, we developed and applied an integrative multi-scale computational approach to model the envelope structure of SARS-CoV-2, focusing on studying the dynamic nature and molecular interactions of its most abundant, but largely understudied, M (membrane) protein. The molecular dynamics simulations allowed us to test the envelop stability under different configurations and revealed that M dimers agglomerated into large, filament-like, macromolecular assemblies with distinct molecular patterns formed by Ms transmembrane and intra-virion (endo) domains. These results were in agreement with the experimental data, demonstrating a generic and versatile integrative approach to model the structure of a virus de novo, providing insights into critical roles of structural proteins in the viral assembly and integration, and proposing new targets for the antiviral therapies.

sentences: Designing fisheries restricted areas using standard fishery survey data: a novel multilevel spatio-temporal approach Marine Protected Areas (MPAs) are a promising management tool for the conservation and recovery of marine ecosystems, as well as fisheries management. MPAs are generally established as permanent closures but marine systems are dynamic, which has generated debate in favour of more dynamic designs. As a consequence, the identification of priority areas should assess their persistence in space and time. Here, we develop a step-by-step approach to assess the spatiotemporal dynamics of fisheries management priority areas using standard fishery-independent survey data. To do so, we fit Bayesian hierarchical spatiotemporal SDM models to different commercially important demersal species and use the resulting maps to fit different spatial prioritisation configurations. We use these results to assess the spatiotemporal dynamics of fisheries priority areas. The proposed method is illustrated through a western Mediterranean case study using fishery-independent trawl survey data on six commercially important species collected over 17 years. We identified two fisheries priority area patterns in the study area, each predominant during a different time-period of the study, asserting the importance of regularly re-assessing MPA designs.

sentences: Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods pose as an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5 position, which allows 5-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.

sentences: Single-cell monitoring of dry mass and dry density reveals exocytosis of cellular dry contents in mitosis Cell mass and composition change with cell cycle progression. Our previous work characterized buoyant mass accumulation dynamics in mitosis (Miettinen et al., 2019), but how dry mass and cell composition change in mitosis has remained unclear. To better understand mitotic cell growth and compositional changes, we develop a single-cell approach for monitoring dry mass and the density of that dry mass every [~]75 seconds with 1.3% and 0.3% measurement precision, respectively. We find that suspension grown mammalian cells lose dry mass and increase dry density following mitotic entry. These changes display large, non-genetic cell-to-cell variability, and the changes are reversed at metaphase-anaphase transition, after which dry mass continues accumulating. The change in dry density causes buoyant and dry mass to differ specifically in early mitosis, thus reconciling existing literature on mitotic cell growth. Mechanistically, the dry composition changes do not require mitotic cell swelling or elongation. Instead, cells in early mitosis increase lysosomal exocytosis, and inhibition of exocytosis prevents the dry composition from changing. Overall, our work provides a new approach for monitoring single-cell dry mass and composition and reveals that mitosis is coupled to extensive exocytosis-mediated secretion of cellular contents.

sentences: Covariance-based information processing in reservoir computing systems In biological neuronal networks, information representation and processing are achieved through plasticity learning rules that have been empirically characterized as sensitive to second and higher-order statistics in spike trains. However, most models in both computational neuroscience and machine learning aim to convert diverse statistical properties in inputs into first-order statistics in outputs, like in modern deep learning networks. In the context of classification, such schemes have merit for inputs like static images, but they are not well suited to capture the temporal structure in time series. In contrast, the recently developed covariance perceptron uses second-order statistics by mapping input covariances to output covariances in a consistent fashion. Here, we explore the applicability of covariance-based perceptron readouts in reservoir computing networks to classify synthetic multivariate time series structured at different statistical orders (first and second). We show that the second-order framework outperforms or matches the classical mean paradigm in terms of accuracy. Our results highlight a nontrivial relationship between input and reservoir properties in generating the output reservoir activity, which suggests an important role for recurrent connectivity in transforming information representations in biologically inspired architectures. Finally, we solve a speech recognition task for the classification of spoken digits to further demonstrate the potential of covariance-based decoding for real data.

sentences: Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen Microorganisms survive and compete by stochastically acquiring mutations that enhance fitness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes experiencing strong selective pressures. Infections caused by Cryptococcus neoformans are responsible for [~]15% of AIDS-related deaths and associated with high mortality rates, attributable to a dearth of antifungal drugs and drug resistance. We identified two hypermutator C. neoformans clinical isolates in which Cnl1 transposon insertions were responsible for drug resistance. Whole-genome sequencing revealed both hypermutator genomes harbor a nonsense mutation in the RNAi component ZNF3 and hundreds of Cnl1 elements organized into massive subtelomeric arrays on every chromosome. QTL mapping identified a significant locus associated with hypermutation that included znf3. CRISPR-mediated editing of the znf3 nonsense mutation abolished hypermutation and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from RNAi loss and a significant burden of Cnl1 elements.

sentences: Modelling conformational state dynamics and its role on infection for SARS-CoV-2 Spike protein variants The SARS-CoV-2 Spike protein needs to be in an open-state conformation to interact with ACE2 as part of the viral entry mechanism. We utilise coarse-grained normal-mode analyses to model the dynamics of Spike and calculate transition probabilities between states for 17081 Spike variants. Our results correctly model an increase in open-state occupancy for the more infectious D614G via an increase in flexibility of the closed-state and decrease of flexibility of the open-state. We predict the same effect for several mutations on Glycine residues (404, 416, 504, 252) as well as residues K417, D467 and N501, including the N501Y mutation, explaining the higher infectivity of the B.1.1.7 and 501.V2 strains. This is, to our knowledge, the first use of normal-mode analysis to model conformational state transitions and the effect of mutations thereon. The specific mutations of Spike identified here may guide future studies to increase our understanding of SARS-CoV-2 infection mechanisms and guide public health in their surveillance efforts.

sentences: A causal role for the right frontal eye fields in value comparison Recent studies have suggested close functional links between visual attention and decision making. This suggests that the corresponding mechanisms may interface in brain regions known to be crucial for guiding visual attention - such as the frontal eye field (FEF). Here, we combined brain stimulation, eye tracking and computational approaches to explore this possibility. We show that inhibitory transcranial magnetic stimulation (TMS) over the right FEF has a causal impact on decision-making, reducing the effect of gaze dwell time on choice while also increasing reaction times. We computationally characterize this putative mechanism by using the attentional drift diffusion model (aDDM), which reveals that FEF inhibition reduces the relative discounting of the non-fixated option in the comparison process. Our findings establish an important causal role of the right FEF in choice, elucidate the underlying mechanism, and provide support for one of the key causal hypotheses associated with the aDDM.

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In vivo antigen expression regulates CD4 T cell differentiation and vaccine efficacy against Mycobacterium tuberculosis infection New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to IFN-{gamma} or nutrient/oxygen deprivation of in vitro infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analysed their corresponding CD4 T cell phenotype and vaccine-protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination and, against the overexpressing strain, vaccination with MPT70 conferred similar protection as ESAT-6. Together our data indicate that high in vivo antigen expression drives T cells towards terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less-differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune-balance in favor of the host.

ImportanceTuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current COVID-19 pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunisation with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.

sentences: Sizing up spotted lanternfly nymphs for instar determination and growth allometry A major ongoing research effort seeks to understand the behavior, ecology and control of the spotted lanternfly (SLF) (Lycorma delicatula), a highly-invasive pest in the U.S. and South Korea. These insects undergo four nymphal stages (instars) before reaching adulthood, and appear to shift host plant preferences, feeding, dispersal and survival patterns, anti-predator behaviors, and response to traps and chemical controls, with each stage. However, categorizing SLF lifestage is challenging for the first three instars, which have the same coloration and shape, because no comprehensive allometric datasets exist. We present a dataset of body mass and length for SLF nymphs throughout a growing season and compare our results with published ranges of instar body lengths based on small samples. An ontogenetic allometric analysis found that SLF nymph body mass scales isometrically with body length (exponent c = 3.05 [2.95,3.15]). An analysis using two clustering methods also revealed that first through third instar body mass and length fell into distinct clusters (Dyars rule), supporting using these two metrics to stage nymphs during a single growing season. The ranges for 2nd and 3rd instars were not consistent between our results and those from earlier studies for diverse locations. Using previously-published data, we also found that tarsal claw and arolia (adhesive footpad) dimensions scale in proportion to body length and mass, respectively, indicating that adhesive ability does not decrease with age, as posited in some previous studies. Conversely, mouthpart dimensions do not correlate with body length, consistent with predictions that these features should reflect preferred host plant characteristics rather than body size. We suggest extending these methods to study how SLF instar development depends on factors such as hatch date, host plant, temperature, and geographic location, using citizen scientist networks to collect morphometric data for a wide range of locations and environmental conditions.

sentences: Effects of pen enrichment on leg health of fast and slower-growing broiler chickens Pen enrichment for broiler (meat-type) chickens is one of the potential strategies to stimulate locomotion and consequently contribute to leg health and welfare. This study was designed to evaluate effects of using a plethora of pen enrichments (barrier perches, angular ramps, horizontal platforms, large distance between feed and water and providing live Black Soldier fly larvae in a dustbathing area) on tibia characteristics, locomotion, leg health and home pen behaviour of fast and slower-growing broiler chickens. The experiment was set up as a 2 x 2 factorial arrangement with a total of 840 male broiler chickens in a complete randomized design (7 replicates per treatment and 30 chickens per replicate) with the following treatments: 1) pen enrichment (enriched pen or non-enriched pen); 2) broiler strain (fast-growing Ross 308 or slower-growing Hubbard JA 757). Home pen behaviour and use of enrichment were observed. At approximately 1400 and 2200 gram body weight, two chickens per pen were randomly selected and slaughtered, to investigate tibia morphological, biophysical and mechanical characteristics and leg health. Pen enrichment positively affected tibia biophysical characteristics, e.g., osseous volume ({Delta}=1.8 cm3, P=0.003), total volume ({Delta}=1.4 cm3, P=0.03) and volume fraction ({Delta}=0.02 %, P=0.002), in both fast and slower-growing chickens, suggesting that pen enrichment particularly affects ossification and mineralization mechanisms. Accordingly, locomotion and active behaviours were positively influenced by pen enrichment. However, pen enrichment resulted in lower body weight gain in both strains, which might be due to higher activity or lower feed intake as a result of difficulties of crossing the barrier perches. Regarding the strain, slower-growing chickens showed consistently more advanced tibia characteristics and more active behaviour than fast-growing chickens. It can be concluded that pen enrichment may lead to more activity and better bone development in both fast and slower-growing chickens.

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Information-Content-Informed Kendall-tau Correlation: Utilizing Missing Values Almost all correlation measures currently available are unable to handle missing values. Typically, missing values are either ignored completely by removing them or are imputed and used in the calculation of the correlation coefficient. In both cases, the correlation value will be impacted based on a perspective that missing data represents no useful information. However, missing values occur in real data sets for a variety of reasons. In omics data sets that are derived from analytical measurements, the primary reason for missing values is that a specific measurable phenomenon falls below the detection limits of the analytical instrumentation. These missing data are not missing at random, but represent some information by their "missingness." Therefore, we propose an information-content-informed Kendall-tau (ICI-Kt) correlation coefficient that allows missing values to carry explicit information in the determination of concordant and discordant pairs. With both simulated and real data sets from RNA-seq experiments, we demonstrate that the ICI-Kt allows for the inclusion of missing data values as interpretable information. Moreover, our implementation of ICI-Kt uses a mergesort-like algorithm that provides O(nlog(n)) computational performance. Finally, we show that approximate ICI-Kt correlations can be calculated using smaller feature subsets of large data sets with significant time savings, which has practical computational value when feature sizes are very large.

The ICI-Kt correlation calculation is available in an R package and Python module on GitHub at https://github.com/moseleyBionformaticsLab/ICIKendallTau and https://github.com/moseleyBionformaticsLab/icikt, respectively.

sentences: Targeted RNA-seq improves efficiency, resolution, and accuracy of allele specific expression for human term placentas Genomic imprinting is an epigenetic mechanism that results in allele specific expression (ASE) based on parent of origin. It is known to play a role in the prenatal and postnatal allocation of maternal resources in mammals. ASE detected by whole transcriptome RNA-seq (wht-RNAseq) has been widely used to analyze imprinted genes using reciprocal crosses in mice to generate large numbers of informative SNPs. Studies in humans are more challenging due to the paucity of SNPs and the poor preservation of RNA in term placentas and other tissues. Targeted RNA-seq (tar-RNAseq) can potentially mitigate these challenges by focusing sequencing resources on the regions of interest in the transcriptome. Here we compared tar-RNAseq and wht-RNAseq in a study of ASE in known imprinted genes in placental tissue collected from a healthy human cohort in Mali, West Africa. As expected, tar-RNAseq substantially improved the coverage of SNPs. Compared to wht-RNAseq, tar-RNAseq produced on average four times more SNPs in twice as many genes per sample and read depth at the SNPs increased 4-fold. In previous research on humans, discordant ASE values for SNPs of the same gene have limited the ability to accurately quantify ASE. We show that tar-RNAseq reduces this limitation as it unexpectedly increased the concordance of ASE between SNPs of the same gene, even in cases of degraded RNA. Studies aimed at discovering associations between individual variation in ASE and phenotypes in mammals and flowering plants will benefit from the improved power and accuracy of tar-RNAseq.

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Pan-cancer landscape of AID-related mutations, composite mutations and its potential role in the ICI response Activation-induced cytidine deaminase, AICDA or AID, is a driver of somatic hypermutation and class-switch recombination in immunoglobulins. In addition, this deaminase belonging to the APOBEC family, may have off-target effects genome-wide, but its effects at pan-cancer level are not well elucidated. Here, we used different pan-cancer datasets, totaling more than 50,000 samples analyzed by whole-genome, whole-exome or targeted sequencing. AID synergizes initial hotspot mutations by a second composite mutation. Analysis of 2.5 million cells, normal and oncogenic, revealed AICDA expression activation after oncogenic transformation and cell cycle regulation loss. AID mutational load was found to be independently associated with favorable outcome in immune-checkpoint inhibitors (ICI) treated patients across cancers after analyzing 2,000 samples. Finally, we found that AID related neoepitopes, resulting from mutations at more frequent hotspots if compared to other mutational signatures, enhance CXCL13/CCR5 expression, immunogenicity and T-cell exhaustion, which may increase ICI sensitivity.

In BriefA combined bulk and single cell multi-omic analysis of over 50,000 patients and 2.5 million cells across 80 tumor types reveals oncogenic acquired AICDA expression inducing composite mutations and clonal immunogenic neoepitopes that are associated with favorable outcome in patients treated by immune-checkpoint inhibitors.

Highlights* Pan-cancer analysis of AID mutations using > 50,000 samples, 2,000 ICI treated cases and 2.5 million cells with genome, exome and transcriptome data
* Oncogenic transient AICDA expression induces mutations mainly during transcription of its off-target genes in virtually all cancers
* AID is implicated in composite mutations on weakly functional alleles and immunogenic clonal neoepitopes at hotspots with greater positive selection
* AID mutational load predicts response and is associated with favorable outcome in ICI treated patients

sentences: Mechanistic insights of radiation-induced endothelial senescence impelling glioblastoma genomic instability at relapse Despite aggressive clinical protocol, all glioblastoma (GBM) recur at the initial site within the irradiated peritumoral microenvironment. Whereas irradiated microenvironment has been recently proposed to accelerate GBM relapse, molecular and cellular mechanisms remain unknown. Here, using relevant in vitro and in vivo models, we decipher how radiation-induced endothelial senescence drives the emergence of aggressive GBM cells. Secretome (SASP) of radiation-induced senescent (RIS) endothelium enhances genomic instability and intratumoral heterogeneity in irradiated GBM cells. In-depth molecular studies revealed that CXCL5 and CXCL8, from the SASP, activate CXCR2 receptor on tumor cells leading to increased DNA hyper-replication, micronuclei formation and aneuploidy. Importantly, through CXCL5/8-CXCR2 axis activation, this SASP increases GBM aggressiveness in vivo. Both chemokines were detected in relapsing, but not primary, GBM biopsies and positively correlated with worst patient outcome. In conclusion, we identify new molecular and preclinical insights of relapsing GBM aggressiveness where RIS vascular niches fuel aggressive tumor emergence.

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TPR1, a novel rifampin derivative demonstrates efficacy alone and in combination with doxycycline against the NIAID Category A Priority Pathogen F. tularensis ObjectivesFrancisella tularensis is a highly virulent and contagious gram-negative intracellular bacterium that causes the disease tularemia in mammals and is classified as a Category A priority pathogen. The high infectivity, difficulty of obtaining a durable cure of disseminated disease and the emergence of drug resistance warrants investigation of new drugs that can be used alone and in combination with existing standard of care clinical drugs.

MethodsWe utilized a systematic analysis of antibacterial potency, extent of dissemination by analysis of bacterial burden in a secondary vital organ, and survival rates to assess the efficacy of a novel rifampicin derivative, TPR1. The efficacy of TPR1 was evaluated alone and in combination with the standard of care drug, doxycycline, against Type A F. tularensis Schu S4 using a lethal pulmonary model of infection in mice.

ResultsTPR1 has an MIC value range of 0.125-4 mg/L against reference laboratory strain SchuS4 and a panel of clinical strains. TPR1 alone reduced the bacterial burden in the lungs and spleen at 40 mg/kg and 80 mg/kg, and no antagonism was observed when co-administered with doxycycline. Dosing at 40 mg/kg doxycycline reduced the bacterial burden by 1 Log10 CFU in the lungs and 4 Log10 CFU spleen in comparison to untreated controls. Co-administration of TPR1 and doxycycline demonstrated efficacy upon treatment withdrawal after 4 days of treatment, and 100% survival.

ConclusionsSignificantly, TPR1 demonstrated efficacy when delivered alone and in combination with doxycycline, which provides compelling evidence of a superior treatment strategy that would normally rely on a single chemotherapeutic for efficacy. In addition, this work substantiates the use of rifampicin derivatives as a platform for the development of novel treatments to other bacterial agents in addition to tularemia.

sentences: Biological mechanisms of aging predict age-related disease multimorbidities in patients Genetic, environmental and pharmacological interventions into the aging process can confer resistance to a multiple age-related diseases in laboratory animals, including rhesus monkeys. These findings imply that mechanisms of aging might contribute to patterns of multimorbidity in humans, and hence could be targeted to prevent multiple conditions simultaneously. To address this question, we text mined 917,645 literature abstracts followed by manual curation, and found strong, non-random associations between age-related diseases and aging mechanisms, confirmed by gene set enrichment analysis of GWAS data. Integration of these associations with clinical data from 3.01 million patients showed that age-related diseases associated with each of five aging mechanisms were more likely than chance to be present together in patients. Genetic evidence revealed that innate and adaptive immunity, the intrinsic apoptotic signalling pathway and activity of the ERK1/2 pathway played a significant role across multiple aging mechanisms and multiple, diverse age-related diseases. Mechanisms of aging therefore contribute to multiple age-related diseases and to patterns of human age-related multimorbidity, and could potentially be targeted to prevent more than one age-related condition in the same patient.

sentences: A Fully Implantable Wireless Bidirectional Neuromodulation System for Mice Novel research in the field of bioelectronic medicine requires systems that pair high-performance neurostimulation and bio-signal acquisition hardware with advanced software signal processing and control algorithms. Although mice are the most commonly used animal in medical research, the size, weight, and power requirements of such systems either preclude their use or impose significant constraints on experimental design. Here, we describe a fully-implantable neuromodulation system suitable for use in mice, measuring 2.2 cm3 and weighing 2.8 g. A bidirectional wireless interface allows simultaneous readout of multiple physiological signals and complete control over stimulation parameters, and a wirelessly rechargeable battery provides a lifetime of up to 5 days on a single charge. The device was successfully implanted (N=12) and a functional neural interface (capable of inducing acute bradycardia) is demonstrated with functional lifetimes exceeding to three weeks. The design utilizes only commercially-available components and 3D-printed packaging, with the goal of accelerating discovery and translation of future bioelectronic therapeutics.

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Top-down contribution to motor network reorganization during action preparation BACKGROUNDIt is unclear whether the Bereitschaftspotential (BP) recorded in humans during action preparation mirrors motor areas activation escalation, or if its early and late phases reflect the engagement of different functional networks.

OBJECTIVEHere, we aimed at recording the TMS evoked-potentials (TEP) stimulating the supplementary motor area (SMA) to assess whether and how cortical excitability and functional connectivity of this region change as the BP increases. We hypothesize that, at later stages, the SMA functional network should become more connected to regions relevant for the implementation of the final motor plan.

METHODSWe performed TMS-EEG recordings on fourteen healthy subjects during the performance of a visuomotor Go/No-go task, eliciting and recording cortical activity and functional connectivity at -700 ms and -300 ms before the onset of visual stimuli over the SMA.

RESULTSWhen approaching stimulus onset, and thus BP peak, the SMA increased its functional connectivity with movement-related structures in the gamma and alpha bands, indicating a regional top-down preparation to implement the motor act. Beta-band connectivity, instead, was maintained constant for the whole BP time-course, being potentially related to sustained attention required by the experimental task.

CONCLUSIONThese findings reveal that the BP is not a mere result of increased activation of the SMA, but the functional networks in which this region is involved qualitatively changes over time, becoming more related to the execution of the motor act.

sentences: NON-LINEAR SUSCEPTIBILITY TO INTERFERENCES IN DECLARATIVE MEMORY FORMATION After encoding, memories go through a labile state followed by a stabilization process known as consolidation. Once consolidated they can enter a new labile state after the presentation of a reminder of the original memory, followed by a period of restabilization (reconsolidation). During these periods of lability the memory traces can be modified. Currently, there are studies that show a rapid stabilization after 30 min, while others show that stabilization occurs after longer periods (e.g. 6 h). Here we investigate the effect of an interference treatment on declarative memory consolidation, comparing distinct time intervals after acquisition. On day 1, participants learned a list of non-syllable pairs (List 1). Immediately after, 30 min, 3 h or 8 h later, they received an interference list (List 2) that acted as an amnesic agent. On day 2 (48 h after training) participants had to recall List 1 first, followed by List 2. We found that the List 1 memory was susceptible to interference when the List 2 was administered immediately or 3 h after learning; however, shortly after acquisition (e.g. 30 min) the List 1 memory becomes transiently protected against interference. We propose the possibility that this rapid memory protection could be induced by a fast and transient neocortical integration (where the memory is transiently protected) becoming partially independent from the hippocampus followed by a hippocampal re-engagement where the memory becomes susceptible to interferences again. Our results open a discussion about the contribution of molecular and systemic aspects to memory consolidation.

sentences: An automated microfluidic platform integrating functional vascularized organoids-on-chip The development of vascular networks on-chip is crucial for the long-term culture of three-dimensional cell aggregates such as organoids, spheroids, tumoroids, and tissue explants. Despite the rapid advancement of microvascular network systems and organoid technology, vascularizing organoids-on-chips remains a challenge in tissue engineering. Moreover, most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical settings to operate. Considering these constraints, we developed an innovative platform to establish and monitor the formation of endothelial networks around model spheroids of mesenchymal and endothelial cells as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 15 days on-chip. Importantly, these networks were functional, demonstrating intravascular perfusion within the spheroids or vascular organoids connected to neighbouring endothelial beds. This microphysiological system thus represents a viable organ-on-chip model to vascularize biological tissues and should allow to establish perfusion into organoids using advanced microfluidics.

sentences: Reverse engineering environmental metatranscriptomes clarifies best practices for eukaryotic assembly Diverse communities of microbial eukaryotes in the global ocean provide a variety of essential ecosystem services, from primary production and carbon flow through trophic transfer to cooperation via symbioses. Increasingly, these communities are being understood through the lens of omics tools, which enable high-throughput processing of communities of multiple species. Metatranscriptomics in particular offers an understanding of real-time gene expression in microbial eukaryotic communities, providing a window into community metabolic activity. However, these approaches are rarely validated. A systematic validation of metatranscriptome assembly and annotation methods is necessary to appropriately assess (1) the fidelity of our community composition measurements and (2) the confidence of novel taxonomic and functional content discovered with metatranscriptomics. Here, we present euk rhythmic, an automated and customizable multi-assembler workflow for eukaryotic metatranscriptome assembly, annotation, and analysis, and validate the ability of the pipeline to recapitulate simulated eukaryotic community-level expression data treated as a gold standard. Finally, we apply this pipeline to two previously published metatranscriptomic datasets, showing both recapitulation of previous results as well as enhanced taxonomic and functional gene discovery.

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Electrophysiological correlates of mood and reward dynamics in human adolescents Despite its omnipresence in everyday interactions and its importance for mental health, mood and its neuronal underpinnings are poorly understood. Computational models can help identify parameters affecting self-reported mood during mood induction tasks. Here we test if computationally modelled dynamics of self-reported mood during monetary gambling can be used to identify trial-by-trial variations in neuronal activity. To this end, we shifted mood in healthy (N=24) and depressed (N=30) adolescents by delivering individually tailored reward prediction errors whilst recording magnetoencephalography (MEG) data. Following a pre-registered analysis, we hypothesize that expectation (defined by previous reward outcomes) would be predictive of beta-gamma oscillatory power (25-40Hz), a frequency shown to modulate to reward feedback. We also hypothesize that trial variations in the evoked response to the presentation of gambling options and in source localized responses to reward feedback. Through our multilevel statistical analysis, we found confirmatory evidence that beta-gamma power is positively related to reward expectation during mood shifts, with possible localized sources in the posterior cingulate cortex. We also confirmed reward prediction error to be predictive of trial-level variations in the response of the paracentral lobule and expectation to have an effect on the cerebellum after presentation of gambling options. To our knowledge, this is the first study to relate fluctuations in mood on a minute timescale to variations in neural oscillations with noninvasive electrophysiology.

Significance StatementBrain mechanisms underlying mood and its relationship with changes in reward contingencies in the environment are still elusive but could have a strong impact on our understanding and treatment of debilitating mood disorders. Building on a previously proposed computational mood model we use multilevel statistical models to find relationship between trial-by-trial variations in model components of mood and neural responses to rewards measured with non-invasive electrophysiology (MEG). Through confirmatory analysis we show that it is possible to observe relationships between trial variations in neural responses and computational parameters describing mood dynamics. Identifying the dynamics of mood and the neural processes it affects could pave the way for more effective neuromodulation treatments.

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A system for multiplexed selection of aptamers with exquisite specificity without counter-selection Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counter-selection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counter-selection parameters. As a result, counter-selection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel, while also eliminating the need for counter-selection. We employ a platform based on a modified benchtop sequencer to conduct a massively-parallel aptamer screening process that enables the selection of highly-specific aptamers against multiple structurally similar molecules in a single experiment, without any counter-selection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes, and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes.

Significance statementAptamers offer the exciting potential to discriminate between structurally similar small molecules. However, generating such highly specific aptamers has been proven challenging using the conventional process of counter-selection. In this work, we describe a high-throughput screening platform that can characterize the specificity of millions of aptamers towards a group of structurally related molecules in a single experiment and generate exquisitely specific aptamers without any counter-selection. As exemplars, we generated aptamers with high affinity and specificity towards three structurally related kynurenine metabolites using our platform. Our platform can be readily adapted to other small molecule targets and should therefore accelerate the development of aptamer reagents with exquisite specificity.

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Alternative splicing changes are associated with pre-birth adaptation during lung development Gas exchanges are ensured by lung alveoli, which are mainly composed by epithelial alveolar type 1 (AT1), alveolar type 2 (AT2) and capillary endothelial cells (ECs). Alveologenesis starts during late embryonic development and continues after birth and relies on extensive biochemical crosstalk between these cell types. How this crosstalk is modulated to anticipate and accommodate the radical changes occurring at birth is still unclear.

We investigated the alternative splicing (AS) changes occurring during lung development at the embryonic to postnatal transition by performing RNAseq of mouse lungs at distinct developmental stages. We found that most of the AS changes occur at the embryonic to postnatal transition. In addition, we identified hnRNP A1, Cpeb4 and Elavl2/HuB as putative splicing regulators of this transition. We show that the AS of a major pro- angiogenic chemokine, vascular endothelial growth factor A (VEGFA), is differentially regulated at this transition. Remarkably, we found that there is a switch from the predominance of Vegfa 164 to Vegfa 188 just before birth specifically in AT1 cells, whilst in other cell populations Vegfa does not undergo AS changes. Moreover, we identified a novel Vegfa isoform generated by the retention of intron 5, Vegfa i5.

Our results reveal a cell type-specific regulation of Vegfa AS that may constitute a pre- birth adaptation mechanism of the epithelial-endothelial crosstalk, which may be fundamental for the adaptation to breathing and may have implications for pathological conditions.

sentences: Apurinic/apyrimidinic nuclease 1 drives genomic evolution contributing to chemoresistance and tumorigenesis in solid tumor Genomic instability fuels genomic alterations that befit cancer cells with necessary adaptations to keep proliferating and overcome the impact of host anti-tumor immunity and cytotoxic therapy. Since DNA breaks are required for genomic rearrangements to take place, we hypothesized that dysregulated nuclease activity mediates genomic instability in cancer. Using an integrated genomics protocol, we identified a four gene deoxyribonuclease signature correlating with genomic instability in six human cancers which included adenocarcinomas of esophagus (EAC), lung, prostate, stomach, pancreas and triple negative breast cancer. Functional screens confirmed the role of these nucleases in genomic instability and growth of cancer cells. Apurinic/apyrimidinic nuclease 1 (APE1), identified as top nuclease in functional screen, was further investigated in five cell lines representing four solid tumors (EAC, lung, prostate and breast cancer). We demonstrate that chemical as well as transgenic suppression of APE1 impaired growth/colony formation and increased cytotoxicity of chemotherapeutic agent, whereas inhibited spontaneous as well as chemotherapy-induced DNA breaks, homologous recombination (HR) activity and genomic instability in all cancer cell types tested. Treatment with APE1 inhibitor also impaired tumor growth and significantly increased efficacy of a chemotherapeutic agent in a subcutaneous mouse model of EAC. Overexpression of APE1 in normal esophageal epithelial cells increased DNA breaks and HR activity, leading to massive mutational, copy number as well as karyotypic instability. Evaluation of by whole genome sequencing identified HR as the top mutational process activated by APE1. Normal cells overexpressing APE1 grew as tumors in mice and tumors removed from mice displayed additional karyotypic changes, providing evidence of genomic instability in vivo. Overall, our data demonstrate that elevated APE1 dysregulates HR activity, G2/M checkpoint and genome stability thus contributing to tumorigenesis and chemoresistance in cancer. Therefore, inhibitors of APE1 have potential to inhibit growth and increase cytotoxicity of chemotherapeutic agents while minimizing spontaneous as well as chemotherapy-induced genomic damage and instability in EAC and other solid tumors.

sentences: Rapid and strain-specific resistance evolution of Staphylococcus aureus against inhibitory molecules secreted by Pseudomonas aeruginosa Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and there is evidence that their interactions negatively affect disease outcome in patients. At the molecular level, interactions between the two bacterial taxa are well-described, with P. aeruginosa usually being the dominant species suppressing S. aureus through a variety of inhibitory molecules. However, in polymicrobial infections, the two species interact over prolonged periods of time, and S. aureus might evolve resistance against inhibitory molecules deployed by P. aeruginosa. Here, we used experimental evolution to test this hypothesis by exposing three different S. aureus strains (Cowan I, 6850, JE2) to the growth-inhibitory supernatant of P. aeruginosa PAO1 over 30 days. We found that all three S. aureus strains rapidly evolved resistance against inhibitory molecules and show that (i) adaptations were strain-specific; (ii) resistance evolution affected the expression of virulence traits; and (iii) mutations in membrane transporters were the most frequent evolutionary targets. Our work indicates that adaptations of S. aureus to co-infecting pathogens could increase virulence and decrease antibiotic susceptibility, because both virulence traits and membrane transporters involved in drug resistance were under selection. Thus, pathogen co-evolution could exacerbate infections and compromise treatment options.

sentences: SpecSeg: cross spectral power-based segmentation of neurons and neurites in chronic calcium imaging datasets Imaging calcium signals in neurons of awake, behaving animals using single- or multi-photon microscopy facilitates the study of coding in large neural populations. Such experiments produce massive datasets requiring powerful methods to extract responses from hundreds of neurons. We present SpecSeg, a new open-source toolbox for 1) segmentation of regions of interest (ROIs) representing neuronal structures, 2) inspection and manual editing of ROIs, 3) neuropil correction and signal extraction and 4) matching of ROIs in sequential recordings. SpecSeg uses a novel method for ROI registration, based on temporal cross-correlations of low-frequency components derived by Fourier analysis, of each pixel with its neighbors. The approach is insightful and enables ROI detection around neurons or neurites. It works for single- (miniscope) and multi-photon microscopy data, eliminating the need for separate toolboxes. SpecSeg thus provides an efficient and user-friendly approach for analyzing calcium responses in neuronal structures imaged over prolonged periods of time.

sentences: PIKfyve inhibition blocks endolysosomal escape of α-synuclein fibrils and spread of α-synuclein aggregation The inter-cellular prion-like propagation of -synuclein aggregation is emerging as an important mechanism driving the progression of neurodegenerative diseases including Parkinsons disease and multiple system atrophy (MSA). To discover therapeutic strategies reducing the spread of -synuclein aggregation, we performed a genome-wide CRISPR interference screen in a human cell-based model. We discovered that inhibiting PIKfyve dramatically reduced -synuclein aggregation induced with both recombinant -synuclein fibrils and fibrils isolated from MSA patient brain. While PIKfyve inhibition did not affect fibril uptake or -synuclein clearance or secretion, it reduced -synuclein trafficking from the early endosome to the lysosome, thereby limiting fibril escape from the lysosome and reducing the amount of fibrils that reach cytosolic -synuclein to induce aggregation. These findings point to the endolysosomal transport of fibrils as a critical step in the propagation of -synuclein aggregation and a potential therapeutic target.

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Pulse-Width Modulation-based TMS mimics effects of conventional TMS on human primary motor cortex AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSObjectiveC_ST_ABSWe developed a novel transcranial magnetic stimulation (TMS) device to generate flexible stimuli and patterns. The system synthesizes digital equivalents of analog waveforms, relying on the filtering properties of the nervous system. Here, we test the hypothesis that the novel pulses can mimic the effect of conventional pulses on the cortex.

ApproachA second-generation programmable TMS (pTMS2) stimulator with magnetic pulse shaping capabilities using pulse-width modulation (PWM) was tested. A computational and an in-human study on twelve healthy participants compared the neuronal effects of conventional and modulation-based stimuli.

Main resultsBoth the computational modeling and the in-human stimulation showed that the PWM-based system can synthesize pulses to effectively stimulate the human brain, equivalent to conventional stimulators. The comparison includes motor threshold, MEP latency and input-output curve measurements.

SignificancePWM stimuli can fundamentally imitate the effect of conventional magnetic stimuli while adding considerable flexibility to TMS systems, enabling the generation of highly configurable TMS protocols.

HO_SCPLOWIGHLIGHTSC_SCPLOWO_LIThe PWM method promises the implementation of flexible neurostimulation
C_LIO_LIPWM magnetic pulses were well tolerated by the participants without adverse events
C_LIO_LIRMTs and MEPs were compared for PWM and conventional stimuli
C_LIO_LIPWM-equivalent of conventional pulses has relatively similar effects on the cortex
C_LIO_LIThe use of digital synthesis techniques to create novel patterns is a promising method for future neuromodulation
C_LI

sentences: CRISPR Del/Rei: A simple, flexible and efficient pipeline for scarless genome editing Scarless genome editing of induced pluripotent stem cells (iPSCs) is crucial for the precise modeling of genetic disease. Here we present CRISPR Del/Rei, a two-step deletion-reinsertion strategy with high editing efficiency and simple PCR-based screening that generates isogenic clones in ~2 months. We apply our strategy to edit iPSCs at 3 loci with only rare off target editing.

sentences: Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To better understand the underlying pathobiology, we used the nitrofen rat model of CDH and characterized the proteome of hypoplastic CDH lungs at the alveolar stage (E21). Amongst the 218 significantly altered proteins between CDH and control lungs were Tenascin C, CREBBP, LYN and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen and human fetal CDH lungs. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially reversed the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3 associated cytokines (IL-15, IL-9, IL-2) are increased in fetal tracheal aspirates of CDH survivors compared to non-survivors after fetoscopic tracheal occlusion. Using pathway analysis for significantly altered proteins in our proteomic analysis, we observed an enrichment in inflammatory response associated with Epstein Barr Virus and cytokine signaling in nitrofen CDH lungs. However, we were unable to detect EBV mRNA via in-situ Hybridization in human CDH lungs. With our unbiased proteomics approach, we show for the first time that inflammatory processes are likely underlying the pathogenesis of abnormal lung development in CDH.

sentences: Activation of G-protein coupled estradiol receptor 1 in the dorsolateral striatum attenuates preference for cocaine and saccharin in male but not female rats There are sex differences in the response to psychomotor stimulants, where females exhibit a greater response than males, due to the presence of the gonadal hormone estradiol (E2). Extensive research has shown that E2 enhances drug-seeking and the rewarding properties of cocaine for females. The role of E2 in male drug-seeking, however, is not well understood. The current study investigated pharmacological manipulation of E2 receptors in the dorsolateral striatum (DLS) on preference for cocaine in gonad-intact male and female rats. In males, activation of G-protein coupled E2 receptor 1 (GPER1), via administration of ICI 182,780 or G1, attenuated conditioned place preference for 10mg/kg cocaine, while inhibition of GPER1, via G15, enhanced preference at a 5mg/kg cocaine dose. Similarly, GPER1 activation, via G1, prevented males from forming a preference for 0.1% saccharin (SACC) versus plain water. Surprisingly, activation of GPER1 did not alter preference for cocaine or SACC in females. These studies also examined the quantity of E2 receptor mRNA in the dorsal striatum, using qPCR. No sex differences in relative mRNA expression of ER, ER{beta}, and GPER1 were observed. However, there was greater GPER1 mRNA, relative to ER and ER{beta}, in both males and females. The results presented here indicate that E2, acting via GPER1, may be protective against drug preference in male rats.

sentences: Non-parallel morphological divergence following colonization of a new host plant Divergent ecological selection may diversify conspecific populations evolving into different niches, and may lead to speciation if gene flow is ceased, e.g, due to reinforcement leading to character displacement. Adaptation to the same niche is expected to be parallel. Whether selection against maladaptive hybridization in secondary sympatry also results in parallel divergence in traits that are not directly related to the ecological niches remains an empirical challenge, although such character displacement can be decisive for if ecotypes develop into new species. Here, we use a host shift in the phytophagous peacock fly Tephritis conura, with both host races represented in two geographically separate areas East and West of the Baltic Sea to investigate convergence in morphological adaptations. We asked i) if there are consistent morphological adaptations to a host plant shift and ii) if the response to secondary sympatry with the alternate host race is parallel across contact zones. We found surprisingly low and variable, albeit significant, divergence between host races. Only one trait, the length of the female ovipositor, which serves an important function in the interaction with the hosts, was consistently different. Instead, co-existence with the other host race significantly affected the degree of morphological divergence, but the divergence was largely driven by different traits in different contact zones. Thus, local stochastic fixation or reinforcement could generate trait divergence, and additional evidence is needed to conclude whether divergence is locally adaptive.

sentences: Discovery of viral myosin genes with complex evolutionary history within plankton Nucleocytoplasmic large DNA viruses (NCLDVs) infect diverse eukaryotes and form a group of viruses with capsids encapsulating large genomes. Recent studies are increasingly revealing a spectacular array of functions encoded in their genomes, including genes for energy metabolisms, nutrient uptake, as well as cytoskeleton. Here, we report the discovery of genes homologous to myosins, the major eukaryotic motor proteins previously unrecognized in the virosphere, in environmental genomes of NCLDVs from the surface of the oceans. Interestingly, these genes were often accompanied by kinesin genes in the environmental genomes, suggesting a role of these viral proteins in the intracellular viral particle transport. Phylogenetic analyses indicate that most viral myosins (named "virmyosins") belong to the Imitervirales order, except for one belonging to the Phycodnaviridae family. On the one hand, the phylogenetic positions of virmyosin-encoding Imitervirales are scattered within the Imitervirales. On the other hand, Imitervirales virmyosin genes form a monophyletic group in the phylogeny of diverse myosin sequences. Furthermore, phylogenetic trends for the virmyosin genes and viruses containing them were incongruent. Based on these results, we argue that multiple transfers of myosin homologs have occurred not only from eukaryotes to viruses but also between viruses, supposedly during co-infections of the same host.

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BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic, however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.

Graphical Abstract

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sentences: A Plasmid System with Tunable Copy Number Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our massively parallel assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as tool for the optimization of synthetic biological systems.

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Extent of Molecular Chaperone Association Might Determine Fates of Membraneless Organelles during Aging in C. elegans Proteome imbalance can lead to protein misfolding and aggregation which is associated with pathologies. Protein aggregation can also be an active, organized process and can be exploited by cells as a survival strategy. In adverse conditions, it is beneficial to deposit the proteins in a condensate rather degrading and resynthesizing. Membraneless organelles (MLOs) are biological condensates formed through liquid-liquid phase separation (LLPS), involving cellular components such as nucleic acids and proteins. LLPS is a regulated process, which when perturbed, can undergo a transition from a physiological liquid condensate to pathological solid-like protein aggregates.

To understand how the MLO-associated proteins (MLO-APs) behave during aging, we performed a comparative meta-analysis with age related proteome of C. elegans. We found that the MLO-APs are highly abundant throughout the lifespan. Interestingly, they are aggregating more in long-lived mutant worms compared to the age matched wildtype worms. GO term analysis revealed that the cell cycle and embryonic development are among the top enriched processes in addition to RNP components in insoluble proteome. Considering antagonistic pleotropic nature of these developmental genes and post mitotic status of C. elegans, we assume that these proteins phase transit during post development. As the organism ages, these MLO-APs either mature to become more insoluble or dissolve in uncontrolled manner. However, in the long-lived daf-2 mutant worms, the MLOs may attain protective states due to extended availability and association of molecular chaperones.

sentences: Autophagy receptor NDP52 alters DNA conformation to modulate RNA Polymerase II transcription NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear function for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We found that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene-expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover novel nuclear roles for NDP52 in gene expression and DNA structure regulation.

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Reparative macrophages regulate fibrosis in the infarcted myocardium by attenuating apoptosis and senescence of fibroblasts RATIONALEAppropriate fibrotic tissue formation after myocardial infarction (MI) is crucial to maintenance of the hearts structure. Reparative or M2-like macrophages play a vital role in post-MI fibrosis by activating cardiac fibroblasts. However, the mechanism by which post-MI cardiac fibrosis is regulated is not fully understood.

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

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

CONCLUSIONSM2-like macrophage-mediated regulation of Nrg1/ErbB signaling, have a substantial effect on fibrotic tissue formation in the infarcted adult mouse heart, is critical for suppressing the progression of senescence and apoptosis of cardiac fibroblasts.

sentences: Community science designed ribosomes with beneficial phenotypes Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

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Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks Complex gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression is sufficiently perturbed it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map connections between regulatory genes and their downstream targets. However, we lack comprehensive knowledge of the upstream regulators of most genes. Here we developed an approach for systematic discovery of upstream regulators of critical immune factors - IL2RA, IL-2, and CTLA4 - in primary human T cells. Then, we mapped the network of these regulators target genes and enhancers using CRISPR perturbations, RNA-Seq, and ATAC-Seq. These regulators form densely interconnected networks with extensive feedback loops. Furthermore, this network is highly enriched for immune-associated disease variants and genes. These results provide insight into how immune-associated disease genes are regulated in T cells and broader principles about the structure of human gene regulatory networks.

HighlightsO_LIA systematic approach to identify upstream regulators of key immune genes in primary human cells
C_LIO_LIComprehensive RNA-Seq and ATAC-Seq perturbation maps after KO of individual discovered regulators
C_LIO_LIAnalysis uncovers a highly interconnected regulatory network of enhancers and genes in T cells
C_LIO_LIThis network is highly enriched for immune disease variants and genes shedding light on the trans-regulatory connections among key immune genes in health and disease
C_LI

sentences: Non-additive biotic interactions improve predictions of tropical tree growth and impact community size structure Growth in individual size or biomass is a key demographic component in population models, with wide-ranging applications from quantifying species performance across abiotic or biotic conditions to assessing landscape-level dynamics under global change. In forest ecology, the responses of tree growth to biotic interactions are widely held to be crucial for understanding forest diversity, function, and structure. To date, most studies on plant-plant interactions only examine the additive competitive or facilitative interactions between species pairs; however, there is increasing evidence of non-additive, higher-order interactions (HOIs) impacting species demographic rates. When HOIs are present, the dynamics of a multi-species community cannot be fully understood or accurately predicted solely from pairwise outcomes because of how additional species  interfere with the direct, pairwise interactions. Such HOIs should be particularly prevalent where species show nonlinear functional responses to resource availability and resource-acquisition traits themselves are density dependent. With this in mind, we used data from a tropical secondary forest--a system that fulfills both of these conditions--to build a ontogenetic diameter-growth model for individuals across ten woody-plant species. We allowed both direct and indirect interactions within communities to influence the species-specific growth parameters in a generalized Lotka-Volterra model. Specifically, indirect interactions entered the model as higher-order quadratic terms, i.e. non-additive effects of conspecific and heterospecific neighbour size on the focal individuals growth. For the whole community and for four out of ten focal species, the model that included HOIs had more statistical support than the model that included only direct interactions, despite the former containing a far greater number of parameters. HOIs had comparable effect sizes to direct interactions, and tended to further reduce the diameter growth rates of most species beyond what direct interactions had already reduced. In a simulation of successional stand dynamics, the inclusion of HOIs lead to rank swaps in species diameter hierarchies, even when community-level size distributions remained qualitatively similar. Our study highlights the implications, and discusses possible mechanisms, of non-additive density dependence in highly diverse and light-competitive tropical forests.

sentences: Investigating the active substance and mechanism of Jing-Fu-Kang granules via mass spectrometry technology and network pharmacology method Jing-Fu-Kang granules (JFKG) is a famous Chinese patent medicine for the treatment of cervical spondylosis around the China, whereas the active substance and mechanism are not completely investigated clearly. In the current study, a rapid separation and identification method using UPLC-QE-Orbitrap-MS was established, 97 chemical constituents from JFKG were identified, and 16 prototype components from plasma samples after administration of JFKG were observed within 16 min. The structures of typical compounds were preliminarily speculated by comparing the retention time and fragmentation pattern. Furthermore, multiple databases were used to integrate the compound targets of JFKG, and the disease targets related to cervical spondylosis. After the intersection of the two sets of targets, a protein-protein interaction (PPI) network and a TCM-component-target-pathway-disease network were established, then using the DAVID database to perform gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis on the common targets to find related pathways. Finally, a total of 531 common targets and 136 pathways were found to participate in the mechanism. Our findings will help to further confirm the mechanism of JFKG for relieving cervical spondylosis, which will improve the scientific rationality of JFKG in clinical use, and can also assist in guiding doctors.

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A new oomycete metabarcoding method using the rps10 gene Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed for rapid and accurate characterization of oomycete communities using DNA metabarcoding. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as a locus useful for oomycete metabarcoding and provide primers predicted to amplify all oomycetes based on available reference sequences from a wide range of taxa. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a mock community using Illumina MiSeq. Amplified sequence variants (ASVs) and operational taxonomic units (OTUs) were identified per community. Both the sequence and predicted taxonomy of ASVs and OTUs were compared to the known composition of the mock community. Both rps10 and ITS yielded ASVs with sequences matching 21 of the 24 species in the mock community and matching all 24 when allowing for a 1 bp difference. Taxonomic classifications of ASVs included 23 members of the mock community for rps10 and 17 for ITS1. Sequencing results for the environmental samples suggest the proposed rps10 locus results in substantially less amplification of non-target organisms than the ITS1 method. The amplified rps10 region also has higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete metabarcoding. The rps10 barcode and methods described herein provide an effective tool for metabarcoding oomycetes using short-read sequencing.

Interpretive summaryOomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause diseases in plants and animals. Improved methods are needed to rapidly characterize the diversity of oomycete species found in environmental samples. We have identified the mitochondrial 40S ribosomal protein S10 gene (rps10) as being useful for oomycete community sequencing. We evaluated its utility relative to a popular barcode, the internal transcribed spacer 1 (ITS1), by sequencing environmental samples and a community we synthesized in the laboratory. The amplified rps10 region is predicted to have a higher taxonomic resolution than ITS1, allowing for greater discrimination of closely related species. We present a new website with a searchable rps10 reference database for species identification and all protocols needed for oomycete community sequencing. The rps10 barcode and methods described herein provide an effective tool for characterizing oomycetes using environmental DNA sequencing.

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Detection and characterization of replication origins defined by DNA polymerase epsilon BackgroundAlthough the process of DNA replication is highly conserved the location of origins of replication (ORI) may vary from one tissue to the next or one round of replication to the next in eukaryotes, suggesting flexibility in the choice of locations to initiate replication. Lists of human ORI therefore vary widely in number and location and there are no methods available to compare them.

ResultsHere we report the genome-wide localization of ORI in POLE-mutated human tumors using whole genome sequencing data. Mutations accumulated after many rounds of replication of unsynchronized dividing cell populations in tumors allow to identify constitutive origins, which we show are shared with high-fidelity between individuals and tumor types. Using a Smith-Waterman-like dynamic programming approach, we compared replication origin positions obtained from multiple different methods. The comparison allowed us to define a consensus set of replication origins, identified consistently by multiple ORI detection methods.

ConclusionsMany DNA features co-localized with the consensus set of ORI, including chromatin loop anchors, G-quadruplexes, S/MARs and CpGs. Among all features, the H2A.Z histone exhibited the most significant association. Our results show that mutation-based detection of replication origins is a viable approach to determining their location and associated sequence features.

sentences: Predicting protein domain temperature adaptation across the prokaryote-eukaryote divide Protein thermostability is important for fitness but difficult to measure across the proteome. Fortunately, protein thermostability is correlated with prokaryote optimal growth temperatures (OGTs), which can be predicted from genome features. Models that can predict temperature sensitivity across the prokaryote-eukaryote divide would help inform how eukaryotes adapt to elevated temperatures, such as those predicted by climate change models. In this study we test whether prediction models can cross the prokaryote-eukaryote divide to predict protein stability in both prokaryotes and eukaryotes. We compare models built using a) the whole proteome, b) Pfam domains, and c) individual amino acid residues. Proteome-wide models accurately predict prokaryote optimal growth temperatures (r2 up to 0.93), while site-specific models demonstrate that nearly half of the proteome is associated with optimal growth temperature in both Archaea and Bacteria. Comparisons with the small number of eukaryotes with temperature sensitivity data suggest that site-specific models are the most transferable across the prokaryote-eukaryote divide. Using the site-specific models, we evaluated temperature sensitivity for 323,850 amino acid residues in 2,088 Pfam domain clusters in Archaea and Bacteria species separately. 59.0% of tested residues are significantly associated with OGT in Archaea and 75.2% of tested residues are significantly associated with OGT in Bacteria species at a 5% false discovery rate. These models make it possible to identify which Pfam domains and amino acid residues are involved in temperature adaptation and facilitate future research questions about how species will fare in the face of increasing environmental temperatures.

sentences: From heterogeneous datasets to predictive models of embryonic development Modern studies of embryogenesis are increasingly quantitative, powered by rapid advances in imaging, sequencing, and genome manipulation technologies. Deriving mechanistic insights from the complex datasets generated by these new tools requires systematic approaches for data-driven analysis of the underlying developmental processes. Here we use data from our work on signal-dependent gene repression in the Drosophila embryo to illustrate how computational models can compactly summarize quantitative results of live imaging, chromatin immunoprecipitation, and optogenetic perturbation experiments. The presented computational approach is ideally suited for integrating rapidly accumulating quantitative data and for guiding future studies of embryogenesis.

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Host kinase CSNK2 is a target for inhibition of pathogenic β-coronaviruses including SARS-CoV-2 Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine {beta}-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in {beta}-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-{beta}-coronavirus drugs.

GRAPHICAL ABSTRACT

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sentences: Opponent vesicular transporters regulate the strength of glutamatergic neurotransmission in a C. elegans sensory circuit At chemical synapses, neurotransmitters are packaged into synaptic vesicles that release their contents in response to depolarization. Despite its central role in synaptic function, regulation of the machinery that loads vesicles with neurotransmitters remains poorly understood. We find that synaptic glutamate signaling in a C. elegans chemosensory circuit is regulated by antagonistic interactions between the canonical vesicular glutamate transporter EAT-4/VGLUT and another vesicular transporter, VST-1. Loss of VST-1 strongly potentiates glutamate release from chemosensory BAG neurons and disrupts chemotaxis behavior. Analysis of the circuitry downstream of BAG neurons shows that excess glutamate release disrupts behavior by inappropriately recruiting RIA interneurons to the BAG-associated chemotaxis circuit. Our data indicate that in vivo the strength of glutamatergic synapses is controlled by regulation of neurotransmitter packaging into synaptic vesicles via functional coupling of VGLUT and VST-1.

sentences: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding Phosphatidylserine (PS) has been shown to be a critical lipid factor in the assembly and spread of numerous lipid enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA approved drug, fendiline, to reduce PS clustering subsequently reducing virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were employed including imaging, viral budding and viral entry assays. Fendiline reduced the PS content in mammalian cells and PS in the plasma membrane, reducing the ability of VP40 to form new virus particles. Further, particles that do form from fendiline treated cells have altered particle morphology and decreased infectivity capacity. These complementary studies reveal the mechanism by which filovirus matrix proteins cluster PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

sentences: Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa. Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.

sentences: From collocations to call-ocations: using linguistic methods to quantify animal call combinations Emerging data in a range of non-human animal species have highlighted a latent ability to combine certain pre-existing calls together into larger structures. Currently, however, there exists no objective quantification of call combinations. This is problematic because animal calls can co-occur with one another simply through chance alone. One common approach applied in language sciences to identify recurrent word combinations is collocation analysis. Through comparing the co-occurrence of two words with how each word combines with other words within a corpus, collocation analysis can highlight above chance, two-word combinations. Here, we demonstrate how this approach can also be applied to non-human animal communication systems by implementing it on a pseudo dataset. We argue collocation analysis represents a promising tool for identifying non-random, communicatively relevant call combinations in animals.

sentences: Unique protein features of SARS-CoV-2 relative to other Sarbecoviruses Defining the unique properties of SARS-CoV-2 protein sequences, has potential to explain the range of Coronavirus Disease 2019 (COVID-19) severity. To achieve this we compared proteins encoded by all Sarbecoviruses using profile Hidden Markov Model similarities to identify protein features unique to SARS-CoV-2. Consistent with previous reports, a small set of bat and pangolin-derived Sarbecoviruses show the greatest similarity to SARS-CoV-2 but unlikely to be the direct source of SARS-CoV-2. Three proteins (nsp3, spike and orf9) showed differing regions between the bat Sarbecoviruses and SARS-CoV-2 and indicate virus protein features that might have evolved to support human infection and/or transmission. Spike analysis identified all regions of the protein that have tolerated change and revealed that the current SARS-CoV-2 variants of concern (VOCs) have sampled only a fraction (~31%) of the possible spike domain changes which have occurred historically in Sarbecovirus evolution. This result emphasises the evolvability of these coronaviruses and potential for further change in virus replication and transmission properties over the coming years.

sentences: Multi-omics Data Integration by Generative Adversarial Network Accurate disease phenotype prediction plays an important role in the treatment of heterogeneous diseases like cancer in the era of precision medicine. With the advent of high throughput technologies, more comprehensive multi-omics data is now available that can effectively link the genotype to phenotype. However, the interactive relation of multi-omics datasets makes it particularly challenging to incorporate different biological layers to discover the coherent biological signatures and predict phenotypic outcomes. In this study, we introduce omicsGAN, a generative adversarial network (GAN) model to integrate two omics data and their interaction network. The model captures information from the interaction network as well as the two omics datasets and fuse them to generate synthetic data with better predictive signals. Large-scale experiments on The Cancer Genome Atlas (TCGA) breast cancer, lung cancer, and ovarian cancer datasets validate that (1) the model can effectively integrate two omics data (e.g., mRNA and microRNA expression data) and their interaction network (e.g., microRNA-mRNA interaction network). The synthetic omics data generated by the proposed model has a better performance on cancer outcome classification and patients survival prediction compared to original omics datasets. (2) The integrity of the interaction network plays a vital role in the generation of synthetic data with higher predictive quality. Using a random interaction network does not allow the framework to learn meaningful information from the omics datasets; therefore, results in synthetic data with weaker predictive signals.

sentences: DomeVR: A setup for experimental control of an immersive dome virtual environment created with Unreal Engine 4 1Immersive virtual reality (VR) environments are a powerful tool to explore cognitive processes ranging from memory and navigation to visual processing and decision making - and to do so in a naturalistic yet controlled setting. As such, they have been employed across different species, and by a diverse range of research groups. Unfortunately, designing and implementing behavioural tasks in such environments often proves complicated. To tackle this challenge, we created DomeVR, an immersive VR environment built using Unreal Engine 4 (UE4). UE4 is a powerful game engine with photo-realistic graphics containing a visual scripting language designed for use by non-programmers. As a result, virtual environments are easily created using drag-and-drop elements. DomeVR aims to make these features accessible to neuroscience experiments. This includes a logging and synchronization system to solve timing uncertainties inherent in UE4; an interactive GUI for scientists to observe subjects during experiments and adjust task parameters on the fly, and a dome projection system for full task immersion in non-human subjects. These key features are modular and can easily be added individually into other UE4 projects. Finally, we present proof-of-principle data highlighting the functionality of DomeVR in three different species: human, macaque and mouse.

sentences: An Open-Access Simultaneous Electrocardiogram and Phonocardiogram Database The electro-phono-cardiogram (EPHNOGRAM) project focused on the development of low-cost and low-power devices for recording simultaneous electrocardiogram (ECG) and phonocardiogram (PCG) data, with auxiliary channels for capturing environmental audio noise, which could be used for PCG quality enhancement through signal processing. The current database, recorded by version 2.1 of the developed hardware, has been acquired from 24 healthy adults aged between 23 and 29 (average: 25.4 {+/-} 1.9 years) in 30 min stress-test sessions during resting, walking, running and biking conditions, using indoor fitness center equipment. The dataset also contains several 30 s sample records acquired during rest conditions. This data is useful for simultaneous multi-modal analysis of ECG and PCG. It provides interesting insights into the inter-relationship between the mechanical and electrical mechanisms of the heart, under rest and physical activity. The database is provided online on PhysioNet.

sentences: Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter Given its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.

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Systems analysis of gut microbiome influence on metabolic disease in HIV and high-risk populations Poor metabolic health, characterized by insulin resistance and dyslipidemia, is higher in people living with HIV and has been linked with inflammation, anti-retroviral therapy (ART) drugs, and ART-associated lipodystrophy (LD). Metabolic disease is associated with gut microbiome composition outside the context of HIV but has not been deeply explored in HIV infection nor in high-risk men who have sex with men (HR-MSM), who have a highly altered gut microbiome composition. Furthermore, the contribution of increased bacterial translocation and associated systemic inflammation that has been described in HIV-positive and HR-MSM individuals has not been explored. We used a multi-omic approach to explore relationships between impaired metabolic health, defined using fasting blood markers, gut microbes, immune phenotypes and diet. Our cohort included ART-treated HIV positive MSM with and without LD, untreated HIV positive MSM, and HR-MSM. For HIV positive MSM on ART, we further explored associations with the plasma metabolome. We found that elevated plasma lipopolysaccharide binding protein (LBP) was the most important predictor of impaired metabolic health and network analysis showed that LBP formed a hub joining correlated microbial and immune predictors of metabolic disease. Taken together, our results suggest the role of inflammatory processes linked with bacterial translocation and interaction with the gut microbiome in metabolic disease among HIV positive and negative MSM.

Importance StatementThe gut microbiome in people living with HIV (PLWH) is of interest as chronic infection often results in long term comorbidities. Metabolic disease is prevalent in PLWH even in well-controlled infection and has been linked with the gut microbiome in previous studies, but little attention has been given to PLWH. Furthermore, integrated analyses that consider gut microbiome together with diet, systemic immune activation, metabolites, and demographics have been lacking. In a systems-level analysis of predictors of metabolic disease in PLWH and men who are at high risk of acquiring HIV, we found that increased LBP, an inflammatory marker indicative of compromised intestinal barrier function, was associated with worse metabolic health. We also found impaired metabolic health associated with specific dietary components, gut microbes, and host and microbial metabolites. This work lays the framework for mechanistic studies aimed at targeting the microbiome to prevent or treat metabolic endotoxemia in HIV-infected individuals.

sentences: A Post-hurricane Quantitative Assessment of the Red-bellied Racer (Alsophis rufiventris) on Saba and Comparison with St. Eustatius We estimated occupancy, abundance (lambda), detection probability, density/ha and abundance of a regionally endemic snake in the Colubrid family on the Dutch Caribbean island of Saba in 2021, four years after hurricanes Irma and Maria impacted the island. Line transect surveys were conducted at 74 sites covering 6.7 ha. The proportion of sites occupied was estimated at 0.74 (min 0.48, max 0.90), with occupancy varying between vegetation types and across elevational gradients. Similarly, lambda was estimated at 1.61 (min 0.7, max 3.7) but varied between vegetation types and elevational gradients. Detection probability was estimated at 0.15 (min 0.10, max 0.21). Using Distance sampling, we estimated 10.9 (min 7.3, max 16.2) racers/ha, with a total population estimate of 4,917 (min 2,577, max 6,362) across the entire study region (438.6 ha.) Based on anecdotal observations from Saban residents and prior literature describing the pre-hurricane population as "abundant" (at least 2.0 racers/hour), we posit that the population experienced a hurricane-induced decline but may have since recovered, though not to previous levels (1.28 racers/hour). Nevertheless, our results suggest that racer densities on Saba are currently higher than those on St. Eustatius. Despite this, given the species extremely limited extant range and the presence of invasive species on both islands, prevention of local extirpation should be a high conservation priority.

sentences: CNS antigen-specific control of early B-lineage development in the meninges The random V(D)J recombination of immunoglobulins (Ig) loci often creates autoreactive B cell progenitors expressing self-recognized B-cell receptors (BCRs)1, which are eliminated or inactivated through an autoantigen-dependent central tolerance checkpoint to prevent autoimmune reactions2,3, a process thought to be restricted to the bone marrow (BM) in the adult mammals4. Here we report that early developing B cells are also present in the meninges of mice at all ages. Single cell RNA-sequencing (scRNA-seq) analysis revealed a consecutive trajectory of meningeal developing B cells in mice and non-human primates (NHPs). Parabiosis together with lineage tracing of hematopoietic stem cells (HSCs) showed that meningeal developing B cells are continuously replenished from the HSC-derived progenitors via a circulationindependent route. Importantly, autoreactive immature B cells which recognize myelin oligodendrocyte glycoprotein (MOG)5, a central nervous system (CNS)-specific antigen, are eliminated from the meninges but not BM. Furthermore, genetic deletion of MOG restored the self-reactive B cells in the meninges. Thus, we propose that meninges function as a unique reservoir for B cell development, allowing in situ negative selection of CNS-antigen-autoreactive B cells to ensure a local non-self-reactive immune repertoire.

sentences: Long-term translocation explains population genetic structure of a recreationally fished iconic species in Japan: Combining current knowledge with reanalysis Ayu (Plecoglossus altivelis altivelis) is an important freshwater fisheries resource and popular recreational fishing species in Japan, lives for only one year, and has a single breeding season. To supplement increased recreational fishing demand for this species, huge numbers of wild-born landlocked juveniles have been translocated from Lake Biwa into most Japanese rivers for more than 100 generations. Hatchery-reared juveniles born from captive-reared parents for more than 30 generations have also been extensively released. Previous studies have reported that landlocked and amphidromous forms of Ayu easily hybridise, but survival of landlocked larvae could be very low in seawater, leading to a general consensus among scientists, hatchery managers, and commercial and recreational fishers that the reproductive success of landlocked Ayu is very low, or even 0 in translocated rivers. Despite this, limited information exists regarding the reproductive success of landlocked Ayu in translocated rivers, and no study has evaluated the effects of translocation on population structure. Demonstrating that hybridisation occurs between the two forms is central to future management and conservation of this specie. To address this issue, a comprehensive literature search was undertaken, and three published genetic data sets are analysed. Analyses provide strong evidence for very high gene flow between populations, but population structure has been retained in several regions, and several populations are nested. Allele frequencies are similar in amphidromous and landlocked forms. Genetic diversity is homogeneous in amphidromous populations. Bayesian admixture analysis infers widespread introgression in Japanese rivers, with a mean introgression proportion of 24 {+/-} 8%. Maximum likelihood admixture graphs detect two migration events from Lake Biwa to anadromous populations. Analyses consistently indicate that hybridisation between translocated landlocked juveniles and native amphidromous Ayu occurs throughout Japanese rivers.

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Measuring the unknown: an estimator and simulation study for assessing case reporting during epidemics The fraction of cases reported, known as  reporting, is a key performance indicator in an outbreak response, and an essential factor to consider when modelling epidemics and assessing their impact on populations. Unfortunately, its estimation is inherently difficult, as it relates to the part of an epidemic which is, by definition, not observed.

We introduce a simple statistical method for estimating reporting, initially developed for the response to Ebola in Eastern Democratic Republic of the Congo (DRC), 2018-2020. This approach uses transmission chain data typically gathered through case investigation and contact tracing, and uses the proportion of investigated cases with a known, reported infector as a proxy for reporting. Using simulated epidemics, we study how this method performs for different outbreak sizes and reporting levels. Results suggest that our method has low bias, reasonable precision, and despite sub-optimal coverage, usually provides estimates within close range (5-10%) of the true value.

Being fast and simple, this method could be useful for estimating reporting in real-time in settings where person-to-person transmission is the main driver of the epidemic, and where case investigation is routinely performed as part of surveillance and contact tracing activities.

Author summaryWhen responding to epidemics of infectious diseases, it is essential to estimate how many cases are not being reported. Unfortunately reporting, the proportion of cases actually observed, is difficult to estimate during an outbreak, as it typically requires large surveys to be conducted on the affected populations. Here, we introduce a method for estimating reporting from case investigation data, using the proportion of cases with a known, reported infector. We used simulations to test the performance of our approach by mimicking features of a recent Ebola epidemic in the Democratic Republic of the Congo. We found that despite some uncertainty in smaller outbreaks, our approach can be used to obtain informative ballpark estimates of reporting under most settings. This method is simple and computationally inexpensive, and can be used to inform the response to any epidemic in which transmission events can be uncovered by case investigation.

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Fragment-based computational design of antibodies targeting structured epitopes De novo design methods hold the promise of reducing the time and cost of antibody discovery, while enabling the facile and precise targeting of predetermined epitopes. Here we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterisation showed that all designs are highly stable, and bind their intended targets with affinities in the nanomolar range without any in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as our method yields similar predictions when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to pre-selected epitopes.

summaryA combinatorial method can rapidly design nanobodies for predetermined epitopes, which bind with KDs in the nanomolar range.

sentences: Expansion of RNA sequence diversity and RNA editing rates throughout human cortical development Post-transcriptional modifications by RNA editing are essential for neurodevelopment, yet their developmental and regulatory features remain poorly resolved. We constructed a full temporal view of base-specific RNA editing in the developing human cortex, from early progenitors through fully mature cells found in the adult brain. Developmental regulation of RNA editing is characterized by an increase in editing rates for more than 10,000 selective editing sites, shifting between mid-fetal development and infancy, and a massive expansion of RNA hyper-editing sites that amass in the cortex through postnatal development into advanced age. These sites occur disproportionally in 3UTRs of essential neurodevelopmental genes. These profiles are preserved in non-human primate and murine models, illustrating evolutionary conserved regulation of RNA editing in mammalian cortical development. RNA editing levels are commonly genetically regulated (editing quantitative trait loci, edQTLs) consistently across development or predominantly during prenatal or postnatal periods. Both consistent and temporal-predominant edQTLs co-localize with risk loci associated with neurological traits and disorders, including attention deficit hyperactivity disorder, schizophrenia, and sleep disorders. These findings expand the repertoire of highly regulated RNA editing sites in the brain and provide insights of how epitranscriptional sequence diversity by RNA editing contributes to neurodevelopment.

sentences: Cellular heterogeneity and molecular reprogramming of host response during influenza acute lung injury Acute lung injury (ALI) caused by influenza A virus (IAV or influenza) manifests from dysregulated cellular interactions between hematopoietic and non-hematopoietic cells that develop into a pathologic host response. However, the lungs diverse cellular framework that dictates the pathologic host response and acute lung injury remains incompletely understood. We performed a single-cell RNA-seq (scRNA-seq) analysis of total lung cells in mice from severe influenza to examine the cellular heterogeneity and cell-specific regulation of host response. We observed that IAV infection resulted in significant myelopoiesis, predominantly monocyte, and macrophage subsets, which constituted over 50% of total immune cells. IAV infection resulted in the significant loss of endothelial and fibroblast cells, representing the most predominant non-hematopoietic cells and crucial to regulating inflammatory response and barrier integrity. We also show the cell-cell communication dynamics of interferon and chemokine signaling and global regulation of these responses in transition from homeostatic to IAV infection state. These data highlight a robust application of scRNA-seq technology in establishing the atlas of cellular heterogeneity from its homeostatic transition to infection state and the host response regulation in IAV-mediated lung pathology.

sentences: Beneficial and Detrimental Effects of Nonspecific Binding During DNA Hybridization DNA strands have to sample numerous states to find the alignment that maximizes Watson-Crick-Franklin base pairing. This process depends strongly on sequence, which affects the stability of the native duplex as well as the prevalence of non-native inter- and intra-molecular helices. We present a theory which describes DNA hybridization as a three stage process: diffusion, registry search, and zipping. We find that non-specific binding affects each of these stages in different ways. Mis-registered intermolecular binding in the registry search stage helps DNA strands sample different alignments and accelerates the hybridization rate. Non-native intramolecular structure affects all three stages by rendering portions of the molecule inert to intermolecular association, limiting mis-registered alignments to be sampled, and impeding the zipping process. Once inregister base-pairs are formed, the stability of the native structure is important to hold the molecules together long enough for non-native contacts to break.

sentences: Long-term population decline of a genetically homogenous continental-wide top Arctic predator Genetic analysis can provide valuable information for conservation programs by unraveling the demographic trajectory of populations, by estimating effective population size, or by inferring genetic differentiation between populations. Here, we investigated the genetic differentiation within the Snowy Owl (Bubo scandiacus), a species identified as vulnerable by the IUCN, to (i) quantify connectivity among wintering areas, (ii) to evaluate current genetic diversity and effective population size and (iii) to infer changes in the historical effective population size changes from the last millennia to the recent past. The Snowy Owl, a highly mobile top predator, breeds across the Arctic tundra which is a region especially sensitive to current climate change. Using SNP-based analyses on Snowy Owls sampled across the North American nonbreeding range, we found an absence of genetic differentiation among individuals located up to 4,650 km apart. Our results suggest high genetic intermixing and effective dispersal at the continental scale despite documented philopatry to nonbreeding sites in winter. Reconstructing the population demographic indicated that North American Snowy Owls have been steadily declining since the Last Glacial Maximum ca 20,000 years ago and concurrently with global increases in temperature. Conservation programs should now consider North American Snowy Owls as a single, genetically homogenous continental-wide population which is most likely sensitive to the long-term global warming occurring since the Last Glacial Maximum.

sentences: A western representative of an eastern clade: phylogeographic history of the gypsophilous plant Nepeta hispanica The preference of certain plant species for gypsum soils leads to disjunct population structures that are thought to generate island-like dynamics potentially influencing biogeographic patterns at multiple evolutionary scales. Here, we study the evolutionary and biogeographic history of Nepeta hispanica, a western Mediterranean plant associated with gypsum soils and displaying a patchy distribution with populations very distant from each other. Three approaches were used: (a) interspecific phylogenetic analyses based on nuclear DNA sequences of the ITS region to unveil the relationships and times of divergence between N. hispanica and its closest relatives; ((b)phylogeographic analyses using plastid DNA regions trnS-trnG and psbJ-petA to evaluate the degree of genetic isolation between populations of N. hispanica, their relationships and their genetic diversity; and (c) ecological niche modelling to evaluate historical distributional changes. Results reveal that N. hispanica belongs to an eastern Mediterranean and Asian clade diversified in arid environments since the Miocene-Pliocene. This species represents the only extant lineage of this clade that colonized the western Mediterranean, probably through the northern Mediterranean coast (southern Europe). Present Iberian populations display a high plastid genetic diversity and, even if geographically distant from each other, they are highly connected according to the distribution of plastid haplotypes and lineages. This can be explained by a scenario involving a complex history of back-and-forth colonisation events, facilitated by a relative stability of suitable conditions for the species across the Iberian Peninsula throughout the Quaternary.

sentences: Eye movements elevate crowding in idiopathic infantile nystagmus syndrome Idiopathic infantile nystagmus syndrome is a disorder characterised by involuntary eye movements, which leads to decreased acuity and visual function. One such function is visual crowding; a process whereby objects that are easily recognised in isolation become impaired by nearby flankers. Crowding typically occurs in the peripheral visual field, though elevations in foveal vision have been reported in congenital nystagmus, similar to those found with amblyopia. Here we examine whether the elevated foveal crowding with nystagmus is driven by similar mechanisms to those documented in amblyopia - long-term neural changes associated with a sensory deficit - or by the momentary displacement of the stimulus through nystagmus eye movements. We used a Landolt-C orientation identification task to measure threshold gap sizes with and without either horizontally or vertically placed pairs of flanking Landolt-Cs. Because nystagmus is predominantly horizontal, crowding should be stronger with horizontal flankers if eye movements cause the interference, whereas a sensory deficit would more likely be equivalent for the two dimensions. Consistent with an origin in eye movements, we observe elevations in nystagmic crowding that are above that of typical vision, and stronger with horizontal than vertical flankers and not found in amblyopic or typical vision. We further demonstrate the same pattern of performance can be obtained in typical vision with stimulus movement that simulates nystagmus. Consequently, we propose that the origin of nystagmic crowding lies in the eye movements, either through image smear of the target and flanker elements or through relocation of the stimulus into peripheral retina.

sentences: METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs A subset of eukaryotic tRNAs is methylated in the anticodon loop to form the 3-methylcytosine (m3C) modification. In mammals, the number of tRNAs containing m3C has expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. Whereas the enzymes catalyzing m3C formation in nuclear-encoded cytoplasmic tRNAs have been identified, the proteins responsible for m3C modification in mt-tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the Methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase along with mt-tRNAs containing m3C. Human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells can be rescued by re-expression of wildtype METTL8 but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mitochondrial tRNAs and uncover a potential role for m3C modification in mitochondrial tRNA structure.

sentences: Monitoring the effects of chemical stimuli on live cells with metasurface-enhanced infrared reflection spectroscopy Infrared spectroscopy has found wide applications in the analysis of biological materials. A more recent development is the use of engineered nanostructures - plasmonic metasurfaces - as substrates for metasurface-enhanced infrared reflection spectroscopy (MEIRS). Here, we demonstrate that strong field enhancement from plasmonic metasurfaces enables the use of MEIRS as a highly informative analytic technique for real-time monitoring of cells. By exposing live cells cultured on a plasmonic metasurface to chemical compounds, we show that MEIRS can be used as a label-free phenotypic assay for detecting multiple cellular responses to external stimuli: changes in cell morphology, adhesion, lipid composition of the cellular membrane, as well as intracellular signaling. Using a focal plane array detection system, we show that MEIRS also enables spectro-chemical imaging at the single-cell level. The described metasurface-based all-optical sensor opens the way to a scalable, high-throughput spectroscopic assay for live cells.

sentences: Detailed measurements and simulations of electric field distribution of two TMS coils cleared for obsessive compulsive disorder in the brain and in specific regions associated with OCD The FDA cleared deep transcranial magnetic stimulation (Deep TMS) with the H7 coil for obsessive-compulsive disorder (OCD) treatment, following a double-blinded placebo-controlled multicenter trial. Two years later the FDA cleared TMS with the D-B80 coil on the basis of substantial equivalence. In order to investigate the induced electric field characteristics of the two coils, these were placed at the treatment position for OCD over the prefrontal cortex of a head phantom, and the field distribution was measured. Additionally, numerical simulations were performed in eight Population Head Model repository models with two sets of conductivity values and three Virtual Population anatomical head models and their homogeneous versions. The H7 was found to induce significantly higher maximal electric fields (p<0.0001, t=11.08) and to stimulate two to five times larger volumes in the brain (p<0.0001, t=6.71). The rate of decay of electric field with distance is significantly slower for the H7 coil (p < 0.0001, Wilcoxon matched-pairs test). The field at the scalp is 306% of the field at a 3 cm depth with the D-B80, and 155% with the H7 coil. The H7 induces significantly higher intensities in broader volumes within the brain and in specific brain regions known to be implicated in OCD (dorsal anterior cingulate cortex (dACC), dorsolateral prefrontal cortex (dlPFC), inferior frontal gyrus (IFG), orbitofrontal cortex (OFC) and pre-supplementary motor area (pre-SMA)) compared to the D-B80. Significant field [≥] 80 V/m is induced by the H7 (D-B80) in 15% (1%) of the dACC, 78% (29%) of the pre-SMA, 50% (20%) of the dlPFC, 30% (12%) of the OFC and 15% (1%) of the IFG. Considering the substantial differences between the two coils, the clinical efficacy in OCD should be tested and verified separately for each coil.

sentences: The effects of a synthetic and biological surfactant on the community composition and metabolic activity of a freshwater biofilm Surfactants are used to control microbial biofilms in industrial and medical settings. Their known toxicity on aquatic biota and their longevity in the environment has encouraged research on biodegradable alternatives such as rhamnolipids. While previous research has investigated the effects of biological surfactants on single species biofilms, there remains a lack of information regarding the effects of synthetic and biological surfactants in freshwater ecosystems. We conducted a mesocosm experiment to test how the surfactant sodium dodecyl sulphate (SDS) and the biological surfactant rhamnolipid altered community composition and metabolic activity of freshwater biofilms. Biofilms were cultured in the flumes using lake water from Lake Lunz in Austria, under high (300 ppm) and low (150 ppm) concentrations of either surfactant over a four-week period. Our results show that both surfactants significantly affected microbial diversity. Up to 36% of microbial operational taxonomic units were lost after surfactant exposure. Rhamnolipid exposure also increased the production of the extracellular enzymes, leucine aminopeptidase and glucosidase, while SDS exposure reduced leucine aminopeptidase and glucosidase. This study demonstrates that exposure of freshwater biofilms to chemical and biological surfactants caused a reduction of microbial diversity and changes in biofilm metabolism, exemplified by shifts in extracellular enzyme activities.

sentences: Design and validation of the first family of photo-activatable ligands for melatonin receptors Melatonin is a neurohormone released in a circadian manner with peak levels during the night. In mammals, melatonin mediates its effects mainly through G protein-coupled MT1 and MT2 receptors. Drugs acting on melatonin receptors are indicated for circadian rhythm- and sleep-related disorders and major depression. Pharmacological tools to study the activation of these receptors with high temporal resolution are lacking. Here, we synthesized a family of light-activatable caged melatonin compounds by covalently attaching o-nitrobenzyl (o-NB) or coumarin photocleavable groups to the N-position of melatonin. All caged compounds showed the expected decrease in binding affinity for MT1 and MT2. Among them, the o-NB derivative MCS-0382 showed the best uncaging and biological properties upon light activation with a significant increase (2.5 log left-shift) in affinity and potency in melatonin receptor binding and signaling, respectively. Generation of biologically active melatonin from MCS-0382 was further demonstrated by its ability to modulate the excitation of SCN neurons in rat brain slices. MCS-0382 is now available to study melatonin effects in a temporally controlled manner in cellular and physiological settings.

sentences: Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors we call SyNthetic Intramembrane Proteolysis Receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the potential transformative utility of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.

sentences: Diversification through sexual selection on gustatorial courtship traits in dwarf spiders Sexual dimorphism can evolve under sexual selection or ecological factors. Sexually dimorphic male prosomal modifications are associated with gustatorial courtship in erigonines. The modifications vary from moderate elevations to bizarre shapes. Males transfer substances from these structures to females, which affect mate acceptance and fecundity. Here, we explore lability of these traits by investigating if modified prosomata are inherently linked to secretory glands, if glands evolved prior to prosomal modifications, and the possibility of convergent evolution and cryptic differentiation, aiming at assessing the possible role of this trait complex in speciation. We reconstructed the positions of glands and the musculature in the anterior part of prosomata of 76 erigonines and three outgroups using micro-CT. We incorporated these characters into an existing morphological character matrix and reanalyzed the phylogeny. Our results support the possession of glands as the ancestral state. The manifold modifications of the prosomal shape have evolved convergently. Differences in glandular positions between species with modified/unmodified prosomata suggest high lability of these traits. Cases of gland loss suggest considerable costs of gustatorial courtship. Our findings demonstrate divergent evolutionary patterns of these traits, and a likely facilitating effect of this type of sexual selection on speciation.

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PCRedux: A Data Mining and Machine Learning Toolkit for qPCR Experiments MotivationQuantitative Real-time PCR (qPCR) is a widely used -omics method for the precise quantification of nucleic acids, in which the result is associated with the presence/absence or quantity of a specific nucleic acid sequence. As the amount of qPCR data increases worldwide, the manual assessment of results becomes challenging and difficult to reproduce. To overcome this, some automatable characteristics of amplification curves have been described in the literature, often with an appropriate "rule of thumb".

ResultsWe developed PCRedux to analyze and calculate 90 numerical qPCR amplification curve descriptors (  features") from large datasets of qPCR amplification curves that are aimed for interpretable machine learning and development of decision support systems. In a case study of a diverse dataset with 3181 positive, negative and ambiguous amplification curves, as assessed by three human raters, we demonstrate a sensitivity >99 % and specificity >97 % in detecting positive and negative amplification. PCRedux is unique as it goes beyond traditional qPCR analysis to capture curvature properties that improve the characterization and classification of amplification curves. The calculation of the features is reproducible and objective, since R is used as a controllable working environment. PCRedux is not a black box, but open source software following on the principle of mathematically interpretable features. These can be combined with user-defined labels for automatic multi-category classification and regression in machine learning.

Availabilityhttps://cran.r-project.org/package=PCRedux. Web server: http://shtest.evrogen.net/PCRedux-app/. Documentation: https://PCRuniversum.github.io/PCRedux/.

sentences: In vivo visualization of propagating α-synuclein pathologies in mouse and marmoset models by a bimodal imaging probe, C05-05 Deposition of intracellular -synuclein fibrils is implicated in neurodegenerative parkinsonian disorders, while high-contrast in vivo detection of -synuclein depositions has been unsuccessful in animal models and humans. Here, we have developed a bimodal imaging probe, C05-05, for visualizing -synuclein inclusions in the brains of living animals modeling -synuclein propagation. In vivo optical and PET imaging of a mouse model demonstrated visualization of -synuclein aggregates by C05-05, revealing a dynamic propagation of fibrillogenesis along neural pathways followed by disruptions of these structures. Moreover, longitudinal 18F-C05-05-PET of a marmoset model captured widespread dissemination of fibrillary pathologies accompanied by neurodegeneration detected by dopamine transporter PET. In addition, in vitro assays demonstrated the high-affinity binding of 18F-C05-05 to -synuclein versus other protein pathologies in human brain tissues. Collectively, we propose a new imaging technology enabling etiological and therapeutic assessments of -synuclein pathogenesis at non-clinical levels, highlighting the applicability of C05-05 to clinical PET.

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Application of sequence semantic and integrated cellular geography approach to study biogenesis of exonic circular RNA BackgroundConcurrent existence of lncRNA and circular RNA at both nucleus and cytosol within a cell at different proportion is well reported. Secondly, information on genes transcribing both circular and lncRNAs along with total number of RBP binding sites for both of these RNA types is extractable from databases. This study showed how these apparently unconnected pieces of reports could be put together to build a model leading to biogenesis of circular RNA.

ResultsAs a result of this study, a model was built under the premises that, sequences with special semantics were molecular precursors in biogenesis of circular RNA which occurred through catalytic role of some specific RBPs. The model outcome was further strengthened by fulfillment of three logical lemmas which were extracted and assimilated in this work using a novel data analytic approach, Integrated Cellular Geography. Result of the study was found to be in well agreement with proposed model. Furthermore this study also indicated that biogenesis of circular RNA was a post-transcriptional event.

ConclusionsOverall, this study provides a novel systems biology based model under the paradigm of Integrated Cellular Geography which can assimilate independently performed experimental results and data published by global researchers on RNA biology to provide important information on biogenesis of exonic circular RNAs considering lncRNAs as precursor molecule.

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Ankyrin repeats in context with human population variation Ankyrin protein repeats bind to a wide range of substrates and are one of the most common protein motifs in nature. Here, we collate a high-quality alignment of 7,407 ankyrin repeats and examine for the first time, the distribution of human population variants from large-scale sequencing of healthy individuals across this family. Population variants are not randomly distributed across the genome but are constrained by gene essentiality and function. Accordingly, we interpret the population variants in context with evolutionary constraint and structural features including secondary structure, accessibility and protein-protein interactions across 383 three-dimensional structures of ankyrin repeats. We find five positions that are highly conserved across homologs and also depleted in missense variants within the human population. These positions are significantly enriched in intra-domain contacts and so likely to be key for repeat packing. In contrast, a group of evolutionarily divergent positions are found to be depleted in missense variants in human but significantly enriched in protein-protein interactions. Our analysis also suggests the domain has three, not two surfaces, each with different patterns of enrichment in protein-substrate interactions and missense variants. Our findings will be of interest to those studying or engineering ankyrin-repeat containing proteins as well as those interpreting the significance of disease variants.

Author SummaryComparison of variation at each position of the amino acid sequence for a protein across different species is a powerful way to identify parts of the protein that are important for its structure and function. Large-scale DNA sequencing of healthy people has recently made it possible to study normal genetic variation within just one species. Our work combines information on genetic differences between over 100,000 people with in-depth analysis of all available three-dimensional structures for Ankyrin repeats which are a widespread family of binding proteins formed by units with similar amino acid sequence that are found in tandem. Our combined analysis identifies sites critical for ankyrin stability as well as the positions most important for substrate interactions and hence function. Although focused only on the Ankyrins, the principles developed in our work are general and can be applied to any protein family.

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HLF Expression Defines the Human Hematopoietic Stem Cell State Hematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and are able to regenerate all blood lineages following transplantation.

Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. While a number of transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported.

To identify genes with the most selective expression in human HSCs, we profiled population- and single-cell transcriptomes of fresh and ex vivo cultured cord blood derived HSPCs as well as peripheral blood, adult bone marrow and fetal liver. Based on these analyses, we propose the master transcription factor HLF (Hepatic Leukemia Factor) as one of the most specific HSC marker genes.

To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells on the basis of a single engineered parameter.

Most importantly, HLF-expressing cells comprise all of the stem cell activity in culture and in vivo during serial transplantation.

Taken together, these results experimentally establish HLF as a defining gene of the human hematopoietic stem cell state and outline a new approach to continuously mark these cells with high fidelity.

Key PointsO_LIIn the human blood system, HLF expression is specific to stem cell populations in primary anatomical sites and during ex vivo expansion.
C_LIO_LICRISPR/rAAV6-mediated integration of a genomic HLF-reporter allows selective and stable genetic labeling of human HSCs ex vivo and in vivo.
C_LI

sentences: LOSS OF PLASMA MEMBRANE LIPID ASYMMETRY CAN INDUCE ORDERED DOMAIN (RAFT) FORMATION In some cases lipids in one leaflet of an asymmetric artificial lipid vesicle can suppress formation of ordered lipid domains (rafts) in the opposing leaflet. Whether suppression of domain formation might occur in plasma membranes was studied using plasma membrane vesicles (PMVs) from RBL-2H3 cells. Membrane domain formation and order was assessed by FRET and fluorescence anisotropy. Ordered domains in PMV prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ~37{degrees}C, while ordered domains in symmetric vesicles formed from extracted PMV lipids were stable to 55{degrees}C, indicating that stability of ordered domains was substantially less in intact PMV. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting that intact PMV have some degree of lipid asymmetry. This was confirmed by annexin binding showing that NEM PMV are much more asymmetric than PMV formed by dithiothreitol/paraformaldehyde treatment. Stabilization of ordered domain formation, and increased membrane order at low temperature was also observed after detergent solubilization of PMV followed by membrane reconstitution via dilution from detergent, which also should destroy asymmetry, even though membrane proteins remained associated with the reconstituted vesicles. Similar changes in domain formation and membrane order were observed after detergent reconstitution of artificial asymmetric lipid vesicles. PMV ordered domain stability was not increased by digesting peripheral domains of PMV proteins with proteinase K. We conclude loss of PMV lipid asymmetry can induce ordered domain formation. Dynamic control of asymmetry may regulate ordered domain formation in plasma membranes.

sentences: The NK receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells Natural killer cells (NK) are a first line of immune defense to eliminate infected, transformed and stressed cells by releasing cytotoxic granules1. NK activation is controlled by the balance of signals transmitted by activating and inhibitory receptors but activating receptor engagement is required to trigger cytotoxicity. The activating receptor NKp46, encoded by the NCR1 gene, is expressed by virtually all NK cells and is the most evolutionarily ancient NK receptor. NKp46 plays a major role in NK recognition of cancer cells, since NKp46 blocking antibodies potently inhibit NK killing of many cancer targets2,3. Although a few viral, fungal and soluble host ligands4 have been identified, the endogenous cell-surface ligand of this important activating NK receptor is unknown. Here we show that NKp46 recognizes and is activated by the P-domain of externalized calreticulin (ecto-CRT). CRT, normally localized to the ER, translocates to the cell surface during ER stress and is a hallmark of chemotherapy-treated dying cancer cells that induce an immune response (immunogenic cell death, ICD)5. NKp46 caps with ecto-CRT in NK immune synapses formed with ecto-CRT-bearing target cells. ER stress, induced by ZIKV infection, ICD-causing chemotherapy drugs and some senescence activators, externalizes CRT and triggers NKp46 signaling. NKp46-mediated killing is inhibited by CRT knockout or knockdown or anti-CRT antibodies and is enhanced by ectopic expression of GPI-anchored CRT. NCR1/Ncr1-deficient human and mouse NK are impaired in killing ZIKV-infected, ER-stressed, and senescent cells and cancer cells that endogenously or ectopically express ecto-CRT. Importantly, NKp46 recognition of ecto-CRT controls the growth of B16 melanoma and RAS-driven lung cancer in mouse models and enhances tumor-infiltrating NK degranulation and cytokine secretion. Thus, ecto-CRT is a danger-associated molecular pattern (DAMP) that is an endogenous NKp46 ligand that promotes innate immune elimination of ER-stressed cells.

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A data-informed mean-field approach to mapping of cortical parameter landscapes Constraining the many biological parameters that govern cortical dynamics is computa-tionally and conceptually difficult because of the curse of dimensionality. This paper addresses these challenges by proposing (1) a novel data-informed mean-field (MF) approach to efficiently map the parameter space of network models; and (2) an organizing principle for studying parameter space that enables the extraction biologically meaningful relations from this high-dimensional data. We illustrate these ideas using a large-scale network model of the Macaque primary visual cortex. Of the 10-20 model parameters, we identify 7 that are especially poorly constrained, and use the MF algorithm in (1) to discover the firing rate contours in this 7D parameter cube. Defining a "biologically plausible" region to consist of parameters that exhibit spontaneous Excitatory and Inhibitory firing rates compatible with experimental values, we find that this region is a slightly thickened codimension-1 submanifold. An implication of this finding is that while plausible regimes depend sensitively on parameters, they are also robust and flexible provided one compensates appropriately when parameters are varied. Our organizing principle for conceptualizing parameter dependence is to focus on certain 2D parameter planes that govern lateral inhibition: Intersecting these planes with the biologically plausible region leads to very simple geometric structures which, when suitably scaled, have a universal character independent of where the intersections are taken. In addition to elucidating the geometry of the plausible region, this invariance suggests useful approximate scaling relations. Our study offers, for the first time, a complete characterization of the set of all biologically plausible parameters for a detailed cortical model, which has been out of reach due to the high dimensionality of parameter space.

Author SummaryCortical circuits are characterized by a high degree of structural and dynamical complexity, and this biological reality is reflected in the large number of parameters in even semi-realistic cortical models. A fundamental task of computational neuroscience is to understand how these parameters govern network dynamics. While some neuronal parameters can be measured in vivo, many remain poorly constrained due to limitations of available experimental techniques. Computational models can address this problem by relating difficult-to-measure parameters to observable quantities, but to do so one must overcome two challenges: (1) the computational expense of mapping a high dimensional parameter space, and (2) extracting biological insights from such a map. This study aims to address these challenges in the following ways: First, we propose a parsimonious data-informed algorithm that efficiently predicts spontaneous cortical activity, thereby speeding up the mapping of parameter landscapes. Second, we show that lateral inhibition provides a basis for conceptualizing cortical parameter space, enabling us to begin to make sense of its geometric structure and attendant scaling relations. We illustrate our approach on a biologically realistic model of the monkey primary visual cortex.

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MetaboListem and TABoLiSTM: Two Deep Learning Algorithms for Metabolite Named Entity Recognition Reviewing the metabolomics literature is becoming increasingly difficult because of the rapid expansion of relevant journal literature. Text-mining technologies are therefore needed to facilitate more efficient literature review. Here we contribute a standardised corpus of full-text publications from metabolomics studies and describe the development of two new metabolite named entity recognition (NER) methods. We introduce two deep learning methods for metabolite NER based on Bidirectional Long Short-Term Memory (BiLSTM) networks incorporating different transfer learning techniques. Our first model (MetaboListem) follows prior methodology using GloVe word embeddings. Our second model exploits BERT and BioBERT for embedding and is named TABoLiSTM (Transformer-Affixed BiLSTM). The methods are trained on a novel corpus annotated using rule-based methods, and evaluated on manually annotated metabolomics articles. MetaboListem (F1 score 0.890, precision 0.892, recall 0.888) and TABoLiSTM (BioBERT version: F1 score 0.909, precision 0.926, recall 0.893) have achieved state-of-the-art performance on metabolite NER. A corpus with >1,200 full-text Open Access metabolomics publications and >116,000 annotated metabolites was created. This work demonstrates that deep learning algorithms are capable of identifying metabolite names accurately and efficiently in text. The proposed corpus and NER algorithms can be used for metabolomics text-mining tasks such as information retrieval, document classification and literature-based discovery.

AvailabilityThe corpus and NER algorithms are freely available with detailed instructions from Github at https://github.com/omicsNLP/MetaboliteNER.

sentences: Intraflagellar transport-A deficiency attenuates ADPKD in a renal tubular- and maturation-dependent manner Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multi-protein complexes. Deletion of different IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult Autosomal Dominant (AD) PKD mouse models, while deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, here we investigate the impact of global deletion of an IFT-A gene, Thm1, in juvenile and adult ADPKD mouse models. Both juvenile and adult models exhibited increased kidney weight:body weight (KW/BW) ratios, renal cysts, inflammation, lengthened renal cilia, and increased levels of the nutrient sensor, O-linked {beta}-N-acetylglucosamine (O-GlcNAc). Thm1 deletion in juvenile ADPKD mice reduced KW/BW ratios and cortical collecting duct cystogenesis, but increased proximal tubular and glomerular dilations and did not reduce inflammation, cilia lengths, and O-GlcNAc signaling. In contrast, Thm1 deletion in adult ADPKD mice markedly attenuated renal cystogenesis, inflammation, cilia lengths, and O-GlcNAc. Thus, unlike IFT-B genes, the role of Thm1 deletion in ADPKD mouse models is development-specific. Unlike an IFT-A adaptor gene, deleting Thm1 in juvenile ADPKD mice is partially ameliorative. Our studies suggest that different microenvironmental factors found in distinct nephron segments and between developing and mature kidneys modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a novel feature and critical regulator of certain key ADPKD pathological processes.

sentences: MetA is a thermal fuse that arrests growth and protects Escherichia coli at elevated temperatures Adaptive stress resistance in microbes is mostly attributed to the expression of stress response genes, such as heat shock proteins, which prevent deterioration of cellular material. Here, we report a novel response of E. coli to heat stress: induction of a growth-arrested state, caused by degradation of an enzyme in the methionine biosynthesis pathway (MetA). While MetA degradation is detrimental for proliferation, we show that the resulting growth arrest has a direct benefit for survival at high temperatures; it protects cells when temperatures rise beyond 50{degrees}C, increasing the survival chances by over 1000-fold. Using a combination of experiments and mathematical modelling, we show that degradation of MetA leads to the coexistence of growing and non-growing cells, allowing microbes to bet-hedge between continued growth if conditions remain bearable and survival if conditions worsen. We test our model experimentally and verify quantitatively how protein expression, degradation rates and environmental stresses affect the partitioning between growing and non-growing cells. Because growth arrest can be abolished with simple mutations, such as point mutations of MetA and knock-outs of proteases, we interpret the breakdown of methionine synthesis as a system that has evolved to disintegrate at high temperature and shut off growth, analogous to  thermal fuses used in engineering to shut off electricity when the device could be damaged by overheating.

sentences: SARS-CoV-2 bearing a mutation at the S1/S2 cleavage site exhibits attenuated virulence and confers protective immunity Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by host furin protease. Proteolytic cleavage activates the spike protein and influences both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogensis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited a low growth property. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, indicating the attenuated variant nature of S gene mutants. We reproduced the attenuated growth of S gene mutants in primary differenciated human airway epithelial cells. This transient infection was enough to induce protective neutralizing antibodies crossreacting with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of SARS-CoV-2 and highlights the potential benefits of S gene mutants as potential immunogens.

sentences: Stimulus-independent neural coding of event semantics: Evidence from cross-sentence fMRI decoding Multivariate neuroimaging studies indicate that the brain represents word and object concepts in a format that readily generalises across stimuli. Here we investigated whether this was true for neural representations of simple events described using sentences. Participants viewed sentences describing four events in different ways. Multivariate classifiers were trained to discriminate the four events using a subset of sentences, allowing us to test generalisation to novel sentences. We found that neural patterns in a left-lateralised network of frontal, temporal and parietal regions discriminated events in a way that generalised successfully over changes in the syntactic and lexical properties of the sentences used to describe them. In contrast, decoding in visual areas was sentence-specific and failed to generalise to novel sentences. In the reverse analysis, we tested for decoding of syntactic and lexical structure, independent of the event being described. Regions displaying this coding were limited and largely fell outside the canonical semantic network. Our results indicate that a distributed neural network represents the meaning of event sentences in a way that is robust to changes in their structure and form. They suggest that the semantic system disregards the surface properties of stimuli in order to represent their underlying conceptual significance.

sentences: Comparing lipid remodeling in mouse adipose and liver tissue with quantitative Raman microscopy Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, non-chronic dietary intervention (a one-week interval) on WAT and BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that one week of HFD intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after one week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance.

sentences: Synergistic epistasis of the deleterious effects of transposable elements The replicative nature and generally deleterious effects of transposable elements (TEs) give rise to an outstanding question about how TE copy number is stably contained in host populations. Classic theoretical analyses predict that, when the decline in fitness due to each additional TE insertion is greater than linear, or when there is synergistic epistasis, selection against TEs can result in a stable equilibrium of TE copy number. While several mechanisms are predicted to yield synergistic deleterious effects of TEs, we lack empirical investigations of the presence of such epistatic interactions. Purifying selection with synergistic epistasis generates repulsion linkage between deleterious alleles and, accordingly, an underdispersed distribution for the number of deleterious mutations among individuals. We investigated this population genetic signal in an African Drosophila melanogaster population and found evidence for synergistic epistasis among TE insertions, especially those expected to have large fitness impacts. Curiously, even though ectopic recombination has long been predicted to generate nonlinear fitness decline with increased TE copy number, TEs predicted to suffer higher rates of ectopic recombination are not more likely to be underdispersed. On the other hand, underdispersed TE families are more likely to show signatures of deleterious epigenetic effects and stronger ping-pong signals of piRNA amplification, a hypothesized source from which synergism of TE-mediated epigenetic effects arises. Our findings set the stage for investigating the importance of epistatic interactions in the evolutionary dynamics of TEs.

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Philympics 2021: Prophage Predictions Perplex Programs Most bacterial genomes contain integrated bacteriophages--prophages--in various states of decay. Many are active and able to excise from the genome and replicate, while others are cryptic prophages, remnants of their former selves. Over the last two decades, many computational tools have been developed to identify the prophage components of bacterial genomes, and it is a particularly active area for the application of machine learning approaches. However, progress is hindered and comparisons thwarted because there are no manually curated bacterial genomes that can be used to test new prophage prediction algorithms.

Here, we present a library of gold-standard bacterial genome annotations that include manually curated prophage annotations, and a computational framework to compare the predictions from different algorithms. We use this suite to compare all extant stand-alone prophage prediction algorithms to identify their strengths and weaknesses.

We provide a FAIR dataset for prophage identification, and demonstrate the accuracy, precision, recall, and f1 score from the analysis of seven different algorithms for the prediction of prophages. We discuss caveats and concerns in this analysis and how those concerns may be mitigated.

sentences: Identification of a Novel SARS-CoV-2 Delta-Omicron Recombinant Virus in the United States Recombination between SARS-CoV-2 virus variants can result in different viral properties (e.g., infectiousness or pathogenicity). In this report, we describe viruses with recombinant genomes containing signature mutations from Delta and Omicron variants. These genomes are the first evidence for a Delta-Omicron hybrid Spike protein in the United States.

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Elucidation of fibril helix structure responsible for swimming in Spiroplasma using electron microscopy Spiroplasma, known pathogens and commensals of arthropods and plants, are helical-shaped bacteria lacking the peptidoglycan layer. They swim by alternating between left- and right-handed cell helicity, which is driven by an internal structure called the ribbon. This system is unrelated to flagellar motility that is widespread in bacteria. The ribbon comprises the bacterial actin homolog MreB and fibril, the protein specific to Spiroplasma. Here, we isolated the ribbon and its core, the fibril filament, and using electron microscopy, found that the helicity of the ribbon and the cell is linked to the helicity of the fibril. Single particle analysis using the negative-staining method revealed that the three-dimensional structures of the fibril filament comprise a repeated ring structure twisting along the filament axis. The handedness of these structures were verified by the structures reconstituted through tomogram from quick-freeze deep-etch replica electron microscopy. Based on these observations, we propose a scheme for the helicity-switching mechanism in which the twists caused by the conformational changes in the fibril filament are accumulated, transmitted to the ribbon, and then propel the cells by rotating the cell body like a screw.

Significance StatementSpiroplasma are commensal and pathogenic bacteria widespread globally in animals and plants. A few species are also recognized as male-killing bacteria of insects. Their special swimming mechanism is caused by helicity switching, which could be the simplest swimming mechanism, because helicity shift can be caused by contraction and extension of paired filaments. This mechanism has attracted research attention for many years because of the possible application in the field of nano actuators; however, the details of this mechanism remain to be clarified. Here, we propose a model for the swimming mechanism based on the structure of the core of the Spiroplasma ribbon.

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A flexible and versatile system for multicolor fiber photometry and optogenetic manipulation Fiber photometry is a technique of growing popularity in neuroscientific research. It is widely used to infer brain activity by recording calcium dynamics in genetically defined populations of neurons. Aside from the wide variety of calcium indicators, other genetically encoded biosensors have recently been engineered to measure membrane potential, neurotransmitter release, pH, or various cellular metabolites, such as ATP or cAMP. Due to the spectral characteristics of these molecular tools, different assemblies of optical hardware are usually needed to reveal the full potential of different biosensors. In addition, the combination of multiple biosensors in one experiment often requires the investment in more complex equipment, which limits the flexibility of the experimental design. Such constraints often hamper a straightforward implementation of new molecular tools, evaluation of their performance in vivo, and design of new experimental paradigms - especially if the financial budget is a limiting factor. Here, we propose a novel approach for fiber photometry recordings, based on a multimode optical fused-fiber coupler (FFC) for both light delivery and collection. Recordings can readily be combined with optogenetic manipulations in a single device without the requirement for dichroic beam-splitters. In combination with a multi-color light source and appropriate emission filters, our approach offers remarkable flexibility in experimental design and facilitates the implication of new molecular tools in vivo at minimal cost. The ease of assembly, operation, characterization, and customization of this platform holds the potential to foster the development of experimental strategies for multicolor fused fiber photometry (FFP) combined with optogenetics far beyond its current state.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/484590v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@13abbdcorg.highwire.dtl.DTLVardef@10384d7org.highwire.dtl.DTLVardef@67938org.highwire.dtl.DTLVardef@1005aee_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIFused Fiber Photometry (FFP) enables straightforward monitoring and manipulation of brain activity
C_LIO_LIFFP allows monitoring of indicators with virtually any spectral characteristics
C_LIO_LIFFP is compatible with simultaneous optogenetic manipulation
C_LIO_LIEasy assembly, characterization, and customization
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sentences: The usefulness of sparse k-means in metabolomics data: Anexample from breast cancer data In processing metabolomics data, multidimensional quantitative data from thousands of metabolites are often sparse, that is, only a small fraction of metabolites are relevant to the phenotype of interest. Clustering is therefore used to discover subtypes from omics data. Sparse processing, which selects important metabolites from the total omics data, is an effective clustering technique. This study investigated the effectiveness of sparse k-means for metabolomics data. Specifically, sparse k-means was used to cluster blood lipid metabolite data of breast cancer patients in two studies: (1) before and after menopause, and (2) pre- and postoperative chemotherapy. In both cases, sparse k-means showed comparable discrimination accuracy with fewer metabolites than k-means. Furthermore, when the L1 norm values were varied, no significant changes were observed. The mean silhouette coefficients of sparse k-means and k-means were (1) 0.38 {+/-} 0.14 (S.D.) and 0.17 {+/-} 0.01, (2) 0.38 {+/-} 0.07 and 0.17 {+/-} 0.01, indicating that feature selection using sparse k-means can improve clustering results. In addition, metabolite selection using sparse k-means was consistent regardless of the test data or the constrained value of the L1 norm, indicating robustness.

sentences: BugSplit: highly accurate taxonomic binning of metagenomic assemblies enables genome-resolved metagenomics A large gap remains between sequencing a microbial community and characterizing all of the organisms inside of it. Here we develop a novel method to taxonomically bin metagenomic assemblies through alignment of contigs against a reference database. We show that this workflow, BugSplit, bins metagenome-assembled contigs to species with a 33% absolute improvement in F1-score when compared to alternative tools. We perform nanopore mNGS on patients with COVID-19, and using a reference database predating COVID-19, demonstrate that BugSplits taxonomic binning enables sensitive and specific detection of a novel coronavirus not possible with other approaches. When applied to nanopore mNGS data from cases of Klebsiella pneumoniae and Neisseria gonorrhoeae infection, BugSplits taxonomic binning accurately separates pathogen sequences from those of the host and microbiota, and unlocks the possibility of sequence typing, in silico serotyping, and antimicrobial resistance prediction of each organism within a sample. BugSplit is available at https://bugseq.com/academic.

sentences: Tropical land use alters functional diversity of soil food webs and leads to monopolization of the detrital energy channel Agricultural expansion is among the main threats to biodiversity and functions of tropical ecosystems. Conversion of rainforest into plantations erodes biodiversity with little-explored consequences for food-web structure and energetics of belowground communities, and associated ecosystem functions and services. We used a unique combination of approaches in stable isotope analysis and food web energetics to analyze in a comprehensive way consequences of the conversion of rainforest into plantations on the structure of and channeling of energy through soil animal food webs in Sumatra, Indonesia. Across the 23 animal groups studied, the channeling of energy shifted towards freshly-fixed plant carbon in plantations, indicating fast energy channeling as opposed to slow energy channeling in rainforests. Earthworms as major detritivores stayed unchanged in their trophic niche and monopolized the detrital pathway in plantations. Functional diversity metrics of soil food webs reflected the reduced amount of litter, tree density and species richness in plantations, providing guidelines how to improve the complexity of the structure of and channeling of energy through soil food webs. Our results highlight the strong restructuring of soil food webs with the conversion of rainforest into plantations threatening soil functioning and ecosystem stability in the long term.

sentences: Extinction risk, reconstructed catches, and management of chondrichthyan fishes in the Western Central Atlantic Ocean Chondrichthyan fishes are among the most threatened vertebrates on the planet because many species have slow life histories that are outpaced by intense fishing. The Western Central Atlantic Ocean, which includes the greater Caribbean, is a hotspot of chondrichthyan biodiversity and abundance, but is historically characterized by extensive shark and ray fisheries and a lack of sufficient data for effective management and conservation. To inform future research and management decisions, we analyzed patterns in chondrichthyan extinction risk, reconstructed catches, and regulations in this region. We summarized the extinction risk of 180 sharks, rays, and chimaeras using contemporary IUCN Red List assessments and found that over one-third (35.6%) were assessed as Vulnerable, Endangered, or Critically Endangered largely due to fishing. Reconstructed catches from 1950 to 2016 reached their peak in 1992, then declined by 40.2% through the end of the series. The United States, Venezuela, and Mexico were responsible for most catches and hosted large proportions of the regional distributions of threatened species; these countries therefore held the greatest responsibility for chondrichthyan management. The abundance and resolution of fisheries landings data were poor in much of the region, and national-level regulations varied widely across jurisdictions. Deepwater fisheries represent an emerging threat, although many deepwater chondrichthyans currently find refuge beyond the depths of most fisheries. Regional collaboration as well as effective and enforceable management informed by more complete fisheries data, particularly from small-scale fisheries, are required to protect and recover threatened species and ensure sustainable fisheries.

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MeShClust v3.0: High-quality clustering of DNA sequences using the mean shift algorithm and alignment-free identity scores BackgroundTools for accurately clustering biological sequences are among the most important tools in computational biology. Two pioneering tools for clustering sequences are CD-HIT and UCLUST, both of which are fast and consume reasonable amounts of memory; however, there is a big room for improvement in terms of cluster quality. Motivated by this opportunity for improving cluster quality, we applied the mean shift algorithm in MeShClust v1.0. The mean shift algorithm is an instance of unsupervised learning. Its strong theoretical foundation guarantees the convergence to the true cluster centers. Our implementation of the mean shift algorithm in MeShClust v1.0 was a step forward; however, it was not the original algorithm. In this work, we make progress toward applying the original algorithm while utilizing alignment-free identity scores in a new tool: MeShClust v3.0.

ResultsWe evaluated CD-HIT, MeShClust v1.0, MeShClust v3.0, and UCLUST on 22 synthetic sets and five real sets. These data sets were designed or selected for testing the tools in terms of scalability and different similarity levels among sequences comprising clusters. On the synthetic data sets, MeShClust v3.0 outperformed the related tools on all sets in terms of cluster quality. On two real data sets obtained from human microbiome and maize transposons, MeShClust v3.0 outperformed the related tools by wide margins, achieving 55%--300% improvement in cluster quality. On another set that includes degenerate viral sequences, MeShClust v3.0 came third. On two bacterial sets, MeShClust v3.0 was the only applicable tool because of the long sequences in these sets. MeShClust v3.0 requires more time and memory than the related tools; almost all personal computers at the time of this writing can accommodate such requirements. MeShClust v3.0 can estimate an important parameter that controls cluster membership with high accuracy.

ConclusionsThese results demonstrate the high quality of clusters produced by MeShClust v3.0 and its ability to apply the mean shift algorithm to large data sets and long sequences. Because clustering tools are utilized in many studies, providing high-quality clusters will help with deriving accurate biological knowledge.

sentences: Genome size evolution in the beetle genus Diabrotica Diabrocite corn rootworms are one of the most economically significant pests of maize in the United States and Europe and an emerging model for insect-plant interactions. Genome sizes of several species in the genus Diabrotica were estimated using flow cytometry along with that of Acalymma vittatum as an outgroup. Genome sizes ranged between 1.56 - 1.64 gigabase pairs (Gb) and between 2.26-2.59 Gb, respectively, for the Diabrotica subgroups fucata and virgifera; the Acalymma vittatum genome size was around 1.65 Gb. This result indicated that a substantial increase in genome size occurred in the ancestor of the virgifera group. Further analysis of fucata group and virgifera group genome sequencing reads indicated that the genome size difference between the Diabrotica subgroups could be attributed to a higher content of transposable elements, mostly miniature inverted-transposable elements (MITEs) and gypsy-like long terminal repeat (LTR) retroelements.

sentences: Phenotypic plasticity explains apparent reverse evolution of fat synthesis in parasitoid insects Over the last few decades, numerous examples have been described where a trait that was once lost during the course of evolution had been regained. Here, we argue that such reverse evolution can also become apparent when trait expression is plastic in response to the environment. We tested this hypothesis for the loss and regain of fat synthesis in parasitic wasps. Wasps from lineages that supposedly regained lipogenic ability ~80 million years ago were grown under a fat-poor or fat-rich environment. In line with our hypothesis, it turned out that fat synthesis had not been lost and regained, but was only switched on in low-fat environments. Functional protein domains of key lipogenesis genes were also found in other parasitoid species, suggesting that plasticity of fat synthesis may be more widespread in parasitoids. Individual-based simulations then revealed that a switch for plastic expression can remain functional in the genome for thousands of generations, even if it is only used sporadically. The evolution of plasticity may thus also explain other examples of apparent reverse evolution.

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spongEffects: ceRNA modules offer patient-specific insights into the miRNA regulatory landscape MotivationCancer is one of the leading causes of death worldwide. Despite significant improvements in prevention and treatment, mortality remains high for many cancer types. Hence, innovative methods that use molecular data to stratify patients and identify biomarkers are needed. Promising biomarkers can also be inferred from competing endogenous RNA (ceRNA) networks that capture the gene-miRNA-gene regulatory landscape. Thus far, the role of these biomarkers could only be studied globally but not in a sample-specific manner. To mitigate this, we introduce spongEffects, a novel method that infers subnetworks (or modules) from ceRNA networks and calculates patient- or sample-specific scores related to their regulatory activity.

ResultsWe show how spongEffects can be used for downstream interpretation and machine learning tasks such as tumor classification and for identifying subtype-specific regulatory interactions. In a concrete example of breast cancer subtype classification, we prioritize modules impacting the biology of the different subtypes. In summary, spongEffects prioritizes ceRNA modules as biomarkers and offers insights into the miRNA regulatory landscape. Notably, these module scores can be inferred from gene expression data alone and can thus be applied to cohorts where miRNA expression information is lacking.

Availabilityhttps://bioconductor.org/packages/devel/bioc/html/SPONGE.html spongEffects scores calculated for TCGA datasets: https://doi.org/10.6084/m9.figshare.19328885.v1

Contactmarkus.list@tum.de

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Recruitment of transcriptional effectors by Cas9 creates cis regulatory elements and demonstrates distance-dependent transcriptional regulation It is essential to regulate the expression of genes, such as those encoding the proteins of the cardiac sarcomere. This regulation is often mediated by cis regulatory elements termed enhancers and repressors that recruit transcription factors to gene-distal sites. However, the relationship between transcription factors recruitment to gene-distant sites and the regulation of gene expression is not fully understood. Specifically, it is unclear if such recruitment to any genomic site is sufficient to form an enhancer or repressor at the site, and what is the relationship between the cis regulatory elements position and its ability to control the transcription of distant genes. Using dead Cas9 to recruit either viral or endogenous transcription factor activation domains, we demonstrate that targeting  naive genomic sites lacking open chromatin or active enhancer marks is sufficient to alter the chromatin signature of the target site, the distant gene promoter, and significantly induce the distant gene expression, even across chromatin insulating loci. The magnitude of induction is affected by the distance between the activation site and the cognate gene in a non-linear manner. Dead Cas9 mediated recruitment of repression domains behave similarly to activation in that targeting of non-regulatory regions could repress gene expression with a nonlinear distance dependence and across chromatin insulating loci. These findings expand the models of enhancer generation and function by showing that an arbitrary genomic site can become a regulatory element and interact epigenetically and transcriptionally with a distant promoter. They also provide new fundamental insights into the rules governing gene expression.

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A novel TREM2 splice isoform lacking the ligand binding is expressed in brain and shares localization BackgroundGenetic variants in TREM2 are strongly associated with Alzheimers Disease (AD) risk but alternative splicing in TREM2 transcripts has not been comprehensively described.

ObjectiveRecognizing that alternative splice variants can result in reduced gene expression and/or altered function, we sought to fully characterize splice variation in TREM2.

MethodsHuman blood and anterior cingulate autopsy tissue from 61 donors were used for end-point and quantitative PCR and Western blotting to identify and quantify novel TREM2 isoforms.

ResultsIn addition to previously described transcripts lacking exon 3 or exon 4, or retaining part of intron 3, we identified novel isoforms lacking exon 2, along with isoforms lacking multiple exons. Isoforms lacking exon 2 were predominant at approximately 10% of TREM2 mRNA in the brain. Expression of TREM2 and frequency of exon 2 skipping did not differ between AD samples and non-AD controls (p = 0.1268 and p = 0.4909, respectively). Further, these novel splice isoforms were also observed across multiple tissues with similar frequency (range 5.3 - 13.0%). We found that the exon 2 skipped isoform D2-TREM2 is translated to protein and localizes similarly to full-length TREM2 protein, that both proteins are primarily retained in the Golgi complex, and that D2-TREM2 is expressed in AD and non-AD brain.

ConclusionSince the TREM2 ligand binding domain is encoded by exon 2, and skipping this exon retains reading frame while conserving localization, we hypothesize that D2-TREM2 acts as an inhibitor of TREM2 and targeting TREM2 splicing may be a novel therapeutic pathway for AD.

sentences: Procyanidin C1 is a natural agent with senolytic activity against aging and age-related diseases Aging causes functional decline of multiple organs and increases the risk of age-related pathologies. In advanced lives, accumulation of senescent cells, which develop the senescence-associated secretory phenotype (SASP), promotes chronic inflammation and causes diverse conditions. Here we report the frontline outcome of screening a natural product library with human primary stromal cells as an experimental model. Multiple candidate compounds were assayed, and grape seed extract (GSE) was selected for further investigation due to its leading capacity in targeting senescent cells. We found procyanidin C1 (PCC1), a polyphenolic component, plays a critical role in mediating the antiaging effects of GSE. PCC1 blocks the SASP expression when used at low concentrations. Importantly, it selectively kills senescent cells upon application at higher concentrations, mainly by enhancing production of reactive oxygen species (ROS) and disturbing mitochondrial membrane potential, processes accompanied by upregulation of Bcl-2 family pro-apoptotic factors Puma and Noxa in senescent cells. PCC1 depletes senescent cells in treatment-damaged tumor microenvironment (TME) and enhances therapeutic efficacy when combined with chemotherapy in preclinical assays. Intermittent administration of PCC1 to both senescent cell-implanted mice and naturally aged animals alleviated physical dysfunction and prolonged post-treatment survival, thus providing substantial benefits in late life stage. Together, our study identifies PCC1 as a distinct natural senolytic agent, which may be exploited to delay aging and control age-related pathologies in future medicine.

sentences: Growing Glycans in Rosetta: Accurate de novo glycan modeling, density fitting, and rational sequon design Carbohydrates and glycoproteins modulate key biological functions. Computational approaches inform function to aid in carbohydrate structure prediction, structure determination, and design. However, experimental structure determination of sugar polymers is notoriously difficult as glycans can sample a wide range of low energy conformations, thus limiting the study of glycan-mediated molecular interactions. In this work, we expanded the RosettaCarbohydrate framework, developed and benchmarked effective tools for glycan modeling and design, and extended the Rosetta software suite to better aid in structural analysis and benchmarking tasks through the SimpleMetrics framework. We developed a glycan-modeling algorithm, GlycanTreeModeler, that computationally builds glycans layer-by-layer, using adaptive kernel density estimates (KDE) of common glycan conformations derived from data in the Protein Data Bank (PDB) and from quantum mechanics (QM) calculations. After a rigorous optimization of kinematic and energetic considerations to improve near-native sampling enrichment and decoy discrimination, GlycanTreeModeler was benchmarked on a test set of diverse glycan structures, or "trees". Structures predicted by GlycanTreeModeler agreed with native structures at high accuracy for both de novo modeling and experimental density-guided building. GlycanTreeModeler algorithms and associated tools were employed to design de novo glycan trees into a protein nanoparticle vaccine that are able to direct the immune response by shielding regions of the scaffold from antibody recognition. This work will inform glycoprotein model prediction, aid in both X-ray and electron microscopy density solutions and refinement, and help lead the way towards a new era of computational glycobiology.

sentences: Inhibition of the IL-17A axis Protects against Immune-related Adverse Events while Supporting Checkpoint Inhibitor Anti-tumor Efficacy Checkpoint inhibitor (ICI) immunotherapy leverages the bodys own immune system to attack cancer cells but leads to unwanted autoimmune side effects in up to 60% of patients. Such immune related adverse events (IrAE) may lead to treatment interruption, permanent organ dysfunction, hospitalization and premature death. Thyroiditis is one of the most common IrAE, but the cause of thyroid IrAE remains unknown. Here we present a novel mouse model in which checkpoint inhibitor therapy leads to multi-organ autoimmune infiltrates and show that activation and infiltration of Type 3 immune cells including IL17A+ ROR{gamma}t+ CD4+ (T helper 17 or Th17) and gamma delta 17 ({gamma}{delta}T17) T cells promote thyroid IrAE development. IL-17A+ T cells were similarly found in thyroid specimens from cancer patients treated with ICI who developed thyroid IrAE. Furthermore, antibody-based inhibition of IL-17A, a clinically available therapy, significantly reduced thyroid IrAE development in ICI-treated mice. Finally, combination of IL-17A neutralization with ICI treatment in multiple tumor models did not reduce ICI anti-tumor efficacy. These studies suggest that targeting Th17 and {gamma}{delta}17 function may reduce IrAE without impairing ICI anti-tumor efficacy and may be a generalizable strategy to address IL17-mediated IrAE.

sentences: ZFP36L1 regulates Fgf21 mRNA turnover and modulates alcoholic hepatic steatosis and inflammation in mice Zinc finger protein 36 like 1 (ZFP36L1) enhances the turnover of mRNAs containing AU-rich elements (AREs) in their 3untranslated regions (3UTR). The physiological and pathological functions of ZFP36L1 in liver, however, remain largely unknown. To investigate the role of ZFP36L1 in liver physiology and pathology, we generated liver-specific ZFP36L1-deficient (Zfp36l1flox/flox /Cre+; L1LKO) mice. Under normal conditions, the L1LKO mice and their littermate controls (Zfp36l1flox/flox/Cre-; L1FLX) appeared normal. When fed a Lieber-DeCarli liquid diet containing alcohol, L1LKO mice were significantly protected from developing alcohol-induced hepatic steatosis and inflammation compared to L1FLX mice. Serum ALT levels were significantly increased in alcohol-fed L1FLX versus alcohol-fed L1LKO mice. RNA-Seq analysis revealed 584 differentially-expressed transcripts in L1FLX alcohol-fed mice, many of which were inflammatory mediators, compared to only 159 in alcohol-fed L1LKO mice. Most importantly, fibroblast growth factor 21 (Fgf21) mRNA was significantly increased in the livers of alcohol-fed L1LKO mice but not in the alcohol-fed control group. The Fgf21 mRNA contains three AREs in its 3UTR, and Fgf21 3UTR was directly regulated by ZFP36L1 in luciferase reporter assays. Steady state levels of Fgf21 mRNA were significantly decreased by wildtype ZFP36L1, but not by a non-binding zinc-finger ZFP36L1 mutant. Finally, wildtype ZFP36L1, but not the ZFP36L1 mutant, bound to Fgf21 3UTR ARE RNA probe. Our results demonstrate that ZFP36L1 inactivation protects against alcohol-induced hepatic steatosis and liver injury, possibly by stabilizing Fgf21 mRNA. Our findings suggest that the modulation of ZFP36L1 may be beneficial in the prevention or treatment of human alcoholic liver disease.

sentences: A Role for Synaptonemal Complex in Meiotic Mismatch Repair During meiosis a large subset of interhomolog recombination repair intermediates form within the physical context of the synaptonemal complex (SC), a protein-rich structure assembled at the interface of aligned homologous chromosomes. However, the functional relationship between SC structure and homologous recombination remains poorly defined. In prior work we determined that tripartite SC is dispensable for recombination in S. cerevisiae; SC central element proteins Ecm11 and Gmc2 instead limit the number of recombination events. Here we report that while dispensable for recombination per se, SC central element proteins influence the processing of interhomolog recombination intermediates in a manner that minimizes errors in mismatch correction. Failure to correct mis-paired bases within heteroduplex at meiotic recombination sites leads to genotypically sectored colonies (post meiotic segregation events) arising from mitotic proliferation of mismatch-containing spores. We discovered an increase in post-meiotic segregation at the THR1 locus in cells lacking Ecm11 or Gmc2, or in the SC-deficient but crossover recombination-proficient zip1[{Delta}21-163] mutant. High-throughput sequencing of octad meiotic products revealed a genome-wide increase in recombination events with uncorrected mismatches in ecm11 mutants relative to wild type. Meiotic cells missing Ecm11 also display longer gene conversion tracts, but tract length alone does not account for the higher frequency of uncorrected mismatches. Interestingly, the per-nucleotide mismatch frequency is elevated in ecm11 mutants when analyzing all gene conversion tracts, but is similar between wild type and ecm11 if one considers only those events with uncorrected mismatches. Our data suggest that a subset of recombination events is similarly susceptible to mismatch repair errors in both wild type and ecm11 strains, but in ecm11 mutants many more recombination events fall into this inefficient repair category. Finally, we observe elevated post-meiotic segregation at THR1 in mutants with a dual deficiency in MutS{gamma}-mediated crossover recombination and SC assembly, but not in the mlh3 mutant, which lacks MutS{gamma} crossovers but has abundant SC. We propose that SC structure promotes efficient mismatch repair of joint molecule recombination intermediates resolved via both MutS{gamma}-associated and MutS{gamma}-independent pathways, and is the molecular basis for elevated post-meiotic segregation in both MutS{gamma} crossover-proficient (ecm11, gmc2) and MutS{gamma} crossover-deficient (msh4, zip3) strains.

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Two transcriptionally distinct pathways drive female development in a reptile with both genetic and temperature dependent sex determination How temperature determines sex remains unknown. A recent hypothesis proposes that conserved cellular mechanisms (calcium and redox;  CaRe status) sense temperature and identify genes and regulatory pathways likely to be involved in driving sexual development. We take advantage of the unique sex determining system of the model organism, Pogona vitticeps, to assess predictions of this hypothesis. P. vitticeps has ZZ male: ZW female sex chromosomes whose influence can be overridden in genetic males by high temperatures, causing male-to-female sex reversal. We compare a developmental transcriptome series of ZWf females and temperature sex reversed ZZf females. We demonstrate that early developmental cascades differ dramatically between genetically driven and thermally driven females, later converging to produce a common outcome (ovaries). We show that genes proposed as regulators of thermosensitive sex determination play a role in temperature sex reversal. Our study greatly advances the search for the mechanisms by which temperature determines sex.

Author SummaryIn many reptiles and fish, environment can determine, or influence, the sex of developing embryos. How this happens at a molecular level that has eluded resolution for half a century of intensive research. We studied the bearded dragon, a lizard that has sex chromosomes (ZZ male and ZW female), but in which that temperature can override ZZ sex chromosomes to cause male to female sex reversal. This provides an unparalleled opportunity to disentangle, in the same species, the biochemical pathways required to make a female by these two different routes. We sequenced the transcriptomes of gonads from developing ZZ reversed and normal ZW dragon embryos and discovered that different sets of genes are active in ovary development driven by genotype or temperature. Females whose sex was initiated by temperature showed a transcriptional profile consistent with the recently-proposed Calcium-Redox hypotheses of cellular temperature sensing. These findings are an important for understanding how the environment influences the development of sex, and more generally how the environment can epigenetically modify the action of genes.

sentences: DeepREAL: A Deep Learning Powered Multi-scale Modeling Framework Towards Predicting Out-of-distribution Receptor Activity of Ligand Binding Drug discovery has witnessed intensive exploration of the problem of drug-target physical interactions over two decades, however, a strong drug binding affinity to a single target often fails to translate into desired clinical outcomes. A critical knowledge gap needs to be filled for correlating drug-target interactions with phenotypic responses: predicting the receptor activities or function selectivity upon the ligand binding (i.e., agonist vs. antagonist) on a genome-scale and for novel chemicals. Two major obstacles compound the difficulty on this direction: known data of receptor activity is far too scarce to train a robust model in light of genome-scale applications, and real-world applications need to deploy a model on data from various shifted distributions. To address these challenges, we have developed an end-to-end deep learning framework, DeepREAL, for multi-scale modeling of genome-wide receptor activities of ligand binding. DeepREAL utilizes self-supervised learning on tens of millions of protein sequences and pre-trained binary interaction classification to solve the data distribution shift and data scarcity problems. Extensive benchmark studies that simulate real-world scenarios demonstrate that DeepREAL achieves state-of-the-art performance in out-of-distribution settings.

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Spergulin-A strives intra-macrophage leishmanicidal activity by regulating the host P2X7R-P38MAPK axis Current drugs are inadequate for the treatment of visceral leishmaniasis an immunosuppressive ailment caused by Leishmania donovani. Regrettably, there is no plant-origin antileishmanial drug present. P2X7R is constitutively present on macrophage surfaces and can be a putative therapeutic target in intra-macrophage pathogens with function attributes towards inflammation, host cell apoptosis, altered redox, and phagolysosomal maturation by activating p38MAPK. Here we demonstrated that the initial interaction of Spergulin-A (SpA), a triterpenoid saponin with RAW 264.7 macrophages was mediated through P2X7R involving the signaling cascade intermediates Ca++, P38MAPK, and NF-{kappa}{beta}. P38MAPK involvement is shown to have specific and firm importance in leishmanial killing with increased NF-{kappa}Bp65. Phago-lysosomal maturation by Sp A also campaigns for another contribution of P2X7R. In vivo evaluation of the anti-leishmanial activity of Sp A was monitored through expression analyses of P2X7R, P38MAPK, and NF-{kappa}{beta} in murine spleen and bone-marrow macrophages and advocated Sp A of being a natural compound of leishmanicidal functions which acted through the P2X7R-P38MAPK axis.

SIGNIFICANCE OR IMPORTANCEPreciously, this manuscript demonstrated previously unreported initial interaction of Spergulin-A, a triterpenoid saponin isolated from Glinus oppositifolius with macrophages through P2X7R involving the signaling cascade intermediates Ca++, P38MAPK, and NF-{kappa}{beta}. Signaling interaction is shown to have specific importance in the leishmanial killing. Phago-lysosomal maturation also campaigns for another contribution of P2X7R. In vivo evaluation was monitored through P2X7R, P38MAPK, and NF-{kappa}{beta} in murine spleen and bone-marrow macrophages and advocated Sp A of being a natural compound of leishmanicidal functions which acted through the P2X7R-P38MAPK axis. The result supports that Spergulin-A can provide new lead molecules for the development of alternative drugs against VL. We feel very strongly that this work can be very interesting as it describes a detailed evaluation of leishmanicidal effect by Sp A and thus has every potential to attract a lot of workers especially in the fields of pharmacology, drug development, immunology, as well as parasitology.

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Ultraviolet screening by slug tissue and tight packing of plastids protect photosynthetic sea slugs from photoinhibition O_LIOne of the main unsolved questions regarding photosynthetic sea slugs is how the slug plastids handle photoinhibition of Photosystem II. Photoinhibition has not been studied in detail in these animals although resilience against photoinhibition might obviously explain the longevity of plastids inside animal cytosol.
C_LIO_LILight response and action spectrum of photoinhibition were measured from the slug Elysia timida and its prey alga Acetabularia acetabulum. Plastid packing in the slugs and algae was compared with spectroscopic and microscopic methods. The importance of plastid concentration was also estimated by measuring photoinhibition from starved slugs.
C_LIO_LICompared to A. acetabulum, E. timida is highly resistant against photoinhibition. The resilience of the slugs is even more pronounced in the UV-region, as the slug tissue screens UV radiation. The plastids in the slug tissue are tightly packed, and the outer plastids protect the inner ones from photoinhibition.
C_LIO_LIThe sea slug E. timida protects its plastids from photoinhibition by screening UV radiation and packing the plastids tightly in its tissues. Both mechanisms enhance the longevity of the plastids in slug cytosol and ameliorate the need for repair of photoinhibited Photosystem II.
C_LI

sentences: Novel tuberculosis combination regimens of two and three-months therapy duration The tuberculosis treatment duration is 4-6 months, but can be 18-20 months with multidrug resistance. Ultrashort regimens that cure regardless of resistance status ["pan-tuberculosis regimens"] would be a major step towards global tuberculosis control. Starting with seven drugs [bedaquiline, delamanid, pretomanid, OPC-167832, sutezolid, moxifloxacin, and pyrazinamide] in clinical testing, we calculated that there were thousands of possible novel combinations to evaluate. We used mathematical and pharmacokinetics/pharmacodynamic-based hollow fiber modeling to reduce this complexity to nine combinations, which we tested. Each regimens fast and slow growth bacteria kill-slope trajectories and times-to-extinction were estimated and translated to minimum required therapy duration in patients. Sputum microbial kill trajectories of two combination regimens that have undergone clinical testing were correctly predicted by this approach. Four pan-tuberculosis regimens were predicted to achieve relapse-free cure after 2-3 months therapy in patients. The kill-slopes were used to design expedited clinical trials that minimize patients risks.

sentences: Pre-trauma memory contextualization as predictor for PTSD-like behavior in male rats While many people experience potentially threatening events during their life, only a minority develops posttraumatic stress disorder (PTSD). The identification of individuals at risk among those exposed to trauma is crucial for PTSD prevention in the future. Since re-experiencing trauma elements outside of the original trauma-context is a core feature of PTSD, we investigate if the ability to bind memories to their original encoding context (i.e. memory contextualisation) predicts PTSD vulnerability. We hypothesize that pre-trauma neutral memory contextualization (under stress) negatively relates to PTSD-like behavior, in a prospective design using the cut-off behavioral criteria rat model for PTSD. 72 male Sprague Dawley rats were divided in two experimental groups to assess the predictive value of 1) memory contextualization without acute stress (NS-group) and 2) memory contextualization during the recovery phase of the acute stress-response (S-group) for susceptibility to PTSD-like behavior. A powerful extension to regression analysis-path analysis-was used to test this specific hypothesis, together with secondary research questions. Following traumatic predator scent stress, 19.4% of the rats displayed PTSD-like behavior. Results showed a negative relation between pre-trauma memory contextualization and PTSD-like behavior, but only in the NS-group. Pre-trauma memory contextualization was positively related to fear association in the trauma environment, again only in the NS group. If the predictive value of pre-trauma contextualization of neutral information under non-stressful conditions for PTSD susceptibility is replicated in prospective studies in humans, this factor would supplement already known vulnerability factors for PTSD and improve the identification of individuals at risk among the trauma exposed, especially those at high trauma risk such as soldiers deployed on a mission.

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Rules for the self-assembly of ESCRT-III on endosomes We, the authors, have withdrawn this manuscript. During our work on the revision of this manuscript for eLife, it became clear that several key results of the manuscript were not reproducible by my lab. The rules for ESCRT-III assembly and the model that we have proposed in this manuscript are no longer supported by the data. Currently we are performing additional experiments to test some of our conclusions further. Therefore, we do not wish this work to be cited.

We sincerely apologize to the scientific community for any inconvenience resulting from this manuscript.

If you have any questions, please contact the corresponding author.

sentences: Identification of a fully dechlorinated product of chlordecone in soil microcosms and enrichment cultures Anaerobic microcosms constructed with soil from Guadeloupe, amended with electron donor (ethanol and acetone) and incubated for more than a decade, transformed chlordecone (CLD) into a suite of progressively more dechlorinated products, including a fully dechlorinated carboxylated indene product. This fully dechlorinated transformation product has never before been observed and indicates that complete dechlorination of CLD is possible. The carboxylated indene was detected by LC-MS and structure was confirmed by LC-MS/MS using a Q-Exactive Orbitrap mass spectrometer.

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Rapid in vivo development of resistance to daptomycin in vancomycin-resistant Enterococcus faecium due to genomic rearrangements BackgroundDaptomycin is a cyclic lipopeptide used in the treatment of vancomycin-resistant Enterococcus faecium (VREfm). However, the development of daptomycin-resistant VREfm challenges the treatment of nosocomial VREfm infections. Resistance mechanisms of daptomycin are not fully understood. Here we analysed the genomic changes leading to a daptomycin-susceptible VREfm isolate becoming resistant after 40 days of daptomycin and linezolid combination therapy.

MethodsThe two isogenic VREfm isolates (daptomycin-susceptible and daptomycin-resistant) were analysed using whole genome sequencing with Illumina and Nanopore.

ResultsWhole genome comparative analysis identified the loss of a 46.5 kb fragment and duplication of a 29.7 kb fragment in the daptomycin-resistant isolate, with many implicated genes involved in cell wall synthesis. Two plasmids of the daptomycin-susceptible isolate were also found integrated in the chromosome of the resistant isolate. One nonsynonymous SNP in the rpoC gene was identified in the daptomycin-resistant isolate.

ConclusionsDaptomycin resistance developed through chromosomal rearrangements leading to altered cell wall structure. Such novel types of resistance mechanisms can only be identified by comparing closed genomes of isogenic isolates.

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Standardised empirical dispersal kernels emphasise the pervasiveness of long-distance dispersal in European birds O_LIDispersal is a key life-history trait for most species and essential to ensure connectivity and gene flow between populations and facilitate population viability in variable environments. Despite the increasing importance of range shifts due to global change, dispersal has proved difficult to quantify, limiting empirical understanding of this phenotypic trait and wider synthesis.
C_LIO_LIHere we aim to estimate and compare empirical dispersal kernels for European breeding birds considering average dispersal, natal (before first breeding) and breeding dispersal (between subsequent breeding attempts), and test whether different dispersal properties are phylogenetically conserved.
C_LIO_LIWe standardised and analysed data from an extensive volunteer-based bird ring-recoveries database in Europe (EURING) by accounting for biases related to different censoring thresholds in reporting between countries and to migratory movements. Then, we fitted four widely used probability density functions in a Bayesian framework to compare and provide the best statistical descriptions of the average, the natal and the breeding dispersal kernels for each bird species.
C_LIO_LIThe dispersal movements of the 234 European bird species analysed were statistically best explained by heavy-tailed kernels, meaning that while most individuals disperse over short distances, long-distance dispersal is a feature in almost all bird species. The overall phylogenetic signal in both median and long dispersal distances was low (Pagels {lambda} < 0.40), implying a high degree of taxonomic generality in our findings. As expected in birds, natal dispersal was 5 Km greater as an average than breeding dispersal for most species (88% species analysed).
C_LIO_LIOur comprehensive analysis of empirical kernels indicates that long-distance dispersal is common among European breeding bird species and across life stages. The dispersal estimates offer a first guide to selecting appropriate dispersal kernels in range expansion studies and provide new avenues to improve our understanding of the mechanisms and rules underlying dispersal events.
C_LI

sentences: Secreted SARS-CoV-2 ORF8 modulates the cytokine expression profile of human macrophages Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-{kappa}B signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.

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Dynamics of Functional Network Organization Through Graph Mixture Learning Understanding the organizational principles of human brain activity at the systems level remains a major challenge in network neuroscience. Here, we introduce a fully data-driven approach based on graph learning to extract meaningful repeating network patterns from regionally-averaged time-courses. We use the Graph Laplacian Mixture Model (GLMM), a generative model that treats functional data as a collection of signals expressed on multiple underlying graphs. By exploiting covariance between activity of brain regions, these graphs can be learned without resorting to structural information. To validate the proposed technique, we first apply it to task fMRI with a known experimental paradigm. The probability of each graph to occur at each time-point is found to be consistent with the task timing, while the spatial patterns associated to each epoch of the task are in line with previously established activation patterns using classical regression analysis. We further on apply the technique to resting state data, which leads to extracted graphs that correspond to well-known brain functional activation patterns. The GLMM allows to learn graphs entirely from the functional activity that, in practice, turn out to reveal high degrees of similarity to the structural connectome. We compared similarity of the default mode network estimated from different task data and comparing them to each other and to structure. Using different metrics, a similar distinction between high- and low-level cognitive tasks arises.

Overall, this method allows us to infer relevant functional brain networks without the need of structural connectome information. Moreover, we find that these networks correspond better to structure compared to traditional methods.

sentences: Anti-inflammatory effects of WFS1 in pancreatic beta-cells Wolfram syndrome is a rare genetic disorder characterized by juvenile-onset diabetes mellitus, optic nerve atrophy, hearing loss, diabetes insipidus, and progressive neurodegeneration. Pathogenic variants in the WFS1 gene are the main causes of Wolfram syndrome. WFS1 encodes a transmembrane protein localized to the endoplasmic reticulum (ER) and regulates the unfolded protein response (UPR). Loss of function of WFS1 leads to dysregulation of insulin production and secretion, ER calcium depletion, and cytosolic calpains activation, resulting in activation of apoptotic cascades. Although the terminal UPR has been shown to induce inflammation that accelerates {beta}-cell dysfunction and death in diabetes, the contribution of {beta}-cell inflammation to the development of diabetes in Wolfram syndrome has not been fully understood. Here we show that WFS1-deficiency enhances the gene expression of inflammatory cytokines and chemokines, leading to cytokine-induced ER-stress and cell death in pancreatic {beta}-cells. PERK and IRE1 pathways mediate high glucose-induced inflammation in a {beta}-cell model of Wolfram syndrome. M1-macrophage infiltration and hypervascularization are seen in the pancreatic islets of Wfs1 whole-body knockout mice, demonstrating that WFS1 regulates anti-inflammatory responses in {beta}-cells. Our results demonstrate that inflammation plays an essential role in the progression of {beta}-cell death and diabetes in Wolfram syndrome and reveal that the pathways involved in ER stress-mediated inflammation provide potential therapeutic targets for the treatment of Wolfram syndrome.

sentences: ConvDip: A convolutional neural network for better M/EEG Source Imaging 1The EEG is a well-established non-invasive method in neuroscientific research and clinical diagnostics. It provides a high temporal but low spatial resolution of brain activity. In order to gain insight about the spatial dynamics of the EEG one has to solve the inverse problem, i.e. finding the neural sources that give rise to the recorded EEG activity. The inverse problem is ill-posed, which means that more than one configuration of neural sources can evoke one and the same distribution of EEG activity on the scalp. Artificial neural networks have been previously used successfully to find either one or two dipoles sources. These approaches, however, have never solved the inverse problem in a distributed dipole model with more than two dipole sources. We present ConvDip, a novel convolutional neural network (CNN) architecture that solves the EEG inverse problem in a distributed dipole model based on simulated EEG data. We show that (1) ConvDip learned to produce inverse solutions from a single time point of EEG data and (2) outperforms state-of-the-art methods on all focused performance measures. It is more flexible when dealing with varying number of sources, produces less ghost sources and misses less real sources than the comparison methods. It produces plausible inverse solutions for real EEG recordings from human participants. (4) The trained network needs less than 40 ms for a single prediction. Our results qualify ConvDip as an efficient and easy-to-apply novel method for source localization in EEG data, with high relevance for clinical applications, e.g. in epileptology and real time applications.

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easyfm : An easy software suite for file manipulation of Next Generation Sequencing data on desktops Storing and manipulating Next Generation Sequencing (NGS) file formats is an essential but difficult task in biological data analysis. The easyfm (easy file manipulation) toolkit (https://github.com/TaekAndBrendan/easyfm) makes manipulating commonly used NGS files more accessible to biologists. It enables them to perform end-to-end reproducible data analyses using a free standalone desktop application (available on Windows, Mac and Linux). Unlike existing tools (e.g. Galaxy), the Graphical User Interface (GUI)-based easyfm is not dependent on any high-performance computing (HPC) system and can be operated without an internet connection. This specific benefit allow easyfm to seamlessly integrate visual and interactive representations of NGS files, supporting a wider scope of bioinformatics applications in the life sciences.

Author summaryThe analysis and manipulation of NGS data for understanding biological phenomena is an increasingly important aspect in the life sciences. Yet, most methods for analysing, storing and manipulating NGS data require complex command-line tools in HPC or web-based servers and have not yet been implemented in comprehensive, easy-to-use software. This is a major hurdle preventing more general application in the field of NGS data analysis and file manipulation. Here we present easyfm, a free standalone Graphical User Interface (GUI) software with Python support that can be used to facilitate the rapid discovery of target sequences (or users interest) in NGS datasets for novice users. For user-friendliness and convenience, easyfm was developed with four work modules and a secondary GUI window (herein secondary window), covering different aspects of NGS data analysis (mainly focusing on FASTA files), including post-processing, filtering, format conversion, generating results, real-time log, and help. In combination with the executable tools (BLAST+ and BLAT) and Python, easyfm allows the user to set analysis parameters, select/extract regions of interest, examine the input and output results, and convert to a wide range of file formats. To help augment the functionality of existing web-based and command-line tools, easyfm, a self-contained program, comes with extensive documentation (hosted at https://github.com/TaekAndBrendan/easyfm) including a comprehensive step-by-step guide.

sentences: A small fraction of progenitors differentiate into mature adipocytes due to constraints on the cell structure change Differentiating 3T3-L1 pre-adipocytes are a mixture of non-identical culture cells. It is vital to identify the cell types that respond to the induction stimulus to understand the pre-adipocyte potential and the mature adipocyte behavior. To test this hypothesis, we deconvoluted the gene expression profiles of the cell culture of MDI-induced 3T3-L1 cells. Then we estimated the fractions of the sub-populations and their changes in time. We characterized the sub-populations based on their specific expression profiles. Initial cell cultures comprised three distinct phenotypes. A small fraction of the starting cells responded to the induction and developed into mature adipocytes. Unresponsive cells were probably under structural constraints or were committed to differentiating into alternative phenotypes. Using the same population gene markers, similar proportions were found in induced human primary adipocyte cell cultures. The three sub-populations had diverse responses to treatment with various drugs and compounds. Only the response of the maturating sub-population resembled that estimated from the profiles of the mixture. We then showed that even at a low division rate, a small fraction of cells could increase its share in a dynamic two-populations model. Finally, we used a cell cycle expression index to validate that model. To sum, pre-adipocytes are a mixture of different cells of which a limited fraction become mature adipocytes.

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Atg8 licenses adipokine nuclear exit Adipokines released from the adipocytes function as a systemic adipometer; they impinge on neural circuits to signal nutrient status. On starvation, adipokines must be retained to signal energy deficit; else, it significantly reduces starvation survival. But how fat cells retain adipokines is unclear. Here, we demonstrate that Atg8, a cell-intrinsic autophagy factor, regulates the starvation-induced acute retention of the Leptin Drosophila ortholog Upd2. We show that on starvation, as a direct consequence of Atg8s lipidation, Upd2 accumulates in the nucleus. We illustrate that Upd2s nuclear retention is critical to fat mobilization and increased starvation resilience. Furthermore, nuclear Upd2 promotes the expression of a secreted innate immune gene signature. This hints at an unanticipated connection between adipokine nuclear retention and increased innate immunity. In conclusion, we propose that, during starvation, Atg8s role is not just limited to autophagy but is critical for withholding adipokines in the nucleus to promote starvation resilience.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/428913v5_ufig1.gif" ALT="Figure 1">
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C_LIO_LIAtg8s lipidation on starvation results in Upd2s nuclear accumulation.
C_LIO_LIUpd2 nuclear retention on starvation increases fat mobilization and post-starvation hunger.
C_LIO_LIOn starvation Upd2 nuclear retention increases expression of a secreted innate immune signature.
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sentences: Omicron variant of SARS-COV-2 gains new mutations in New Zealand and Hong Kong Disclaimer statementThe authors have withdrawn this manuscript because an issue was raised about the SARS- COV-2 genome sequence data used in the study. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

sentences: Strong and opponent contributions of dorsomedial striatal pathways to behavior depends on cognitive demands and task strategy A classic view of the striatum holds that activity in direct and indirect pathways oppositely modulates motor output. Whether this involves direct control of movement, or reflects a cognitive process underlying movement, remains unresolved. Here we find that strong, opponent control of behavior by the two pathways of the dorsomedial striatum (DMS) depends on the cognitive requirements of a task. Furthermore, a latent state model (a hidden markov model with generalized linear model observations) reveals that--even within a single task--the contribution of the two pathways to behavior is state-dependent. Specifically, the two pathways have large contributions in one of two states associated with a strategy of evidence accumulation, compared to a state associated with a strategy of repeating previous choices. Thus, both the demands imposed by a task, as well as the internal state of mice when performing a task, determine whether DMS pathways provide strong and opponent control of behavior.

sentences: Structural and Preliminary Biochemical Characterization of MppQ, a PLP-Dependent Aminotransferase from 	Streptomyces hygroscopicus MppQ is an enzyme of unknown function from Streptomyces hygroscopicus that is involved in the biosynthesis of the nonproteinogenic amino acid L-enduracididine (L-End). Since L-End is a component of several peptides showing high activity against methicillin-resistant Staphylococcus aureus (MRSA), a complete understanding of its biosynthetic pathway is of utmost importance for developing chemoenzymatic routes for syntheses of novel antibiotics. In this work, we report high-resolution X-ray crystal structures of MppQ complexed with pyridoxal-5-phosphate (PLP) and pyridoxamine-5-phosphate (PMP). The structure of MppQ shares a fold with known Type I PLP-dependent aminotransferases, consisting of an N-terminal extension, large domain, and a small domain. We also report the first functional characterization of MppQ, which we incubated with enzymatically produced 2-ketoenduracidine and observed conversion to L-End via mass spectroscopy. Additionally, we have observed that MppQ has a relatively high affinity for 2-ketoarginine, a shunt product in the L-End biosynthetic pathway, indicating a possible role of MppQ in increasing efficiency of L-End biosynthesis by converting 2-ketoarginine back to the starting material, L-arginine.

sentences: Majority of choice-related variability in perceptual decisions is present in early sensory cortex While performing challenging perceptual tasks such as detecting a barely visible target, our perceptual reports vary across presentations of identical stimuli. This perceptual variability is presumably caused by neural variability in our brains. How much of the neural variability that correlates with the perceptual variability is present in the primary visual cortex (V1), the first cortical processing stage of visual information? To address this question, we recorded neural population responses from V1 using voltage-sensitive dye imaging while monkeys performed a challenging reaction-time visual detection task. We found that V1 population responses in the period leading to the decision correspond more closely to the monkeys report than to the visual stimulus. These results, together with a simple computational model that allows one to quantify the captured choice-related variability, suggest that most of this variability is present in V1 as additive noise, and that areas downstream to V1 contain relatively little independent choice-related variability.

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Enterococcus faecalis antagonizes Pseudomonas aeruginosa growth in polymicrobial biofilms Enterococcus faecalis is often co-isolated with Pseudomonas aeruginosa in polymicrobial biofilm-associated infections of wounds and the urinary tract. As a defense strategy, the host innately restricts iron availability at infection sites. Despite their co-prevalence, the polymicrobial interactions of these two pathogens in iron- restricted conditions, such as those found in the host, remains unexplored. Here we show that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase (ldh1) gives rise to L-lactate production during fermentative growth. We find that E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced in iron-restricted conditions, resulting in increased lactic acid exported and consequently, a reduction in pH. Together, our results suggest that E. faecalis synergistically inhibit P. aeruginosa growth by decreasing environmental pH and L-lactate-mediated iron chelation. Overall, this study highlights that the microenvironment in which the infection occurs is important for understanding its pathophysiology.

IMPORTANCEMany infections are polymicrobial and biofilm-associated in nature. Iron is essential for many metabolic processes and plays an important role in controlling infections, where the host restricts iron as a defense mechanism against invading pathogens. However, polymicrobial interactions between pathogens are underexplored in iron- restricted conditions. Here, we explore the polymicrobial interactions between commonly co-isolated E. faecalis and P. aeruginosa within biofilms. We find that E. faecalis modulates the microenvironment by exporting lactic acid which further chelates already limited iron, and also lowers the environmental pH to antagonize P. aeruginosa growth in iron-restricted conditions. Our findings provide insights into polymicrobial interactions between pathogens in an infection-relevant condition and how manipulating the microenvironment can be taken advantage of to better control infections.

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M-Phase oscillations in PP1 activity are essential for accurate progression through mammalian oocyte meiosis Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-Phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-Phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here we report that PP1 is important for M-Phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-Phase regulation.

Summary statementAltering the normal cyclical activity of the phosphatase PP1 in oocytes causes a range of severe meiotic defects, pointing to essential roles for PP1 in M-Phase entry, progression, and exit.

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Machine learning models based on molecular descriptors to predict human and environmental toxicological factors in continental freshwater AO_SCPLOWBSTRACTC_SCPLOWIt is a real challenge for life cycle assessment practitioners to identify all relevant substances contributing to the ecotoxicity. Once this identification has been made, the lack of corresponding ecotoxicity factors can make the results partial and difficult to interpret. So, it is a real and important challenge to provide ecotoxicity factors for a wide range of compounds. Nevertheless, obtaining such factors using experiments is tedious, time-consuming, and made at a high cost. A modeling method that could predict these factors from easy-to-obtain information on each chemical would be of great value. Here, we present such a method, based on machine learning algorithms, that used molecular descriptors to predict two specific endpoints in continental freshwater for ecotoxicological and human impacts. The different tested machine learning algorithms show good performances on a learning database and the non-linear methods tend to outperform the linear ones. The cluster-then-predict approaches usually show the best performances which suggests that these predicted models must be derived for somewhat similar compounds. Finally, predictions were derived from the validated model for compounds with missing toxicity/ecotoxicity factors.

HighlightsO_LICharacterization factors (for human health and ecotoxicological impacts) were predicted using molecular descriptors.
C_LIO_LISeveral linear or non-linear machine learning methods were compared.
C_LIO_LIThe non-linear methods tend to outperform the linear ones using a train and test procedure. Cluster-then-predict approaches often show the best performances, highlighting their usefulness.
C_LIO_LIThis methodology was then used to derive characterization factors that were missing for more than a hundred chemicals in USEtox(R).
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Graphical abstract

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sentences: Brain aging differs with cognitive ability regardless of education Higher general cognitive ability (GCA) is associated with lower risk of neurodegenerative disorders, but neural mechanisms are unknown. GCA could be associated with more cortical tissue, from young age, i.e. brain reserve, or less cortical atrophy in adulthood, i.e. brain maintenance. Controlling for education, we investigated the relative association of GCA with reserve and maintenance of cortical volume, -area and -thickness through the adult lifespan, using multiple longitudinal brain imaging cohorts (n = 3327, 7002 MRI scans, baseline age 20-88 years, followed-up up to 11 years). There were widespread positive relationships between GCA and cortical characteristics (level-level associations). In select regions, higher baseline GCA was associated with less atrophy over time (level-change associations). Relationships remained when controlling for polygenic scores for both GCA and education. Our findings suggest that higher GCA is associated with cortical volumes by both brain reserve and -maintenance mechanisms through the adult lifespan.

sentences: Neural network models for spinal implementation of muscle synergies Muscle synergies have been proposed as functional modules to simplify the complexity of body motor control; however, their neural implementation is still unclear. Converging evidence suggests that output projections of the spinal premotor interneurons (PreM-INs) underlie the formation of muscle synergies, but they exhibit a substantial variation across neurons and exclude standard models assuming a small number of unitary "modules" in the spinal cord. Here we compared neural network models for muscle synergies to seek a biologically plausible model that reconciles previous clinical and electrophysiological findings. We examined three neural network models: one with random connections (non-synergy model), one with a small number of spinal synergies (simple synergy model), and one with a large number of spinal neurons representing muscle synergies with a certain variation (population synergy model). We found that the simple and population synergy models emulate the robustness of muscle synergies against cortical stroke observed in human stroke patients. Furthermore, the size of the spinal variation of the population synergy matched well with the variation in spinal PreM-INs recorded in monkeys. These results suggest that a spinal population with moderate variation is a biologically plausible model for the neural implementation of muscle synergies.

sentences: Expansion and contraction of resource allocation in sensory bottlenecks Topographic sensory representations often do not scale proportionally to the size of their input regions, with some expanded and others contracted. In vision, the foveal representation is magnified cortically, as are the fingertips in touch. What principles drive this allocation, and how should receptor density, e.g. the high innervation of the fovea or the fingertips, and stimulus statistics, e.g. the higher contact frequencies on the fingertips, contribute? Building on work in efficient coding, we address this problem using linear models that maximize information transmission through decorrelation. We introduce a sensory bottleneck to impose constraints on resource allocation and derive the optimal neural allocation. We find that bottleneck width is a crucial factor in resource allocation, inducing either expansion or contraction. Both receptor density and stimulus statistics affect allocation and jointly determine convergence for wider bottlenecks. Furthermore, we show a close match between the predicted and empirical cortical allocations in a well-studied model system, the star-nosed mole. Overall, our results suggest that the strength of cortical magnification depends on resource limits.

sentences: Arabidopsis spliceosome factor SmD3 modulates immunity to Pseudomonas syringae infection SmD3 is a core component of the small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. The role of Arabidopsis SmD3 in plant immunity was assessed by testing sensitivity of smd3a and smd3b mutants to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and its pathogenesis effectors flagellin (flg22), EF-Tu (elf18) and coronatine (COR). Both smd3 mutants exhibited enhanced susceptibility to Pst accompanied by marked changes in the expression of key pathogenesis markers. mRNA levels of these factors were also altered upon treatment with Pseudomonas effectors. We showed that SmD3-b dysfunction impairs mainly stomatal immunity as a result of defects in stomatal development. Our genome-wide transcriptome analysis of the smd3b-1 mutant infected with Pst revealed that lack of SmD3-b deregulates defense against Pst infection at the transcriptional and posttranscriptional levels including defects in splicing and an altered pattern of alternative splicing. Other changes in the smd3b-1 mutant involved enhanced elf18- and flg22-induced callose deposition, reduction of flg22-triggered production of early ROS and boost of secondary ROS caused by Pst infection. Together, our data indicate that SmD3 contributes to the plant immune response possibly via regulation of mRNA splicing of key pathogenesis factors.

sentences: Transcriptomic-based quantification of the epithelial-hybrid-mesenchymal spectrum across biological contexts Epithelial-mesenchymal plasticity (EMP) underlies embryonic development, wound healing, and cancer metastasis and fibrosis. Cancer cells exhibiting EMP often have more aggressive behavior, characterized by drug resistance, and tumor-initiating and immuno-evasive traits. Thus, the EMP status of cancer cells can be a critical indicator of patient prognosis. Here, we compare three distinct transcriptomic-based metrics - each derived using a different gene list and algorithm - that quantify the EMP spectrum. Our results for 96 cancer-related RNA-seq datasets reveal a high degree of concordance among these metrics in quantifying the extent of EMP. Moreover, each metric, despite being trained on cancer expression profiles, recapitulates the expected changes in EMP scores for non-cancer contexts such as lung fibrosis and cellular reprogramming into induced pluripotent stem cells. Thus, we offer a scoring platform to quantify the extent of EMP in vitro and in vivo for diverse biological applications including cancer.

sentences: Macrophage network dynamics depend on haptokinesis for optimal local surveillance Macrophages are key immune cells with important roles for tissue surveillance in almost all mammalian organs. Cellular networks made up of many individual macrophages allow for optimal removal of dead cell material and pathogens in tissues. However, the critical determinants that underlie these population responses have not been systematically studied. Here, we investigated how cell shape and the motility of individual cells influences macrophage network responses in 3D culture settings and in mouse tissues. We show that surveying macrophage populations can tolerate lowered actomyosin contractility, but cannot easily compensate for a lack of integrin-mediated adhesion. Although integrins were dispensable for macrophage chemotactic responses, they were crucial to control cell movement and protrusiveness for optimal surveillance by a macrophage population. Our study reveals that {beta}1 integrins are important for maintaining macrophage shape and network sampling efficiency in mammalian tissues, and sets macrophage motility strategies apart from the integrin-independent 3D migration modes of many other immune cell subsets.

sentences: LKB1 inactivation modulates chromatin accessibility to drive metastatic progression Metastasis is the leading cause of cancer-related deaths, enabling cancer cells to expand to secondary sites and compromise organ function1. Given that primary tumors and metastases often share the same constellation of driver mutations2-4, the mechanisms driving their distinct phenotypes are unclear. Here, we show that inactivation of the frequently mutated tumor suppressor gene, liver kinase B1 (LKB1), has evolving effects throughout lung cancer progression, leading to the differential epigenetic re-programming of early-stage primary tumors compared to late-stage metastases. By integrating genome-scale CRISPR/Cas9 screening with bulk and single-cell multi-omic analyses, we unexpectedly identify LKB1 as a master regulator of chromatin accessibility in lung adenocarcinoma primary tumors. Using an in vivo model of metastatic progression, we further reveal that loss of LKB1 activates the early endoderm transcription factor SOX17 in metastases and a metastatic-like sub-population of cancer cells within primary tumors. SOX17 expression is necessary and sufficient to drive a second wave of epigenetic changes in LKB1-deficient cells that enhances metastatic ability. Overall, our study demonstrates how the downstream effects of an individual driver mutation can appear to change throughout cancer development, with implications for stage-specific therapeutic resistance mechanisms and the gene regulatory underpinnings of metastatic evolution.

sentences: Regulation of layered T cell tolerance mechanisms by the NR4A family is essential to preserve immune homeostasis and suppress autoimmunity The NR4A family of orphan nuclear receptors (Nr4a1-3) plays redundant roles upstream of Foxp3 to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to isolate the functions of this family in other immune cells. Here we take advantage of a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1-/- Nr4a3-/- (DKO) bone marrow develop autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely wild-type origin. This disease differs qualitatively from that seen with Treg-deficiency and is B cell-extrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional and phenotypic features of anergy accumulate in chimeric mice. Despite this, DKO T cells exhibit enhanced IL-2 production, implying a cell-intrinsic role for the NR4A family in peripheral T cell tolerance. These studies reveal roles for the NR4A family in multiple layered T cell tolerance mechanisms and demonstrate that each is essential to preserve immune homeostasis.

sentences: Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes Errors in mitosis that cause chromosome missegregation lead to aneuploidy and micronuclei formation which are associated with cancer. Accurate segregation requires the alignment of all chromosomes by the mitotic spindle at the metaphase plate, and any misalignment must be corrected before anaphase is triggered. The spindle is situated in a membrane-free "exclusion zone", beyond this zone, endomembranes (endoplasmic reticulum, nuclear envelope and other organelles) are densely packed. We asked what happens to misaligned chromosomes that find themselves beyond the exclusion zone? Here we show that such chromosomes become ensheathed in multiple layers of endomembranes. Chromosome ensheathing delays mitosis and increases the frequency of chromosome missegregation. The micronuclei that form following missegregation have a disrupted nuclear envelope with internal endomembranes. We use an induced organelle relocalization strategy in live cells to show that clearance of endomembranes allows for the rescue of chromosomes that were destined for missegregation. Our findings indicate that endomembranes promote the missegregation of misaligned chromosomes that are outside the exclusion zone, and therefore constitute a risk factor for aneuploidy.

sentences: Targeting RNA:Protein Interactions with an Integrative Approach Leads tothe Identification of Potent YB-1 Inhibitors RNA-binding proteins are promising targets for developing new molecules of therapeutic interest. Nevertheless, targeting RNA:Protein interfaces is hampered by the lack of methods able to detect these interactions in cells while being amenable to High Content Screening. Here, we adapt the microtubule bench assay to score small molecules targeting interactions of endogenous mRNA with a specific protein in cells and demonstrate its robustness by targeting YB-1 (YBX-1 gene), a mRNA-binding protein involved in cancer progression and resistance to chemotherapy. The implementation of an integrative approach led to the identification of 22 hits validated by NMR and MD simulations of which 11 were found to significantly interfere with the binding of mRNA to YB-1 in cells at low micromolar concentrations. One of our leads is P1, an FDA-approved PARP-1 inhibitor. This work shows the potential of our integrative approach and paves the way for the development of RNA:Protein Interaction inhibitors.

sentences: ICOS expression is required for maintenance but not the formation of germinal centers in the spleen in response to P. yoelii infection. Inducible T cell co-stimulator (ICOS) plays a key role in the differentiation and maintenance of follicular helper T (Tfh) cells and thus germinal center (GC) formation. Previously, our lab showed in a Plasmodium chabaudi infection model that Icos-/- mice did not form GCs despite a persistent infection and thus a continued antigen (Ag) load. Here, we show that resolution of a primary infection with P. yoelii, was delayed in Icos-/- mice. This phenotype was associated with a reduction in the accumulation of Tfh-like and GC Tfh cells and an early deficiency in Ag-specific antibody (Ab) production. However, Icos-/- mice maintained their ability to form GCs, though they were less frequent in number than in wild-type (WT) mice. Furthermore, while Ab production in Icos-/- mice matched that of WT mice after the infection cleared, the Abs lacked signs of affinity maturation, suggesting functional defects associated with these GCs. Eventually, these GC structures dissipated more rapidly in Icos-/- mice than in WT mice. Moreover, the ability of Icos-/- mice to form these GC structures is not reliant on the high Ag load associated with P. yoelii infections, as GC formation was preserved in Icos-/- mice treated with early with atovaquone. Finally, mice were unable to form secondary GCs in the absence of ICOS after re-challenge. Overall, these data demonstrate the necessity of ICOS in the maintenance of Tfh cells, the formation and maintenance of sufficient numbers of functioning GCs, and the ability to generate new GC structures after re-infection with P. yoelii.

sentences: Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system Segregation Distorter (SD) is a male meiotic drive system in Drosophila melanogaster. Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD+ chromosome. It is well established that distortion results from an interaction between Sd, the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp, on the SD+ chromosome. However, the molecular and cellular mechanisms leading to post-meiotic SD+ sperm elimination remain unclear. Here we show that SD/SD+ males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD+ spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD+ genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. Our analyses of different SD+ chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. We propose that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization.

sentences: Unbreakable DNA tension probes show that cell adhesion receptors detect the molecular force-extension curve of their ligands Integrin receptors transduce the mechanical properties of the extracellular matrix. Past studies using DNA probes showed that integrins sense the magnitude of ligand forces with pN resolution. An open question is whether integrin receptors also sense the force-extension trajectory of their ligands. The challenge in addressing this question pertains to the lack of molecular probes that can control force-extension trajectories independently of force magnitude. To address this limitation, we synthesized two reversible DNA probes that fold with identical self-complementary domains but with different topologies. Thus, these probes unfold at the same steady-state force magnitude but following different kinetic pathways to reach the fully extended ssDNA state. Hairpin-like probes unzip with a low barrier of 14 pN while the pseudo-knot-like probes shear at 59 pN. Confirming that we had created probes with different barriers of unfolding, we quantified platelet integrin forces and measured 50-fold more tension signal with the unzipping probes over the shearing probes. In contrast, fibroblasts opened both probes to similar levels indicating more static forces. Surprisingly, fibroblast mechanotransduction markers, such as YAP levels, fibronectin production, actin organization, and integrin activation were significantly elevated on unzipping probes. This demonstrates that integrin receptors within focal adhesions sense the molecular force-extension profile of their ligands and not only the magnitude of equilibrium mechanical resistance.

sentences: NPAS4 in the medial prefrontal cortex mediates chronic social defeat stress- induced anhedonia and dendritic spine loss Chronic stress can produce reward system deficits (i.e. anhedonia) and other common symptoms associated with depressive disorders, as well as neural circuit hypofunction in the medial prefrontal cortex (mPFC). However, the molecular mechanisms by which chronic stress promotes depressive-like behavior and hypofrontality remain unclear. We show here that the neuronal activity-regulated transcription factor, NPAS4, in the mPFC is regulated by chronic social defeat stress (CSDS), and its required in this brain region for CSDS-induced changes in sucrose preference and natural reward motivation. Interestingly, NPAS4 is not required for CSDS-induced social avoidance or anxiety-like behavior. We also find that mPFC NPAS4 is required for CSDS-induced reduction of pyramidal cell dendritic spine density, revealing a relationship between mPFC dendritic spine changes and anhedonia-like behavior, but not social avoidance behavior. Finally, transcriptomic analysis from the mPFC revealed that NPAS4 influences expression of numerous genes linked to glutamatergic synapses and ribosomal function, as well as many dysregulated genes observed in common neuropsychiatric disorders, including depression. Together our findings reveal an essential role for the activity-regulated transcription factor, NPAS4, in chronic stress-induced mPFC hypofunction and anhedonia.

sentences: Towards a catalog of pome tree architecture genes: the draft 'd'Anjou' genome (Pyrus communis L.) The rapid development of sequencing technologies has led to a deeper understanding of horticultural plant genomes. However, experimental evidence connecting genes to important agronomic traits is still lacking in most non-model organisms. For instance, the genetic mechanisms underlying plant architecture are poorly understood in pome fruit trees, creating a major hurdle in developing new cultivars with desirable architecture, such as dwarfing rootstocks in European pear (Pyrus communis). Further, the quality and content of genomes vary widely. Therefore, it can be challenging to curate a list of genes with high-confidence gene models across reference genomes. This is often an important first step towards identifying key genetic factors for important traits. Here we present a draft genome of P. communis  dAnjou and an improved assembly of the latest P. communis  Bartlett genome. To study gene families involved in tree architecture in European pear and other rosaceous species, we developed a workflow using a collection of bioinformatic tools towards curation of gene families of interest across genomes. This lays the groundwork for future functional studies in pear tree architecture. Importantly, our workflow can be easily adopted for other plant genomes and gene families of interest.

sentences: Inositol hexakisphosphate (IP6) accelerates immature HIV-1 Gag protein assembly towards kinetically-trapped morphologies During the late stages of the HIV-1 lifecycle, immature virions are produced by the concerted activity of Gag polyproteins, primarily mediated by the capsid (CA) and spacer peptide 1 (SP1) domains, which assemble into a spherical lattice, package viral genomic RNA, and deform the plasma membrane. Recently, inositol hexakisphosphate (IP6) has been identified as an essential assembly cofactor that efficiently produces both immature virions in vivo and immature virus-like particles in vitro. To date, however, several distinct mechanistic roles for IP6 have been proposed on the basis of independent functional, structural, and kinetic studies. In this work, we investigate the molecular influence of IP6 on the structural outcomes and dynamics of CA/SP1 assembly using coarse-grained (CG) molecular dynamics (MD) simulations and free energy calculations. Here, we derive a bottom-up, low-resolution, and implicit-solvent CG model of CA/SP1 and IP6, and simulate their assembly under conditions that emulate both in vitro and in vivo systems. Our analysis identifies IP6 as an assembly accelerant that promotes curvature generation and fissure-like defects throughout the lattice. Our findings suggest that IP6 induces kinetically-trapped immature morphologies, which may be physiologically important for later stages of viral morphogenesis and potentially useful for virus-like particle technologies.

sentences: Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis Synucleinopathies including Parkinsons disease and dementia with Lewy bodies are neurodegenerative diseases characterized by the intracellular accumulation of the protein alpha-synuclein called "Lewy pathology." Alpha-synuclein within Lewy pathology is aggregated into protease resistant filamentous structures and is predominantly phosphorylated at serine 129 (PSER129). Lewy pathology has been hypothesized to spread throughout the nervous system as the disease progresses. Cross-sectional studies have shown the olfactory bulb and olfactory tract consistently bare LP for common synucleinopathies, making these structures likely starting points for the spreading process, and thus disease. Here we examined the distribution of PSER129 in non-diseased brain. To do this we used a sensitive tyramide signal amplification (TSA) technique to detect low abundance endogenous PSER129 under ideal antibody binding conditions. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb and several brain regions of the olfactory cortex across the neuroaxis (i.e., olfactory bulb to brain stem). PSER129 was particularly apparent in the mitral cell layer and the outer plexiform layer of the olfactory bulb where it was observed as cytosolic/nuclear puncta or fibers, respectively. PSER129 immunoreactivity in the healthy olfactory bulb was abolished by pretreatment of the tissue with proteinase K, pre-absorption of the primary antibody against the purified PSER129 peptide fragment, or the omission of the PSER129 antibody. Furthermore, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. Dual labeling for the PSER129 and the mitral cell marker TBX21 showed that PSER129 positive structures of the healthy OB were found in mitral cells. We found evidence of the same PSER129 positive structures in the olfactory bulb of non-diseased rats, non-human primates, healthy humans, but not individuals diagnosed with PD. Results suggest biological pathways responsible for alpha-synuclein phosphorylation are constitutively active in OB mitral cells and alpha-synuclein in these cells may be predisposed to pathological aggregation. Pathological seeds originating in mitral cells may act as a source for alpha-synuclein spread competent assemblies that spreads throughout the brain via fibers of the olfactory tract. Future studies should investigate the normal function of alpha-synuclein in the mitral cells of the olfactory bulb, which may give insight into synucleinopathy disease origins.

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Choice of pre-processing pipeline influences clustering quality of scRNA-seq datasets BackgroundSingle-cell RNA sequencing (scRNA-seq) has quickly become one of the most dominant techniques in modern transcriptome assessment. In particular, 10X Genomics Chromium system, with its high throughput approach, turn key and thorough user guide made this cutting-edge technique accessible to many laboratories using diverse animal models. However, standard pre-processing, including the alignment and cell filtering pipelines might not be ideal for every organism or tissue. Here we applied an alternative strategy, based on the pseudoaligner kallisto, on twenty-two publicly available single cell sequencing datasets from a wide range of tissues of eight organisms and compared the results with the standard 10X Genomics Cell Ranger pipeline.

ResultsIn most of the tested samples, kallisto produced higher sequencing read alignment rates and total gene detection rates in comparison to Cell Ranger. Although datasets processed with Cell Ranger had higher cell counts, outside of human and mouse datasets, these additional cells were routinely of low quality, containing low gene detection rates. Thorough downstream analysis of one kallisto processed dataset, obtained from the zebrafish pineal gland, revealed clearer clustering, allowing the identification of an additional photoreceptor cell type that previously went undetected. The finding of the new cluster suggests that the photoreceptive pineal gland is essentially a bi-chromatic tissue containing both green and red cone-like photoreceptors and implies that the alignment and pre-processing pipeline can affect the discovery of biologically-relevant cell types.

ConclusionWhile Cell Ranger favors higher cell numbers, using kallisto results in datasets with higher median gene detection per cell. We could demonstrate that cell type identification was not hampered by the lower cell count, but in fact improved as a result of the high gene detection rate and the more stringent filtering. Depending on the acquired dataset, it can be beneficial to favor high quality cells and accept a lower cell count, leading to an improved classification of cell types.

sentences: Bayesian Additive Regression Trees for Genotype by Environment Interaction Models We propose a new class of models for the estimation of genotype by environment (GxE) interactions in plant-based genetics. Our approach, named AMBARTI, uses semi-parametric Bayesian additive regression trees to accurately capture marginal genotypic and environment effects along with their interaction in a fully Bayesian model. We demonstrate that our approach is competitive or superior to similar models widely used in the literature via both simulation and a real world data set. Furthermore, we introduce new types of visualisation to properly assess both the marginal and interactive predictions from the model. An R package that implements our approach is available at https://github.com/ebprado/ambarti.

sentences: A comprehensive in silico investigation into the nsSNPs of Drd2 gene predicts significant functional consequences in dopamine signaling and pharmacotherapy DRD2 is a neuronal cell surface protein involved in brain development and function. Variations in the Drd2 gene have clinical significance since DRD2 is a pharmacotherapeutic target for treating psychiatric disorders like ADHD and schizophrenia. Despite numerous studies on the disease association of single nucleotide polymorphisms (SNPs) in the intronic regions, investigation into the coding regions is surprisingly limited. In this study, we aimed at identifying potential functionally and pharmaco-therapeutically deleterious non-synonymous SNPs of Drd2. A wide array of bioinformatics tools was used to evaluate the impact of nsSNPs on protein structure and functionality. Out of 260 nsSNPs retrieved from the dbSNP database, initially 9 were predicted as deleterious by 15 tools. Upon further assessment of their domain association, conservation profile, homology models and inter-atomic interaction, the mutant F389V was considered as the most impactful. In-depth analysis of F389V through Molecular Docking and Dynamics Simulation revealed a decline in affinity for its native agonist dopamine and an increase in affinity for the antipsychotic drug risperidone. Remarkable alterations in binding interactions and stability of the protein-ligand complex in simulated physiological conditions were also noted. These findings will improve our understanding of the consequence of nsSNPs in disease-susceptibility and therapeutic efficacy.

sentences: Identification of Japanese Encephalitis Virus Genotype V and Other Mosquito-borne Viruses in Camp Humphreys, Republic of Korea, using Metagenomic Analysis Recent outbreaks of emerging and re-emerging viruses such as Zika, West Nile and Japanese encephalitis (JEV) viruses have shown that timely detection of novel arboviruses with epidemic potential is essential to mitigate human health risks. There have been rising concerns that an emergent JEV genotype (genotype V, GV) is circulating in Asia, against which the current US-FDA-approved JEV vaccine may not be efficacious. To ascertain if JEV GV and other arboviruses are circulating in East Asia, we conducted next-generation sequencing on 260 pools of Culex tritaeniorhynchus and Culex bitaeniorhynchus mosquitoes (6,540 specimens) collected at Camp Humphreys, Republic of Korea (ROK), from mid-May - October 2018. Metagenomic analysis demonstrated a highly abundant and diverse virome with correlates of health and ecological relevance. Additionally, two complete JEV GV genome sequences were obtained from separate mosquito pools, indicating that JEV GV is circulating in the Pyeongtaek area near Seoul, ROK. Retrospective sample and sequence analyses showed that JEV GV was also present in 2016 mosquito pools collected in Seoul, ROK. Sequence-based analysis of JEV GV indicates a divergent genotype that is the most distant from the GIII derived live attenuated SA14-14-2 vaccine strain. A GV E protein investigation and 3D modeling in context to SA14-14-2 indicated likely regions responsible for reduced antibody affinity, including clusters of significant amino acid changes at externally exposed domains. These data highlight the critical need for continued mosquito surveillance as a means of detecting and identifying emerging and re-emerging arboviruses of public health relevance. Importantly, our results emphasize recent concerns that there may be a possible shift in the circulating JEV genotype in East Asia and highlights the critical need for a vaccine proven to be efficacious against this re-emergent virus.

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Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in ovarian cancer enhances therapeutic efficacy BackgroundIn the U.S., more than 50% of ovarian cancer patients die within 5 years of diagnosis, highlighting the need for innovations such as engineered T cell therapies. Mesothelin (Msln) is an attractive immunotherapy target for this cancer, as it is overexpressed by the tumor and contributes to malignant and invasive phenotypes, making antigen loss disadvantageous to the tumor. We previously showed that adoptively transferred T cells engineered to be Msln-specific (TCR1045) preferentially accumulate within established ovarian tumors, delay tumor growth and significantly prolong survival in the ID8VEGF mouse model. However, T cell persistence and anti-tumor activity were not sustained, and we and others have previously detected FasL in the tumor vasculature and the tumor microenvironment (TME) of human and murine ovarian cancers, which can induce apoptosis in infiltrating lymphocytes expressing Fas receptor (Fas).

MethodsTo concurrently overcome this mechanism for potential immune evasion and enhance T cell responses, we generated an immunomodulatory fusion protein (IFP) containing the Fas extracellular binding domain fused to a 4-1BB co-stimulatory domain, rather than the natural death domain. T cells engineered to express TCR1045 alone or in combination with the IFP were transferred into ID8VEGF-tumor bearing mice and evaluated for persistence, proliferation, anti-tumor cytokine production, and therapeutic efficacy.

ResultsRelative to T cells modified only to express TCR1045, T cells engineered to express both TCR1045 and a Fas IFP preferentially persisted in the TME of tumor-bearing mice due to improved T cell proliferation and survival. Moreover, adoptive immunotherapy with IFP+ T cells significantly prolonged survival in tumor-bearing mice, relative to TCR1045 T cells lacking the IFP.

ConclusionsFas/FasL signaling can mediate T cell death in the ovarian cancer microenvironment, as well as induce activation-induced cell death, an apoptotic mechanism responsible for regulating T cell expansion. Upregulation of FasL by tumor cells and tumor vasculature represents a mechanism for protecting growing tumors from attack by tumor-infiltrating lymphocytes. As many solid tumors overexpress FasL, an IFP that converts the Fas-mediated death signal into pro-survival and proliferative signals may provide an opportunity to enhance engineered adoptive T cell therapy against many malignancies.

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TimeCycle: Topology Inspired MEthod for the Detection of Cycling Transcripts in Circadian Time-Series Data MotivationThe circadian rhythm drives the oscillatory expression of thousands of genes across all tissues. The recent revolution in high-throughput transcriptomics, coupled with the significant implications of the circadian clock for human health, has sparked an interest in circadian profiling studies to discover genes under circadian control.

ResultWe present TimeCycle: a topology-based rhythm detection method designed to identify cycling transcripts. For a given time-series, the method reconstructs the state space using time-delay embedding, a data transformation technique from dynamical systems theory. In the embedded space, Takens theorem proves that the dynamics of a rhythmic signal will exhibit circular patterns. The degree of circularity of the embedding is calculated as a persistence score using persistent homology, an algebraic method for discerning the topological features of data. By comparing the persistence scores to a bootstrapped null distribution, cycling genes are identified. Results in both synthetic and biological data highlight Time-Cycles ability to identify cycling genes across a range of sampling schemes, number of replicates, and missing data. Comparison to competing methods highlights their relative strengths, providing guidance as to the optimal choice of cycling detection method.

Availability and ImplementationA fully documented open-source R package implementing Time-Cycle is available at: https://nesscoder.github.io/TimeCycle/.

sentences: Capillary-associated microglia regulate vascular structure and function through PANX1-P2RY12 coupling Microglia are brain-resident immune cells with a repertoire of functions in the developing, mature and pathological brain. Their wide-ranging roles in physiology include the clearance of cellular debris, elimination of excess synapses, regulation of neuronal activity and contributions to blood vessel development. Despite these known roles for microglia, the extent of their interactions with the vasculature and potential regulation of vascular physiology has been insufficiently explored. Here, using in vivo acute and longitudinal two-photon imaging in transgenic mice combined with electron microscopy, fixed tissue immunohistochemistry, pharmacological treatments and laser speckle imaging, we document the steady-state interactions between ramified CX3CR1+ myeloid cell somata and capillaries in the brain. We first confirm that these myeloid cells are bona fide microglia by molecular, morphological and ultrastructural approaches. Then we give a detailed spatio-temporal characterization of these capillary-associated microglia (CAMs) comparing and contrasting them with parenchymal microglia (PCMs) in their static, dynamic and chronic morphological activities including during microglial depletion and repopulation. Molecularly, we identify microglial-specific purinergic P2RY12 receptors as a receptor regulating CAM interactions under the control of released purines from pannexin 1 (PANX1) channels. Furthermore, to elucidate roles for microglia in vascular structure and function, we eliminated microglia and showed that this triggered capillary dilation, blood flow increase, and impaired vasodilative responses. We find that P2RY12-/- and PANX1-/- mice recapitulate these vascular impairments suggesting purines released through PANX1 channels play important roles in activating microglial P2RY12 receptors to regulate neurovascular structure and function.

sentences: Improved Metabolite Prediction Using Microbiome Data-Based Elastic Net Models Microbiome data are becoming increasingly available in large health cohorts yet metabolomics data are still scant. While many studies generate microbiome data, they lack matched metabolomics data or have considerable missing proportions of metabolites. Since metabolomics is key to understanding microbial and general biological activities, the possibility of imputing individual metabolites or inferring metabolomics pathways from microbial taxonomy or metagenomics is intriguing. Importantly, current metabolomics profiling methods such as the HMP Unified Metabolic Analysis Network (HUMAnN) have unknown accuracy and are limited in their ability to predict individual metabolites. To address this gap, we developed a novel metabolite prediction method, and we present its application and evaluation in an oral microbiome study. We developed ENVIM based on the Elastic Net Model (ENM) to predict metabolites using micorbiome data. ENVIM introduces an extra step to ENM to consider variable importance scores and thus achieve better prediction power. We investigate the metabolite prediction performance of ENVIM using metagenomic and metatranscriptomic data in a supragingival biofilm multi-omics dataset of 297 children ages 3-5 who were participants of a community-based study of early childhood oral health (ZOE 2.0) in North Carolina, United States. We further validate ENVIM in two additional publicly available multi-omics datasets generated from studies of gut health and vagina health. We select gene-family sets based on variable importance scores and modify the existing ENM strategy used in the MelonnPan prediction software to accommodate the unique features of microbiome and metabolome data. We evaluate metagenomic and metatranscriptomic predictors and compare the prediction performance of ENVIM to the standard ENM employed in MelonnPan. The newly-developed ENVIM method showed superior metabolite predictive accuracy than MelonnPan using metatranscriptomics data only, metagenomics data only, or both of these two. Both methods perform better prediction using gut or vagina microbiome data than using oral microbiome data for the samples corresponding metabolites. The top predictable compounds have been reported in all these three datasets from three different body sites. Enrichment of prediction some contributing species has been detected.

sentences: Sunk cost sensitivity in mice, rats, and humans on the Restaurant Row and WebSurf tasks cannot be explained by attrition biases alone In a recent bioRxiv preprint, Ott et al. argue that sensitivities to sunk costs that have been reported in two serial foraging tasks (the Restaurant Row task in mice and rats, and the Web-Surf task in humans) may be due to simple consequences of the way that subjects perform these tasks and not due to an actual sensitivity to sunk costs. However, several variants of these tasks have been studied, in which the sensitivity to sunk costs changes. In order to test the Ott et al. model against these experimental observations, we simulated the model under these additional experimental conditions. We find that it is incompatible with the actual data. While we applaud the simplicity of the Ott et al. model, we must reject it as an explanation for the observed sensitivity to sunk costs seen in these tasks. We thus conclude that the alternative explanation - that mice, rats, and humans are sensitive to actual sunk costs in these tasks - is a better explanation for the data.

sentences: Path vectors: a neural code for sequential memory While recalling lists of unrelated items is highly challenging we can recall much longer sequences structured as an episode or story. It is unknown why these patterns have such a striking influence on memory. We introduce a model where the experience of an episode or story is represented as a path through a pre-existing network of cognitive states. We demonstrate that by summing the neural representations of the visited states, this path can be transformed into a simple neural code: a path vector. We show how, by leveraging sparse connectivity and high dimensionality, path vectors provide robust codes for a large set of sequences and can be decoded mechanistically for memory retrieval. Fitting our model to data reveals how human free and serial recall may emerge from adapting coding mechanisms tuned for sequences aligned with existing network paths. We thus posit that sequences such as episodes or stories map more directly to existing cognitive network paths than arbitrary lists, with the latter eliciting paths that tend to interfere and impair recall. Our model suggests that mnemonic strategies like imposing narrative structure on a list act to improve recall by reducing such path interference. This work illuminates a simple bioplausible means for flexibly recruiting existing cognitive structures to encode new memories.

sentences: Brain-wide neuronal activation and functional connectivity are modulated by prior exposure to repetitive learning episodes. Memory storage and retrieval are shaped by past experiences. Prior learning and memory episodes have numerous impacts on brain structure from micro to macroscale. Previous experience with specific forms of learning increases the efficiency of future learning. It is less clear whether such practice effects on one type of memory might also have transferable effects to other forms of memory. Different forms of learning and memory rely on different brain-wide networks but there are many points of overlap in these networks. Enhanced structural or functional connectivity caused by one type of learning may be transferable to another type of learning due to overlap in underlying memory networks. Here, we investigated the impact of prior chronic spatial training on the task-specific functional connectivity related to subsequent contextual fear memory recall in mice. Our results show that mice exposed to prior spatial training exhibited decreased brain-wide activation compared to control mice during the retrieval of a context fear memory. With respect to functional connectivity, we observed changes in several network measures notably an increase in global efficiency. Interestingly, we also observed an increase in network resilience based on simulated targeted node deletion. Overall, this study suggests that chronic learning has transferable effects on the functional connectivity networks of other types of learning and memory. The generalized enhancements in network efficiency and resilience suggest that learning itself may protect brain networks against deterioration.

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Survival Genie, a web platform for survival analysis across pediatric and adult cancers. The genomics data-driven identification of gene signatures and pathways has been routinely explored for predicting cancer survival and making decisions related to targeted treatments. A large number of packages and tools have been developed to correlate gene expression/mutations to the clinical outcome but lack the ability to perform such analysis based on pathways, gene sets, and gene ratios. Furthermore, in this single-cell omics era, the cluster markers from cancer single-cell transcriptomics studies remain an underutilized prognostic option. Additionally, no bioinformatics online tool evaluates the associations between the enrichment of canonical cell types and survival across cancers. Here we have developed Survival Genie, a web tool to perform survival analysis on single-cell RNA-seq (scRNA-Seq) data and a variety of other molecular inputs such as gene sets, genes ratio, tumor infiltrating immune cells proportion, gene expression profile scores, and tumor mutation burden. For a comprehensive analysis, Survival Genie contains 53 datasets of 27 distinct malignancies from 11 different cancer programs related to adult and pediatric cancers. Users can upload scRNA-Seq data or gene sets and select a gene expression partitioning method (i.e., mean, median, quartile, cutp) to determine the effect of expression levels on survival outcomes. The tool provides comprehensive results including box plots of low and high-risk groups, Kaplan-Meier plots with univariate Cox proportional hazards model, and correlation of immune cell enrichment and molecular profile.

The analytical options and comprehensive collection of cancer datasets make Survival Genie a unique resource to correlate gene sets, pathways, cellular enrichment, and single-cell signatures to clinical outcomes to assist in developing next-generation prognostic and therapeutic biomarkers. Survival Genie is open-source and available online at https://bbisr.shinyapps.winship.emory.edu/SurvivalGenie/.

sentences: Systematic analysis uncovers SYK dependency in NF1LoF melanoma cells The loss of function (LoF) of NF1 is the third most frequent mutation that drives hyperactivated RAS and tumor growth in >10% of melanomas. NF1LoF melanoma cells, however, do not show consistent sensitivity to individual MEK, ERK, or PI3K/mTOR inhibitors. Here, we perform a targeted kinase inhibitor screen and identify a tool compound, named MTX-216, to be highly effective in blocking NF1LoF melanoma cells. Single-cell analysis links drug-induced cytotoxicity to effective co-suppression of proliferation marker Ki-67 and the ribosomal S6 phosphorylation, an integrator of multiple RAS-mediated signaling pathways. Using a combination of kinome selectivity assay, transcriptomic analysis, and genetic experiments, we find the anti-tumor efficacy of MTX-216 to be dependent on its ability to inhibit not only PI3K (its nominal target) but also SYK, and suppression of a group of genes that regulate mitochondrial electron transport chain and whose expression is associated with poor survival in NF1LoF melanoma patients. Furthermore, combinations of inhibitors targeting either MEK or PI3K/mTOR with an independent SYK kinase inhibitor or SYK knockdown show favorable effects. These studies provide a path to exploit SYK dependency to selectively block NF1LoF melanoma cells.

sentences: The characteristics of intracellular Ca2+ signals in vivo necessitate a new model for salivary fluid secretion Salivary fluid secretion involves an intricate choreography to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are based on experimental observations in enzymatically isolated cells where the Ca2+ signal invariably propagates globally and thus appears ideally suited to activate spatially separated Cl and K channels. We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. Given this incompatibility to the previous model, a new mathematical model was constructed to explain how salivary secretion can be efficiently stimulated by apically localized Ca2+ signals.

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Corticosterone differentially modulates time-dependent fear generalization following mild or moderate fear conditioning training in rats Stressful and emotionally arousing experiences create strong memories that seem to lose specificity over time. It is uncertain, however, how the stress system contributes to the phenomenon of time-dependent fear generalization. Here, we investigated whether post-training corticosterone (CORT-HBC) injections, given after different training intensities, affect contextual fear memory specificity at several time points. We trained male Wistar rats on the contextual fear conditioning (CFC) task using two footshock intensities (mild CFC, 3 footshocks of 0.3 mA, or moderate CFC, 3x 0.6 mA) and immediately after the training session we administered CORT-HBC systemically. We first tested the animals in a novel context and then in the training context at different intervals following training (2, 14, 28 or 42 days). By measuring freezing in the novel context and then contrasting freezing times shown in both contexts, we inferred contextual fear generalization for each rat, classifying them into Generalizers or Discriminators. Following mild CFC training, the glucocorticoid injection promoted an accurate contextual memory at the recent time point (2 days), and increase the contextual memory accuracy 28 days after training. In contrast, after the moderate CFC training, CORT-HBC facilitated contextual generalization at 14 days, compared to the control group that maintained contextual discrimination at this timepoint. For this training intensity, however, CORT-HBC did not have any effect on recent memory specificity. These findings indicate that treatment with CORT-HBC immediately after the encoding of mild or moderately arousing experiences may differentially modulate memory consolidation and time-dependent fear generalization.

HIGHLIGHTS- Rats were trained in a contextual fear task and tested for fear generalization.
- Corticosterone (CORT) injected after a mild training promoted accuracy up to 28 days.
- CORT injected after a moderate training facilitated time-dependent fear generalization.

sentences: Dynamic Interactions of Fully Glycosylated SARS-CoV-2 Spike Protein with Various Antibodies The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a public health crisis, and the vaccines that can induce highly potent neutralizing antibodies are essential for ending the pandemic. The spike (S) protein on the viral envelope mediates human angiotensin-converting enzyme 2 (ACE2) binding and thus is the target of a variety of neutralizing antibodies. In this work, we built various S trimer-antibody complex structures on the basis of the fully glycosylated S protein models described in our previous work, and performed all-atom molecular dynamics simulations to get insight into the structural dynamics and interactions between S protein and antibodies. Investigation of the residues critical for S-antibody binding allows us to predict the potential influence of mutations in SARS-CoV-2 variants. Comparison of the glycan conformations between S-only and S-antibody systems reveals the roles of glycans in S-antibody binding. In addition, we explored the antibody binding modes, and the influences of antibody on the motion of S protein receptor binding domains. Overall, our analyses provide a better understanding of S-antibody interactions, and the simulation-based S-antibody interaction maps could be used to predict the influences of S mutation on S-antibody interactions, which will be useful for the development of vaccine and antibody-based therapy.

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Structural and functional alterations of neuromuscular synapses in a mouse model of ACTA1 congenital myopathy Mutations in skeletal muscle -actin (Acta1) cause a variety of myopathies. In a mouse model of congenital myopathy, heterozygous Acta1 (H40Y) knock-in (Acta1+/Ki) mice exhibit features of human nemaline myopathy, including premature lethality, severe muscle weakness, reduced mobility, and the presence of nemaline rods in muscle fibers. In this study, we investigated the structure and function of the neuromuscular junction (NMJ) in the Acta1+/Ki mice. We found marked impairments in NMJ structure in the mutant mice, including fragmented endplates and nerve terminals, reduced density of acetylcholine receptors (AChRs) on endplate membranes, reduced nerve occupancy at endplates, and increased numbers of muscle fiber subsynaptic nuclei. We compared the NMJs in three different types of muscles - the extensor digitorum longus (EDL, composed of fast-twitch muscle fibers), soleus (Sol, enriched in slow-twitch fibers) and the triangularis sterni muscle (TS, a mixed fiber type muscle). Among these three types of muscles, EDL was affected to the greatest extent, suggesting that fast-twitch fibers may be most susceptible to NMJ fragmentation in Acta1+/Ki nemaline myopathy.

Electrophysiological analysis of mutant NMJs showed a reduced quantal size (reduced mEPP amplitude), increased mEPP frequency, and increased quantal content, but normal EPP amplitude compared to wild type (WT) NMJs. The results suggest that affected synapses may have undergone homeostatic compensation to maintain normal levels of neurotransmitter release. In addition, paired-pulse facilitation was reduced and synaptic depression under repetitive nerve stimulation was enhanced, indicating shortterm synaptic plasticity was compromised in the mutant mice.

Key pointsO_LIMice heterozygous for an Acta1 (H40Y) knock-in mutation exhibit clinical features of human nemaline myopathy. We report structural and functional alterations of neuromuscular synapses in these mutant mice. The NMJ impairments include endplate fragmentation, reduced endplate nerve occupancy, and increased numbers of subsynaptic nuclei in muscle fibers.
C_LIO_LINeuromuscular synaptic transmission was compromised - demonstrating both increased quantal content and changes in short-term synaptic plasticity.
C_LIO_LIIncreases in spontaneous neurotransmitter release and quantal content suggest homeostatic compensation of synapses to maintain normal transmitter release in the mutant NMJs.
C_LI

sentences: Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. Despite extensive work on the formation and functions of EVs in cultured cells, studies of EVs in vivo have remained scarce. We report here that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. We defined two distinct EV subpopulations, one of which contains the Munc13-4 homologue Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion events. The formation of Stac-positive luminal EVs depends on the tip-cell-specific GTPase Arl3, which is also required for the formation of Stac-positive multivesicular bodies, suggesting that Stac-EVs derive from fusion of Stac-MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-MVB formation and tube fusion. We propose that Stac-MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35, and Stac/Munc13-4.

sentences: Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells expressing a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing a wild type TOR1 allele from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. While consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats were capable of utilizing non-fermentable as well as fermentable carbon sources and of growth during severe acid stress, but were uniquely unable to grow on lactate. Genome-wide expression analysis showed paradoxical simultaneous activation of anabolic- and starvation responses in cells lacking Tor1 N-terminal HEAT repeats, with misregulation of carbon metabolism and of translational machinery biosynthesis. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

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A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation The surface of pollen grains is reinforced by pollen wall components produced non-cell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage-specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types therefore cooperate to coordinate pollen wall deposition through a multilateral molecular dialog.

Significance StatementPollen viability depends on a tough external barrier called the pollen wall. Pollen wall components are produced by tapetum cells which surround developing pollen grains within the anther. Precise coordination of tapetum activity with pollen grain development is required to ensure effective pollen wall formation. Here we reveal that this is achieved through a multidirectional dialogue involving three distinct cell types. We show that peptide precursors from the tapetum are activated by proteases produced stage-specifically in developing pollen grains. Unexpectedly, we found that activated peptides are perceived not in the tapetum, but in the middle layer, which encloses the developing tapetum and pollen grains, revealing an unsuspected role for this enigmatic cell layer in the control of tapetum development.

sentences: Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein-protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co-elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic -- Diffuse B-cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

sentences: Models of the cardiac L-type calcium current: a quantitative comparison The L-type calcium current (ICaL) plays a critical role in cardiac electrophysiology, and models of ICaL are vital tools to predict arrhythmogenicity of drugs and mutations. Five decades of measuring and modelling ICaL have resulted in several competing theories (encoded in mathematical equations). However, the introduction of new models has not typically been accompanied by a data-driven critical comparison with previous work, so that it is unclear where predictions overlap or conflict, or which model is best suited for any particular application. We gathered 71 mammalian ICaL models, compared their structure, and reproduced simulated experiments to show that there is a large variability in their predictions, which was not substantially diminished when grouping by species or other categories. By highlighting the differences in these competing theories, listing major data sources, and providing simulation code, we have laid strong foundations for the development of a consensus model of ICaL.

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Minor deviations from randomness have huge repercussions on the functional structuring of sequence space Approaches based on molecular evolution have organized natural proteins into a hierarchy of families, superfamilies, and folds, which are often pictured as islands in a great sea of unrealized and generally non-functional polypeptides. In contrast, approaches based on information theory have substantiated a mostly random scatter of natural proteins in global sequence space. We evaluate these opposing views by analyzing fragments of a given length derived from either a natural dataset or different random models. For this, we compile distances in sequence space between fragments within each dataset and compare the resulting distance distributions between sets. Even for 100-mers, more than 95% of distances can be accounted for by a random sequence model that incorporates the natural amino acid frequency of proteins. When further accounting for the specific residue composition of the respective fragments, which would include biophysical constraints of protein folding, more than 99% of all distances can be modeled. Thus, while the local space surrounding a protein is almost entirely shaped by common descent, the global distribution of proteins in sequence space is close to random, only constrained by divergent evolution through the requirement that all intermediates connecting two forms in evolution must be functional.

Significance StatementWhen generating new proteins by evolution or design, can the entire sequence space be used, or do viable sequences mainly occur only in some areas of this space? As a result of divergent evolution, natural proteins mostly form families that occupy local areas of sequence space, suggesting the latter. Theoretical work however indicates that these local areas are highly diffuse and do not dramatically affect the statistics of sequence distribution, such that natural proteins can be considered to effectively cover global space randomly, though extremely sparsely. By comparing the distance distribution of natural sequences to that of various random models, we find that they are indeed distributed largely randomly, provided that the amino acid composition of natural proteins is respected.

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Mathematical Modeling of Rhesus Cytomegalovirus (RhCMV) Placental Transmission in Seronegative Rhesus Macaques Approximately 1 in 200 infants is born with congenital cytomegalovirus (CMV), making it the most common congenital infection. About 1 in 5 congenitally-infected babies will suffer long-term sequelae, including sensorineural deafness, intellectual disability, and epilepsy. CMV infection is highly species-dependent, and the Rhesus CMV (RhCMV) infection of rhesus monkey fetuses is the only animal model that replicates essential features of congenital CMV infection in humans, including placental transmission, fetal disease, and fetal loss. To better understand the determinants and dynamics of congenital CMV transmission, we developed a mathematical model for placental transmission, comprising of maternal, placental, and fetal compartments using parameters from literature and experimental data from RhCMV seronegative rhesus macaques inoculated with RhCMV at 7.7-9.0 weeks of pregnancy. The model was then used to study the effect of the timing of inoculation, maternal immune suppression, and hyper-immune globulin infusion on the risk of placental transmission in the context of primary and reactivated chronic maternal CMV infection.

Author summaryCongenital cytomegalovirus (CMV) is the most common congenital infection in humans. Congenial CMV affects 1 in 200 infants, and can result in sensorineural deafness, intellectual disability, epilepsy, and death. The Rhesus CMV (RhCMV) model is the only animal model that replicates essential features of congenital CMV infection and fetal sequelae in humans and provides a critical experimental system to develop mechanistic insight. We propose a novel mathematical model for CMV transmission that integrates viral dynamics in the maternal, placental, and fetal compartments. We calibrate the model using data from RhCMV transmission experiments and show that the model can recapitulate experimental observations of primary versus reactivated chronic CMV infection in pregnancy, primary infection at different stages in pregnancy, and infection in the presence of varying degrees of immune suppression and hyper-immune globulin infusion. Our in-silico model provides a means to rapidly explore mechanistic hypotheses for the physical, viral, and immune determinants of CMV transmission to complement and support expensive and difficult experiments on non-human primates.

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Multiple dimensions underlying the functional organisation of the language network Understanding the different neural networks that support human language is an ongoing challenge for cognitive neuroscience. Which divisions are capable of distinguishing the functional significance of regions across the language network? A key separation between semantic cognition and phonological processing was highlighted in early meta-analyses, yet these seminal works did not formally test this proposition. Moreover, organisation by domain is not the only possibility. Regions may be organised by the type of process performed, as in the separation between representation and control processes proposed within the Controlled Semantic Cognition framework. The importance of these factors was assessed in a series of activation likelihood estimation meta-analyses that investigated which regions of the language network are consistently recruited for semantic and phonological domains, and for representation and control processes. Whilst semantic and phonological processing consistently recruit many overlapping regions, they can be dissociated (by differential involvement of bilateral anterior temporal lobes and superior temporal gyri) only when using both formal analysis methods and sufficient data. Both semantic and phonological regions are further dissociable into control and representation regions, highlighting this as an additional, distinct dimension on which the language network is functionally organised. Furthermore, some of these control regions overlap with multiple-demand network regions critical for control beyond the language domain, suggesting the relative level of domain-specificity is also informative. Multiple, distinct dimensions are critical to understand the role of language regions. Here we present a proposal as to the core principles underpinning the functional organisation of the language network.

SIGNIFICANCE STATEMENTTraditional theories of the organisation of the cortical language network separate areas based on the type of information processed, such as distinct regions representing the meaning of words or their sounds. Here, we re-analyse and update a seminal study, to directly compare these networks. This differentiates some language regions, yet a high degree of overlap is found, suggesting this division alone, does not provide a full mapping of the language network. Additional dimensions, reflecting different kinds of information, are demonstrated to underlie the functional organisation of the language network. First, representation and control processes engage distinct regions in each subdomain. Secondly, comparison outside of language highlights the importance of a  domain-specificity dimension, distinguishing subdomain-specific semantic control and domain-general control regions.

sentences: Novel microRNAs targeting NMDA receptor subunits in animal models of schizophrenia N-methyl-D-aspartate receptors (NMDAR) are downregulated in schizophrenia possibly through microRNAs (miRNAs) that are differentially expressed in this condition. We screened the miRNAs that are altered in schizophrenia against the targets, Grin2A and Grin2B subunits of NMDAR using bioinformatic tools. Among the predicted miRNAs some interacted with the 3-UTR sequences of Grin2A (miR-296, miR-148b, miR-129-2, miR-137) and Grin2B (miR-296, miR-148b, miR-129-2, miR-223) in dual luciferase assays. This was supported by downregulation of the GluN2B protein in primary hippocampal neurons upon overexpressing Grin2B targeting miRNAs. In two models of schizophrenia-pharmacological MK-801 model and neurodevelopmental methylazoxymethanol acetate (MAM) model which showed cognitive deficits - protein levels of GluN2A and GluN2B were downregulated but their transcript levels were upregulated. MiR-296-3p, miR-148b-5p and miR-137 levels showed upregulation in both models which could have interacted with Grin2A/Grin2B transcripts resulting in translational arrest. In MAM model, reciprocal changes in the expression of the 3p and 5p forms of miR-148b and miR-137 were observed. Expression of neuregulin 1 (NRG1), BDNF and CaMKII, genes implicated in schizophrenia, were also altered in these models. This is the first report of downregulation of GluN2A and GluN2B by miR-296, miR-148b and miR-129-2. Mining miRNAs regulating NMDA receptors might give insights into the pathophysiology of this disorder, providing avenues in therapeutics.

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SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential The balance between the mitochondrial respiratory chain activity and the cells needs in ATP ensures optimal cellular function. Cytochrome c is an essential component of the electron transport chain (ETC), which regulates ETC activity, oxygen consumption, ATP synthesis and can initiate apoptosis. The impact of conformational changes in cytochrome c on its function is not understood for lack of access to these changes in intact mitochondria. We have developed a novel sensor that uses unique properties of label-free surface-enhanced Raman spectroscopy (SERS) to identify conformational changes in heme of cytochrome c and to elucidate their role in functioning mitochondria. We verify that molecule bond vibrations assessed by SERS is a reliable indicator of the heme conformation during changes in the inner mitochondrial membrane potential and ETC activity. We have found that cytochrome c heme reversibly switches between planar and ruffled conformations in response to the inner mitochondrial membrane potential and H+ concentration in the intermembrane space to regulate the efficiency of the mitochondrial respiratory chain, thus, adjusting the mitochondrial respiration to the cells consumption of ATP and the overall activity. The ability of the proposed SERS-based sensor to track mitochondrial function opens wide perspectives on cell bioenergetics.

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Dynamic regulation and requirement for ribosomal RNA transcription duringmammalian development Ribosomal RNA (rRNA) transcription by RNA Polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies, which frequently affect craniofacial development. Here, we describe a cellular and molecular mechanism underlying the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCC), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCC particularly sensitive to rRNA synthesis defects. Consistent with this model, NCC-specific deletion of Pol I subunits Polr1a, Polr1c, and associated factor Tcof1 in mice cell-autonomously diminishes rRNA synthesis, which causes an imbalance between rRNA and ribosomal proteins. This leads to increased binding of ribosomal proteins Rpl5 and Rpl11 to Mdm2 and concomitantly diminished binding between Mdm2 and p53. Consequently, p53 protein accumulates, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbates the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins Syndrome and Acrofacial Dysostosis-Cincinnati Type. Altogether, our novel results demonstrate a dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of congenital craniofacial disorders.

Significance statementRNA Polymerase I (Pol I) mediated rRNA transcription is required for protein synthesis in all tissues for normal growth and survival as well as for proper embryonic development. Interestingly, disruptions in Pol I mediated transcription perturb ribosome biogenesis and lead to tissue-specific birth defects, which commonly affect the head and face. Our novel results show that during mouse development, Pol I mediated rRNA transcription and protein translation is tissue-specifically elevated in neural crest cells, which give rise to bone, cartilage, and ganglia of the head and face. Using new mouse models, we further show that neural crest cells are highly sensitive to disruptions in Pol I and that when rRNA synthesis is genetically downregulated, it specifically results in craniofacial anomalies.

sentences: MorphData: Automating the data extraction process of morphological features of microglial cells in ImageJ Microglial cells are the first line of defense within the central nervous system, with morphological characterization being widely used to define their activation status. Most methods to evaluate microglia status are manual, and, therefore, often biased, inaccurate, and time consuming. In fact, the process to collect morphological data starts with the acquisition of photomicrographs from where images of single cells are extracted. Then, the researcher collects the morphological features that characterize each cell. However, a manual data collection process from single cells can take weeks to complete. This work describes an open-source ImageJ plugin, MorphData, which automatizes the data extraction process of morphological features of single microglial cells. The plugin collects, processes, and organizes features associated with cell complexity and ramification. In a computer with limited computing power, MorphData was able to handle 699 single cells in less than 14 minutes. The same process, if performed manually, would take almost 19 working days. Overall, MorphData significantly reduces the time taken to collect morphological data from microglial cells, which can then be used to study, understand, and characterize microglia behavior in the brain of human patients or of animal models of neurological and psychiatric diseases.

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Identification of cells expressing Calcitonins A and B, PDF and ACP in Locusta migratoria using cross-reacting antisera and in situ hybridization This work was initiated because an old publication suggested that electrocoagulation of four paraldehyde fuchsin positive cells in the brain of Locusta migratoria might produce a diuretic hormone, the identity of which remains unknown, since none of the antisera to the various putative Locusta diuretic hormones recognizes these cells. The paraldehyde fuchsin positive staining suggests a peptide with a disulfide bridge and the recently identified Locusta calcitonins have both a disulfide bridge and are structurally similar to calcitonin-like diuretic hormone.

In situ hybridization and antisera raised to calcitonin-A and -B were used to show were these peptides are expressed in Locusta. Calcitonin-A is produced by neurons and neuroendocrine cells that were previously shown to be immunoreactive to an antiserum to pigment dispersing factor (PDF). The apparent PDF-immunoreactivity in these neurons and neuroendocrine cells is due to crossreactivity with the calcitonin-A precursor. As confirmed by both an PDF-precursor specific antiserum and in situ hybridisation, those calcitonin-A expressing cells do not express PDF.

Calcitonin B is expressed by numerous enteroendocrine cells in the midgut as well as the midgut caeca. A guinea pig antiserum to calcitonin A seemed quite specific as it recognized only the calcitonin A expressing cells. However, rabbit antisera to calcitonin-A and-B both crossreacted with neuroendocrine cells in the brain that produce ACP, this is almost certainly due to the common C-terminal dipeptide SPamide that is shared between Locusta calcitonin-A, calcitonin-B and ACP.

sentences: Monitoring rapid evolution of plant populations at scale with Pool-Sequencing The change in allele frequencies within a population over time represents a fundamental process of evolution. By monitoring allele frequencies, we can analyze the effects of natural selection and genetic drift on populations. To efficiently track time-resolved genetic change, large experimental or wild populations can be sequenced as pools of individuals sampled over time using high-throughput genome sequencing (called the Evolve & Resequence approach, E&R). Here, we present a set of experiments using hundreds of natural genotypes of the model plant Arabidopsis thaliana to showcase the power of this approach to study rapid evolution at large scale. First, we validate that sequencing DNA directly extracted from pools of flowers from multiple plants -- organs that are relatively consistent in size and easy to sample -- produces comparable results to other, more expensive state-of-the-art approaches such as sampling and sequencing of individual leaves. Sequencing pools of flowers from 25-50 individuals at ~40X coverage recovers genome-wide frequencies in diverse populations with accuracy r>0.95. Secondly, to enable analyses of evolutionary adaptation using E&R approaches of plants in highly replicated environments, we provide open source tools that streamline sequencing data curation and calculate various population genetic statistics two orders of magnitude faster than current software. To directly demonstrate the usefulness of our method, we conducted a two-year outdoor evolution experiment with A. thaliana to show signals of rapid evolution in multiple genomic regions. We demonstrate how these laboratory and computational Pool-seq-based methods can be scaled to study hundreds of populations across many climates.

sentences: Signaling by the integrated stress response kinase PKR is fine-tuned by dynamic clustering The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the alpha subunit of the eukaryotic initiation factor 2 (eIF2) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKRs sensor domain and by front-to-front interfaces between PKRs kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2 is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2 phosphorylation. Together, these results support a model in which PKR clustering buffers downstream signaling, which may enable proofreading the ISR.

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Using Transfer Learning for Automated Microbleed Segmentation IntroductionCerebral microbleeds are small perivascular haemorrhages that can occur in both grey and white matter brain regions. Microbleeds are a marker of cerebrovascular pathology, and are associated with an increased risk of cognitive decline and dementia. Microbleeds can be identified and manually segmented by expert radiologists and neurologists, usually from susceptibility-contrast MRI. The latter is hard to harmonize across scanners, while manual segmentation is laborious, time-consuming, and subject to inter- and intra-rater variabiltiy. Automated techniques so far have shown high accuracy at a neighborhood ("patch") level at the expense of a high number of false positives voxel-wise lesions. We aimed to develop an automated, more precise microbleeds segmentation tool able to use standardizable MRI contrasts.

MethodsWe first trained a ResNet50 network on another MRI segmentations task (cerberospinal fluid versus background segmentation) using T1-weighted, T2-weighted, and T2* MRI. We then used transfer learning to train the network for the detection of microbleeds with the same contrasts. As a final step, we employed a combination of morphological operators and rules at the local lesion level to remove false positives. Manual segmentations of microbleeds from 78 participants were used to train and validate the system. We assessed the impact of patch size, freezing weights of the initial layers, mini-batch size, learning rate, as well as data augmentation on the performance of the Microbleed ResNet50 network.

ResultsThe proposed method achieved a high performance, with a patch-level sensitivity, specificity, and accuracy of 99.57%, 99.16%, and 99.93%, respectively. At a per lesion level, sensitivity, precision, and Dice similarity index values were 89.1%, 20.1%, and 0.28 for cortical GM; 100%, 100%, and 1.0 for deep GM; and 91.1%, 44.3%, and 0.58 for WM, respectively.

DiscussionThe proposed microbleed segmentation method is more suitable for the automated detection of microbleeds with high sensitivity.

sentences: Sigma70Pred: A highly accurate method for predicting sigma70 promoter in prokaryotic genome Sigma70 factor plays a crucial role in prokaryotes and regulates the transcription of most of the housekeeping genes. One of the major challenges is to predict the sigma70 promoter or sigma70 factor binding site with high precision. In this study, we trained and evaluate our models on a dataset consists of 741 sigma70 promoters and 1400 non-promoters. We have generated a wide range of features around 8000 which includes Dinucleotide Auto-Correlation, Dinucleotide Cross-Correlation, Dinucleotide Auto Cross-Correlation, Moran Auto-Correlation, Normalized Moreau-Broto Auto-Correlation, Parallel Correlation Pseudo Tri-Nucleotide Composition, etc. Our SVM based model achieved maximum accuracy 97.38% with AUROC 0.99 on training dataset, using 200 selected features. In order to check the robustness of our model we have tested our model on the independent dataset made by using latest version of RegulonDB10.8, which included 1134 sigma70 and 638 non-sigma70 promoters, and able to achieve accuracy of 90.41% with AUROC of 0.95. We have developed a method, Sigma70Pred, which is available as webserver and standalone packages at https://webs.iiitd.edu.in/raghava/sigma70pred/. The services are freely accessible.

sentences: Exploring the effects of genetic variation on gene regulation in cancer in the context of 3D genome structure Numerous genome-wide association studies (GWAS) conducted to date revealed genetic variants associated with various diseases, including breast and prostate cancers. Despite the availability of these large-scale data, relatively few variants have been functionally characterized, mainly because the majority of single-nucleotide polymorphisms (SNPs) map to the non-coding regions of the human genome. The functional characterization of these non-coding variants and the identification of their target genes remain challenging. In this communication, we explore the potential functional mechanisms of non-coding SNPs by integrating GWAS with the high-resolution chromosome conformation capture (Hi-C) data for breast and prostate cancers. We show that more genetic variants map to regulatory elements through the 3D genome structure than the 1D linear genome lacking physical chromatin interactions. Importantly, the association of enhancers, transcription factors, and their target genes with breast and prostate cancers tends to be higher when these regulatory elements are mapped to high-risk SNPs through spatial interactions compared to simply using a linear proximity. Finally, we demonstrate that topologically associating domains (TADs) carrying high-risk SNPs also contain gene regulatory elements whose association with cancer is generally higher than those belonging to control TADs containing no high-risk variants. Our results suggest that many SNPs may contribute to the cancer development by affecting the expression of certain tumor-related genes through long-range chromatin interactions with gene regulatory elements. Integrating large-scale genetic datasets with the 3D genome structure offers an attractive and unique approach to systematically investigate the functional mechanisms of genetic variants in disease risk and progression.

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Semi-field evaluation of the space spray efficacy of Fludora Co-Max EW against wild insecticide-resistant Aedes aegypti and Culex quinquefasciatus mosquito populations from Abidjan, Cote d'Ivoire BackgroundSpace spraying of insecticides is still an important mean of controlling Aedes and Culex mosquitoes and arboviral diseases. This study evaluated the space spray efficacy of Fludora Co-Max EW (a combination of flupyradifurone and transfluthrin, with Film Forming Aqueous Spray Technology (FFAST)) against wild, insecticide-resistant Aedes aegypti and Culex quinquefasciatus populations from Abidjan, Cote dIvoire, against K-Othrine EC (deltamethrin-only product), through small-scale field trials.

MethodologyWild Ae. aegypti and Cx. quinquefasciatus mosquito larvae were collected in Abidjan, Cote dIvoire from August to December 2020. Mosquito larvae were reared until adult stage. Emerged adult females were tested against Fludora Co-Max EW and K-Othrine EC using ultra-low volume cold fogging (ULV) and thermal fogging (TF) both outdoors and indoors in Agboville, Cote dIvoire. Cages containing 20 mosquitoes each were placed at 10, 25, 50, 75 and 100 m from the spraying line for outdoor spraying, and at ceiling, mid-height and floor levels for indoor house spraying. Knockdown and mortality were recorded at each checkpoint and compared by treatments.

Principal findingsOverall, Fludora Co-Max EW induced significantly higher knockdown and mortality effects in the wild insecticide-resistant Ae. aegypti and Cx. quinquefasciatus compared with K-Othrine EC. With both species, Fludora Co-Max EW mortality rates were above 80% (up to 100%) for outdoor ULV spray at each distance checkpoint (i.e. 10 to 100 m), and 100% for indoor ULV and TF sprays at all level checkpoints (i.e. ceiling, mid-height and floor). K-Othrine EC induced high mortality indoors (97.9-100%), whereas outdoor mortality rapidly declined in Ae. aegypti from 96.7% to 36.7% with ULV, and 85.0% to 38.3% with TF, from 10 to 100 m. For outdoor Fludora Co-Max EW spray, ULV showed both higher knockdown and killing performance Ae. aegypti and Cx. quinquefasciatus compared with TF. Fludora Co-Max EW performed better against Cx. quinquefasciatus compared with Ae. aegypti.

Conclusion/significanceFludora Co-Max EW induced high mortality and knockdown effects against wild insecticide-resistant Ae. aegypti and Cx. quinquefasciatus Abidjan strains and performed better than K-Othrine EC. The presence of flupyradifurone and transfluthrin (with new and independent modes of action) and FFAST technology in the current Fludora Co-Max EW formulation appears to have broadened its killing capacity. Fludora Co-Max EW is thus an effective adulticide and may be a useful tool for Aedes and Culex mosquito and arbovirus control in endemic areas.

Author SummarySpace spraying of insecticides is an important tool to control Aedes and Culex mosquitoes and prevent the viral diseases (i.e. dengue, yellow fever, etc.) that they transmit. We studied the efficacy of the product Fludora Co-Max EW (a new space spray insecticide) against adult wild insecticide-resistant populations of Aedes aegypti and Culex quinquefasciatus mosquitoes from Abidjan, Cote dIvoire. We compared Fludora Co-Max EW knockdown and mortality effects in these mosquitoes with the local insecticide K-Othrine EC using ultra-low volume (ULV) and thermal fogging (TF) spraying outdoors and indoors. The product Fludora Co-Max EW induced high rates of knockdown and mortality (i.e. 80-100%) in these wild insecticide-resistant mosquitoes and performed better than the product K-Othrine EC. Additionally, ULV sprays of Fludora Co-Max EW demonstrated higher knockdown and killing efficacy at larger distances (i.e. up to 100 m) compared with TF. The higher efficacy of Fludora Co-Max EW may be due to the interaction of two unrelated insecticides, flupyradifurone and transfluthrin, in combination with Film Forming Aqueous Spray Technology (FFAST). Fludora Co-Max EW therefore appears to be an effective and useful tool to control adult populations of wild insecticide-resistant Aedes and Culex mosquitoes and may be recommended for preventing related mosquito-transmitted viral diseases.

sentences: The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 toolkit with three components: (i) a set of optimized S. pyogenes Cas9-guide RNA (gRNA) expression vectors with five selectable markers and the option to either pre-clone or co-transform the gRNAs; (ii) vectors for the one-step construction of integration cassettes expressing an untagged or GFP/RFP/HA-tagged gene of interest at one of three levels, supporting localization and overexpression studies; and (iii) integration cassettes containing moderately expressed GFP- or RFP-tagged compartmental markers for colocalization experiments. These components allow rapid, high efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. To demonstrate the ease of use, we applied our complete set of compartmental markers to co-label all target subcellular compartments with GFP and RFP. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle to simplify yeast research.

sentences: Enhancement of central norepinephrinergic neurotransmission contributes to inhibition of seizure-induced respiratory arrest by targeting the beta-1 adrenergic receptor (β1-AR) locating in the cardiomyocytes in the DBA/1 mouse SUDEP model Sudden unexpected death of epilepsy (SUDEP) is the leading cause of of death in patients with epilepsy. Due to the complicated pathogenesis of SUDEP, however, the exact mechanism of SUDEP remains elusive. Currently, although it is recognized that the seizure-induced respiratory arrest (S-IRA) may be a main cause for SUDEP, other factors resulting in SUDEP cant be excluded e.g arrhythmias. Our previous findings indicated that the incidence of S-IRA and SUDEP evoked by acoustic stimulation or pentetrazol (PTZ) injection was significantly reduced by atomoxetine, a norepinephrine reuptake inhibitor (NRI), suggesting that noradrenergic neurotransmission modulates S-IRA and SUDEP. Given that norepinephrine acts on the target to modulate respiratory and circulation function by targeting adrenergic receptor  and beta (a-AR and {beta}-AR) and the arrhythmias can be contributed to SUDEP. Meanwhile, to further test whether cardiac factors are implicated in S-IRA and SUDEP, we choose esmolol hydrochloride, a selective antagonist of beta-1 adrenergic receptor ({beta}1-AR) to test it in our models. Our findings demonstrated that the lower incidence of S-IRA and SUDEP evoked by acoustic stimulation or PTZ injection in DBA/1 mice by administration with atomoxetine was significantly reversed by intraperitoneal (IP) of esmolol hydrochloride. Whats more, the data of electrocardiogram (ECG) showed that the cardiac arrhythmia including the ventricular tachycardia, ventricular premature beat and atrioventricular block can be evoked by acoustic stimulation or PTZ injection in our model. Administration of atomoxetine significantly reduced these arrhythmias and the incidence of S-IRA and SUDEP in our models. However, administration of esmolol hydrochloride with the dose without affecting ECG and mortality changing of DBA/1 significantly blocking the protective effects of atomoxetine on S-IRA and SUDEP in our models. Thus, the dysfunction of respiratory and circulation may be implicated in the pathogenesis of S-IRA and SUDEP. Enhancing the central norepinephrinergic neurotransmission in the brain contributes to inhibition of seizure-induced respiratory arrest by targeting {beta}1-AR locating in the cardiomyocytes. Our findings will show a new light on decoding the pathogenesis of SUDEP.

sentences: Super-cells untangle large and complex single-cell transcriptome networks The exponential scaling of scRNA-seq data represents an important hurdle for downstream analyses. Here we develop a coarse-graining framework where highly similar cells are merged into metacells. We demonstrate that metacells not only preserve but often improve the results of downstream analyses including visualization, clustering, differential expression, cell type annotation, gene correlation, imputation, RNA velocity and data integration. By capitalizing on the redundancy inherent to scRNA-seq data, metacells significantly facilitate and accelerate the construction and interpretation of single-cell atlases, as demonstrated by the integration of 1.46 million cells from COVID-19 patients in less than two hours on a standard desktop.

sentences: The human ribosomal RNA gene is composed of highly homogenized tandem clusters The structure of the human ribosomal RNA gene clustering region (rDNA) has traditionally been hard to analyze due to its highly repetitive nature. However, the recent development of long-read sequencing technology, such as Oxford Nanopore sequencing, has enabled us to approach the large-scale structure of the genome. Using this technology, we found that human cells have a quite regular rDNA structure. Although each human rDNA copy has some variations in its non-coding region, contiguous copies of rDNA are similar, suggesting that homogenization through gene conversion frequently occurs between copies. Analysis of rDNA methylation by Nanopore sequencing further showed that all of the non-coding regions are heavily methylated, whereas about half of the coding regions are clearly unmethylated. The ratio of unmethylated copies, which are speculated to be transcriptionally active, was lower in individuals with a higher rDNA copy number, suggesting that there is a mechanism that keeps the active copy number stable. Lastly, the rDNA in progeroid syndrome patient cells with reduced DNA repair activity had more unstable copies as compared with control normal cells, although the rate was much lower than previously reported using a Fiber FISH method. Collectively, our results alter the view of rDNA stability and transcription regulation in human cells, indicating the presence of mechanisms for both homogenization to ensure sequence quality and maintenance of active copies for cellular functions.

sentences: Invariant Object Representation Based on Principle of Maximum Dependence Capturing Sensory inputs conveying information about the environment are often noisy and incomplete, yet the brain can achieve remarkable consistency in object recognition. This cognitive robustness is thought to be enabled by transforming the varying input patterns into invariant representations of objects, but how this transformation occurs computationally remains unclear. Here we propose that sensory coding should follow a principle of maximal dependence capturing to encode associations among structural components that can uniquely identify objects. We show that a computational framework incorporating dimension expansion and a specific form of sparse coding can capture structures that contain maximum information about specific objects, allow redundancy coding, and enable consistent representation of object identities. Using symbol and face recognition, we demonstrate that a two-layer system can generate representations that remain invariant under conditions of occlusion, corruption, or high noise.

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Structural basis of polyamine transport by human ATP13A2 (PARK9) Polyamines are small, organic polycations that are ubiquitous and essential to all forms of life. Currently, how polyamines are transported across membranes is not understood. Recent studies have suggested that ATP13A2 and its close homologs, collectively known as P5B-ATPases, are polyamine transporters at endo-/lysosomes. Loss-of-function mutations of ATP13A2 in humans cause hereditary early-onset Parkinsons disease. To understand the polyamine transport mechanism of ATP13A2, we determined high-resolution cryo-EM structures of human ATP13A2 in five distinct conformational intermediates, which together represent a near-complete transport cycle of ATP13A2. The structural basis of the polyamine specificity was revealed by an endogenous polyamine molecule bound to a narrow, elongated cavity within the transmembrane domain. The structures show an atypical transport path for a water-soluble substrate, where polyamines may exit within the cytosolic leaflet of the membrane. Our study provides important mechanistic insights into polyamine transport and a framework to understand functions and mechanisms of P5B-ATPases.

HighlightsCryo-EM structures of human ATP13A2 in five distinct conformations at 2.5-3.7 [A] resolutions.

Unique features of ATP13A2 in comparison to other P-type ATPases.

Structure of the substrate-binding pocket of ATP13A2 and the molecular basis of polyamine binding.

Conformational changes along the transport cycle and proposed model for polyamine transport.

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Topography of corticopontine projections is controlled by postmitotic expression of the area-mapping gene Nr2f1 Axonal projections from layer V neurons of distinct neocortical areas are topographically organized into discrete clusters within the pontine nuclei during the establishment of voluntary movements. However, the molecular determinants controlling corticopontine connectivity are insufficiently understood. Here, we show that an intrinsic cortical genetic program driven by Nr2f1 graded expression is directly implicated in the organization of corticopontine topographic mapping. Transgenic mice lacking cortical expression of Nr2f1 and exhibiting areal organization defects were used as model systems to investigate the arrangement of corticopontine projections. Combining three-dimensional digital brain atlas tools, Cre-dependent mouse lines, and axonal tracing, we show that Nr2f1 expression in postmitotic neurons spatially and temporally controls somatosensory topographic projections, whereas expression in progenitor cells influences the ratio between corticopontine and corticospinal fibres passing the pontine nuclei. We conclude that cortical gradients of area patterning genes are directly implicated in the establishment of a topographic somatotopic mapping from the cortex onto pontine nuclei.

Summary statementCortical gradient expression of the area patterning gene Nr2f1 spatially and temporally controls corticopontine topographic connectivity in layer V projection neurons.

sentences: Epitope Mapping of SARS-CoV-2 Spike Protein Reveals Distinguishable Antibody Binding Activity of Vaccinated and Infected Individuals. A multitude of studies have attempted to characterize the antibody response of individuals to the SARS-CoV-2 virus on a linear peptide level by utilizing peptide microarrays. These studies have helped to identify epitopes that have potential to be used for diagnostic tests to identify infected individuals, however the immunological responses of individuals who have received the currently available mRNA vaccines have not been characterized. We aimed to identify linear peptides of the SARS-CoV-2 spike protein that elicited high IgA or IgG binding activity and compare the immunoreactivity of infected individuals to those who received recommended doses of either the Moderna mRNA-1273 or Pfizer BNT162b2 vaccines by utilizing peptide microarrays. Our results revealed peptide epitopes of significant IgG binding among recently infected individuals, many of which are located near functional domains implicated in the high infectivity of SARS-CoV-2. Vaccinated individuals were found to have less intense antibody binding activity than those acutely infected, yet novel markers of IgG binding were identified in the vaccinated group.

sentences: Urbanization and green corridors influence reproductive success and pollinators of common milkweed Urbanization exerts many pressures on species, yet little is known about how these pressures impact species interactions. Studies of urban plant-pollinator systems provide mounting evidence that urbanization impairs pollinator movement in fragmented urban landscapes, yet the consequences for pollinator-mediated plant reproduction remains unclear. In non-urban areas, habitat corridors can facilitate the movement of organisms including pollinators, but whether these corridors facilitate plant-pollinator interactions in urban areas remains understudied. To examine how urban environments and green corridors influence plant-pollinator interactions, we measured reproductive success in the native plant common milkweed (Asclepias syriaca), and the community structure of its pollinators, for two years along two urban-rural transects in the Greater Toronto Area, Canada, one of which followed a green corridor. We found that urbanization decreased male fitness (i.e., pollen removal), increased fruit set (i.e., mean no. of follicles per inflorescence), and inconsistently affected female fitness (i.e., no. of follicles) in A. syriaca. Urbanization simultaneously decreased pollinator abundance but increased pollinator richness. Proximity to a green corridor inconsistently affected male fitness but increased reproductive effort (i.e., no. of inflorescences) in A. syriaca, while pollinator diversity and richness was lower in corridors. Notably, there were no consistent relationships between pollinator community structure and reproductive success in A. syriaca in both the presence, and absence, of a green corridor. These results demonstrate the complexity with which urbanization, green corridors, and pollinator communities can shape the reproductive investment and fitness of native plant populations.

sentences: Sensitive and specific spectral library searching with COSS and Percolator Maintaining high sensitivity while limiting false positives is a key challenge in peptide identification from mass spectrometry data. Here, we therefore investigate the effects of integrating the machine learning-based post-processor Percolator into our spectral library searching tool COSS. To evaluate the effects of this post-processing, we have used forty data sets from two different projects and have searched these against the NIST and MassIVE spectral libraries. The searching is carried out using two spectral library search tools, COSS and MSPepSearch with and without Percolator post-processing, and using sequence database search engine MS-GF+ as a baseline comparator. The addition of the Percolator rescoring step to COSS is effective and results in a substantial improvement in sensitivity and specificity of the identifications. COSS is freely available as open source under the permissive Apache2 license, and binaries and source code are found at https://github.com/compomics/COSS

sentences: A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV2 at the immune-epithelial interface Infection by SARS-CoV2 provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled by in vitro co-culture the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV2-specific inflammatory gene cluster distinct from that seen in influenza-A or Ebola virus-infected co-cultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV2 proteins (Spike and some non-structural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age-dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV2 induces in epithelia.

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Skin specimen as an alternative to brain tissue for post mortem rabies diagnosis in animals to scale up animal rabies surveillance BackgroundDetection of the virus or some of its specific components using WHO and OIE recommended standard laboratory tests is the only way to get a reliable diagnosis of rabies. Brain tissue is the preferred specimen for post-mortem diagnosis of rabies in both humans and animals. Higher biosecurity requirements, skill and transportation facilities required for collection and transport of brain or whole carcass to the laboratory is one of the reasons for the poor rabies surveillance in animals. Point of care testing with simple, reliable and easy to operate devices would be an ideal approach for providing rapid results.

MethodsThe study evaluated diagnostic performance of two reference tests, DFAT and RTPCR on skin specimen, to assess its suitability as an alternative of brain tissue for post mortem rabies diagnosis in animals. Brain tissue and skin sample belonging to different species of animals (n=90) collected at necropsy were compared using Fluorescent Antibody Test and RT PCR, internationally approved methods for rabies diagnosis.

ResultsValidation of RT-PCR on skin and DFAT on skin in comparison with DFAT on brain as gold standard gave a sensitivity of 98% (95% CI:94.1-100) and 80% (95% CI:71.8-88.2) respectively. Specificity was 100% in both tests.

ConclusionThe findings highlight the potential of skin specimen for improving rabies surveillance in animals especially in resource poor countries.

Author SummaryThe present study was undertaken to define a reliable protocol for diagnosing animal rabies using skin specimen, a superficial tissue sample which is collected in a non-invasive manner for post-mortem diagnosis. Our aim was to design a protocol to replace the classical post-mortem diagnostic method that uses brain biopsy with an ultimate target of stepping up rabies surveillance in animals. Brain tissue and skin sample belonging to different species of animals collected at necropsy were compared using Fluorescent Antibody Test and RT PCR, internationally approved methods for rabies diagnosis. The study established that RT PCR on skin specimen is rapid, sensitive and specific, opening its potential as an ideal rabies surveillance tool overcoming the logistical challenges of carcass transportation to reference laboratories and alleviating biosafety concerns associated with brain collection. The study highlights the potential of skin specimen for improving rabies surveillance in animals especially in resource poor countries.

sentences: Evidence for a force favouring GC over AT at short intronic sites in Drosophila simulans and D. melanogaster Population genetics studies often make use of a class of nucleotide site free from selective pressures in order to make inferences about population size changes or natural selection at other sites. If such neutral sites can be identified, they offer the opportunity to avoid any confounding effects of selection. Here we investigate evolution at putatively neutrally evolving short intronic sites in natural populations of Drosophila melanogaster and D. simulans, in order to understand the properties of spontaneous mutations and the extent of GC-biased gene conversion in these species. Use of data on the genetics of natural populations is advantageous because it integrates information from large numbers of individuals over long timescales. In agreement with direct evidence from observations of spontaneous mutations in Drosophila, we find a bias in the spectrum of mutations towards AT basepairs. In addition, we find that this bias is stronger in the D. melanogaster lineage than the D. simulans lineage. The evidence for GC-biased gene conversion in Drosophila has been equivocal. Here we provide evidence for a weak force favouring GC in both species, which is stronger in D. simulans. Some homologous short intronic sites have diverged in GC content between the two species, which may have been caused by lineage-specific changes in the extent to which different regions of the genome are subject to a GC (or AT)-favouring force.

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Selective ablation of adult GFAP-expressing tanycytes leads to hypogonadotropic hypogonadism in males In adult mammals, neural stem cells emerge in three neurogenic regions, the subventricular zone of the lateral ventricle (SVZ), the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the hypothalamus. In the SVZ and the SGZ, neural stem/progenitor cells (NSPCs) express the glial fibrillary acidic protein (GFAP) and selective ablation of these NSPCs drastically decreases cell proliferation in vitro and in vivo. In the hypothalamus, GFAP is expressed by -tanycytes, which are specialized radial glia-like cells in the wall of the third ventricle. To explore the role of these hypothalamic GFAP-positive tanycytes, we used transgenic mice expressing herpes simplex virus thymidine kinase (HSV-Tk) under the control of the mouse Gfap promoter and 4-week intracerebroventricular infusion of the antiviral agent ganciclovir (GCV) that kills dividing cells expressing Tk. While GCV drastically reduced the number and growth of hypothalamus-derived neurospheres from adult transgenic mice in vitro, it caused hypogonadism in vivo. The selective death of dividing tanycytes expressing GFAP indeed caused a marked decrease in testosterone levels and testicular weight, as well as vacuolization of the seminiferous tubules and loss of spermatogenesis. In addition, GCV-treated GFAP-Tk mice showed impaired sexual behavior, but no alteration in food intake or body weight. Our results also show that the selective ablation of GFAP-expressing tanycytes leads to a sharp decrease in the number of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons and blunted LH secretion. Altogether, our data show that GFAP-expressing tanycytes play a central role in the regulation of male reproductive function.

Main pointsKilling adult hypothalamic GFAP-expressing cells blunts neurosphere formation in vitro and leads to GnRH deficiency and hypogonadism in vivo. This work pinpoints an unreported role of dividing GFAP-expressing tanycytes in reproductive function.

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The gut microbiome is associated with cocaine behavior and predicts addiction vulnerability in adult male rats The gut-brain axis is a bi-directional communication system through which microbial communities in the gut interact with the nervous system, perhaps influencing neuropsychiatric disorders such as drug abuse. This study used behavioral data and biological samples from the Cocaine Biobank to test the hypothesis that the gut microbiota can predict and reflect susceptibility to cocaine reinforcement. Adult male heterogenous rats were catheterized and allowed to self-administer cocaine in short-access sessions (2 hr/day, 10 days, 0.5 mg/kg per intravenous infusion), followed by progressive ratio (PR) tests, long-access sessions (6 hr/day, 14 days), and alternating blocks of PR, long-access, and footshock testing. Fecal samples were collected at three time points and bacterial 16s rRNA genes were sequenced to profile the microbiota. As expected, cocaine-related behavior varied among subjects, such that a quartile split identified low and high responders on each measure, as well as an overall addiction index. Although beta diversity in the microbiota at baseline and after short access did not predict membership in high or low addiction quartiles, linear discriminant analysis (LDA) identified taxa that were more robustly represented in low or high responders. Beta diversity after long access was different among quartiles, as were several specific taxa. Investigation of baseline differences revealed that high relative abundance of Akkermansia muciniphila predicted future low response rates, whereas Ruminococcaceae predicted high response. This study is the first to report that microbiota variability reflects levels of cocaine intake and that microbial profiles might facilitate diagnosis and identify risk factors predictive of drug vulnerability.

Significance StatementMicrobial organisms inhabiting the gut of animals appear to influence organismal function through various signaling pathways, ultimately affecting behavior and disease vulnerability. This experiment investigates links between gut bacteria and vulnerability to addiction-related behaviors in adult male rats. Not only did gut bacterial profiles change as a result of cocaine intake but also gut bacterial profiles before any exposure to cocaine predicted which animals would be high or low addiction-prone individuals. These results suggest that microbial profiles might facilitate diagnosis and identify risk factors predictive of drug addiction.

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Outcome-selective reinstatement is predominantly context-independent, and associated with c-Fos activation in the posterior dorsomedial striatum Research from human and animal studies has found that responding that has been successfully reduced following treatment can return upon exposure to certain contexts. An individual in recovery from alcohol use disorder, for example, might relapse to drinking upon visiting their favourite bar. However, most of these data have been derived from experiments involving a single (active) response, and the context-dependence of returned responding in situations involving choice between multiple actions and outcomes is less well-understood. We thus investigated how outcome-selective reinstatement - a procedure involving choice between multiple actions - was affected by altering the physical context in rats. In Experiment 1, rats were trained over 6 days to press a left lever for one food outcome (pellets or sucrose) and a right lever for the other outcome. Then, rats received an extinction session in either the same context (A) as lever press training, or in a different context (B). Rats were tested immediately (5 minutes) after extinction in Context A or B such that there were four groups in total: AAA, ABB, ABA, and AAB. Reinstatement testing consisted of one food outcome being delivered  freely (i.e. unearned by lever pressing and unsignalled by cues) to the food magazine every 4 minutes in the following order: Sucrose, Pellet, Pellet, Sucrose. Selective reinstatement was considered intact if pellet delivery increased pressing selectively on the pellet lever, and sucrose delivery selectively increased pressing on the sucrose lever. This result (Reinstated > Nonreinstated) was observed for rats in group AAA and ABB, but not rats in groups ABA and AAB. Experiment 2 was conducted identically, except that rats received two extinction sessions over two days and tested one day later. This time, all groups demonstrated intact outcome-selective reinstatement regardless of context. Analysis of c-Fos expression in several brain regions revealed that only c-Fos expression in the posterior dorsomedial striatum (pDMS) was related to intact reinstatement performance. Overall, these results suggest that outcome-selective reinstatement is predominantly context-independent, and that intact reinstatement is related to neuronal activity in the pDMS.

HighlightsO_LIOutcome-selective reinstatement is predominantly context-independent
C_LIO_LIOutcome-selective reinstatement is entirely context-independent after multiple extinction sessions
C_LIO_LIOutcome-selective reinstatement increases c-Fos expression in dorsomedial striatum
C_LIO_LIc-Fos expression in orbitofrontal cortex and dorsal hippocampus is unaffected by selective reinstatement.
C_LI

sentences: Theta activity paradoxically boosts gamma and ripple frequency sensitivity in prefrontal interneurons Fast oscillations in cortical circuits critically depend on GABAergic interneurons. Which interneuron types and populations can drive different cortical rhythms, however, remains unresolved and may depend on brain state. Here, we measured the sensitivity of different GABAergic interneurons in prefrontal cortex under conditions mimicking distinct brain states. While fast-spiking neurons always exhibited a wide bandwidth of around 400 Hz, the response properties of spike-frequency adapting interneurons switched with the background inputs statistics. Slowly fluctuating background activity, as typical for sleep or quiet wakefulness, dramatically boosted the neurons sensitivity to gamma- and ripple-frequencies. A novel time-resolved dynamic gain analysis revealed rapid sensitivity modulations that enable neurons to periodically boost gamma oscillations and ripples during specific phases of ongoing low-frequency oscillations. This mechanism presumably contributes substantially to cross-frequency coupling and predicts these prefrontal interneurons to be exquisitely sensitive to high-frequency ripples, especially during brain states characterized by slow rhythms.

sentences: Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.

sentences: Pseudomonas aeruginosa utilises host-derived polyamines to facilitate antimicrobial tolerance Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the two-component regulatory system sensor kinase gene pmrB, in P. aeruginosa from CF and from experimental infection of mice. Here, we demonstrate that whilst such mutations lower in vitro MICs for multiple antimicrobial classes, this is not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impairs aminoarabinose modification of lipopolysaccharide, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this can be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coats the surface of PmrB-deficient P. aeruginosa, reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine is elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilised.

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Low cost and sustainable hyaluronic acid production in a manufacturing platform based on Bacillus subtilis 3NA strain Hyaluronic acid (HA) is a high value glycosaminoglycan mostly used in health and cosmetic applications. Commercial HA is produced from animal tissues or in toxigenic bacteria of the genus Streptococcus grown in complex media, which are expensive and raise environmental concerns due to the disposal of large amounts of broth with high organic loads. Other microorganisms were proposed as hosts for the heterologous production of HA, but the methods are still costly. The extraordinary capacity of this biopolymer to bind and retain water attracts interest for large scale applications where biodegradable materials are needed, but its high cost and safety concerns are barriers for its adoption.

Bacillus subtilis 3NA strain is prototrophic, amenable for genetic manipulation, GRAS, and can rapidly reach high cell densities in salt-based media. These phenotypic traits were exploited to create a platform for biomolecule production using HA as a proof of concept. First, the 3NA strain was engineered to produce HA; second, a chemically defined medium was formulated using commodity-priced inorganic salts combined at the stoichiometric ratios needed to build the necessary quantities of biomass and HA; and third, a scalable fermentation process, where HA can be produced at the maximum volumetric productivity (VP), was designed.

A comparative economic analysis against other methods indicates that the new process may increase the operating profit of a manufacturing plant by more than 100 %. The host, the culture medium, and the rationale employed to develop the fermentation process described here, introduce an IP free platform that could be adaptable for production of other biomolecules.

Key PointsO_LIA platform for the production of biomolecules was designed based on B. subtilis 3NA, a chemically defined medium and a fermentation process.
C_LIO_LIAs proof of concept, high quality hyaluronic acid was produced with an environmentally friendly process.
C_LIO_LIA techno-economic analysis indicates that the process is more that 100% profitable than current methods.
C_LI

sentences: ProMoCell and ProModb: web services for analyzing interaction-based functionally localized protein modules in a cell The modular organization of a cell which can be determined by its interaction network allows us to understand a mesh of cooperation among the functional modules. Therefore, cellular level identification of functional modules aids in understanding the functional and structural characteristics of the biological network of a cell and also assists in determining or comprehending the evolutionary signal. We develop ProMoCell that performs real-time web scraping for generating clusters of the cellular level functional units of an organism. ProMoCell constructs the Protein Locality Graphs and clusters the cellular level functional units of an organism by utilizing experimentally verified data from various online sources. Also, we develop ProModb, a database service that houses precomputed whole-cell protein-protein interaction network-based functional modules of an organism using ProMoCell. Our web service is entirely synchronized with the KEGG pathway database and allows users to generate spatially localized protein modules for any organism belonging to the KEGG genome using its real-time web scraping characteristics. Hence, the server will host as many organisms as is maintained by the KEGG database. Our web services provide the users a comprehensive and integrated tool for an efficient browsing and extraction of the spatial locality-based protein locality graph and the functional modules constructed by gathering experimental data from several interaction databases and pathway maps. We believe that our web services will be beneficial in pharmacological research, where a novel research domain called modular pharmacology has initiated the study on the diagnosis, prevention, and treatment of deadly diseases using functional modules.

sentences: EEG Alpha Power and Pupil Diameter Reflect Endogenous Auditory Attention Switching and Listening Effort Auditory attention describes a listeners focus on an acoustic source while they ignore other competing sources that might be present. In an environment with multiple talkers and background noise (i.e. the cocktail party effect), auditory attention can be difficult, requiring the listener to expend measurable cognitive effort. A listener will naturally interrupt sustained attention on a source when switching towards another source during conversation. This change in attention is potentially even more taxing than maintaining sustained attention due to the limits of human working memory, and this additional effort required has not been well studied. In this work, we evaluated an attention decoder algorithm for detecting the change in attention and investigated cognitive effort expended during attentional switching and sustained attention. Two variants of endogenous attention switching were explored: the switches either had in-the-moment decision making or a pre-defined attentional switch time. A least-squares, EEG-based, attention decoding algorithm achieved 64.1% accuracy with a 5-second correlation window and illustrated smooth transitions in the attended talker prediction through switches in sustained attention at approximately half of the analysis window size (2.2 seconds). The expended listening effort, as measured by simultaneous electroencephalography (EEG) and pupillometry, was also a strong indicator of switching. Specifically, centrotemporal alpha power [F(2, 18) = 7.473, P = 0.00434] and mean pupil diameter [F(2, 18) = 9.159, P = 0.0018] were significantly different for trials that contained a switch in comparison to sustained trials. We also found that relative attended and ignored talker locations modulate the EEG alpha topographic response. This alpha lateralization was found to be impacted by the interaction between experimental condition and whether the measure was computed before or after the switch [F(2,18) = 3.227, P = 0.0634]. These results suggest that expended listening effort is a promising feature that should be pursued in a decoding context, in addition to speech and location-based features.

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GSK2556286 is a novel antitubercular drug candidate effective in vivo with the potential to shorten tuberculosis treatment As a result of a high-throughput compound screening campaign of Mycobacterium tuberculosis infected macrophages, a new preclinical drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (IC50 = 0.07 {micro}M), is active against extracellular bacteria in cholesterol-containing culture media and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug or extensively-resistant (M/XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens.

One Sentence SummaryGSK2556286 is a novel preclinical drug candidate for the treatment of tuberculosis with a new mode of action potentially able to contribute to the shortening of TB chemotherapy.

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A potential prokaryotic and microsporidian pathobiome that may cause shrimp white feces syndrome (WFS) White feces syndrome (WFS) in shrimp cultivation ponds is characterized by the occurrence of shrimp with abnormal, white intestines (midguts) combined with large floating mats of white, shrimp fecal strings. The etiology for WFS is complex, similar to diarrhea in humans. EHP-WFS is a type of WFS characterized by massive quantities of spores from the microsporidian parasite Enterocytozoon hepatopenaei (EHP) together with mixed, unidentified bacteria in the shrimp hepatopancreas, midgut and fecal strings. However, WFS does not always develop in shrimp with severe EHP infections in controlled laboratory challenges. Further, in EHP-WFS outbreak ponds, some shrimp show white midguts (WG) while others in the same pond show grossly normal midguts (NG). We hypothesized that comparison of the microbial flora between WG and NG from the same EHP-WFS pond would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this hypothesis, we selected a pond exhibiting a severe EHP-WFS outbreak in cultivated Penaeus vannamei and used a combination of microscopic and microbial profiling analyses to compare WG and NG samples. By histology, EHP plasmodia and spores were confirmed in the hepatopancreas (HP) and midgut of WG and NG shrimp, but pathological severity and spore quantity was higher in the WG shrimp. In addition, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium quantity in the HP of WG shrimp was significantly higher (P = 1.08e-5) than in NG shrimp (4,506 vs. 3 copies /100 ng DNA, respectively). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as a potential eukaryote-prokaryote pathobiome that causes EHP-WFS in P. vannamei.

HighlightsO_LIWhite feces syndrome (WFS) shrimp often harbor the microsporidian Enterocytozoon hepatopenaei (EHP)
C_LIO_LIThe hepatopancreas (HP) and midgut of EHP-WFS shrimp had more EHP copies and spores than EHP-non.-WFS shrimp
C_LIO_LIVibrio spp., Propionigenium sp. and EHP dominated in HP microbiomes of EHP-WFS shrimp
C_LIO_LIPropionigenium copy numbers were uniquely high in the HP of EHP-WFS shrimp
C_LIO_LIEHP-WFS shrimp also showed intestinal microbiomes of reduced diversity but more heterogeneity
C_LI

sentences: Reconstruction of genome-scale metabolic model for Hansenula polymorpha using RAVEN toolbox Genome scale metabolic models (GEMs) provide a useful framework for modeling the metabolism of microorganisms. While the applications of GEMs are wide and far reaching, the reconstruction and continuous curation of such models can be perceived as a tedious and time consuming task. Using the RAVEN toolbox, this protocol practically demonstrates how researchers can create their own GEMs using a homology based approach. To provide a complete example, we reconstruct a draft GEM for the industrially relevant yeast Hansenula polymorpha.

sentences: Interleukin-10 suppression enhances T-cell antitumor immunity and responses to checkpoint blockade in chronic lymphocytic leukemia T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1. However, most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated analogue of the Sp1 inhibitor mithramycin (MTMox32E) to suppress CLL IL-10. MTMox32E treatment inhibited mouse and human CLL IL-10 production and maintained T-cell effector function in vitro. In the E-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden and increased CD8+ T-cell proliferation, effector and memory cell prevalence, and interferon-{gamma} production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared to anti-PD-L1 alone. Combination therapy also produced more interferon-{gamma}+, cytotoxic effector KLRG1+, and memory CD8+ T-cells, and fewer exhausted T-cells. Since current therapies for CLL do not target IL-10, this provides a novel strategy to improve immunotherapies.

sentences: ATGs ubiquitination is required for circumsporozoite protein to subvert host innate immunity against malaria liver stage Although exoerythrocytic forms (EEFs) of liver stage malaria parasite in parasitophorous vacuole (PV) encountered with robust host innate immunity, EEFs can still survive and successfully complete infection of hepatocytes, and the underlying mechanism is largely unknown. Here, we showed that sporozoite circumsporozoite protein (CSP) translocated from the parasitophorous vacuole into the hepatocyte cytoplasm significantly inhibited the killing of exo-erythrocytic forms (EEFs) by interferon-gamma (IFN-{gamma}). Attenuation of IFN-{gamma}-mediated killing of EEFs by CSP was dependent on its ability to reduce the levels of autophagy-related genes (ATGs) in hepatocytes. The ATGs downregulation occurred through its enhanced ubiquitination mediated by E3 ligase NEDD4, an enzyme that was upregulated by CSP when it translocated from the cytoplasm into the nucleus of hepatocytes via its nuclear localization signal (NLS) domain. Thus, we have revealed an unrecognized role of CSP in subverting host innate immunity and shed new light for a prophylaxis strategy against liver-stage infection.

sentences: Distinct brainstem to spinal cord noradrenergic pathways differentially regulate spinal neuronal activity Brainstem to spinal cord pathways modulate spinal neuronal activity. We implemented locus coeruleus (LC) targeting strategies by microinjecting CAV-PRS-ChR2 virus in the spinal cord (LC:SC module) or LC (LC:LC module). While activation of both modules inhibited evoked spinal neuronal firing via 1-adrenoceptor-mediated actions, LC:SC opto-activation abolished diffuse noxious inhibitory controls. The LC as a pain generator is likely mechanistically underpinned by maladaptive communication with discrete descending modulatory pathways.

sentences: Co-expression patterns of microglia markers Iba1, TMEM119 and P2RY12 in Alzheimer's disease Microglia have been identified as key players in Alzheimers disease pathogenesis, and other neurodegenerative diseases. Iba1, and more specifically TMEM119 and P2RY12 are gaining ground as presumedly more specific microglia markers, but comprehensive characterization of the expression of these three markers individually as well as combined is currently missing. Here we used a multispectral immunofluorescence dataset, in which over seventy thousand microglia from both aged controls and Alzheimer patients have been analysed for expression of Iba1, TMEM119 and P2RY12 on a single-cell level. For all markers, we studied the overlap and differences in expression patterns and the effect of proximity to {beta}-amyloid plaques. We found no difference in absolute microglia numbers between control and Alzheimer subjects, but the prevalence of specific combinations of markers (phenotypes) differed greatly. In controls, the majority of microglia expressed all three markers. In Alzheimer patients, a significant loss of TMEM119+-phenotypes was observed, independent of the presence of {beta}-amyloid plaques in its proximity. Contrary, phenotypes showing loss of P2RY12, but consistent Iba1 expression were increasingly prevalent around {beta}-amyloid plaques. No morphological features were conclusively associated with loss or gain of any of the markers or any of the identified phenotypes. All in all, none of the three markers were expressed by all microglia, nor can be wholly regarded as a pan- or homeostatic marker, and preferential phenotypes were observed depending on the surrounding pathological or homeostatic environment. This work could help select and interpret microglia markers in previous and future studies.

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Modeling of individual neurophysiological brain connectivity IntroductionComputational models are often used to assess how functional connectivity (FC) patterns emerge from neuronal population dynamics and anatomical connections in the brain. However, group averaged data is often used in this context and it remains unclear whether individual predictions of FC patterns using this approach can be made. Here, we assess the value of using individualized structural data for simulation of individual whole-brain FC.

MethodsThe Jansen and Rit neural mass model was employed, where masses were coupled using individual structural connectivity (SC) obtained from diffusion weighted imaging. Simulated FC was correlated to individual magnetoencephalography-derived empirical FC. FC was estimated using both phase-based (phase lag index (PLI), phase locking value (PLV)) and amplitude-based (amplitude envelope correlation (AEC)) metrics to analyze the goodness-of-fit of different metrics for individual predictions. Prediction of individual FC was compared against the prediction of group averaged FC. We further tested whether SC of a different participant could equally well predict a participants FC pattern.

ResultsThe AEC provided a significantly better match between individually simulated and empirical FC than phase-based metrics. Simulations with individual SC provided higher correlations between simulated and empirical FC compared to using the group-averaged SC. However, using SC from other participants resulted in similar correlations between simulated and empirical FC compared to using participants own SC.

DiscussionThis work underlines the added value of FC simulations based on individual instead of group-averaged SC, and could aid in a better understanding of mechanisms underlying individual functional network trajectories in neurological disease.

Impact statementIn this work, we investigated how well individual empirical functional connectivity can be simulated using the individuals structural connectivity matrix combined with neural mass modeling. Our research highlights the potential added value of using individual simulations of functional connectivity, and could aid in a better understanding of mechanisms underlying individual functional network trajectories in neurological disease. Moreover, individualized prediction of disease trajectories could enhance patient care and may provide better treatment options.

sentences: Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis.

sentences: A preliminary description of the ecological characteristics of wild waterbird Japanese encephalitis virus hosts in high risk landscapes in India Wild reservoirs of Japanese encephalitis virus are under-studied globally, which presents critical knowledge gaps for JEV infection ecology despite decades of received wisdom regarding this high-impact mosquito-borne virus. As a result, ardeid birds, generally understood to be the primary reservoirs for JEV, as well as other waterbirds occupying landscapes at high risk for spillover to humans, are frequently ignored by current surveillance mechanisms and infrastructure. This is particularly true in India, which experiences a high annual burden of human outbreaks. Incorporating wild reservoirs into surveillance of human and livestock populations is therefore essential but will first require a data-driven approach to target individual host species. The current study sought to define a preliminary ecological profile of JEV hosts based on 1) species ecological traits, and 2) species presence and abundance adjusted for the biotic constraints of sympatry. Optimal host species tended to be generalists and demonstrate regionally-increasing populations. While ardeid bird species richness, abundance, and relative abundance did demonstrate the strongest and most consistent associations with the distribution of human JEV outbreaks, this study also identified several individual species among two other bird families in these landscapes, the Anatidae and the Rallidae, which also exhibited an optimal host profile and were strongly associated with the distribution of outbreaks. The findings from this work provide the first data-driven evidence base to inform wildlife sampling for the monitoring of JEV circulation in outbreak hotspots in India and thus identify good preliminary targets for the development of One Health wildlife JEV surveillance.

sentences: Empirical estimation of marine phytoplankton assemblages in coastal and offshore areas using an in situ multi-wavelength excitation fluorometer Phytoplankton assemblages are important for understanding the quality of primary production in marine ecosystems. Here, we describe development of a methodology for monitoring marine phytoplankton assemblages using an in situ multi-wavelength excitation fluorometer (MEX) and its application for seasonal observations in coastal and offshore areas around Japan. The MEX recorded the fluorescence excited with nine light-emitting diodes, temperature, and sensor depth. We prepared reference datasets comprising temperature, MEX fluorescence, and plant-pigment-based chemotaxonomy phytoplankton assemblages. Target MEX fluorescence was decomposed by reference MEX fluorescence using a linear inverse model for calculating coefficients after the reference data were limited by temperature, followed by reconstruction of plant-pigment-based chemotaxonomy of the target MEX fluorescence using the coefficients and the chemotaxonomy assemblages of the reference data. Sensitivity analysis indicated poor estimation of the proportion and/or chlorophyll a-based abundance of chlorophytes, haptophytes, prasinophytes, and prochlorophytes; however, limiting the estimations to five chemotaxonomic groups [diatoms, dinoflagellates, cryptophytes, cyanobacteria (cyanophytes and prochlorophytes), and other eukaryotes (chlorophytes, haptophytes, and prasinophytes)] resulted in positive correlations of both the proportion and abundances, suggesting that the five taxonomic abundances were well-estimated using the MEX. Additionally, MEX observations denoted spatial and seasonal variations of phytoplankton assemblages, with high contributions from other eukaryotes in every area and season, cyanobacteria highly during the summer in surface Kuroshio and Japan Sea waters, and diatoms in the Oyashio and Oyashio-Kuroshio transition areas and the Okhotsk Sea. Furthermore, ratios of water-column-integrated chlorophyll-based abundances to those on the surface at the chemotaxonomy group level were differed among the areas and groups. These findings suggested that phytoplankton-assemblage monitoring in the vertical direction is essential for evaluation of their current biomass, and that the MEX promotes the acquisition of these observations.

sentences: Statistical properties of the MetaCore network of protein-protein interactions The MetaCore commercial database describes interactions of proteins and other chemical molecules and clusters in the form of directed network between these elements, viewed as nodes. The number of nodes goes beyond 40 thousands with almost 300 thousands links between them. The links have essentially bi-functional nature describing either activation or inhibition actions between proteins. We present here the analysis of statistical properties of this complex network applying the methods of the Google matrix, PageRank and CheiRank algorithms broadly used in the frame of the World Wide Web, Wikipedia, the world trade and other directed networks. We specifically describe the Ising PageRank approach which allows to treat the bi-functional type of protein-protein interactions. We also show that the developed reduced Google matrix algorithm allows to obtain an effective network of interactions inside a specific group of selected proteins. This method takes into account not only direct protein-protein interactions but also recover their indirect nontrivial couplings appearing due to summation over all the pathways passing via the global bi-functional network. The developed analysis allows to espablish an average action of each protein being more oriented to activation or inhibition. We argue that the described Google matrix analysis represents an efficient tool for investigation of influence of specific groups of proteins related to specific diseases.

sentences: The elephant retina examined across a range of ages The effect of aging in the human retina has been well documented, as have the signs of age-related retinal disease. Comparative studies in animals allow us to further investigate how the retina ages in different species. The African bush elephant (Loxodonta africana) has a retina comparable to other mammalian species, but with some reported distinctive differences in retinal ganglion cell (RGC) distribution and type. This is a first survey of the retina of Loxodonta africana from individuals aged 2 months to 32 years old. Gross examination, H&E staining and cell counts were used to compare calves (0-5 years), juveniles (6-10 years), sub-adults (11-20 years) and adults (>20 years). Dorsal-ventral eye diameter was shown to be significantly greater with age, whilst no significant changes in photoreceptor number were found. Changes in retinal thickness differed from past findings in elephants and humans, with thickness decreasing, then increasing in adults. Various morphological differences were evident in the samples including the presence of nuclei in the outer plexiform layer (OPL), degeneration of the inner plexiform layer (IPL), interruptions in nuclei columns of the outer nuclear layer (ONL), and larger unidentified cells within the inner nuclear layer (INL). These are initial observations provide some baseline information for a species where this range of samples has not been described previously.

sentences: Bee core venom genes predominantly originated before aculeate stingers evolved Venoms have convergently evolved in all major animal lineages and are ideal candidates to unravel the genomic processes underlying convergent trait evolution. However, few animal groups have been studied in detail, and large-scale comparative genomic analyses to address toxin gene evolution are rare. The hyper-diverse hymenopterans are the most speciose group of venomous animals, but the origin of their toxin genes has been largely overlooked. We combined proteo-transcriptomics with comparative genomics compiling an up-to-date list of core bee venom proteins to investigate the origin of 11 venom genes in 30 hymenopteran genomes including two newly sequenced genomes of stingless bees. We found a more distinct pattern in which toxin genes originated predominantly by single gene co-option, a prevalent mechanism in parasitoid wasps. These are always accompanied by parallel expansion events in other bee and hymenopteran sister gene groups. The short toxic peptides melittin and apamin appear to be unique to bees, a result supported by a novel machine learning approach. Based on the syntenic pattern we propose here Anthophilin1 as a bee-unique gene family that includes apamin and MCDP. It appears that bees co-adapted their venom genes which predominantly already existed before the aculeate ovipositor evolved to a stinger which exclusively injects venom. Our results provide insight into the large-scale evolution of bee venom compounds, and we present here the first study revealing general processes of venom evolution at a comparative genomic level for this mega-diverse Hymenoptera.

sentences: Low-cost and open-source super-resolution fluorescence microscope with autofocus for teaching and research Super-resolution fluorescence microscopy revolutionized optical microscopy by breaking the theoretical resolution limit and thus providing an unprecedented view of biological structure in exquisite detail. Additionally, the relative ease of use and implementation for some super-resolution microscopes provided hope that this technology would be widely available and accessible. Despite initial excitement, super-resolution microscopy is still largely limited to facilities that can afford expensive commercial instruments or with personnel experienced in optical engineering that can construct an instrument. Recently, the open scientific hardware movement has attempted to make scientific instrumentation more widely available and accessible in a similar way to the open software movement. The widespread availability and affordability of prototyping devices, such as three-dimensional printers and interfaces (e.g., Arduino) have all contributed to unlocking the potential of the open hardware movement, turning expensive "black box" hardware into open-source, affordable, and robust instrumentation. Many others have recognized the potential of describing low-cost implementations of super-resolution microscope designs. However, we have found these microscope designs lacking in the documentation and details required to meet the requirements of open-source hardware. In this paper, we attempt to provide the documentation, details, and instructions necessary for the construction of an open-source and low-cost super-resolution microscope at a targeted cost of <$15,000.

sentences: Intranasal Nanoemulsion Adjuvanted S-2P Vaccine Demonstrates Protection in Hamsters and Induces Systemic, Cell-Mediated and Mucosal Immunity in Mice With the rapid progress made in the development of vaccines to fight the SARS-CoV-2 pandemic, almost >90% of vaccine candidates under development and a 100% of the licensed vaccines are delivered intramuscularly (IM). While these vaccines are highly efficacious against COVID-19 disease, their efficacy against SARS-CoV-2 infection of upper respiratory tract and transmission is at best temporary. Development of safe and efficacious vaccines that are able to induce robust mucosal and systemic immune responses are needed to control new variants. In this study, we have used our nanoemulsion adjuvant (NE01) to intranasally (IN) deliver stabilized spike protein (S-2P) to induce immunogenicity in mouse and hamster models. Data presented demonstrate the induction of robust immunity in mice resulting in 100% seroconversion and protection against SARS-CoV-2 in a hamster challenge model. There was a significant induction of mucosal immune responses as demonstrated by IgA- and IgG-producing memory B cells in the lungs of animals that received intranasal immunizations compared to an alum adjuvanted intramuscular vaccine. The efficacy of the S-2P/NE01 vaccine was also demonstrated in an intranasal hamster challenge model with SARS-CoV-2 and conferred significant protection against weight loss, lung pathology, and viral clearance from both upper and lower respiratory tract. Our findings demonstrate that intranasal NE01-adjuvanted vaccine promotes protective immunity against SARS-CoV-2 infection and disease through activation of three arms of immune system: humoral, cellular, and mucosal, suggesting that an intranasal SARS-CoV-2 vaccine may play a role in addressing a unique public health problem and unmet medical need.

sentences: A Pilot Study Using Two Novel fMRI Tasks: Understanding Theory of Mind and Emotion Recognition Among Children With ASD Children with autism spectrum disorder (ASD) struggle with social interactions due to deficits in theory of mind (ToM). In this study, we collected behavioral and neuroimaging data from 9 children with ASD and 19 neurotypical children between the age of 7 and 14 years old, particularly in the area of emotion recognition to better understand those skills needed for meaningful social interaction. The results suggest impaired abilities in multiple ToM metrics and brain deficits associated with ToM-related emotion recognition and processing among children with ASD. Findings from this study are expected to establish connections between behavior and brain activities surrounding ToM in ASD, which may assist the development of neuroanatomical diagnostic criteria and provide a way to measure intervention outcomes.

sentences: Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 12 homeobox genes in 19 distinct neuron classes that are mostly unrelated by location, function and lineage history. 10 of these 19 neuron classes had no homeobox gene function ascribed to them before, while in the other 9 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analysis, 112 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancient feature of animal nervous systems.

sentences: Transcriptome-wide association study in UK Biobank Europeans identifies associations with blood cell traits. Previous genome-wide association studies (GWAS) of hematological traits have identified over 10,000 distinct trait-specific risk loci, but the underlying causal mechanisms at these loci remain incompletely characterized. We performed a transcriptome-wide association study (TWAS) of 29 hematological traits in 399,835 UK Biobank (UKB) participants of European ancestry using gene expression prediction models trained from whole blood RNA-seq data in 922 individuals. We discovered 557 TWAS signals associated with hematological traits distinct from previously discovered GWAS variants, including 10 completely novel gene-trait pairs corresponding to 9 unique genes. Among the 557 associations, 301 were available for replication in a cohort of 141,286 participants of European ancestry from the Million Veteran Program (MVP). Of these 301 associations, 199 replicated at a nominal threshold ( = 0.05) and 108 replicated at a strict Bonferroni adjusted threshold ( = 0.05/301). Using our TWAS results, we systematically assigned 4,261 out of 16,900 previously identified hematological trait GWAS variants to putative target genes. Compared to coloc, our TWAS results show reduced specificity and increased sensitivity to assign variants to target genes.

sentences: Global prevalence of potentially pathogenic short-tandem repeats in an epilepsy cohort This study aims to decipher the role of short tandem repeats (STRs) in epilepsy patients. Whole genome short-read sequencing data of 734 epileptic patients was used to look for known STR expansions associated with increased risk of neurodevelopmental diseases or epilepsy using three different software. Results show one hit of particular interest on ARX gene associated with Early Infantile Encephalopathic Epilepsy that could be causal for one patient with developmental and epileptic encephalopathy. However, we show that the different software do not agree on most of the calls above the threshold and that experimental validation is still needed for diagnostic, although these algorithms could prove useful for pre-selection of samples to be validated.

sentences: Identification of proximal SUMO-dependent interactors using SUMO-ID The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We developed an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allowed us to identify novel interactors of SUMOylated SALL1, a less characterized SUMO substrate. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.

sentences: Paving the way towards understanding the inflammatory pathways triggered by giant viruses in mammalian cells: effect of mimivirus-cell interactions on IκBα degradation Even after two decades since the identification of the first giant virus, the Acanthamoeba polyphaga mimivirus (APMV), it still elude scientists. Their gigantic size and genome are unique in the whole virosphere, and many aspects of their biology are still unknown, including their possible hosts. They are cultivated in laboratories using Acanthamoeba cells as hosts, but little is known about the infectivity of these giant viruses in vertebrate cells. However, there is evidence of the possible involvement of APMV in pneumonia and activation of inflammatory pathways. Among the hundreds of prospected giant viruses members is Tupanvirus, isolated in Brazil. Its particles have a characteristically large size varying between 1.2 to 2 m and are covered by fibrils. In the present work, we aim to study the consequences of the incubation of APMV and Tupanvirus with mammalian cells. These cells express Toll-like receptors (TLR) that are capable of recognizing lipopolysaccharides, favoring the internalization of the antigen and activation of the inflammatory system. We used a lineage of human lung adenocarcinoma cells (A549) to evaluate possible effects of TLR activation by the giant viruses and if we could detect the probable cause of the said giant-virus dependent pneumonia. Our results show that APMV and Tupanvirus (TPV) activate cellular receptors related to the Toll-like 4 type-induced inflammatory response and that the A549 cells are capable of internalizing the latter virus. Therefore, this study brings new insights into the possible interactions established between mimiviruses (here represented by APMV and Tupanvirus) and members of the innate cellular immune response.

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Mechanical activation of mitochondrial energy metabolism during cell differentiation In multicellular lives, differentiation of many types of stem and progenitor cells is often accompanied by a metabolic transition from glycolysis to mitochondrial oxidative phosphorylation. The mechanisms driving this metabolic transition in vivo are largely unknown. Here, we show that, during differentiation of the Drosophila female germline cyst, the surrounding somatic cells compress the cyst and increase the tension of cyst cells membranes. Transmembrane channel-like, an evolutionarily conserved ion channel involved in mechanosensation, maintains cytosolic Ca2+ levels in compressed differentiating cysts. Cytosolic Ca2+ induces transcriptional activation of oxidative phosphorylation through a CaMKI-Fray-JNK signaling relay. Our findings demonstrate a molecular link between cell mechanics and mitochondrial energy metabolism, with implications in other developmentally orchestrated metabolic transitions in mammals.

One-Sentence SummaryMechanical forces from the surrounding tissue activate mitochondrial energy metabolism in differentiating cells in vivo.

sentences: Biophysical fitness landscape of the SARS-CoV-2 Delta variant receptor binding domain Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased number of deaths. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single mutations as well as the double mutation on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than receptor binding, is the main biophysical parameter determining the fitness landscape of the Delta variant RBD and is determined by the L452R mutation.

sentences: Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region The N-terminus of S. aureus staphylocoagulase (SC) triggers activation of host prothrombin (ProT), and the SC{middle dot}ProT* complex cleaves host fibrinogen (Fbg) to form fibrin (Fbn) deposits, a hallmark of SC-positive endocarditis. The C-terminal domain of the prototypical Newman D2 Tager 104 SC contains 1 pseudo-repeat (PR) and 7 repeats (R1[->]R7) that bind Fbg/Fbn Fragment D (Frag D). This work defines affinities and stoichiometries of Frag D binding to single- and multi-repeat C-terminal constructs, using fluorescence equilibrium binding, NMR titration, Ala scanning, and native PAGE. Constructs containing PR and each single repeat bound Frag D with KD ~50 - 130 nM and a 1:1 stoichiometry, indicating a conserved binding site shared between PR and each repeat. NMR titration of PR-R7 with Frag D revealed that residues 22-49, bridging PR and R7, constituted the minimal peptide (MP) required for binding, corroborated by Ala scanning, and binding of labeled MP to Frag D. MP alignment with the PR-repeat and inter-repeat junctions identified conserved residues critical for binding. Labeled PR-(R1[->]R7) bound Frag D with KD ~7 - 32 nM and stoichiometry of 1:5; and PR-R1R2R3, PR-R1R6R7, PR-R3R4R7, and PR-R3R6R7 competed with PR-(R1[->]R7) for Frag D binding, with a 1:3 stoichiometry and KD ~7 - 42 nM. These findings are consistent with binding at the PR-R junctions with modest inter-repeat sequence variability. Circular dichroism of PR-R7 and PR-(R1[->]R7) suggested a largely disordered structure and conformational flexibility, allowing binding of multiple fibrin(ogen) molecules. This property facilitates pathogen localization on host fibrin networks.

sentences: Protist and Fungal Metaxin-Like Proteins: Relationship to Vertebrate Metaxins that Function in Protein Import into Mitochondria Metaxin-like proteins are shown to be encoded in the genomes of a wide range of protists and fungi. The metaxin proteins were originally described in humans and mice, and were experimentally demonstrated to have a role in the import of nascent proteins into mitochondria. In this study, metaxin-like proteins of protists and fungi predicted from genome sequences were identified by criteria including their sequence homology with vertebrate metaxin proteins and the existence of distinctive metaxin protein domains. Protists of diverse taxa, including amoebae, protozoa, phytoplankton, downy mildews, water molds, and algae, were found to possess genes for metaxin-like proteins. With fungi, the important taxonomic divisions (phyla) of Ascomycota, Basidiomycota, Chytridiomycota, Mucoromycota, and Zoopagomycota had species with metaxin-like protein genes. The presence of distinctive GST_N_Metaxin, GST_C_Metaxin, and Tom37 domains in the predicted proteins indicates that the protist and fungal proteins are related to the vertebrate metaxins. However, the metaxin-like proteins are not direct homologs of vertebrate metaxins 1, 2, or 3, but have similarity to each of the three. The alignment of the metaxin-like proteins of a variety of protists with vertebrate metaxins 1 and 2 showed about 26% and 19% amino acid identities, respectively, while for fungal metaxin-like proteins, the identities were about 29% and 23%. The different percentages with the two vertebrate metaxins indicates that the metaxin-like proteins are both metaxin 1-like and, to a lesser degree, metaxin 2-like. The secondary structures of protist and fungal metaxin-like proteins both consist of nine -helical segments, the same as for the vertebrate metaxins, with a negligible contribution from {beta}-strand. Phylogenetic analysis demonstrated that the protist and fungal metaxin-like proteins and the vertebrate metaxins form distinct and separate groups, but that the groups are derived from a common ancestral protein sequence.

sentences: Parametric control of flexible timing through low-dimensional neural manifolds Biological brains possess an unparalleled ability to generalize adaptive behavioral responses from only a few examples. How neural processes enable this capacity to extrapolate is a fundamental open question. A prominent but underexplored hypothesis suggests that generalization is facilitated by a low-dimensional organization of collective neural activity. Here we tested this hypothesis in the framework of flexible timing tasks where dynamics play a key role. Examining trained recurrent neural networks we found that confining the dynamics to a low-dimensional subspace allowed tonic inputs to parametrically control the overall input-output transform and enabled smooth extrapolation to inputs well beyond the training range. Reverse-engineering and theoretical analyses demonstrated that this parametric control of extrapolation relies on a mechanism where tonic inputs modulate the dynamics along non-linear manifolds in activity space while preserving their geometry. Comparisons with neural data from behaving monkeys confirmed the geometric and dynamical signatures of this mechanism.

sentences: Development of a novel hybrid alphavirus-SARS-CoV-2 particle for rapid in vitro screening and quantification of neutralization antibodies, viral variants, and antiviral drugs Timely development of vaccines and antiviral drugs is critical to control the COVID-19 pandemic 1-6. Current methods for quantifying vaccine-induced neutralizing antibodies involve the use of pseudoviruses, such as the SARS-CoV-2 spike protein (S) pseudotyped lentivirus7-14. However, these pseudoviruses contain structural proteins foreign to SARS-CoV-2, and require days to infect and express reporter genes15. Here we describe the development of a new hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) particle for rapid and accurate quantification of neutralization antibodies and viral variants. Ha-CoV-2 is a non-replicating SARS-CoV-2 virus-like particle, composed of SARS-CoV-2 structural proteins (S, M, N, and E) and a RNA genome derived from a fast expressing alphavirus vector 16. We demonstrated that Ha-CoV-2 can rapidly and robustly express reporter genes in target cells within 3-6 hours. We further validated Ha-CoV-2 for rapid quantification of neutralization antibodies, viral variants, and antiviral drugs. In addition, as a proof-of-concept, we assembled and compared the relative infectivity of a panel of 10 Ha-CoV-2 variant isolates (D614G, P.1, B.1.1.207, B.1.351, B.1.1.7, B.1.429, B.1.258, B.1.494, B.1.2, B.1.1298), and demonstrated that these variants in general are 2-10 fold more infectious. Furthermore, we quantified the anti-serum from an infected and vaccinated individual; the one dose vaccination with Moderna mRNA-1273 has greatly increased the anti-serum titer for approximately 6 fold. The post-vaccination serum has also demonstrated various neutralizing activities against all 9 variants tested. These results demonstrated that Ha-CoV-2 can be used as a robust platform for rapid quantification of neutralizing antibodies against SARS-CoV-2 and its variants.

sentences: Connecting neuromodulation, brain development, and life history timing associated with ecological speciation in Rhagoletis pomonella Host shifts are considered a key generator of insect biodiversity. In insects, adaption to new host plants often requires synchronization of growth and development during larval and pupal stages with the new host as well as changes in adult preference for that host after metamorphosis. Neurochemicals such as biogenic amines play key roles in both development and behavior, and therefore provide a potential source for such synchronization. Here, we correlated life history timing, brain development, and corresponding levels of 14 neurochemicals in Rhagoletis pomonella (Diptera: Tephritidae), a species undergoing ecological speciation through an ongoing host shift from hawthorn to apple fruit. These races are known to exhibit differences in both diapause timing as well as adult neurophysiological response with respect to their hosts. In turn, we found that the apple host race exhibited adult brain differentiation during metamorphosis three weeks earlier than the hawthorn host race, which correlated with significantly lower titers of several neurochemicals. These differences in neurotransmitter titres between the races are carried through further developmental stages. In some cases, particularly for biogenic amines, the differences persisted through the eclosion and sexual maturation in the adult fly, the stage at which host preference is exhibited. We thus propose that changes in neuromodulation during specific stages of adult brain development could link differences in life history timing and host preference in these races, providing a new hypothesis regarding the genesis of our planets most specious multicellular taxon.

sentences: Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 g/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

sentences: Cx43 carboxyl terminal domain determines AQP4 and Cx30 endfoot organization and blood brain barrier permeability The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood-brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if blood-brain-barrier integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.

sentences: Impaired developmental microglial pruning of excitatory synapses on CRH-expressing hypothalamic neurons exacerbates stress responses throughout life The developmental origins of stress-related mental illnesses are well-established, and early-life stress/adversity (ELA) is an important risk factor. However, it is unclear how ELA impacts the maturation of salient brain circuits, provoking enduring vulnerability to stress and stress-related disorders. Here we find that ELA increases the number and function of excitatory synapses onto stress-sensitive hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons, and implicate disrupted synapse pruning by microglia as a key mechanism. Microglial process dynamics on live imaging, and engulfment of synaptic elements by microglia, were both attenuated in ELA mice, associated with deficient signaling of the microglial phagocytic receptor Mer. Accordingly, selective chemogenetic activation of ELA microglia increased microglial process dynamics and reduced excitatory synapse density to control levels. Selective early-life microglial activation also mitigated the adrenal hypertrophy and prolonged stress responses in adult ELA mice, establishing microglial actions during development as powerful contributors to experience-dependent sculpting of stress-related brain circuits.

sentences: Mechanical communication-induced cell directional migration and branching connections mediated by calcium channels, integrin β1 and N-cadherin Cell-cell mechanical communications at large spatial scale (above hundreds of micrometers) have been increasingly recognized in recent decade, which shows importance in tissue-level assembly and morphodynamics. The involved mechanosensing mechanism and resulted physiological functions are still to be fully understood. Recent work showed that traction force sensation in the matrix induces cell communications for self-assembly. Here, based on the experimental model of cell directional migration on Matrigel hydrogel containing 0.5 mg/ml type I collagen, we studied the mechano-responsive pathways for cell distant communications. Airway smooth muscle (ASM) cells assembled network structure on the hydrogel, whereas stayed isolated individually when cultured on glass without force transmission. Cell directional migration, or network assembly was significantly attenuated by inhibited actomyosin activity, or inhibition of inositol 1,4,5-trisphosphate receptor (IP3R) calcium channel or SERCA pump on endoplasmic reticulum (ER) membrane, or L-type calcium channel on the plasma membrane. Inhibition of integrin {beta}1 with siRNA knockdown reduced cell directional migration and branching assembly, whereas inhibition of cell junctional N-cadherin with siRNA had little effect on distant attractions but blocked branching assembly. Our work demonstrated that the ER calcium channels and integrin are mechanosensing signals for cell mechanical communications regulated by actomyosin activity, while N-cadherin is responsible for traction force-induced cell stable connections in the assembly.

sentences: Predicting the re-distribution of antibiotic molecules caused byinter-species interactions in microbial communities Microbes associate in nature forming complex communities, but they are often studied in purified form. Here I show that differences in antibiotic sensitivity between co-existing bacterial species can modify the diffusion of antibiotic molecules, and alter the drug efficacy measured in conventional in vitro assays. I developed a model, validated experimentally using an artificial two-species community, showing antibiotic molecules re-distribute evenly across all species if they have similar drug sensitivity. The result is lower drug efficacy with respect to conventional in vitro assays. However, when species have different sensitivities, the re-distribution of antibiotic is uneven and most sensitive species accumulate more drug. The result is increased drug efficacy against these species. Despite this discrepancy with conventional laboratory assays, my data suggests the changes in drug efficacy are predictable and provides insight into how communities can affect the diffusion of other molecules in the environment.

sentences: CONSULT: Accurate contamination removal using locality-sensitive hashing A fundamental question appears in many bioinformatics applications: Does a sequencing read belong to a large dataset of genomes from some broad taxonomic group, even when the closest match in the set is evolutionarily divergent from the query? For example, low-coverage genome sequencing (skimming) projects either assemble the organelle genome or compute genomic distances directly from unassembled reads. Using unassembled reads needs contamination detection because samples often include reads from unintended groups of species. Similarly, assembling the organelle genome needs distinguishing organelle and nuclear reads. While k-mer-based methods have shown promise in read-matching, prior studies have shown that existing methods are insufficiently sensitive for contamination detection. Here, we introduce a new read-matching tool called CONSULT that tests whether k-mers from a query fall within a user-specified distance of the reference dataset using locality-sensitive hashing. Taking advantage of large memory machines available nowadays, CONSULT libraries accommodate tens of thousands of microbial species. Our results show that CONSULT has higher true-positive and lower false-positive rates of contamination detection than leading methods such as Kraken-II and improves distance calculation from genome skims. We also demonstrate that CONSULT can distinguish organelle reads from nuclear reads, leading to dramatic improvements in skims-based mitochondrial assemblies.

sentences: Variation in supplemental carbon dioxide requirements defines lineage-specific antibiotic resistance acquisition in Neisseria gonorrhoeae The evolution of the obligate human pathogen Neisseria gonorrhoeae has been shaped by selective pressures from diverse host niche environments1,2 as well as antibiotics3,4. The varying prevalence of antibiotic resistance across N. gonorrhoeae lineages5 suggests that underlying metabolic differences may influence the likelihood of acquisition of specific resistance mutations6,7. We hypothesized that the requirement for supplemental CO2, present in approximately half of isolates8, reflects one such example of metabolic variation. Here, using a genome-wide association study and experimental investigations, we show that CO2-dependence is attributable to a single substitution in a {beta}-carbonic anhydrase, canB. CanB19E is necessary and sufficient for growth in the absence of CO2, and the hypomorphic CanB19G variant confers CO2-dependence. Furthermore, ciprofloxacin resistance is correlated with CanB19G in clinical isolates, and the presence of CanB19G increases the likelihood of acquisition of ciprofloxacin resistance. Together, our results suggest that metabolic variation has impacted the acquisition of fluoroquinolone resistance.

sentences: selscan 2.0: scanning for sweeps in unphased data Haplotype-based scans to identify recent and ongoing positive selection have become commonplace in evolutionary genomics studies of numerous species across the tree of life. However, the most widely adopted approaches require phased haplotypes to compute the key statistics. Here we release a major update to the selscan software that re-defines popular haplotype-based statistics for use with unphased "multi-locus genotype" data. We provide unphased implementations of iHS, nSL, XP-EHH, and XP-nSL and evaluate their performance across a range of important parameters in a generic demographic history. Source code and executables are available at https://www.github.com/szpiech/selscan.

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Simultaneous quantification of ankle, muscle, and tendon impedance in humans ObjectiveRegulating the impedance of our joints is essential for the effective control of posture and movement. The impedance of a joint is governed mainly by the mechanical properties of the muscle-tendon units spanning it. Many studies have quantified the net impedance of joints but not the specific contributions from the muscles and tendons. The inability to quantify both muscle and tendon impedance limits the ability to determine the causes underlying altered movement control associated with aging, neuromuscular injury, and other conditions that have different effects on muscle and tendon properties. Therefore, we developed a technique to quantify joint, muscle, and tendon impedance simultaneously and evaluated this technique at the human ankle.

MethodsWe used a single degree of freedom actuator to deliver pseudorandom rotations to the ankle while measuring the corresponding torques. We simultaneously measured the displacement of the medial gastrocnemius muscle-tendon junction with B-mode ultrasound. From these experimental measurements, we were able to estimate ankle, muscle, and tendon impedance using non-parametric system identification.

ResultsWe validated our estimates by comparing them to previously reported muscle and tendon stiffness, the position-dependent component of impedance, to demonstrate that our technique generates reliable estimates of these properties.

ConclusionOur approach can be used to clarify the respective contributions from the muscle and tendon to the net mechanics of a joint.

SignificanceThis is a critical step forward in the ultimate goal of understanding how muscles and tendons govern ankle impedance during posture and movement.

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Predicting continuous ground reaction forces from accelerometers during uphill and downhill running: A recurrent neural network solution Ground reaction forces (GRFs) are important for understanding human movement, but their measurement is generally limited to a laboratory. Previous studies used neural networks to predict GRF waveforms during running from wearable device data, but these predictions are limited to the stance phase of level-ground running. We sought to develop a recurrent neural network capable of predicting continuous normal (perpendicular to surface) GRFs across a range of running speeds and slopes from accelerometer data.

19 subjects ran on a force-measuring treadmill at five slopes (0{degrees}, {+/-}5{degrees}, {+/-}10{degrees}) and three speeds (2.5, 3.33, 4.17 m/s) per slope with sacral- and shoe-mounted accelerometers. We then trained a recurrent neural network to predict normal GRF waveforms frame-by-frame. The predicted versus measured GRF waveforms had an average {+/-} SD RMSE of 0.16 {+/-} 0.04 BW and relative RMSE of 6.4 {+/-} 1.5% across all conditions and subjects.

The recurrent neural network predicted continuous normal GRF waveforms across a range of running speeds and slopes with greater accuracy than neural networks implemented in previous studies. This approach may facilitate predictions of biomechanical variables outside the laboratory in near real-time and improves the accurately of quantifying and monitoring external loads experienced by the body when running.

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ifCNV: a novel isolation-forest-based package to detect copy number variations from NGS datasets Copy number variations (CNVs) are an essential component of genetic variation distributed across large parts of the human genome. CNV detection from next-generation sequencing data and artificial intelligence algorithms has progressed in recent years. However, only a few tools have taken advantage of machine learning algorithms for CNV detection, and none propose using artificial intelligence to automatically detect probable CNV-positive samples. Furthermore, in general, most CNV software that is developed for specific data types has sub-optimal reliability for routine practice. In addition, the most developed approach is to use a reference or normal dataset to compare with the samples of interest, and it is well known that selecting appropriate normal samples represents a challenging task which dramatically influences the precision of results in all CNV-detecting tools. With careful consideration of these issues, we propose here ifCNV, a new software based on isolation forests that creates its own reference, available in R and python with customisable parameters. ifCNV combines artificial intelligence using two isolation forests and a comprehensive scoring method to faithfully detect CNVs among various samples. It was validated using datasets from diverse origins (capture and amplicon, germline and somatic), and it exhibits high sensitivity, specificity and accuracy. ifCNV is a publicly available open-source software that allows the detection of CNVs in many clinical situations.

Key pointsO_LICopy number variation detection
C_LIO_LIMachine learning
C_LIO_LILocalisation scoring
C_LIO_LIBenchmark on various clinical situations and on various datasets
C_LIO_LIEasy-to-use R and Python open-source Package
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sentences: Control of G2 phase duration by CDC25B modulates the switch from direct to indirect neurogenesis in the neocortex During development, cortical neurons are produced in a temporally regulated sequence from apical progenitors, directly, or indirectly through the production of intermediate basal progenitors. The balance between these major progenitors types is determinant for the production of the proper number and types of neurons and it is thus important to decipher the cellular and molecular cues controlling this equilibrium. Here we address the role of a cell cycle regulator, the CDC25B phosphatase, in this process. We show that deleting CDC25B in apical progenitors leads to a transient increase of the production of TBR1+ neurons at the expense of TBR2+ basal progenitors in mouse neocortex. This phenotype is associated with lengthening of the G2 phase of the cell cycle, the total cell cycle length being unaffected. Using in utero electroporation and cortical slice cultures, we demonstrate that the defect in TBR2+ basal progenitor production requires interaction with CDK1 and is due to the G2 phase lengthening in CDC25B mutants. Altogether, this study identifies a new role for CDC25B and the length of the G2 phase in direct versus indirect neurogenesis at early stages of the cortical development.

sentences: Comparison of proton transfer paths to the QA and QB sites of the Rb. sphaeroides photosynthetic reaction centers The photosynthetic bacterial reaction centers from purple non-sulfur bacteria use light energy to drive the transfer of electrons from cytochrome c to ubiquinone. Ubiquinone bound in the QA site cycles between quinone, QA, and anionic semiquinone, QA*-, being reduced once and never binding protons. In the QB site, ubiquinone is reduced twice by QA*-, binds two protons and is released into the membrane as the quinol, QH2. The network of hydrogen bonds formed in a molecular dynamics trajectory was drawn to investigate proton transfer pathways from the cytoplasm to each quinone binding site. QA is isolated with no path for protons to enter from the surface. In contrast, there is a complex and tangled network requiring residues and waters that can bring protons to QB. There are three entries from clusters of surface residues centered around HisH126, GluH224, and HisH68. The network is in good agreement with earlier studies, Mutation of key nodes in the network, such as SerL223, were previously shown to slow proton delivery. Mutational studies had also shown that double mutations of residues such as Asp M17 and AspL210 along multiple paths in the network presented here slow the reaction, while single mutations do not. Likewise, mutation of both HisH126 and HisH128, which are at the entry to two paths reduce the rate of proton uptake.

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Mimicking extracellular matrix-mediated mechano-activation by antibodies to control signaling of the adhesion G protein-coupled receptor GPR126/ADGRG6 The adhesion G protein-coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination or peripheral nerve repair. This renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that translates binding of extracellular matrix (ECM) molecules to its N terminus into a metabotropic intracellular signal. To date, the structural requirements and the character of the forces needed for this ECM-mediated receptor activation are largely unknown.

In this study we provide this information by combining classic second messenger detection with single cell atomic force microscopy. We establish a monoclonal antibody targeting the N terminus to stimulate GPR126 and compare it to the activation through its known ECM ligands collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N terminus can be viewed as an allosteric module that can fine-tune receptor activation in a context-specific manner.

sentences: Growth and grain yield of eight maize hybrids is aligned with water transport, stomatal conductance, and photosynthesis in a semi-arid irrigated system There is increasing interest in understanding how trait networks can be manipulated to improve the performance of crop species. Working towards this goal, we have identified key traits linking the acquisition of water, the transport of water to the sites of evaporation and photosynthesis, stomatal conductance, and growth across eight maize hybrid lines grown under well-watered and water-limiting conditions in Northern Colorado. Under well-watered conditions, well-performing hybrids exhibited high leaf-specific conductance, low operating water potentials, high rates of midday stomatal conductance, high rates of net CO2 assimilation, greater leaf osmotic adjustment, and higher end-of-season growth and grain yield. This trait network was similar under water-limited conditions with the notable exception that linkages between water transport, midday stomatal conductance, and growth were even stronger than under fully-watered conditions. The results of this experiment suggest that similar trait networks might confer improved performance under contrasting climate and soil conditions, and that efforts to improve the performance of crop species could possibly benefit by considering the water transport pathway within leaves, as well as within the whole-xylem, in addition to root-level and leaf-level traits.

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MAP4K4 determines cancer cell phenotype by controlling the plasma membrane-associated proteome The composition of the plasma membrane (PM)-associated proteome of tumor cells determines cell-cell and cell-matrix interactions and the response to environmental cues. Whether the PM-associated proteome impacts the phenotype of Medulloblastoma (MB) tumor cells and how it adapts in response to growth factor cues is poorly understood. Using a spatial proteomics approach, we observed that hepatocyte growth factor (HGF)-induced activation of the receptor tyrosine kinase c-MET in MB cells changes the abundance of transmembrane and membrane-associated proteins. The depletion of MAP4K4, a pro-migratory effector kinase downstream of c-MET, leads to a specific decrease of the adhesion and immunomodulatory receptor CD155 and of components of the fast-endophilin-mediated endocytosis (FEME) machinery in the PM-associated proteome of HGF-activated MB cells. The decreased surface expression of CD155 or of the FEME effector Endophilin A1 reduces growth and invasiveness of MB tumor cells in the tissue context. These data thus describe a novel function of MAP4K4 in the control of the PM-associated proteome of tumor cells and identified two downstream effector mechanisms controlling proliferation and invasiveness of MB cells.

Graphical abstractc-MET activation upon HGF stimulation induces c-MET internalization and induces downstream MAP4K4 activity. (1) MAP4K4 is required downstream of activated c-MET for the maintenance of surface presentation of CD155 in activated cells. CD155 expression is required for MB cell migration, invasion and proliferation in the tissue context. (2) MAP4K4 is required downstream of activated c-MET to maintain membrane depolarization, possibly by regulating the surface localization of several ion channels and transporters. (3) MAP4K4 is required downstream of activated c-MET cause PM-proximal localization of FEME effector CIP4, FBP17 and CIN85. The FEME effector endophilin A is necessary for MB cell migration, invasion and dissemination.

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Corpus callosal microstructure predicts bimanual motor performance in chronic stroke survivors: A preliminary cross-sectional study AO_SCPLOWBSTRACTC_SCPLOWMicrostructural changes in the corpus callosum are associated with more severe motor impairment in the paretic hand, poor recovery, and general disability. Considering its role in bimanual coordination, we suspected that these microstructural changes across the callosum may also be reflected in the performance of ecologically valid routine bimanual tasks. Thus, the purpose of this study was to determine if callosal microstructure predicts bimanual motor performance in chronic stroke survivors by examining the regions of the corpus callosum connecting both the sensorimotor and non-sensorimotor cortices. We examined the relationship between the fractional anisotropy across the CC and movement times for two self-initiated and self-paced bimanual tasks in 41 chronic stroke survivors. Using publicly available control datasets (n = 52), matched closely for acquisition parameters, we also explored the effect of stroke and age on callosal microstructure. There were two main findings: First, callosal microstructure was significantly associated with bimanual performance in chronic stroke survivors. Notably, a significant relationship was observed not only with the primary sensorimotor regions, but also regions of the premotor/supplementary motor and prefrontal regions. Second, chronic stroke survivors presented with significantly lower mean FA, compared to neurologically intact adults. We conclude that in mild-to-moderate chronic stroke survivors with relatively localized lesions to the motor areas, callosal microstructure can be expected to change in not only the primary sensorimotor region, but also more anteriorly in the secondary motor regions and the genu and is associated with performance on cooperative bimanual tasks.

SignificanceA goal of rehabilitation after stroke is to promote the return to pre-stroke levels of upper limb function and use, predominantly characterized by coordinated bimanual activities. In this study, we find that in the chronic phase of stroke, microstructural disorganization within the corpus callosum predicts motor performance on real-world bimanual tasks and lends important insight into the indirect, remote effects of stroke.

HO_SCPLOWIGHLIGHTSC_SCPLOWO_LIWe provide initial evidence that corpus callosal microstructure predicts performance on two self-initiated and self-paced bimanual tasks.
C_LIO_LIAssociations were strongest for fibers connecting the primary sensorimotor cortices followed by the pre- and supplementary motor, and prefrontal cortices.
C_LIO_LISignificant reductions in fractional anisotropy were observed in stroke survivors for all regions of the corpus callosum.
C_LI

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The birth-and-death evolution of cytochrome P450 genes in bees The birth-and-death model of multigene family evolution describes how families can expand by duplication and contract by gene deletion and formation of pseudogenes. The phylogenetic stability of a gene is thought to be related to the degree of functional importance. However, it is unclear how much evolution of a gene in a gene family is driven by adaptive versus neutral processes. The cytochrome P450s are one of the most diverse and well-studied multigene families, involved in both physiological and xenobiotic functions. Bees have a high toxin exposure due to their diet of nectar and pollen, as well as the resin-collecting behavior exhibited by some bees. Here, we describe the P450s of the orchid bee Euglossa dilemma. Orchid bees are a neotropical clade in which males form perfume bouquets used in courtship displays by collecting a diverse set of volatile compounds, resulting in high chemical compound exposure. We conducted phylogenetic and selection analyses across ten bee species encompassing three bee families. We do not find a relationship between the ecology of a bee species and its P450 repertoire. Our analyses reveal that P450 clades can be classified into stable and unstable clades, and that genes involved in xenobiotic metabolism are more likely to belong to unstable clades. Furthermore, we find that unstable clades are under more dynamic evolutionary pressures, with signals of adaptive evolution detected, suggesting that both gene duplication and positive selection driving sequence divergence have played a role in the diversification of bee P450s. Our works highlights the complexity of multigene family evolution which does not always follow generalized predictions.

Significance statementGene family evolution is characterized by deletion and duplication, but it is unclear if this is driven by adaptive or neutral evolutionary processes. To investigate the dynamics of gene family evolution we analyze the P450s of ten bee species, a well-studied gene family involved in both physiological and detoxification roles. We find that genes involved in detoxification are more likely to belong to unstable clades, exhibit dynamic evolutionary pressures, and show signals of adaptive evolution. However, we do not find evidence for a relationship between resin collection or sociality, and the P450 repertoire of a bee species. Our findings highlight the complexity of multigene family evolution which does not always follow generalized predictions.

sentences: Evidence for evolutionary adaptation of mixotrophic nanoflagellates to warmer temperatures Mixotrophs, organisms that combine photosynthesis and heterotrophy to gain energy, play an important role in global biogeochemical cycles. Metabolic theory predicts that mixotrophs will become more heterotrophic with rising temperatures, potentially creating a positive feedback loop that accelerates carbon dioxide accumulation in the atmosphere. Studies testing this theory have focused on phenotypically plastic (short-term) thermal responses of mixotrophs. However, as small organisms with short generation times and large population sizes, mixotrophs may rapidly evolve in response to climate change. Here we present data from a 3-year experiment quantifying the evolutionary response of two mixotrophic nanoflagellates to temperature. We found evidence for adaptive evolution through increasing growth rates in the obligately mixotrophic strain, but not in the facultative mixotroph. All lineages showed trends of increased carbon use efficiency, flattening of thermal reaction norms, and a return to homeostatic gene expression. Generally, mixotrophs evolved reduced photosynthesis and higher grazing with increased temperatures, suggesting that evolution may act to exacerbate mixotrophs effects on global carbon cycling.

sentences: Shell shape as an indicator of phenotypic stocks of Tehuelche scallop (Aequipecten tehuelchus) in Northern Patagonia, Argentina Tehuelche scallop, Aequipecten tehuelchus, is a commercially exploited species in Northern Patagonia, Argentina. Without genetic differentiation at the species level, A. tehuelchus presents three morphotypes: tehuelchus, madrynensis, and a non-common variant felipponei. The main goal of this study was to analyze the shell shape variation of Tehuelche scallop to differentiate and identify the phenotypic stocks. The shape differences between and within the two main morphotypes (tehuelchus and madrynensis) were assessed using geometric morphometrics in nine localities. The shell shape presented variability at geographic scale, with the morphologic traits that maximized the differentiation among localities between the tehuelchus and madrynensis morphotypes. Scallops from madrynensis morphotype presented higher and circular shell discs with smaller auricles than those from tehuelchus morphotype. Morphometric differentiation was also detected among localities of each morphotype, wherein most of the variability was related to the disc circularity and the symmetry of the auricles. The presence of morphologic variation in San Matias and San Jose gulfs, wherein a single genetic pool is shared, evidenced the plastic nature of the species. Given the distribution of this resource in distinct provincial jurisdictions, the differentiation of phenotypic stocks has relevance in the context of fishery management, especially if zoning and rotational strategies are implemented.

sentences: Nanobody Mediated Macromolecular Crowding Induces Membrane Fission and Remodeling in the African Trypanosome The dense Variant Surface Glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the hosts antibody response. To explore the epitope space of VSGs, we generated anti-VSG nanobodies and combined high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody caused rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endo-and exocytosis is mechanistically linked to T. brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrated that this loss of motility was accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells.

sentences: Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the  clade and the {beta} clade. Most  clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in {beta} clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_{beta}IB1). A structural comparison of available PLDs structures reveals a conserved aromatic cage in the  clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular-dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the  clade, Loxosceles intermedia IA1 (Li_IA) and Loxosceles laeta III1 (Ll_III1), and one from the {beta} clade, St_{beta}IB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_{beta}IB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the  clade PLDs. Here, we confirmed the membrane binding site of  and {beta} clade PLDs on choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the  clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

sentences: Reciprocity between retrograde signal and putative metalloprotease reconfigures plastidial metabolism and structure Reconfiguration of the plastidial proteome in response to environmental inputs is central to readjustment of its metabolic and structural states. This is necessary for the functionality of this metabolic hub, and the maintenance of organismal integrity. This report establishes the role of the plastidial retrograde signaling metabolite, MEcPP, in increasing the abundance of the putative plastidial metalloprotease (VIR3), and the ensuing decline of VIR3 target enzymes, ascorbate peroxidase and glyceraldehyde 3-phophate dehydrogenase B. The decreased abundance of these enzymes is linked to increased levels of their substrates: H2O2, an elicitor of salicylic acid production and stromule formation; and G3P the substrate for MEcPP synthesis. High-light treatment of wild type plants recapitulated the VIR3-associated reconfiguration of the plastidial metabolic and structural states. These results identify a previously unrecognized link between the stress-induced plastidial retrograde signaling metabolite and a putative zinc-binding metalloprotease. Moreover, the data reveal that the reciprocity between these two components, results in the reconfiguration of the metabolic and structural states of the plastid, deemed necessary to maintain cellular integrity and to shape adaptive responses.

sentences: An "individualist" model of an active genome in a developing embryo The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the whole genome. Using Drosophila embryos undergoing the first wave of genome activation, we detected discrete "speckles" of RNA Polymerase II (Pol II), and showed that they overlap with transcribing loci. We characterized the spatial distribution of Pol II speckles and quantified how this distribution changes in the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda. Although the number and size of Pol II speckles were reduced, indicating that Zelda promotes Pol II speckle formation, we observed a uniform distribution of distances between active genes in the nuclei of both wildtype and zelda mutant embryos. This suggests that the topologically associated domains identified by Hi-C studies do little to spatially constrain groups of transcribed genes at this time. We provide evidence that linear genomic distance between transcribed genes is the primary determinant of measured physical distance between the active loci. Furthermore, we show active genes can have distinct Pol II pools even if the active loci are in close proximity. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, our data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems.

sentences: The development of efficient communication in the human connectome. The structure of the human connectome develops from childhood throughout adolescence to middle age, but how this affects the speed of neuronal signaling is not well described. To understand the maturation process of transmission speed across the connectome, we stimulated electrocorticographic (ECoG) electrode pairs in 75 human subjects of 4 to 51 years old. We then measured the latency of cortico-cortical evoked responses in other electrodes. Each year, responses got faster, until transmission speed reached a minimum latency at the age of 29-44 years old. Responses during middle age were about two times faster compared to childhood. These increases in speed were observed in both long- and short-range connections between and within frontal, central, parietal, and temporal areas. These findings indicate that long-range and short-range communication between brain regions increase in efficiency well throughout adolescence.

sentences: Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops. The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the {beta} subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimise conflicts between transcription and other processes on DNA including replication.

sentences: Genomic epidemiological models describe pathogen evolution across fitness valleys Genomics is fundamentally changing epidemiological research. However, exploring hypotheses about pathogen evolution in different epidemiological contexts poses new challenges. Models intertwining pathogen epidemiology and genomic evolution can help understand processes such as the emergence of novel pathogen genotypes with higher transmission or resistance to treatment. In this work, we present Opqua, a computational framework for flexible simulation of pathogen epidemiology and evolution. We use Opqua to study determinants of evolution across fitness valleys. We confirm that competition can limit evolution in high transmission environments and find that low transmission, host mobility, and complex pathogen life cycles facilitate reaching new adaptive peaks through population bottlenecks and decoupling selective pressures. The results show the potential of genomic epidemiological modeling as a tool in infectious disease research.

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Correlational networking guides the discovery of cryptic natural product biosynthetic enzymes Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on cryptic enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by cryptic proteases required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and cryptic proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5,209 correlations between precursors and cryptic proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of novel class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of cryptic lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Sex differences in behavioral responding and dopamine release during early Pavlovian learning Learning associations between cues and rewards requires the mesolimbic dopamine system. The dopamine response to cues signals differences in reward value in well-trained animals. However, these value-related dopamine responses are absent during early training sessions when cues signal differences in the reward rate. These findings suggest cue-evoked dopamine release conveys differences between outcomes only after extensive training, though it is unclear if this is unique to when cues signal differences in reward rate, or if this is also evident when cues signal differences in other value-related parameters such as reward size. To address this, we utilized a Pavlovian conditioning task in which one audio cue was associated with a small reward (one pellet) and another audio cue was associated with a large reward (three pellets). We performed fast-scan cyclic voltammetry to record changes in dopamine release in the nucleus accumbens of male and female rats throughout learning. While female rats exhibited higher levels of conditioned responding, a faster latency to respond, and elevated post-reward head entries relative to male rats, there were no sex differences in the dopamine response to cues. Multiple training sessions were required before cue-evoked dopamine release signaled differences in reward size. Reward-evoked dopamine release scaled with reward size, though females displayed lower reward-evoked dopamine responses relative to males. Conditioned responding related to the decrease in the peak reward-evoked dopamine response and not to cue-evoked dopamine release. Collectively these data illustrate sex differences in behavioral responding as well as in reward-evoked dopamine release during Pavlovian learning.

sentences: Multi-influential interactions alters behaviour and cognition through six main biological cascades in Down syndrome mouse models Down syndrome (DS) is the most common genetic form of intellectual disability caused by the presence of an additional copy of human chromosome 21 (Hsa21). To provide novel insights into genotype-phenotype correlations, we used standardized behavioural tests, magnetic resonance imaging (MRI) and hippocampal gene expression to screen several DS mouse models for the mouse chromosome 16 region homologous to Hsa21. First, we unravelled several genetic interactions between different regions of chromosome 21 and how they contribute significantly to altering the outcome of the phenotypes in brain cognition, function and structure. Then, in-depth analysis of misregulated expressed genes involved in synaptic dysfunction highlighted 6 biological cascades centred around DYRK1A, GSK3{beta}, NPY, SNARE, RHOA and NPAS4. Finally, we provide a novel vision of the existing altered gene-gene crosstalk and molecular mechanisms targeting specific hubs in DS models that should become central to better understanding of DS and improving the development of therapies.

sentences: Curtobacterium glycinis sp. nov. from Glycine max, Curtobacterium gossypii sp. nov. from Gossypium hirsutum and Curtobacterium oryzae sp. nov. from Oryza sativa, three new Curtobacterium species and endophytes from agricultural crops Three new Curtobacterium species from healthy tissues of agricultural crop plants in the United States are reported. They are Curtobacterium glycinis sp. nov. from soybean in Missouri, Curtobacterium gossypii from cotton in Puerto Rico and corn in Missouri, and Curtobacterium oryzae sp. nov. from rice in Texas.

sentences: PEP7 is a ligand for receptor kinase SIRK1 to regulate aquaporins and root growth Plant receptor kinases constitute a large protein family that regulate various aspects of development and responses to external biotic and abiotic cues. Functional characterization of this protein family and particularly the identification of their ligands remains a major challenge in plant biology. Previously, we identified plasma membrane-intrinsic SUCROSE INDUCED RECEPTOR KINASE 1 (SIRK1) and QIAN SHOU KINASE 1 (QSK1) as a receptor / co-receptor pair involved in regulation of aquaporins in response to osmotic conditions induced by sucrose. Here, we identified a member of the Elicitor Peptide (PEP) family, namely PEP7, as the specific ligand of receptor kinase SIRK1. PEP7 binds to the extracellular domain of SIRK1 with a binding constant of 1.44{+/-}0.79 {micro}M and is secreted to the apoplasm specifically in response to sucrose treatment. Stabilization of a signaling complex involving SIRK1, QSK1 and aquaporins as substrates is mediated by alterations in the external sucrose concentration or by PEP7 application. Moreover, the presence of PEP7 induces the phosphorylation of aquaporins in vivo and enhance water influx into protoplasts. The loss-of-function mutant of SIRK1 is not responsive to external PEP7 treatment regarding kinase activity, aquaporin phosphorylation and water influx activity. Our data indicate that the PEP7/SIRK1/QSK1 complex represents a crucial perception and response module mediating sucrose-controlled water flux in plants.

sentences: Genomic architecture and evolutionary dynamics of a social niche polymorphism in the California harvester ant, Pogonomyrmex californicus Social insects vary considerably in their social organization both between and within species. In the California harvester ant, Pogonomyrmex californicus (Buckley 1867), colonies are commonly founded and headed by a single queen (haplometrosis, primary monogyny). However, in some populations in California (USA), unrelated queens cooperate not only during founding (pleometrosis) but throughout the life of the colony (primary polygyny). The genetic architecture and evolutionary dynamics of this complex social niche polymorphism (haplometrosis vs pleometrosis) have remained unknown. Here, we provide a first analysis of its genomic basis and evolutionary history. We discover a recently evolved (< 200 k years), 8 Mb non-recombining region segregating with the observed social niche polymorphism, showing characteristics of a supergene comparable to those underlying social polymorphisms in other ant species. However, we also find remarkable differences to the other so far described social supergenes. Particularly, four additional genomic regions not in linkage with the supergene show signatures of a selective sweep in the pleometrotic population. Within these regions, we find for example genes crucial for epigenetic regulation via histone modification (chameau) and DNA methylation (dnmt1). Our results suggest that social morph in this species is a polygenic trait including an incipient supergene that evolved less than 200 000 years ago.

sentences: Genetic mapping of sex and self-incompatibility determinants in the androdioecious plant Phillyrea angustifolia AO_SCPLOWBSTRACTC_SCPLOWThe diversity of mating and sexual systems in angiosperms is spectacular, but the factors driving their evolution remain poorly understood. In plants of the Oleaceae family, an unusual self-incompatibility (SI) system has been discovered recently, whereby only two distinct homomorphic SI specificities segregate stably. To understand the role of this peculiar SI system in preventing or promoting the diversity of sexual phenotypes observed across the family, an essential first step is to characterize the genetic architecture of these two traits. Here, we developed a high-density genetic map of the androdioecious shrub P. angustifolia based on a F1 cross between a hermaphrodite and a male parent with distinct SI genotypes. Using a double restriction-site associated digestion (ddRAD) sequencing approach, we obtained reliable genotypes for 196 offspring and their two parents at 10,388 markers. The resulting map comprises 23 linkage groups totaling 1,855.13 cM on the sex-averaged map. We found strong signals of association for the sex and SI phenotypes, that were each associated with a unique set of markers on linkage group 12 and 18 respectively, demonstrating inheritance of these traits as single, independent, mendelian factors. The P. angustifolia linkage map shows robust synteny to the olive tree genome overall. Two of the six markers strictly associated with SI in P. angustifolia have strong similarity with a recently identified 741kb chromosomal region fully linked to the SI phenotype on chromosome 18 of the olive tree genome, providing strong cross-validation support. The SI locus stands out as being markedly rearranged, while the sex locus has remained relatively more collinear between the two species. This P. angustifolia linkage map will be a useful resource to investigate the various ways by which the sex and SI determination systems have co-evolved in the broader phylogenetic context of the Oleaceae family.

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The Asian gypsy moth (Lymantria dispar L.) populations: resistance of eggs to extreme winter temperatures Gypsy moth Lymantria dispar (GM) is a polyphagous insect and one of the most significant pests in the forests of Eurasia and North America. Accurate information on GM cold hardiness is needed to improve methods for the prediction of population outbreaks, as well as for forecasting possible GM range displacements due to climate change.

As a result of laboratory and field studies, we found that the lower lethal temperature (at which all L. dispar asiatica eggs die) range from -29.0 {degrees}C to -29.9 {degrees}C for three studied populations, and no egg survived cooling to -29.9 {degrees}C. These limits agree to within one degree with the previously established cold hardiness limits of the European subspecies L. dispar dispar, which is also found in North America. This coincidence indicates that the lower lethal temperature of L. dispar is conservative.

Thus, we found that the Siberian populations of GM inhabit an area where winter temperatures go beyond the limits of egg physiological tolerance, because temperature often fall below -30 {degrees}C. Apparently, it is due to the flexibility of ovipositional behavior that L. dispar asiatica survives in Siberia: the lack of physiological tolerance of eggs is compensated by choosing warm biotopes for oviposition. One of the most important factors contributing to the survival of GM eggs in Siberia is the stability of snow cover.

SummaryWithin the geographical range of Siberian gypsy moth populations, extreme temperatures go beyond the limits of the physiological tolerance of wintering eggs (-29.9 {degrees}C), and their survival depends on the choice of warm biotopes for oviposition.

sentences: Ataxin-2 is essential for neurodevelopment in Drosophila and is a major regulator of the cytoskeleton. Ataxin-2 (Atx2) is a highly conserved RNA binding protein. Atx2 undergoes polyglutamine expansion leading to Amyotrophic Lateral Sclerosis (ALS) or Spinocerebellar Ataxia type 2 (SCA2). However, the physiological functions of Atx2 in neurons remain unknown. Here, using the powerful genetics of Drosophila, we show that Atx2 is essential for normal neuronal cytoskeletal dynamics and organelle trafficking. Upon neuron-specific Atx2 loss, the microtubule and actin networks were abnormally stabilized and cargo transport was drastically inhibited. Depletion of Atx2 caused multiple morphological defects in the nervous system of 3rd instar larvae. These include reduced brain size, impaired optic lobe innervation and decreased dendrite outgrowth. Defects in the nervous system caused loss of the ability to crawl and lethality at the pupal stage. Taken together, these data mark Atx2 as a major regulator of cytoskeletal dynamics and denote Atx2 as an essential gene in neurodevelopment, as well as a neurodegenerative factor.

sentences: Fructose Metabolism Contributes to the Warburg effect Fructose metabolism is increasingly recognized as a preferred energy source for cancer cell proliferation. However, it remains unclear why cancer cells favor fructose metabolism and how they acquire a sufficient amount of fructose. Here we report that cancer cells convert glucose into fructose through intra- and extracellular polyol pathways. The fructose metabolism bypasses normal aerobic respirations self-control to supply excessive metabolites to glycolysis and promotes the Warburg effect. Inhibition of fructose production drastically suppressed glycolysis and ATP production in cancer. Furthermore, we determined that a glucose transporter, SLC2A8/GLUT8, exports intracellular fructose to other cells in the tumor microenvironment. Taken together, our study suggests that the Warburg effect is achieved by means of fructose metabolism, instead of glucose metabolism alone.

sentences: Midkine noncanonically suppresses AMPK activation through disrupting the LKB1-STRAD-Mo25 complex Midkine (MDK), an extracellular growth factor, regulates signal transduction and cancer progression by interacting with receptors, and it can be internalized into the cytoplasm by endocytosis. However, its intracellular function and signaling regulation remain unclear. Here, we show that intracellular MDK interacts with LKB1 and STRAD to disrupt the LKB1-STRAD-Mo25 complex. Consequently, MDK decreases the activity of LKB1 to dampen both the basal and stress-induced activation of AMPK by glucose starvation or treatment of 2-DG. We also found that MDK accelerates cancer cell proliferation by inhibiting the activation of the LKB1-AMPK axis. In human cancers, compared to other well-known growth factors, MDK expression is most significantly upregulated in cancers, especially in liver, kidney and breast cancers, correlating with clinical outcomes and inversely correlating with PRKAA1 (encoding AMPK1) expression and phosphorylated AMPK levels. Our study elucidates an inhibitory mechanism for AMPK activation, which is mediated by the intracellular MDK through disrupting the LKB1-STRAD-Mo25 complex.

sentences: Network design principle for robust oscillatory behaviors with respect to biological noise Oscillatory behaviors, which are ubiquitous in transcriptional regulatory networks, are often subject to inevitable biological noise. Thus a natural question is how transcriptional regulatory networks can robustly achieve accurate oscillation in the presence of biological noise. Here, we search all two- and three-node transcriptional regulatory network topologies for those robustly capable of accurate oscillation against the parameter variability (extrinsic noise) or stochasticity of chemical reactions (intrinsic noise). We find that, no matter what source of the noise is applied, the topologies containing the repressilator with positive auto-regulation show higher robustness of accurate oscillation than those containing the activator-inhibitor oscillator, and additional positive auto-regulation enhances the robustness against noise. Nevertheless, the attenuation of different sources of noise is governed by distinct mechanisms: the parameter variability is buffered by the long period, while the stochasticity of chemical reactions is filtered by the high amplitude. Furthermore, we analyze the noise of a synthetic human nuclear factor {kappa}B (NF-{kappa}B) signaling network by varying three different topologies, and verify that the addition of a repressilator to the activator-inhibitor oscillator, which leads to the emergence of high-robustness motif--the repressilator with positive auto-regulation, improves the oscillation accuracy in comparison to the topology with only an activator-inhibitor oscillator. These design principles may be applicable to other oscillatory circuits.

sentences: Development of a vaccine against the newly emerging COVID-19 virus based on the receptor binding domain displayed on virus-like particles The ongoing coronavirus COVID-19 pandemic is caused by a new coronavirus (SARS-CoV-2) with its origin in the city of Wuhan in China. From there it has been rapidly spreading to many cities inside and outside China. Nowadays more than 33 millions with deaths surpassing 1 million have been recorded worldwide thus representing a major health issue. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here we demonstrated that recombinantly expressed receptor binding domain (RBD) of the spike protein homologous to SARS binds to ACE2, the viral receptor. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus (CuMVTT) resulted in a vaccine candidate that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized COVID-19 virus in vitro.

sentences: Competition for water and species coexistence in phenologically structured annual plant communities Both competition for water and phenological variation are important determinants of plant community structure, but ecologists lack a synthetic theory for how they affect coexistence outcomes. We developed an analytically tractable model of water competition for Mediterranean annual communities and demonstrate that variation in phenology alone can maintain high diversity in spatially homogenous assemblages of water-limited plants. We modeled a system where all water arrives early in the season and species vary in their ability to grow under drying conditions. As a consequence, species differ in growing season length, and compete by shortening the growing season of their competitors. This model replicates and offers mechanistic explanations for qualitative patterns observed in prior empirical studies of how phenology influences coexistence among Mediterranean annuals. Additionally, we found that a decreasing, concave-up tradeoff between growth rate and access to water can theoretically maintain infinite diversity under simple but realistic assumptions. High diversity is possible because: 1) later plants escape competition after their earlier-season competitors have gone to seed and 2) early-season species are more than compensated for their shortened growing season by a growth-rate advantage. Together, these mechanisms provide an explanation for how annual plant species might coexist when competing only for water.

sentences: Breeding displacement in gray wolves (Canis lupus): Three males usurp breeding position and pup rearing from a neighboring pack in Yellowstone National Park Gray Wolves (Canis lupus) are territorial, group living carnivores that live in packs typically consisting of a dominant breeding pair and their offspring. Breeding tenures are relatively short and competitive, with vacancies usually occurring following a breeders death, and are often filled by unrelated immigrants or by relatives of the previous breeder. The frequency and conditions of active breeder displacements are poorly understood. Position changes in the dominance hierarchy are common yet rarely documented in detail. We describe a turnover in male breeding position in a wolf pack by males from a neighboring pack in mid-summer 2016 in Yellowstone National Park. Over the course of two months, three males from the Mollies pack displaced the breeding male of the neighboring Wapiti Lake pack, joined the packs two adult females, and subsequently raised the previous males four approximately three-month old pups. In the four years following the displacement (2017 to 2020), at least one of the intruding males has successfully bred with the dominant female and most years with a subordinate female (who was one of the pups at the time of displacement). The pack reared pups to adulthood each year. Male breeding displacements are likely influenced by male-male competition and female mate choice. These changes are the result of individuals competing to improve breeding position and may lead to increased pack stability and greater reproductive success. We report in detail on the behavior of a closely observed breeding displacement and we discuss the adaptive benefits of the change.

sentences: Are microbes growing on flowers evil? Effects of old flower microbes on fruit set in a wild ginger with one-day flowers, Alpinia japonica (Zingiberaceae) Flowers are colonized and inhabited by diverse microbes. Plants rapidly replace flowers with short lifespan, and old flowers senesce. This may contribute to avoiding adverse effects of the microbes. In this study, we investigate if the flower microbial community on old flowers impedes fruit and seed production in a wild ginger with one-day flowers. We inoculated newly opened flowers with old flower microbes, and monitored the effects on fruit and seed set. We also assessed prokaryotic communities on the flowers using amplicon sequencing. We found six bacterial amplicon sequence variants (ASVs) whose proportions were increased on the inoculated flowers. These ASVs were also found on flower buds and flowers that were bagged by net or paper during anthesis, suggesting that they had been present in small numbers prior to flowering. Fruit set was negatively associated with the proportions of these ASVs, while seed set was not. The results suggest that old flowers harbor microbial communities different from those at anthesis, and that the microbes abundant on old flowers negatively affect plant reproduction. Though the short lifespan of flowers has gotten little attention, it might be an essential defense mechanism to cope with antagonistic microbes that rapidly proliferate on the flowers.

sentences: Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination Class switch recombination generates antibody distinct isotypes critical to a robust adaptive immune system and defects are associated with auto-immune disorders and lymphomagenesis. Transcription is required during class switch to recruit the cytidine deaminase AID--an essential step for the formation of DNA double-strand breaks--and strongly induces the formation of R loops within the immunoglobulin heavy chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here we report that cells lacking two enzymes involved in R loop removal-- Senataxin and RNase H2--exhibit increased R loop formation and genome instability at the immunoglobulin heavy chain locus without impacting class switch recombination efficiency, transcriptional activity, or AID recruitment. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking Senataxin or RNase H2B alone. We propose that Senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis.

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Permutation tests for hypothesis testing with animal social data: problems and potential solutions O_LIPermutation tests are widely used to test null hypotheses with animal social network data, but suffer from high rates of type I and II error when the permutations do not properly simulate the intended null hypothesis.
C_LIO_LITwo common types of permutations each have limitations. Pre-network (or datastream) permutations can be used to control "nuisance effects" like spatial, temporal, or sampling biases, but only when the null hypothesis assumes random social structure. Node (or node-label) permutation tests can test null hypotheses that include nonrandom social structure, but only when nuisance effects do not shape the observed network.
C_LIO_LIWe demonstrate one possible solution addressing these limitations: using pre-network permutations to adjust the values for each node or edge before conducting a node permutation test. We conduct a range of simulations to estimate error rates caused by confounding effects of social or non-social structure in the raw data.
C_LIO_LIRegressions on simulated datasets suggest that this "double permutation" approach is less likely to produce elevated error rates relative to using only node permutations, pre-network permutations, or node permutations with simple covariates, which all exhibit elevated type I errors under at least one set of simulated conditions. For example, in scenarios where type I error rates from pre-network permutation tests exceed 30%, the error rates from double permutation remain at 5%.
C_LIO_LIThe double permutation procedure provides one potential solution to issues arising from elevated type I and type II error rates when testing null hypotheses with social network data. We also discuss alternative approaches that can provide robust inference, including fitting mixed effects models, restricted node permutations, testing multiple null hypotheses, and splitting large datasets to generate replicated networks. Finally, we highlight ways that uncertainty can be explicitly considered and carried through the analysis.
C_LI

sentences: Individual Molecular Motors use Low Forces to bypass Roadblocks during Collective Cargo Transport A cargo encounters many obstacles during its transport by molecular motors as it moves throughout the cell. Multiple motors on the cargo exert forces to steer the cargo to its destination. Measuring these forces is essential for understanding intracellular transport. Using kinesin as an example, we measured the force exerted by multiple stationary kinesins in vitro, driving a common microtubule. We find that individual kinesins generally exert less than a piconewton (pN) of force, even while bypassing obstacles, whether these are artificially placed 20-100 nm particles or tau, a Microtubule Associated Protein. We demonstrate that when a kinesin encounters an obstacle, the kinesin either becomes dislodged and then re-engages or switches protofilaments while the other kinesins continue to apply their (sub-)pN forces. By designing a high-throughput assay involving nanometer-resolved multicolor-fluorescence and a force-sensor able to measure picoNewtons of force, our technique is expected to be generally useful for many different types of molecular motors.

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Cell-to-cell variations in subcellular localisation of V2R are independent of its expression level G protein coupled receptors (GPCRs) translate the actions of hormones into intracellular signalling events. Mutations in GPCRs can prevent their correct expression and trafficking to the cell surface and cause disease. We use single cell measurements in HEK293 cells to show that the balance between endoplasmic reticulum (ER) and cell surface localisation of the Vasopressin 2 receptor (V2R) varies significantly from cell to cell. We find that mutations in the V2R affect the proportion of cells able to send this GPCR to the cell surface but do not prevent all cells in the population from correctly trafficking the mutant receptors. These findings reveal that the ability of cells to correctly traffic V2R to the cell surface depends not only on the expressed V2R mutant but also on the individual cell environment.

Significance statementMissense mutations in the Vasopressin 2 Receptor (V2R) cause Nephrogenic Diabetes Insipidus. Some of these mutations prevent correct expression and trafficking of V2R to the cell surface resulting in a loss-of-function. We show -using single cell measurements-that the balance between endoplasmic reticulum and cell surface localisation of the V2R varies significantly from cell to cell, independent from its expression level. Mutations affect the proportion of cells able to send V2R to the cell surface but do not prevent all cells in the population from correctly trafficking the mutant receptors. Hence, the ability of cells to correctly traffic V2R to the cell surface depends not only on expressed V2R mutant but also on the cell environment.

sentences: Neurogrid simulates cortical cell-types, active dendrites, and top-down attention A central challenge for systems neuroscience and artificial intelligence is to understand how cognitive behaviors arise from large, highly interconnected networks of neurons. Digital simulation is linking cognitive behavior to neural activity to bridge this gap in our understanding at great expense in time and electricity. A hybrid analog-digital approach, whereby slow analog circuits, operating in parallel, emulate graded integration of synaptic currents by dendrites while a fast digital bus, operating serially, emulates all-or-none transmission of action potentials by axons, may improve simulation efficacy. Due to the latters serial operation, this approach has not scaled beyond millions of synaptic connections (per bus). This limit was broken by following design principles the neocortex uses to minimize its wiring. The resulting hybrid analog-digital platform, Neurogrid, scales to billions of synaptic connections, between up to a million neurons, and simulates cortical models in real-time using a few watts of electricity. Here, we demonstrate that Neurogrid simulates cortical models spanning five levels of experimental investigation: biophysical, dendritic, neuronal, columnar, and area. Bridging these five levels with Neurogrid revealed a novel way active dendrites could mediate top-down attention.

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Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered domains (rafts) Using efficient methyl-alpha-cyclodextrin mediated lipid exchange, we studied the effect of altering plasma membrane outer leaflet phospholipid composition upon the activity of insulin receptor (IR) in mammalian cells. After substitution of endogenous lipids with lipids having an ability to form liquid ordered (Lo) domains (sphingomyelins) or liquid disordered (Ld) domains (unsaturated phosphatidylcholines (PCs)), we found that the propensity of lipids to form ordered domains is required for high IR activity. Additional substitution experiments using a series of saturated PCs showed that IR activity increased substantially with increasing acyl chain length. Increasing acyl chain length increases both bilayer width and the propensity to form ordered domains. To distinguish the effects of membrane width and domain formation, we incorporated purified IR into alkyl maltoside micelles with increasing hydrocarbon lengths. IR activity increases with increased chain length, but more modestly than by increasing lipid acyl chain length in cells. This suggests that the ability to form Lo domains as well as wide bilayer width contributes to increased IR activity. Inhibition of phosphatases with sodium orthovanadate showed that some of the lipid dependence of IR activity upon lipid structure reflected protection from phosphatases by lipids that support Lo domain formation. The results are consistent with a model in which a combination of bilayer width and ordered domain formation modulate IR activity via effects upon IR conformation and accessibility to phosphatases.

SignificanceThis study shows how methyl--cyclodextrin mediated lipid exchange can be used to probe the influence of lipid structure upon the functioning of a transmembrane receptor. Plasma membranes having a propensity to form Lo domains are required to support a high level of IR activity. The studies indicate this may reflect an effect of lipid environment upon IR domain localization, which in turn alters its conformation and vulnerability to phosphatases. Alterations in lipid composition could conceivably regulate IR activity in vivo.

sentences: Structural basis of drug recognition by the multidrug transporter ABCG2 ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (E1S) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.

sentences: SARS-CoV-2 spike protein unlikely to bind to integrins via the Arg-Gly-Asp (RGD) motif of the Receptor Binding Domain: evidence from structural analysis and microscale accelerated molecular dynamics The Receptor Binding Domain (RBD) of SARS-CoV-2 virus harbors a sequence of Arg-Gly-Asp tripeptide named RGD motif, which has also been identified in extracellular matrix proteins that bind integrins as well as other disintegrins and viruses. Accordingly, integrins have been proposed as host receptors for SARS-CoV-2. The hypothesis was supported by sequence and structural analysis. However, given that the microenvironment of the RGD motif imposes structural hindrance to the protein-protein association, the validity of this hypothesis is still uncertain. Here, we used normal mode analysis, accelerated molecular dynamics microscale simulation, and protein-protein docking to investigate the putative role of RGD motif of SARS-CoV-2 RBD for interacting with integrins. We found, by molecular dynamics, that neither RGD motif nore its microenvironment show any significant conformational shift in the RBD structure. Highly populated clusters were used to run a protein-protein docking against three RGD-binding integrin types, showing no capability of the RBD domain to interact with the RGD binding site. Moreover, the free energy landscape revealed that the RGD conformation within RBD could not acquire an optimal geometry to allow the interaction with integrins. Our results highlighted different structural features of the RGD motif that may prevent its involvement in the interaction with integrins. We, therefore, suggest, in the case where integrins are confirmed to be the direct host receptors for SARS-CoV-2, a possible involvement of other residues to stabilize the interaction.

sentences: Mutation-specific reporter for the optimization and enrichment of prime editing We present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) and any prime editor protein, including novel variants with flexible PAM recognition. Successful reporter editing enriches for genomic editing. FluoPEER can be employed for efficient correction of patient cells and to elucidate cellular mechanisms needed for successful prime editing.

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DNA damage induces senescent phenotypes in mouse NIT1 beta cells and human islets ObjectiveType 1 Diabetes (T1D) is characterized by progressive loss of insulin-producing pancreatic {beta} cells as a result of autoimmune destruction. In addition to {beta} cell death, recent work has shown that subpopulations of {beta} cells acquire dysfunction during T1D. We previously reported that some {beta} cells adopt a senescent fate involving a DNA damage response (DDR) during the pathogenesis of T1D, however, the question of how senescence develops in {beta} cells has not been investigated.

MethodsHere, we tested the hypothesis that unrepaired DNA damage triggers {beta} cell senescence using culture models including the mouse NIT1 {beta} cell line derived from the T1D-susceptible nonobese diabetic (NOD) strain, human donor islets and EndoC {beta} cells. DNA damage was chemically induced using etoposide or bleomycin and cells or islets were analyzed by a combination of molecular assays for senescence phenotypes including Western blotting, qRT-PCR, Luminex assays, flow cytometry and histochemical staining. RNA-seq was carried out to profile global transcriptomic changes in human islets undergoing DDR and senescence. Insulin ELISAs were used to quantify glucose stimulated insulin secretion from chemically-induced senescent islets and cells in culture.

ResultsSub-lethal DNA damage in NIT1 cells led to several classical hallmarks of senescence including sustained DDR activation, growth arrest, enlarged flattened morphology and a senescence-associated secretory phenotype (SASP) resembling what occurs in primary {beta} cells during T1D in NOD mice. Some of these phenotypes differed between NIT1 cells and the MIN6 {beta} cell line derived from a non-T1D susceptible mouse strain. RNA-seq analysis of human islets undergoing DDR and early senescence revealed a coordinated p53-p21 transcriptional program and upregulation of prosurvival signaling and SASP genes, which were confirmed in independent islet preparations at the protein level. Importantly, chemically induced DNA damage also led to DDR activation and senescence phenotypes in the EndoC-{beta}H5 human {beta} cell line, confirming that this response can occur directly in human {beta} cells. Finally, DNA damage and senescence in both mouse {beta} cell lines and human islets led to decreased insulin content.

ConclusionsTaken together, these findings suggest that some of the phenotypes of {beta} cell senescence during T1D can be modeled by chemically induced DNA damage in mouse {beta} cell lines and human islets and {beta} cells in culture. These culture models will be useful tools to understand some of the mechanisms of {beta} cell senescence in T1D.

HighlightsO_LIDNA damage induces senescent phenotypes in mouse {beta} cell lines
C_LIO_LIDNA damage induces a p53-p21 transcriptional program and senescent phenotypes in human islets and EndoC cells
C_LIO_LIDNA damage and senescence leads to decreased insulin content
C_LIO_LIDNA damage models some aspects of {beta} cell senescence in Type 1 Diabetes
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sentences: Structural basis for APE1 processing DNA damage in the nucleosome Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determined that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3 - 6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site identified that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identified contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

sentences: Systematic analysis of C. elegans embryo behavior reveals a stereotyped developmental progression and a rhythmic pattern of behavioral quiescence Systematic analysis of rich behavioral recordings is being used to uncover how circuits encode complex behaviors. Here we apply the approach to embryos. What are the first embryonic behaviors and how do they evolve as early neurodevelopment ensues? To address these questions, we present a systematic description of behavioral maturation for Caenorhabditis elegans embryos. Posture libraries were derived from a genetically encoded motion capture suit imaged with light-sheet microscopy and annotated using custom semi-automated tracking software (Multiple Hypothesis Hypergraph Tracking; MHHT). Analysis of cell trajectories, postures, and behavioral motifs revealed a stereotyped developmental progression. Early movement is dominated by flipping between dorsal and ventral coiling, which gradually slows into a period of reduced motility. Late-stage embryos exhibit sinusoidal waves of dorsoventral bends, prolonged bouts of directed motion, and a rhythmic pattern of pausing, which we designate slow wave twitch (SWT). Synaptic transmission is required for late-stage motion but not for early flipping or the intervening inactive phase. A high-throughput behavioral assay and calcium imaging revealed that SWT is elicited by the rhythmic activity of a quiescence-promoting neuron (RIS). Similar periodic quiescent states are seen prenatally in divergent animals and may play an important role in promoting normal developmental outcomes.

sentences: De novo chromosome-length assembly of the mule deer (Odocoileus hemionus) genome The mule deer (Odocoileus hemionus) is an ungulate species that ranges from western Canada to central Mexico. Mule deer are an essential source of food for many predators, are relatively abundant, and commonly make broad migration movements. A clearer understanding of the mule deer genome can help facilitate knowledge of its population genetics, movements, and demographic history, aiding in conservation efforts. While mule deer are excellent candidates for population genomic studies because of their large population size, continuous distribution, and diversity of habitat, few genomic resources are currently available for this species. Here, we sequence and assemble the mule deer genome into a highly contiguous chromosome-length assembly for use in future research using long-read sequencing and Hi-C. We also provide a genome annotation and compare demographic histories of the mule deer and white-tail deer using PSMC. We expect this assembly to be a valuable resource in the continued study and conservation of mule deer.

sentences: Identification of sample mix-ups and mixtures in microbiome data in Diversity Outbred mice In a Diversity Outbred mouse project with genotype data on 500 mice, including 297 with microbiome data, we identified three sets of sample mix-ups (two pairs and one trio) as well as at least 15 microbiome samples that appear to be mixtures of pairs of mice. The microbiome data consisted of shotgun sequencing reads from fecal DNA, used to characterize the gut microbial communities present in these mice. These sequence reads included sufficient reads derived from the host mouse to identify the individual. A number of microbiome samples appeared to contain a mixture of DNA from two mice. We describe a method for identifying sample mix-ups in such microbiome data, as well as a method for evaluating sample mixtures in this context.

sentences: In vitro determination of inhibitory effects by humic substances complexing Zn and Se on SARS-CoV-2 virus replication Humic substances are well known human nutritional supplement materials and play important performance-enhancing roles as animal feed additives, too. For decades, ingredients of humic substances have also been proven to carry potent antiviral effects against different viruses. Here, the antiviral activity of a humic substance containing ascorbic acid, Se- and Zn2+ ions intended as a nutritional supplement material was investigated against SARS-CoV-2 virus B1.1.7 Variant of Concern ("Alpha Variant") in a VeroE6 cell line. Results show that this combination has a significant in vitro antiviral effect at a very low concentration range of its intended active ingredients. Even picomolar concentration ranges of humic substances, vitamin C and Zn/Se ions in the given composition were enough to achieve fifty percent viral replication inhibition in the applied SARS-CoV-2 virus inhibition test.

sentences: Context-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing Klebsiella pneumoniae Genomic surveillance can inform effective public health responses to pathogen outbreaks. However, integration of non-local data is rarely done. We investigate two large hospital outbreaks of a carbapenemase-carrying Klebsiella pneumoniae strain in Germany and show the value of contextual data. By screening more than ten thousand genomes, 500 thousand metagenomes, and two culture collections using in silico and in vitro methods, we identify a total of 415 closely related genomes reported in 28 studies. We identify the relationship between the two outbreaks through time-dated phylogeny, including their respective origin. One of the outbreaks presents extensive hidden transmission, with descendant isolates only identified in other studies. We then leverage the genome collection from this meta-analysis to identify genes under positive selection. We thereby identify an inner membrane transporter (ynjC) with a putative role in colistin resistance. Contextual data from other sources can thus enhance local genomic surveillance at multiple levels and should be integrated by default when available.

sentences: Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62 Autophagy is an essential degradation program required to maintain cell homeostasis. Amongst its many functions is the engulfment and destruction of cytosolic pathogens, termed Xenophagy. Not surprisingly, many pathogens have adapted various strategies to circumvent or co-opt autophagic degradation during infection. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Despite the fact that these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that over-expression of the xenophagy receptors p62, NDP52 and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 was found to initially target cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 during infection was dependent upon p62 NLS2, and VACV kinase mediated phosphorylation of associated p62 residues T269/S272. These results indicate that VACV actively targets the xenophagy receptor p62 during the early stages of infection to avoid destruction, and further suggest that poxviruses exhibit a unique multi-layered control of autophagy in order to facilitate cytoplasmic replication.

sentences: MOLECULAR VIDEOGAMING: SUPER-RESOLVED TRAJECTORY-BASED NANOCLUSTERING ANALYSIS USING SPATIO-TEMPORAL INDEXING Single-molecule localization microscopy (SMLM) techniques are emerging as vital tools to unravel the nanoscale world of living cells. However, current analysis methods primarily focus on defining spatial nanoclusters based on detection density, but neglect important temporal information such as cluster lifetime and recurrence in "hotspots" on the plasma membrane. Spatial indexing is widely used in videogames to effectively detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to perform SMLM data analysis and determine whether the bounding boxes of individual molecular trajectories overlap, as a measure of their potential membership in nanoclusters. Extending the spatial indexing into the time dimension allows unique resolution of spatial nanoclusters into multiple spatiotemporal clusters. We have validated this approach using synthetic and SMLM-derived data. Quantitative characterization of recurring nanoclusters allowed us to demonstrate that both syntaxin1a and Munc18-1 molecules transiently cluster in hotspots on the neurosecretory plasma membrane, offering unprecedented insights into the dynamics of these protein which are essential to neuronal communication. This new analytical tool, named Nanoscale Spatiotemporal Indexing Clustering (NASTIC), has been implemented as a free and open-source Python graphic user interface.

sentences: Pan-cancer survey of tumour mass dormancy and underlying mutational processes Tumour mass dormancy is the key intermediate step between immune surveillance and cancer progression, yet due to its transitory nature it has been difficult to capture and characterise. Little is understood of its prevalence across cancer types and of the mutational background that may favour such a state. While this balance is finely tuned internally by the equilibrium between cell proliferation and cell death, the main external factors contributing to tumour mass dormancy are immunological and angiogenic. To understand the genomic and cellular context in which tumour mass dormancy may develop, we comprehensively profiled signals of immune and angiogenic dormancy in 9,631 cancers from the Cancer Genome Atlas and linked them to tumour mutagenesis. We find evidence for immunological and angiogenic dormancy-like signals in 16.5% of bulk sequenced tumours, with a frequency of up to 33% in certain tissues. Mutations in the CASP8 and HRAS oncogenes were positively selected in dormant tumours, suggesting an evolutionary pressure for controlling cell growth/apoptosis signals. By surveying the mutational damage patterns left in the genome by known cancer risk factors, we found that ageing-induced mutations were relatively depleted in these tumours, while patterns of smoking and defective base excision repair were linked with increased tumour mass dormancy. Furthermore, we identified a link between APOBEC mutagenesis and dormancy, which comes in conjunction with immune exhaustion and may partly depend on the expression of the angiogenesis regulator PLG as well as interferon and chemokine signals. Tumour mass dormancy also appeared to be impaired in hypoxic conditions in the majority of cancers. The microenvironment of dormant cancers was enriched in cytotoxic and regulatory T cells, as expected, but also in macrophages and showed a reduction in inflammatory Th17 signals. Finally, tumour mass dormancy was linked with improved patient survival outcomes. Our analysis sheds light onto the complex interplay between dormancy, exhaustion, APOBEC activity and hypoxia, and sets directions for future mechanistic explorations.

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Differential effects of soil-based agricultural diversification on arthropods and crop yield O_LIAgricultural diversification often promotes biodiversity and ecosystem services by increasing habitat diversity. However, responses to agricultural diversification are context dependent, differentially impacting functional groups of service-providing organisms and crop yields.
C_LIO_LIConservation and no tillage are promoted as agricultural diversification practices that increase soil heterogeneity and habitat diversity. Here we investigated whether soil tillage practices in canola crop fields altered arthropod biodiversity or yield, and how effects of field-scale diversification compared to landscape-scale habitat context. We focused on effects of high, medium, or no tillage on five functional groups with unique diets and reproductive strategies: (i) herbivores, (ii) kleptoparasites, (iii) parasitoids, (iv) pollinators, and (v) predators.
C_LIO_LIEffects of agricultural diversification on arthropod abundance and diversity varied across functional groups. Pollinators responded to on-farm soil diversification, benefiting from medium tillage. Predators and herbivores responded most strongly to landscape-scale habitat composition and were more abundant in landscapes with more semi-natural habitat. However, variation in arthropod communities had little effect on canola crop yield, which was lowest in fields with no tillage.
C_LIO_LIPolicy implications: Our results indicate that natural history differences among arthropod functional groups mediate how habitat availability affects biodiversity. Crop yields, however, showed no response to biodiversity of ecosystem service providers. Our research highlights the need to determine the contexts in which soil diversification practices meet a multi-faceted goal of simultaneously supporting natural biodiversity, ecosystem services, and crop yield.
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sentences: Variation in adaptation across geographic ranges is consistent with strong selection in marginal climates and legacies of range expansion Every species experiences limits to its geographic distribution. Some evolutionary models predict that populations at range edges are less well-adapted to their local environments due to drift, expansion load, or swamping gene flow from the range interior. Alternatively, populations near range edges might be uniquely adapted to marginal environments. In this study, we use a database of transplant studies that quantify performance at broad geographic scales to test how local adaptation, site quality, and population quality change from spatial and climatic range centers towards edges. We find that populations from poleward edges perform relatively poorly, both on average across all sites (15% lower population quality) and when compared to other populations at home (31% relative fitness disadvantage), consistent with these populations harboring high genetic load. Populations from equatorial edges also perform poorly on average (18% lower population quality) but, in contrast, outperform foreign populations (16% relative fitness advantage), suggesting that populations from equatorial edges have strongly adapted to unique environments. Finally, we find that populations from sites that are thermally extreme relative to the species niche demonstrate strong local adaptation, regardless of their geographic position. Our findings indicate that both nonadaptive processes and adaptive evolution contribute to variation in adaptation across species ranges.

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EMoMiS: A Pipeline for Epitope-based Molecular Mimicry Search in Protein Structures with Applications to SARS-CoV-2 MotivationEpitope-based molecular mimicry occurs when an antibody cross-reacts with two different antigens due to structural and chemical similarities. Molecular mimicry between proteins from two viruses can lead to beneficial cross-protection when the antibodies produced by exposure to one also react with the other. On the other hand, mimicry between a protein from a pathogen and a human protein can lead to auto-immune disorders if the antibodies resulting from exposure to the virus end up interacting with host proteins. While cross-protection can suggest the possible reuse of vaccines developed for other pathogens, cross-reaction with host proteins may explain side effects. There are no computational tools available to date for a large-scale search of antibody cross-reactivity.

ResultsWe present a comprehensive Epitope-based Molecular Mimicry Search (EMoMiS) pipeline for computational molecular mimicry searches. EMoMiS, when applied to the SARS-CoV-2 Spike protein, identified eight examples of molecular mimicry with viral and human proteins. These findings provide possible explanations for (a) differential severity of COVID-19 caused by cross-protection due to prior vaccinations and/or exposure to other viruses, and (b) commonly seen COVID-19 side effects such as thrombocytopenia and thrombophilia. Our findings are supported by previously reported research but need validation with laboratory experiments. The developed pipeline is generic and can be applied to find mimicry for novel pathogens. It has applications in improving vaccine design.

AvailabilityThe developed Epitope-based Molecular Mimicry Search Pipeline (EMoMiS) is available from https://biorg.cs.fiu.edu/emomis/.

Contactgiri@cs.fiu.edu

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ALG-097111, a potent and selective SARS-CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo efficacy in a Syrian Hamster model O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 M). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.

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Spontaneous alpha-band oscillations bias subjective contrast perception Perceptual decisions depend both on the features of the incoming stimulus and on the ongoing brain activity at the moment the stimulus is received. Specifically, trial-to-trial fluctuations in cortical excitability have been linked to fluctuations in the amplitude of pre-stimulus alpha oscillations ({approx}8-13 Hz), which are in turn are associated with fluctuations in subjects tendency to report the detection of a stimulus. It is currently unknown whether alpha oscillations bias post-perceptual decision making, or even bias subjective perception itself. To answer this question, we used a contrast discrimination task in which subjects reported which of two gratings - one in each hemifield - was perceived as having a stronger contrast. Our EEG analysis showed that subjective contrast was reduced for the stimulus in the hemifield represented in the hemisphere with relatively stronger pre-stimulus alpha amplitude, reflecting reduced cortical excitability. Furthermore, the strength of this spontaneous hemispheric lateralization was strongly correlated with the magnitude of individual subjects biases, suggesting that the spontaneous patterns of alpha lateralization play a role in explaining the intersubject variability in contrast perception. These results indicate that spontaneous fluctuations in cortical excitability, indicted by patterns of pre-stimulus alpha amplitude, affect perceptual decisions by altering the phenomenological perception of the visual world.

Significance StatementOur moment to moment perception of the world is shaped by the features of the environment surrounding us, as much as by the constantly evolving states that characterize our brain activity. Previous research showed how the ongoing electrical activity of the brain can influence whether a stimulus has accessed conscious perception. However, evidence is currently missing on whether these electrical brain states can be associated to the subjective experience of a sensory input. Here we show that local changes in patterns of electrical brain activity preceding visual stimulation can bias our phenomenological perception. Importantly, we show that the strength of these variations can help explaining the great inter-individual variability in how we perceive the visual environment surrounding us.

sentences: Impact of hedonic value of stimuli on sampling dynamics during a preference test The decision of whether to continue with a current action or to stop and consider alternatives is ever present in the life of an animal. Such continuous-time decision making lies at the heart of food preference tests whose outcomes are typically quantified by a single variable, the total amount consumed. However, the dynamics that give rise to such a quantity in terms of durations of bouts of sampling at a stimulus before pauses, and the impact of alternative stimuli on those bout durations and subsequent actions following a pause, can contain a richness of behavior that is not captured in a single palatability measure. Here we carry out multiple analyses of these dynamics, with a particular focus on assessing how the hedonic value of one taste stimulus impacts the behavior of a rat sampling a second taste stimulus during a preference test. We find evidence for an explicit competitive interaction between bout durations, such that the more palatable a stimulus the longer the bout durations when the rat samples the stimulus and the shorter the bout durations at the alternative. Such competition is reproduced in a model of a neural circuit that could underlie the continuous decision of when to end a sampling bout. We find that the competitive impact on bout durations is relatively short-lived whereas a competitive impact on the choice of which stimulus to approach following a pause persists. Such a discrepancy in the timescales for the decay of the impact of the alternative stimulus suggests different neural processes are involved in the choice of which stimulus to approach versus the choice of how long to sample from it. Since these two choices together combine to determine net consumption and therefore the inferred palatability or preference of a gustatory stimulus, our results suggest that palatability is not a unitary quantity but the result of at least two distinct, context-dependent neural processes.

sentences: Hydrophobic mismatch effect is a key factor in protein transport on the Tat pathway The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we assess the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.

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Diverse arsenic-containing lipids in the surface ocean Arsenic is present at nanomolar levels throughout the surface ocean, and microbes assimilate this toxic element due to its similarity to inorganic phosphorus. Although the concentration and characterization of dissolved arsenic has been a focus of ocean studies, the size of the particulate arsenic pool and its partitioning into organic molecules within the microbial community is not known. We measured the particulate pool of arsenic in five surface samples from the open ocean and determined the contribution of arsenic-containing lipids to this pool. Here we show that the accumulation of arsenic into lipids is a widespread phenomenon in the surface ocean. Particulate arsenic concentrations were 15 to 42 pmol L-1 with 7-20% of the particulate arsenic pool in the form of arsenolipids. We characterized these arsenolipids and found that arsenosugar phospholipids dominated the arsenolipid pools in our samples with a minor component of arsenohydrocarbons and other unidentified lipids. A significant portion of the arsenosugar phospholipids (up to 35%) were present as previously undescribed mixed acyl ether lipids, suggesting a bacterial source.

Scientific significance statementMarine microbes experience constant exposure to the toxic element arsenic. However, there are no baseline measurements of how much arsenic accumulates in microbial communities nor do we know the full spectrum of arsenic containing biomolecules produced in marine systems. In culture-based studies there is strong evidence that phytoplankton synthesize arsenic-containing lipids, but these lipids have not been observed in natural communities of marine microbes. Here we make measurements of bulk particulate arsenic at five sites in the surface open ocean and show that a significant portion of this particulate arsenic is present as complex arsenic-containing lipids. We characterize this arsenolipid pool chemically and quantitatively to show a variety of chemically distinct and quantitatively significant lipids that expand our understanding of marine arsenic biogeochemistry.

Data availability statementMass spectrometry data and its associated metadata will be available on Dryad with publication. Scripts for data processing and figure generation found on github at https://github.com/kheal/particulate_As_data_analysis.

sentences: A trade-off between stress resistance and tolerance underlies the adaptive response to hydrogen peroxide In response to environmental stress, cellular defense strategies may be divided into two categories: those, as in homeostatic systems, that seek to maintain cell proliferation by degrading the stressor (i.e., resistance); and those that ensure cell survival (i.e. tolerance), even if this is often at the expense of cell proliferation. In this study, we have explored the genetic bases of the antagonism between resistance and tolerance during the response to hydrogen peroxide (H2O2) in budding yeast. We show that inactivation of protein kinase A (PKA) by H2O2 signaling induces an abrupt transition from normal homeostatic function to a stress-tolerant state by protecting the growth machinery, hence maximizing cellular fitness in a changing environment. This model system paves the way for developing antiproliferative strategies that target both resistance and tolerance mechanisms to prevent relapse.

sentences: CAR modulates plasma membrane nano-organization and immune signaling downstream of RALF1-FERONIA ligand-receptor signaling system The highly selective segregation of molecules at the plasma membrane (PM) to form domains is a widespread phenomenon. But how distinct signaling platforms are established and maintained by specific signals remains unclear. Here, we show that the receptor of Rapid Alkalinization Factor 1 (RALF1), the FERONIA (FER) receptor kinase, physically interacts with C2 domain ABA-related (CAR) proteins to control the nano-organization of the PM and to regulate extracellular signal transduction in Arabidopsis. During this process, the RALF1-FER pathway upregulates CAR protein synthesis, which then controls the membrane lipid order. This might act as a rapid feedforward loop to stabilize FER in PM nanodomains. FER then interacts with and phosphorylates CARs, reducing their lipid-binding ability, which might break the feedback regulation and downregulate CAR activity at latter time point. Similar to fer mutant, a pentuple car14569 mutant inhibits flg22-induced FLS2-BAK1 complex formation, which ultimately impacts plant immunity. Together, we proposed that the FER-CAR module controls the dynamics of the PM nano-organization during RALF signaling through a self-contained amplifying loop including both positive and negative feedbacks.

sentences: Detecting expressed cancer somatic mutations from single-cell RNA sequencing data Identifying expressed somatic mutations directly from single-cell RNA sequencing (scRNA-seq) data is challenging but highly valuable. Computational methods have been attempted but no reliable methods have been reported to identify somatic mutations with high fidelity. We present RESA -- Recurrently Expressed SNV Analysis, a computational framework that identifies expressed somatic mutations from scRNA-seq data with high precision. We test RESA in multiple cancer cell line datasets, where RESA demonstrates average area under the curve (AUC) of 0.9 on independently held out test sets, and achieves average precision of 0.71 when evaluated by bulk whole exome, which is substantially higher than previous approaches. In addition, RESA detects a median of 201 mutations per cell, 50 times more than what was reported in experimental technologies with simultaneous expression and mutation profiling. Furthermore, applying RESA to scRNA-seq from a melanoma patient, we demonstrate that RESA recovers the known BRAF driver mutation of the sample and melanoma dominating mutational signatures, identifies mutation associated expression signatures, reveals nondriver perturbed and stage specific cancer hallmarks, and unveils the complex relationship between genomic and transcriptomic intratumor heterogeneity. Therefore, RESA could provide novel views in the study of intratumor heterogeneity and relate genetic alterations to transcriptional changes at single cell level.

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A mosaic bulk-solvent model improves density maps and the fit between model and data Bulk solvent is a major component of bio-macromolecular crystals and therefore contributes significantly to diffraction intensities. Accurate modeling of the bulk-solvent region has been recognized as important for many crystallographic calculations, from computing of R-factors and density maps to model building and refinement. Owing to its simplicity and computational and modeling power, the flat (mask-based) bulk-solvent model introduced by Jiang & Brunger (1994) is used by most modern crystallographic software packages to account for disordered solvent. In this manuscript we describe further developments of the mask-based model that improves the fit between the model and the data and aids in map interpretation. The new algorithm, here referred to as mosaic bulk-solvent model, considers solvent variation across the unit cell. The mosaic model is implemented in the computational crystallography toolbox and can be used in Phenix in most contexts where accounting for bulk-solvent is required. It has been optimized and validated using a sufficiently large subset of the Protein Data Bank entries that have crystallographic data available.

SynopsisA mosaic bulk-solvent method models disordered solvent more accurately than current flat bulk solvent model. This improves the fit between the model and the data, improves map quality and allows for the solution of problems previously inaccessible.

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Exploring the natural origins of SARS-CoV-2 The lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2, and the large geographical distance between Wuhan and where the closest evolutionary related coronaviruses circulating in horseshoe bats (Sarbecoviruses) have been identified, is fuelling speculation on the natural origins of SARS-CoV-2. We have comprehensively analysed phylogenetic relations between SARS-CoV-2, and the related bat and pangolin Sarbecoviruses sampled so far. Determining the likely recombination events reveals a highly reticulate evolutionary history within this group of coronaviruses. Clustering of the inferred recombination events is non-random with evidence that Spike, the main target for humoral immunity, is beside a recombination hotspot likely driving antigenic shift in the ancestry of bat Sarbecoviruses. Coupled with the geographic ranges of their hosts and the sampling locations, across southern China, and into Southeast Asia, we confirm horseshoe bats, Rhinolophus, are the likely SARS-CoV-2 progenitor reservoir species. By tracing the recombinant sequence patterns, we conclude that there has been relatively recent geographic movement and co-circulation of these viruses ancestors, extending across their bat host ranges in China and Southeast Asia over the last 100 years or so. We confirm that a direct proximal ancestor to SARS-CoV-2 is yet to be sampled, since the closest relative shared a common ancestor with SARS-CoV-2 approximately 40 years ago. Our analysis highlights the need for more wildlife sampling to (i) pinpoint the exact origins of SARS-CoV-2s animal progenitor, and (ii) survey the extent of the diversity in the related Sarbecoviruses phylogeny that present high risk for future spillover.

HighlightsO_LIThe origin of SARS-CoV-2 can be traced to horseshoe bats, genus Rhinolophus, with ranges in both China and Southeast Asia.
C_LIO_LIThe closest known relatives of SARS-CoV-2 exhibit frequent transmission among their Rhinolophus host species.
C_LIO_LISarbecoviruses have undergone extensive recombination throughout their evolutionary history.
C_LIO_LIAccounting for the mosaic patterns of these recombinants is important when inferring relatedness to SARS-CoV-2.
C_LIO_LIBreakpoint patterns are consistent with recombination hotspots in the coronavirus genome, particularly upstream of the pike open reading frame with a coldspot in S1.
C_LI

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Automated Modeling of Protein Accumulation at DNA Damage Sites using qFADD.py Cells are exposed to a plethora of influences that can cause damage to DNA and alter the genome, often with detrimental consequences for health. Cells mitigate this damage through a variety of repair protein pathways, and accurate measurement of the accumulation, action, and dissipation timescales of these repair proteins is required to fully understand the DNA damage response. Recently, we described the Q-FADD (Quantitation of Fluorescence Accumulation after DNA Damage) method, which enhances the analytical power of the widely used laser microirradiation technique. In that study, Q-FADD and its preprocessing operations required licensed software and a significant amount of user overhead to find the model of best fit. Here, we present "qFADD.py", an open-source implementation of the Q-FADD algorithm that is available as both a stand-alone software package and on a publicly accessible webserver (https://qfadd.colorado.edu/). Furthermore, we describe significant improvements to the fitting and preprocessing methods that include corrections for nuclear drift and an automated grid-search for the model of best fit. To improve statistical rigor, the grid-search algorithm also includes automated simulation of replicates. As an example, we discuss the recruitment dynamics of the signaling protein PARP1 to DNA damage sites, and we show how to compare different populations of qFADD.py models.

Statement of SignificanceCells are constantly bombarded by factors that can alter or damage their genome, and they have evolved a variety of proteins that can identify and fix this damage. To fully understand how these proteins interact in repair pathways, we need robust methods to quantify the timescales between the initial identification of the DNA damage event and the subsequent protein-protein interactions that lead to repair. Laser microirradiation is a popular method for studying these repair protein cascades in vivo, and methods for quantifying the timescales of recruitment in these experiments have historically been simple to implement but lacking in physical interpretation. Here, we present qFADD.py, the next iteration of the Q-FADD method, which uses Monte Carlo diffusion models to interpret repair protein recruitment timescales to sites of DNA damage. By moving towards automated fitting procedures with minimal bias from the user, qFADD.py provides a statistically robust but low-effort means to analyze laser microirradiation experiments through a biophysical framework.

sentences: Myeloid-to-mesenchymal NGF-p75 signaling coordinates skeletal cell migration during repair Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to re-innervation and osteogenesis is unknown. Here, we found that NGF is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to impact their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.

sentences: Modulation in Alpha Band Activity Reflects Syntax Composition: An MEG Study of Minimal Syntactic Binding Successful sentence comprehension requires the binding, or composition, of multiple words into larger structures to establish meaning. Using magnetoencephalography, we investigated the neural mechanisms involved in binding at the syntax level, in a task where contributions from semantics were minimized. Participants were auditorily presented with minimal sentences that required binding (pronoun and pseudo-verb with the corresponding morphological inflection; "she grushes") and pseudo-verb wordlists that did not require binding ("cugged grushes"). Relative to no binding, we found that syntactic binding was associated with a modulation in alpha band (8-12 Hz) activity in left-lateralized language regions. First, we observed a significantly smaller increase in alpha power around the presentation of the target word ("grushes") that required binding (-0.05s to 0.1s), which we suggest reflects an expectation of binding to occur. Second, during binding of the target word (0.15s to 0.25s), we observed significantly decreased alpha phase-locking between the left inferior frontal gyrus and the left middle/inferior temporal cortex, which we suggest reflects alpha-driven cortical disinhibition serving to strengthen communication within the syntax composition neural network. Together, our findings highlight the critical role of rapid spatial-temporal alpha band activity in controlling the allocation, transfer and coordination of the brains resources during syntax composition.

sentences: The YPhi Motif Defines the Structure-Activity Relationships of Human 20S Proteasome Activators The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesized and screened [~]120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (Y{Phi}) motif. To gain further insight, we created a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A.A cryo-EM structure of PA26E102A-h20S identified key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrated that the Y{Phi} sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.

sentences: Eye movements reveal spatiotemporal dynamics of visually-informed planning in navigation Goal-oriented navigation is widely understood to depend upon internal maps. Although this may be the case in many settings, humans tend to rely on vision in complex, unfamiliar environments. To study the nature of gaze during visually-guided navigation, we tasked humans to navigate to transiently visible goals in virtual mazes of varying levels of difficulty, observing that they took near-optimal trajectories in all arenas. By analyzing participants eye movements, we gained insights into how they performed visually-informed planning. The spatial distribution of gaze revealed that environmental complexity mediated a striking trade-off in the extent to which attention was directed towards two complimentary aspects of the world model: the reward location and task-relevant transitions. The temporal evolution of gaze revealed rapid, sequential prospection of the future path, evocative of neural replay. These findings suggest that the spatiotemporal characteristics of gaze during navigation are significantly shaped by the unique cognitive computations underlying real-world, sequential decision making.

sentences: Mesophotic gorgonian corals evolve multiple times and faster than deep and shallow lineages 1.Mesophotic Coral Ecosystems (MCEs) develop on a unique environment, where abrupt environmental changes take place. Using a time-calibrated molecular phylogeny (mtDNA: mtMutS), we examined the lineage membership of mesophotic gorgonian corals (Octocorallia: Cnidaria) in comparison to shallow and deep-sea lineages of the wider Caribbean-Gulf of Mexico and the Tropical Eastern Pacific. Our results show mesophotic gorgonians originating multiple times from old deep-sea octocoral lineages, whereas shallow-water species comprise younger lineages. The mesophotic gorgonian fauna in the studied areas are related to their zooxanthellate shallow-water counterparts in only two clades (Gorgoniidae and Plexauridae), where the shallow-deep gradient could serve as a driver of diversification. Interestingly, mesophotic clades have diversified faster than either shallow or deep clades. One of this groups with fast diversification is the family Ellisellidae, a major component of the mesophotic gorgonian coral assemblage worldwide.

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Platinum nanoparticles inhibit intracellular ROS generation and protect against Cold Atmospheric Plasma-induced cytotoxicity Platinum nanoparticles (PtNPs) have been investigated for their antioxidant abilities in a range of biological and other applications. The ability to reduce off-target CAP cytotoxicity would be useful in Plasma Medicine, however, little has been published to date about the ability of PtNPs to reduce or inhibit the effects of CAP. Here we investigate whether PtNPs can protect against CAP-induced cytotoxicity in cancerous and non-cancerous cell lines. PtNPs were shown to dramatically reduce intracellular reactive species (RONS) production in human U-251 MG cells. However, RONS generation was unaffected by PtNPs in medium without cells. PtNPs protect against CAP induced mitochondrial membrane depolarization, but not cell membrane permeabilization which is a CAP-induced RONS-independent event. PtNPs act as potent intracellular scavengers of reactive species and can protect both cancerous U-251 MG cells and non-cancerous HEK293 cells against CAP induced cytotoxicity. PtNPs may be useful as a catalytic antioxidant for healthy tissue and for protecting against CAP-induced tissue damage.

Graphical AbstractPtNPs are potent catalase and superoxide dismutase mimetics which makes them strong antioxidant candidates for the protection of cells against oxidative stress. CAP was generated using a Dielectric Barrier Device (DBD) system with a voltage output of 75 kV at a frequency of 50 Hz. A range of concentrations of 3nm uncoated PtNPs combined with CAP were examined in human U-251 MG Glioblastoma (GBM) cells and non-cancerous human embryonic kidney HEK293 cells. The protective effects of PtNPs against CAP were explored using several biochemical indicators of oxidative stress and cytotoxicity.

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sentences: Asymmetric directed functional connectivity within the frontoparietal motor network during motor imagery and execution Both imagery and execution of motor controls consist of interactions within a neuronal network, including frontal motor-related regions and posterior parietal regions. To reveal neural representation in the frontoparietal motor network, two approaches have been proposed thus far: one is decoding of actions/modes related to motor control from the spatial pattern of brain activity; another is to estimate directed functional connectivity, which means a directed association between two brain regions within motor regions. However, directed connectivity among multiple regions of the motor network during motor imagery (MI) or motor execution (ME) has not been investigated. Here, we attempted to characterize the directed functional connectivity within the frontoparietal motor-related networks between the MI and ME conditions. We developed a delayed sequential movement and imagery task to evoke brain activity associated with data under ME and MI via functional magnetic resonance imaging scanning. We applied a causal discovery approach, linear non-Gaussian acyclic causal model, to identify directed functional connectivity among the frontoparietal motor-related brain regions for each condition. We demonstrated higher directed functional connectivity from the contralateral dorsal premotor cortex (dPMC) to the primary motor cortex (M1) in ME than in MI. We mainly identified significant direct effects of the dPMC and ventral premotor cortex (vPMC) to the parietal regions. In particular, connectivity from the dPMC to the superior parietal lobule in the same hemisphere showed significant positive effects across all conditions. Contrastingly, interlateral connectivities from the vPMC to the superior parietal lobule showed significantly negative effects across all conditions. Finally, we found positive effects from A1 to M1 in the same hemisphere, such as the audio-motor pathway. These results indicated that the sources of motor command originated in d/vPMC influenced M1 and parietal regions as achieving ME and MI. Additionally, sequential sounds may functionally facilitate temporal motor processes.

sentences: Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter VcINDY, the sodium-dependent dicarboxylate transporter from Vibrio cholerae, is responsible for C4- and C5- carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predictions of transport cycle mechanistic models. Our data provide the first direct evidence that VcINDY undergoes stochastic, elevator-type conformational motions that enable substrate translocation. Kinetic analysis suggests that the two protomers of the VcINDY homodimer undergo those motions in a non-cooperative manner, and thus catalyze two independent transport reactions. The relative substrate independence of those motions provides evidence that the VcINDY transport cycle maintains strict co-substrate coupling using a cooperative binding mechanism. Finally, thermodynamic modeling provides insight into how such a cooperative binding mechanism provides a generalized approach to optimizing transport for many secondary active transporters.

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High performance single-cell gene regulatory network inference atscale: The Inferelator 3.0 MotivationGene regulatory networks define regulatory relationships between transcription factors and target genes within a biological system, and reconstructing them is essential for understanding cellular growth and function. Methods for inferring and reconstructing networks from genomics data have evolved rapidly over the last decade in response to advances in sequencing technology and machine learning. The scale of data collection has increased dramatically; the largest genome-wide gene expression datasets have grown from thousands of measurements to millions of single cells, and new technologies are on the horizon to increase to tens of millions of cells and above.

ResultsIn this work, we present the Inferelator 3.0, which has been significantly updated to integrate data from distinct cell types to learn context-specific regulatory networks and aggregate them into a shared regulatory network, while retaining the functionality of the previous versions. The Inferelator is able to integrate the largest single-cell datasets and learn cell-type specific gene regulatory networks. Compared to other network inference methods, the Inferelator learns new and informative Saccharomyces cerevisiae networks from single-cell gene expression data, measured by recovery of a known gold standard. We demonstrate its scaling capabilities by learning networks for multiple distinct neuronal and glial cell types in the developing Mus musculus brain at E18 from a large (1.3 million) single-cell gene expression dataset with paired single-cell chromatin accessibility data.

AvailabilityThe inferelator software is available on GitHub (https://github.com/flatironinstitute/inferelator) under the MIT license and has been released as python packages with associated documentation (https://inferelator.readthedocs.io/).

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Facilitation of neural responses to targets moving against optic flow For the human observer, it can be difficult to follow the motion of small objects, especially when they move against background clutter. In contrast, insects efficiently do this, as evidenced by their ability to capture prey, pursue conspecifics, or defend territories, even in highly textured surrounds. We here recorded from target selective descending neurons (TSDNs) which likely subserve these impressive behaviors. To simulate the type of background optic flow that would be generated by the pursuers own movements through the world, we used the coherent motion of a perspective distorted sparse dot field. We show that hoverfly TSDN responses to target motion are suppressed when such background optic flow moves in the same direction as the target. Indeed, the neural responses are strongly attenuated against both translational sideslip as well as rotational yaw. More strikingly, we show that TSDNs are facilitated by background optic flow in the opposite direction to the target, if the target moves horizontally. Furthermore, we show that a small, frontal spatial window of background optic flow is enough to fully facilitate or attenuate TSDN responses to target motion. We argue that the TSDN response facilitation could be beneficial in modulating corrective turns during target pursuit.

Significance statementTarget detection in visual clutter is a difficult computational task that insects, with their poor resolution compound eyes and small brains, do successfully and with extremely short behavioral delays. We here show that the responses of target selective descending neurons are attenuated by background motion in the same direction as target motion, but facilitated by opposite direction background motion. This finding is important for understanding conspecific pursuit behavior, since these descending neurons likely control behavioral output. The facilitation that we describe would come into effect if the hoverfly is subjected to background motion in one direction, but the target it is pursuing moves in the opposite direction, and could therefore be used to modulate gaze stabilizing corrective turns.

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Single molecule studies reveal branched pathways for activator-dependent pre-initiation complex assembly RNA polymerase II (Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.

HIGHLIGHTSSingle molecule microscopy reveals unexpected dynamics of RNA Pol II and GTFs

Multiple Pol IIs cluster on UAS/enhancer-bound activators before binding the core promoter

Pol II, TFIIF, and TFIIE, but not TFIIH, can pre-assemble at the UAS/enhancer

Activators increase the rates of Pol II and GTF association with DNA

eTOC BlurbSingle-molecule microscopy experiments by Baek et al. show that RNA polymerase II and basal transcription factors TFIIF and TFIIE preassemble on UAS/enhancer-bound activators, poised for loading into initiation complexes with TFIIH at the core promoter. Transcription activators kinetically enhance factor recruitment, creating a localized cluster of polymerases at the UAS/enhancer.

sentences: The Autism Risk Factor CHD8 Is a Chromatin Activator and Functionally Dependent on the ERK-MAPK Pathway Effector ELK1 The chromodomain helicase DNA-binding protein CHD8 is among the most frequently found de-novo mutations in autism spectrum disorder (ASD)1-4. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is believed to be a chromatin regulator, but mechanisms for its genomic targeting is also unclear. To elucidate the role of CHD8 in human neurons, we generated conditional loss-of-function alleles in pluripotent stem cells. Chromatin accessibility and transcriptional profiling showed that CHD8 is a potent chromatin opener and transcriptional activator of its direct neuronal targets, including a distinct group of ASD genes. We found the chromatin targeting of CHD8 to be highly context-dependent. In human neurons, CHD8 was preferentially bound at promoter sequences which were significantly enriched in ETS motifs. Indeed, the chromatin state of ETS motif-containing promoters was preferentially affected upon loss of CHD8. Among the many ETS transcription factors, we found ELK1 to be the best correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our ES cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motifcontaining sites. These findings imply the functional cooperativity between ELK1, a key downstream factor of the MAPK/ERK pathway, and CHD8 on chromatin involvement in human neurons. THEREFORE, the MAPK/ERK/ELK1 axis may also play a role in the pathogenesis caused by CHD8 mutations 5.

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STUB1 is an intracellular checkpoint for interferon gamma sensing Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance.

Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFN{gamma}) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFN{gamma} response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition.

Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models - with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFN{gamma} sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.

sentences: Claudin5 protects the peripheral endothelial barrier in an organ and vessel type-specific manner The pathogenesis of numerous diseases is characterised by disruption of the junctions that form the endothelial cell (EC) barrier, the composition of which may differ greatly between organs. However, the expression level variability and precise contribution of different junctional proteins is poorly understood. Here, we focus on organs with continuous endothelium to identify structural and functional in vivo characteristics of the EC barrier. Assembly of multiple single-cell RNAseq datasets into a single integrated database revealed the variability in EC barrier patterning. Across tissues Claudin5 exhibited diminishing expression along the arteriovenous axis, which correlates with EC barrier integrity. Functional analysis identified tissue-specific differences in leakage patterning and response to agonist-induced leakage. We uncover that Claudin5 loss enhances agonist-induced leakage in an organotypic, vessel type-specific and size-selective manner in an inducible, EC-specific, knock-out mouse. Mechanistically, Claudin5 loss induces no change in junction ultrastructure but alters composition, with concomitant loss of zonula occludens-1 (ZO-1) expression and upregulation of VE-Cadherin. These findings uncover the organ-specific organisation of the EC barrier and distinct importance of Claudin5 in different vascular beds and will aid our ability to modify EC barrier stability in a targeted, organ-specific manner.

sentences: G-Protein coupled Purinergic P2Y12 receptor interacts and internalizes TauRD-mediated by membrane-associated actin cytoskeleton remodelling in microglia In Alzheimers disease, the microtubule-associated protein, Tau misfolds to form aggregates and filaments in the intra- and extracellular region of neuronal cells. Microglial cells are the resident brain macrophage cells that are involved in constant surveillance and are activated by the extracellular deposits. Purinergic receptors are involved in chemotactic migration of microglial cells towards the site of inflammation. In our recent study, we found that microglial P2Y12 receptor has been involved in phagocytosis of full-length Tau species such as monomers, oligomers and aggregates by actin-driven chemotaxis. In this study, we have showed the interaction of repeat-domain of Tau (TauRD) with microglial P2Y12 receptor and analysed the corresponding residues for interaction by various in-silico approaches. In cellular studies, TauRD was found to interact with microglial P2Y12R and induces its cellular expression as confirmed by coimmunoprecipitation and western blot analysis respectively. Similarly, immunofluorescence microscopic studies emphasized that TauRD were phagocytosed by microglial P2Y12R via the membrane-associated actin remodelling as filopodia extension. Furthermore, the P2Yl2R-mediated TauRD internalization has activated the microglia with an increase in the Iba1 level and TauRD become accumulated at peri-nuclear region as localized with Iba1. Altogether, microglial P2Y12R interacts with TauRD and mediates directed migration and activation for its internalization.

sentences: Within-cycle instantaneous frequency profiles report oscillatory waveform dynamics. Non-sinusoidal waveform is emerging as an important feature of neuronal oscillations. However, the role of single cycle shape dynamics in rapidly unfolding brain activity remains unclear. Here, we develop an analytical framework that isolates oscillatory signals from time-series using masked Empirical Mode Decomposition to quantify dynamical changes in the shape of individual cycles (along with amplitude, frequency and phase) using instantaneous frequency. We show how phase-alignment, a process of projecting cycles into a regularly sampled phase-grid space, makes it possible to compare cycles of different durations and shapes.  Normalised shapes can then be constructed with high temporal detail whilst accounting for differences in both duration and amplitude. We find that the instantaneous frequency tracks non-sinusoidal shapes in both simulated and real data. Notably, in local field potential recordings of mouse hippocampal CA1, we find that theta oscillations have a stereotyped slow-descending slope in the cycle-wise average, yet exhibiting high variability on a cycle-by-cycle basis. We show how Principal Components Analysis allows identification of motifs of theta cycle waveform that have distinct associations to cycle amplitude, cycle duration and animal movement speed. By allowing investigation into oscillation shape at high temporal resolution, this analytical framework will open new lines of enquiry into how neuronal oscillations support moment-by-moment information processing and integration in brain networks.

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Cortical facilitation of somatosensory inputs using gravity-related tactile information in patients with bilateral vestibular loss A few years after their bilateral vestibular loss, individuals usually show a motor repertoire that is almost back to normal. This recovery is thought to involve an up-regulation of the visual and proprioceptive information that compensates for the lack of vestibular information. Here, we investigated whether plantar tactile inputs, which provide body information relative to the ground and to the Earth-vertical, contribute to this compensation. More specifically, we tested the hypothesis that somatosensory cortex response to electric stimulation of the plantar sole in standing adults will be greater in patients (n = 10) with bilateral vestibular loss than in an aged-matched healthy group (n = 10). Showing significant greater somatosensory evoked potentials (i.e., P1N1) in patients than in controls, the electroencephalographic recordings supported this hypothesis. Furthermore, we found evidence that increasing the differential pressure between both feet, by adding a 1 kg mass at each pending wrist, enhanced the internal representation of body orientation and motion relative to a gravitational reference frame. The large decreased in alpha/beta power in the right posterior parietal cortex (and not in the left) is in line with this assumption. Finally, our behavioral analyses showed smaller body sway oscillations for patients, likely originated from a tactile-based control strategy. Conversely, healthy subjects showed smaller head oscillations suggesting a vestibular-based control strategy, the head serving as a reference for balance control.

HighlightsO_LISomatosensory cortex excitability is greater in patients with bilateral vestibular loss than in aged-matched healthy individuals
C_LIO_LITo control balance, healthy individuals "locked" the head while vestibular patients "locked" their pelvis
C_LIO_LIFor vestibular patients, increasing loading/unloading mechanism enhances the internal representation of body state in the posterior parietal cortex
C_LI

sentences: Tracking chromatin state changes using μMap photo-proximity labeling Interactions between biomolecules, particularly proteins, underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects.1,2 Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health.1 However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study.3 Chromatin remodelers, modifying enzymes, interactors, and transcription factors can all be redirected by subtle changes to the microenvironment, causing global changes in protein expression levels and subsequent physiology. Here, we describe the Chroma-Map method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment for analysis via quantitative chemoproteomics.4 We demonstrate this concept on nine different nuclear proteins with varied function and in each case, elucidating their microenvironments. Additionally, we show that this short-range proximity labeling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. Chroma-Map improves our fundamental understanding of nuclear protein-protein interactions, as well as the effects that small molecule therapeutics have on the local chromatin environment, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.

sentences: Novel functional sequences uncovered through a bovine multi-assembly graph Linear reference genomes are typically assembled from single individuals. They are unable to reflect the genetic diversity of populations and lack millions of bases. To overcome such limitations and make non-reference sequences amenable to genetic investigations, we build a multi-assembly graph from six reference-quality assemblies from taurine cattle and their close relatives. We uncover 70,329,827 bases that are missing in the bovine linear reference genome. The missing sequences encode novel transcripts that are differentially expressed between individual animals. Reads which were previously poorly or unmapped against the bovine reference genome now align accurately to the non-reference sequences. We show that the non-reference sequences contain polymorphic sites that segregate within and between breeds of cattle. Our efforts to uncover novel functional sequences from a multi-assembly graph pave the way towards the transition to a more representative bovine reference genome.

sentences: The DNA-based global positioning system--a theoretical framework for large-scale spatial genomics We present GPS-seq, a theoretical framework that enables massively scalable, optics-free spatial transcriptomics. GPS-seq combines data from high-throughput sequencing with manifold learning to obtain the spatial transcriptomic landscape of a given tissue section without optical microscopy. In this framework, similar to technologies like Slide-seq and 10X Visium, tissue samples are stamped on a surface of randomly-distributed DNA-barcoded spots (or beads). The transcriptomic sequences of proximal cells are fused to DNA barcodes, enabling the recovery of a transcriptomic pixel image by high-throughput sequencing. The barcode spots serve as "anchors" which also capture spatially diffused "satellite" barcodes, and therefore allow computational reconstruction of spot positions without optical sequencing or depositing barcodes to pre-specified positions. In theory, it could generate 100 mm x 100 mm spatial transcriptomic images with 10-20 m resolution by localizing 108 DNA-barcoded pixels with a single Illumina NovaSeq run. The general framework of GPS-seq is also compatible with standard single-cell (or single-nucleus) capture methods, and any modality of single-cell genomics, such as sci-ATAC-seq, could be transformed into spatial genomics in this strategy. We envision that GPS-seq will lead to breakthrough discoveries in diverse areas of biology by enabling organ-scale imaging of multiple genomic statuses at single-cell resolution for the first time.

sentences: Regulatory dissection of the severe COVID-19 risk locus introgressed by Neanderthals Individuals infected with the SARS-CoV-2 virus present with a wide variety of phenotypes ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe COVID-19 phenotype. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the severe COVID-19 phenotype. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform an locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify 4 introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. These variants likely drive the locus impact on severity by putatively modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

sentences: Multiplexed Single-Molecule Epigenetic Analysis of Plasma-Isolated Nucleosomes for Cancer Diagnostics The analysis of cell-free DNA (cfDNA) in plasma represents a rapidly advancing field in medicine, providing information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed EPINUC, a single-molecule multi-parametric assay to comprehensively profile the Epigenetics of Plasma Isolated Nucleosomes, DNA methylation and cancer-specific protein biomarkers. Our system allows high-resolution detection of six active and repressive histone modifications, their ratios and combinatorial patterns, on millions of individual nucleosomes by single-molecule imaging. In addition, it provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins such as mutant p53. Applying this analysis to a cohort of plasma samples detected colorectal cancer at high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue-of-origin of the tumor. EPINUC provides multi-layered clinical-relevant information from limited liquid biopsy material, establishing a novel approach for cancer diagnostics.

sentences: Emotional states as distinct configurations of functional brain networks The conceptualization of emotional states as patterns of interactions between large-scale brain networks has recently gained support. Yet, few studies have directly examined the brains network structure during emotional experiences. Here, we investigated the brains functional network organization during experiences of sadness, amusement, and neutral states elicited by movies, in addition to a resting-state. We tested the effects of the experienced emotion on individual variability in the brains functional connectome. Next, for each state, we defined a community structure of the brain and quantified its segregation and integration. Our results show that emotional states increase the similarity between and within individuals in the brains functional connectome. Second, in the brains modular organization, sadness, relative to amusement, was associated with higher modular integration and increased connectivity of cognitive control networks: the salience and fronto-parietal networks. Last, in both the functional connectome and the emotional report, we found higher similarity among women. Our results suggest that the experience of emotion is linked to a reconfiguration of whole-brain distributed, not emotion-specific, functional networks and that the brains topological structure carries information about the subjective emotional experience.

sentences: In vitro analysis of the effects of acetaminophen, ibuprofen and aspirin on motility of female adult Onchocerca volvulus worms In view of the very ambitious global timelines for elimination of onchocerciasis in 2030, the search for alternative antifilarials cannot depend on drug development from scratch, and repurposed drugs offer cheaper and faster alternatives. Previous studies had demonstrated the presence and potential expression of amidase, an enzyme that can be targeted by repurposed analgesics, in Onchocerca volvulus worms. The aim of this study was to determine the effects of acetaminophen, ibuprofen and aspirin on the motility of adult O. volvulus worms. In total, thirty (30) female O. volvulus worms were exposed to acetaminophen, ibuprofen and aspirin in concentrations of either 5mg/ml, 2.5mg/ml, 1.25mg/ml or 0.63mg/ml control in duplicates. Worm motility was observed and recorded using the WormAssay software and a darkfield imaging apparatus starting on day 2 of incubation and ending on day 8. Acetaminophen, ibuprofen and aspirin inhibited O. volvulus motility by 2-fold compared to control in the first 24 hours of drug exposure. However, an extended exposure of the worms to these test drugs rather improved the motility of the worms. The study has demonstrated that a 24-hour exposure of O. volvulus worms to the analgesic drugs studied, results in significant inhibition in worm motility compared with the control group, but extended duration of exposure led to an enhancement in motility of the previously immobile worms. This finding supports the idea that aspirin and ibuprofen may have some longevity enhancing properties. Further research on the utility of these analgesics as possible anti-filarial drugs is thus warranted.

sentences: An open source statistical and data processing toolbox for wide-field optical imaging in mice Wide-field optical imaging (WOI) produces concurrent hemodynamic and cell-specific calcium recordings across the entire cerebral cortex. There have been multiple studies using WOI to image mouse models with various environmental or genetic manipulations to understand various diseases. Despite the obvious utility of pursuing mouse WOI alongside human functional magnetic resonance imaging (fMRI), and the multitude of analysis toolboxes in the fMRI literature, there is not an available open-source, user-friendly data processing and statistical analysis toolbox for WOI data. Here, we present our MATLAB toolbox for pre-processing WOI data, as described and adapted to combine processing techniques from multiple WOI groups. Additionally, we provide multiple data analysis packages and translate two commonly used statistical approaches from the fMRI literature to the WOI data. To illustrate the utility, we demonstrate the ability of the processing and analysis framework to detect a well-established deficit in a mouse model of stroke. Additionally, we evaluate resting state data in healthy mice.

sentences: Spinal neurons innervating multiple local and distant motor pools Motor neurons control muscle contractions, and their recruitment by premotor circuits is tuned to produce accurate motor behaviours. To understand how these circuits coordinate movement across and between joints, it is necessary to understand whether spinal neurons pre-synaptic to motor pools, project to more than one motor neuron population. Here, we used modified rabies virus tracing in mice to investigate premotor INs projecting to synergist flexor or extensor motor neurons, as well as those projecting to antagonist pairs of muscles controlling the ankle joint. We show that similar proportions of premotor neurons diverge to agonist and antagonist motor pools. Divergent premotor neurons were seen throughout the spinal cord, with decreasing numbers but increasing proportion with distance from the hindlimb enlargement. In the cervical cord, divergent long descending propriospinal neurons were found in contralateral lamina VIII, had large somata, were excitatory, projected to both lumbar and cervical motoneurons, and were at least in part of the V0 class. We conclude that distributed spinal premotor neurons coordinate activity across multiple motor pools and that there are spinal neurons mediating co-contraction of antagonist muscles.

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Mycena species can be opportunist-generalist plant root invaders Recently, several saprotrophic genera have been found to invade/interact with plant roots in laboratory growth experiments, and this coincides with reports of abundant saprotrophic fungal sequences in plant roots. However, it is uncertain if this reflects field phenomena, and if reports on coincidentally amplified saprotrophs are simply coincidental.

We investigated root invasion by presumed saprotrophic fungi by focusing on the large genus Mycena in 1) a systematic analysis of the occurrence of saprotrophic fungi in new and previously published ITS1/ITS2 datasets generated from roots of 10 mycorrhizal plant species, and 2) we analysed natural abundances of 13C/15N stable isotope signatures of fungal/plant communities from five comparable field locations to examine the trophic status of Mycena species.

Mycena was the only saprotrophic genus consistently found in 9 of 10 plant host roots, with high within-host variation in Mycena sequence proportions (0-80%) recovered. Mycena carpophores displayed isotopic signatures consistent with published 13C/15N profiles of both saprotrophic or mutualistic lifestyles, with considerable intraspecific variation, resembling the patterns seen in growth experiments. These results indicate that multiple Mycena species opportunistically invade the roots of a range of plant species, possibly forming a spectrum of interactions. This potentially challenges our general understanding of fungal ecology.

Originality significance statementThis is the first study to apply a dual approach of systematic metabarcoding of plant roots and stable isotope signatures on dried field material to the large and common saprotrophic fungal genus Mycena. This is significant as it shows that members of this genus, normally not expected to be found inside plant roots at all, are in fact associated eith multiple plant hosts. The study furthermore shows that species in this genus may occupy different ecological roles in the field besides being saprotrophic. That a large and common fungal genus known to be a quantitatively important litter decayer can be an opportunistic root invader and interact with host plants is of interest to all mycologists and ecologists working on plant-fungus/microb symbiosis.

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DeepUMQA: Ultrafast Shape Recognition-based Protein Model Quality Assessment using Deep Learning MotivationProtein model quality assessment is a key component of protein structure prediction. In recent research, the voxelization feature was used to characterize the local structural information of residues, but it may be insufficient for describing residue-level topological information. Design features that can further reflect residue-level topology when combined with deep learning methods are therefore crucial to improve the performance of model quality assessment.

ResultsWe developed a deep-learning method, DeepUMQA, based on Ultrafast Shape Recognition (USR) for the residue-level single-model quality assessment. In the framework of the deep residual neural network, the residue-level USR feature was introduced to describe the topological relationship between the residue and overall structure by calculating the first moment of a set of residue distance sets and then combined with 1D, 2D, and voxelization features to assess the quality of the model. Experimental results on test datasets of CASP13, CASP14, and CAMEO show that USR could complement the voxelization feature to comprehensively characterize residue structure information and significantly improve the model assessment accuracy. DeepUMQA outperformed the state-of-the-art single-model quality assessment methods, including ProQ2, ProQ3, ProQ3D, Ornate, VoroMQA, and DeepAccNet.

AvailabilityThe source code and executable are freely available at https://github.com/iobio-zjut/DeepUMQA.

Contactzgj@zjut.edu.cn

sentences: Use of an enclosed elk population to assess two non-invasive methods for estimating population size Non-invasive genetic sampling and spatially explicit capture-recapture (SCR) models are used increasingly to estimate abundance of wildlife populations, but have not been adequately tested on gregarious animals such as elk (Cervus canadensis), for which correlated space use and movements violate model assumptions of independence. To evaluate the robustness and accuracy of SCR, and to assess the utility of an alternative non-invasive method for estimating density of gregarious ungulates, we utilized a tule elk (Cervus canadensis nannodes) population of known size within a fenced enclosure on the San Luis National Wildlife Refuge in central California. We evaluated fecal genetic SCR to camera trap-based random encounter model (REM) approaches to density estimation based on comparison to the true abundance. We also subsampled the dataset to explore the effects of varying search effort and elk density on the precision and accuracy of results. We found that SCR outperformed REM methods in the full datasets, and reliably provided accurate (relative bias <10%) and reasonably precise (relative standard error [≤]20%) estimates of density at moderately low to high densities (6-17 elk/km2), when the subsampling scenarios yielded a minimum average of 20 recaptures. We also found that the number of samples used to construct detection histories was a reliable predictor of precision, and could be used to establish minimum sampling requirements in future population surveys of elk. Although field-testing in free-ranging populations is needed, our results suggest that non-invasive genetic SCR is a promising tool for future population studies and monitoring of elk and potentially other gregarious ungulates. In contrast, the REM estimate of density was highly inaccurate, imprecise, and highly sensitive to camera parameters.

sentences: Interphotoreceptor matrix proteoglycans IMPG1 and IMPG2 proteolyze in the SEA domain and reveal localization mutual dependency The interphotoreceptor matrix (IPM) is a specialized extracellular mesh of molecules surrounding the inner and outer segments of the photoreceptor neurons. The interphotoreceptor matrix proteoglycan 1 and 2 (IMPG1 and IMPG2) are major components of the IPM. Both proteoglycans possess SEA (Sperm protein, Enterokinase and Agrin) domains, which are described to support protein proteolysis. Interestingly, humans with mutations in the SEA domains of IMPG1 and IMPG2 are associated with retinitis pigmentosa (RP) vision disease. This work investigates the SEA-mediated proteolysis of IMPG1 and IMPG2 and its significance to IPM physiology. Western blot relative molecular mobility analysis of IMPG1 from retina confirmed protein proteolysis. Point mutations in the SEA domain of IMPG1 inhibit protein proteolysis, as shown in cell culture assays. Likewise, IMPG2 also proteolyzes at the SEA domain generating two subunits: a membrane-attached and an extracellular peptide, as seen by western blot and immunohistochemical assays. Importantly, the extracellular portion of IMPG2 traffics toward the outer segment IPM by a mechanism dependent on IMPG1. These results demonstrate that proteolysis is part of the maturation of IMPG1 and IMPG2, which is important for protein localization and linked to vision diseases. Moreover, the results indicate interdependency between IMPG1 and IMPG2, which helps understand the molecular mechanisms involved in the development of vision diseases in patients with defective IMPG molecules.

sentences: Highly potent anti-SARS-CoV-2 multivalent DARPin therapeutic candidates Globally accessible therapeutics against SARS-CoV-2 are urgently needed. Here, we report the generation of the first anti-SARS-CoV-2 DARPin molecules with therapeutic potential as well as rapid large-scale production capabilities. Highly potent multivalent DARPin molecules with low picomolar virus neutralization efficacies were generated by molecular linkage of three different monovalent DARPin molecules. These multivalent DARPin molecules target various domains of the SARS-CoV-2 spike protein, thereby limiting possible viral escape. Cryo-EM analysis of individual monovalent DARPin molecules provided structural explanations for the mode of action. Analysis of the protective efficacy of one multivalent DARPin molecule in a hamster SARS-CoV-2 infection model demonstrated a significant reduction of pathogenesis. Taken together, the multivalent DARPin molecules reported here, one of which has entered clinical studies, constitute promising therapeutics against the COVID-19 pandemic.

sentences: Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction Precise control of gene expression underpins normal development. This relies on mechanisms that enable communication between gene promoters and other regulatory elements. In embryonic stem cells (ESCs), the CDK-Mediator (CDK-MED) complex has been reported to physically link gene regulatory elements to enable gene expression and also prime genes for induction during differentiation. Here we discover that CDK-MED contributes little to 3D genome organisation in ESCs, but has a specific and essential role in controlling interactions between inactive gene regulatory elements bound by Polycomb repressive complexes (PRCs). These interactions are established by the canonical PRC1 (cPRC1) complex but rely on CDK-MED, which facilitates binding of cPRC1 to its target sites. Importantly, through separation of function experiments, we reveal that this collaboration between CDK-MED and cPRC1 in creating long-range interactions does not function to prime genes for induction during differentiation. Instead, we discover that priming relies on an interaction-independent mechanism whereby the CDK module supports core Mediator engagement with gene promoters to enable gene activation.

sentences: Dynamic interplay between reward and voluntary attention determines stimulus processing in visual cortex Reward enhances stimulus processing in the visual cortex, but the mechanisms through which this effect occurs remain unclear. Reward prospect can both increase the deployment of voluntary attention and increase the salience of previously neutral stimuli. In this study we orthogonally manipulated reward and voluntary attention while human participants performed a global motion detection task. We recorded steady-state visual evoked potentials (SSVEPs) to simultaneously measure the processing of attended and unattended stimuli linked to different reward probabilities, as they compete for attentional resources. The processing of the high rewarded feature was enhanced independently of voluntary attention, but this gain diminished once rewards were no longer available. Neither the voluntary attention nor the salience account alone can fully explain these results. Instead, we propose how these two accounts can be integrated to allow for the flexible balance between reward-driven increase in salience and voluntary attention.

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Group-walk, a rigorous approach to separate FDR analysis by TDC Target-decoy competition (TDC) is a commonly used method for false discovery rate (FDR) control in the analysis of tandem mass spectrometry data. This type of competitionbased FDR control has recently gained significant popularity in other fields after Barber and Candes laid its theoretical foundation in a more general setting that included the feature selection problem. In both cases, the competition is based on a head-to-head comparison between an (observed) target score and a corresponding decoy (knockoff) score. However, the effectiveness of TDC depends on whether the data is homogeneous, which is often not the case: in many settings, the data consists of groups with different score profiles or different proportions of true nulls. In such cases, applying TDC while ignoring the group structure often yields imbalanced lists of discoveries, where some groups might include relatively many false discoveries and other groups include relatively very few. On the other hand, as we show, the alternative approach of applying TDC separately to each group does not rigorously control the FDR.

We developed Group-walk, a procedure that controls the FDR in the target-decoy / knockoff setting while taking into account a given group structure. Group-walk is derived from the recently developed AdaPT -- a general framework for controlling the FDR with sideinformation. We show using simulated and real datasets that when the data naturally divides into groups with different characteristics Group-walk can deliver consistent power gains that in some cases are substantial. These groupings include the precursor charge state (4% more discovered peptides at 1% FDR threshold), the peptide length (3.6% increase) and the mass difference due to modifications (26% increase).

Group-walk is available at https://cran.r-project.org/web/packages/groupwalk/index.html

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Development of thalamus mediates paternal age effect on offspring reading: A preliminary investigation The importance of (inherited) genetic impact in reading development is well-established. De novo mutation is another important contributor that is recently gathering interest as a major liability of neurodevelopmental disorders, but has been neglected in reading research to date. Paternal age at childbirth (PatAGE) is known as the most prominent risk factor for de novo mutation, which has been repeatedly shown by molecular genetic studies. As one of the first efforts, we performed a preliminary investigation of the relationship between PatAGE, offsprings reading, and brain structure in a longitudinal neuroimaging study following 51 children from kindergarten through third grade. The results showed that greater PatAGE was associated significantly with worse reading, explaining an additional 9.5% of the variations after controlling for a number of confounds--including familial factors and cognitive-linguistic reading precursors. Moreover, this effect was mediated by volumetric maturation of the left posterior thalamus from ages 5 to 8. Complementary analyses indicated the PatAGE-related thalamic region was most likely located in the pulvinar nuclei and related to the dorsal attention network by using brain atlases, public datasets, and offsprings diffusion imaging data. Altogether, these findings provide novel insights into neurocognitive mechanisms underlying the PatAGE effect on reading acquisition during its earliest phase and suggest promising areas of future research.

HighlightsO_LIPaternal age at childbirth (PatAGE) is negatively correlated with offsprings reading abilities.
C_LIO_LIPatAGE is related to volumetric maturation of the left posterior thalamus.
C_LIO_LIThalamic development mediates the PatAGE effect on reading.
C_LIO_LIThe PatAGE-related thalamic area is more likely to connect with the dorsal attention network.
C_LI

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Discrimination of melanoma cell lines with Fourier Transform Infrared (FTIR) spectroscopy Among skin cancers, melanoma is the lethal form and the leading cause of death in humans. Melanoma begins in melanocytes and is curable at early stages. Thus, early detection and evaluation of its metastatic potential are crucial for effective clinical intervention. Fourier transform infrared (FTIR) spectroscopy has gained considerable attention due to its versatility in detecting biochemical and biological features present in the samples. Changes in these features are used to differentiate between samples at different stages of the disease. Previously, FTIR spectroscopy has been mostly used to distinguish between healthy and diseased conditions. With this study, we aim to discriminate between different melanoma cell lines based on their FTIR spectra. Formalin-fixed paraffin embedded samples from three melanoma cell lines (IPC-298, SK-MEL-30 and COLO-800) were used. Statistically significant differences were observed in the prominent spectral bands of three cell lines along with shifts in peak positions. A partial least square discriminant analysis (PLS-DA) model built for the classification of three cell lines showed an overall accuracy of 92.6% with a sensitivity of 85%, 95.75%, 96.54%, and specificity of 97.80%, 92.14%, 98.64% for the differentiation of IPC-298, SK-MEL-30, and COLO-800, respectively. The results suggest that FTIR spectroscopy can differentiate between different melanoma cell lines and thus potentially characterize the metastatic potential of melanoma.

Sources of FundingThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

sentences: The evolution of coordination: A phylogenetic meta-analysis and systematic review To solve many cooperative problems, humans must have evolved the ability to solve physical problems in their environment by coordinating their actions. There have been many studies conducted across multiple different species regarding coordinating abilities. These studies aim to provide data which will help illuminate the evolutionary origins of cooperative problem solving and coordination. However, it is impossible to make firm conclusions about the evolutionary origins of coordinating abilities without a thorough comparative analysis of the existing data. Furthermore, there may be certain aspects of the literature that make it very difficult to confidently address evolutionary and meta-analytic questions. This study aimed to rectify this by using meta-analysis, phylogenetic analysis, and systematic review to analyse the data already obtained across multiple studies, and to assess the reliability of this data. We found that many studies did not provide the information necessary for meta analysis, or were not comparable enough to other studies to be included in analyses, meaning meta analyses were underpowered or could not be conducted due to low samples of both studies and different species. Overall, we found that many studies reported small positive effects across studies, but the standard errors of these effects frequently traversed zero.

sentences: Muscarinic receptors mediate motivation via preparatory neural activity in humans Motivation depends on dopamine, but might be modulated by acetylcholine which influences dopamine release in the striatum, and amplifies motivation in animal studies. A corresponding effect in humans would be important clinically, since anticholinergic drugs are frequently used in Parkinsons disease, a condition that can also disrupt motivation. Reward and dopamine make us more ready to respond, as indexed by reaction times (RT), and move faster, sometimes termed vigour. These effects may be controlled by preparatory processes that can be tracked using EEG. We measured vigour in a placebo-controlled, double-blinded study of trihexyphenidyl (THP), a muscarinic antagonist, with an incentivised eye movement task and EEG. Participants responded faster and with greater vigour when incentives were high, but THP blunted these motivation effects, suggesting that muscarinic receptors facilitate invigoration by reward. Preparatory EEG build-up (contingent negative variation; CNV) was strengthened by high incentives and by muscarinic blockade. The amplitude of preparatory activity predicted both vigour and RT, although over distinct scalp regions. Frontal activity predicted vigour, whereas a larger, earlier, central component predicted RT. Indeed the incentivisation of RT was partly mediated by the CNV, though vigour was not. Moreover, the CNV mediated the drugs effect on dampening incentives, suggesting that muscarinic receptors underlie the motivational influence on this preparatory activity. Taken together, these findings show that a muscarinic blocker used to treat Parkinsons disease impairs motivated action in healthy people, and that medial frontal preparatory neural activity mediates this for RT.

sentences: The effect of different treatment durations with indole-3-acetic acid (IAA)-producing endophytic Bacillus spp. on tomato 'Aicha' under saline conditions This study aimed to investigate the effect of indole-3-acetic acid (IAA)-producing endophytes from saline environments on survival and growth of tomato under salt stress, using different treatment times. Twenty-five endophytic bacteria were isolated from halophytes (Atriplex halimus L., Tamarix aphylla L.) issued from saline regions (Algeria). Five strains were selected based on tolerance to NaCl. A polyphasic taxonomic study was conducted. The selected strains were assigned to the Bacillus cereus group. Quantification of IAA production was done by high-performance liquid chromatography (HPLC). An in vitro test of growth promotion/protection of tomato  Aicha was conducted under saline conditions (50 mM, 75 mM, 100 mM, and 125 mM), for different durations of seed biopriming (1h, 3h, and 12h). Endophytes led to a significant amelioration of salt stress in tomato  Aicha at all salinity levels, depending on treatment times, indicating the necessity of optimizing this crucial factor.

sentences: Structure of human Nav1.5 reveals the fast inactivation-related segments as a mutational hotspot for the Long QT Syndrome Nav1.5 is the primary voltage-gated Na+ (Nav) channel in the heart. Mutations of Nav1.5 are associated with various cardiac disorders exemplified by the type 3 long QT syndrome (LQT3) and Brugada syndrome (BrS). E1784K is a common mutation that has been found in both LQT3 and BrS patients. Here we present the cryo-EM structure of the human Nav1.5-E1784K variant at an overall resolution of 3.3 [A]. Structural mapping of 91 and 178 point mutations that are respectively associated with LQT3 and BrS reveals a unique distribution pattern for LQT3 mutations. Whereas the BrS mutations spread evenly on the structure, LQT3 mutations are mainly clustered to the segments in repeats III and IV that are involved in gating, voltage-sensing, and particularly inactivation. A mutational hotspot involving the fast inactivation segments is identified and can be mechanistically interpreted by our "door wedge" model for fast inactivation. The structural analysis presented here, with a focus on the impact of disease mutations on inactivation and late sodium current, establishes a structure-function relationship for the mechanistic understanding of Nav1.5 channelopathies.

sentences: Daam1 negatively regulates USP10 activity The differentiation of fibroblasts into pathological myofibroblasts during wound healing is in part characterized by increased cell surface expression of v-integrins. Our previous studies found that the deubiquitinase (DUB) USP10 removes ubiquitin from v-integrins, leading to cell surface integrin accumulation, subsequent TGF{beta}1 activation, and myofibroblast differentiation. In this study, a yeast-two hybrid screen elucidated a novel binding partner for USP10, the formin, Daam1. The USP10/Daam1 interaction was also supported by proximity ligation assay (PLA) activity. Treatment with TGF{beta}1 significantly increased USP10 and Daam1 protein expression, PLA signal, and co-localization to actin stress fibers. Furthermore, Daam1 siRNA knockdown significantly reduced a) co-precipitation of USP10 and Daam1 on purified actin stress fibers, and b) {beta}1- and {beta}5-integrin ubiquitination resulting in increased v-, {beta}1-, and {beta}5-integrin total protein levels, v integrin recycling to the cell surface, and extracellular fibronectin (FN) organization. Together, our data suggest that Daam1 negatively regulates USP10s DUB activity and subsequently maintains integrin protein homeostasis.

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Chronic toxicity of three neonicotinoid insecticides and their mixture on two daphniid species: Daphnia magna and Ceriodaphnia dubia Neonicotinoid insecticides represent nearly a quarter of the global market and are widely used in agriculture but also for lawn, garden care, and pest control. They are highly water-soluble, persistent in soil, and may enter the aquatic compartment via spray drift, runoff, or leaching, and contribute to downstream aquatic toxicity. Although insects appear to be the most sensitive group to neonicotinoids, other groups, such as crustaceans and birds, may also be affected. Furthermore, most studies focus on single-insecticide exposure and very little is known concerning the impact of neonicotinoid mixtures on aquatic invertebrates. The present study was designed to test potential toxicological effects of an environmentally relevant mixture of imidacloprid, clothianidin, and thiamethoxam on populations of Ceriodaphnia dubia and Daphnia magna under controlled conditions. Chronic toxicity tests were conducted in the laboratory, and survival and reproduction were measured for both species under exposure to nominal concentrations of imidacloprid (0.256 {micro}g/L), clothianidin (3.11 {micro}g/L), thiamethoxam (1.49 {micro}g/L), and a mixture of the three compounds at the same concentrations of the individual compounds. The neonicotinoids did not affect the survival of C. dubia and D. magna founders. Reproduction of C. dubia was affected only by the mixture. All three individual insecticides as well as the mixture caused a significant reduction in the reproduction of D. magna. Our results highlight the complexity of pesticide toxicity and show that traditional toxicological approaches such as acute mortality studies, especially tests with single compounds, can underestimate negative impacts that occur in the environment.

HighlightsO_LINeonicotinoids are currently the most frequently used insecticides worldwide.
C_LIO_LIAn environmentally relevant mixture of three neonicotinoids was evaluated on two daphniid species.
C_LIO_LIThe mixture negatively affected the reproduction of C. dubia and Daphnia magna.
C_LIO_LITraditional toxicological approaches with single compounds may underestimate the effects occurring in the environment at low concentrations.
C_LI

sentences: Tie-lines reveal interactions driving heteromolecular condensate formation Phase separation of biomolecules give rise to membraneless organelles that contribute to the spatiotemporal organisation of the cell. In most cases, such biomolecular condensates contain multiple components, but the manner in which interactions between components control the stability of condensates remained challenging to elucidate. Here, we develop an approach to determine tie-line gradients in ternary liquid-liquid phase separation (LLPS) systems, based on measurements of the dilute phase concentration of only one component. We show that the sign of the tie-line gradient is related to the cross-interaction energy between the polymers in the system and discriminates between competitive and cooperative phase separation. Using this approach, we studied the interaction between protein Fused in Sarcoma (FUS) and polyethylene glycol (PEG) polymer chains, and measured positive tie-line gradients. Our results show that PEG drives LLPS through an associative interaction with FUS and is not an inert crowder. We further studied the interaction between PolyA RNA (3.0{+/-}0.5kDa) and the protein G3BP1, and using the tie-line gradient as a reporter for the stoichiometry of polymers in the condensate we determined a G3BP1-to-PolyA RNA molar ratio of 1:4 in the dense phase. Our framework for measuring tie-line gradients opens up a route for the characterisation of interaction types and compositions in ternary LLPS systems.

sentences: cAMP controls a trafficking mechanism that directs the neuron specificity and subcellular placement of electrical synapses Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here we show that a transcriptional program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. For these studies, we use live cell imaging to visualize electrical synapses in vivo and a novel optogenetic assay to confirm that they are functional. In VA motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. We show that cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments in an intact nervous system establish that cAMP regulates gap junction trafficking for the biogenesis of electrical synapses.

sentences: PHAROH lncRNA regulates c-Myc translation in hepatocellular carcinoma via sequestering TIAR Hepatocellular carcinoma, the most common type of liver malignancy, is one of the most lethal forms of cancer. We identified a long non-coding RNA, Gm19705, that is over-expressed in hepatocellular carcinoma and mouse embryonic stem cells. We named this RNA Pluripotency and Hepatocyte Associated RNA Overexpressed in HCC, or PHAROH. Depletion of PHAROH impacts cell proliferation and migration, which can be rescued by ectopic expression of PHAROH. RNA-seq analysis of PHAROH knockouts revealed that a large number of genes with decreased expression contain a c-Myc motif in their promoter. C-MYC is decreased at the protein level, but not the mRNA level. RNA-antisense pulldown identified nucleolysin TIAR, a translational repressor, to bind to a 71-nt hairpin within PHAROH, sequestration of which increases c-MYC translation. In summary, our data suggest that PHAROH regulates c-MYC translation by sequestering TIAR and as such represents a potentially exciting diagnostic or therapeutic target in hepatocellular carcinoma.

sentences: Use of untargeted magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and Real-Time-PCR Untargeted magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Untargeted magnetic beads recovered more M. smegmatis cells from PBS suspension than the typical centrifugation method; 96.31% of 1.68 x 104 CFU/mL viable M. smegmatis were recovered by beads and 0% by centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p<0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of higher sensitivity. Magnetic separation of MAP cells from milk, urine, and feces specimens were detected by F57 and IS900 sequences. Ct values demonstrated that beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 x 105 and 2 x 103 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of Johnes disease and other mycobacterial diseases such as tuberculosis.

sentences: Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape Zoonotic pandemics, like that caused by SARS-CoV-2, can follow the spillover of animal viruses into highly susceptible human populations. Their descendants have adapted to the human host and evolved to evade immune pressure. Coronaviruses acquire substitutions more slowly than other RNA viruses, due to a proofreading polymerase. In the spike glycoprotein, we find recurrent deletions overcome this slow substitution rate. Deletion variants arise in diverse genetic and geographic backgrounds, transmit efficiently, and are present in novel lineages, including those of current global concern. They frequently occupy recurrent deletion regions (RDRs), which map to defined antibody epitopes. Deletions in RDRs confer resistance to neutralizing antibodies. By altering stretches of amino acids, deletions appear to accelerate SARS-CoV-2 antigenic evolution and may, more generally, drive adaptive evolution.

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Hydrogen peroxide modulates lignin and silica deposits in sorghum roots Hydrated silica (SiO2{middle dot}nH2O) aggregates in the root endodermis of grasses. Application of soluble silicates (Si) to roots is associated with variations in the balance of reactive oxygen species (ROS), increased tolerance to a broad range of stresses affecting ROS levels, and early lignin deposition. In sorghum (Sorghum bicolor L.), silica aggregation is patterned in an active silicification zone (ASZ) by a special type of lignin. Since lignin polymerization is mediated by ROS, we studied the formation of root lignin and silica under varied conditions of ROS by modulating hydrogen peroxide (H2O2) concentration in the growth solution. Sorghum seedlings were grown hydroponically and supplemented with Si, H2O2, and KI, a salt that catalyzes H2O2 decomposition. Lignin and silica deposits in the endodermis were studied by histology, scanning electron and Raman microscopies. Cell wall composition was quantified by thermal gravimetric analysis. We found that the endodermal H2O2 concentration regulated the extent of ASZ lignin deposition along the root, but not its patterning in spots. Our results show that ASZ lignin is necessary for root silica aggregation in sorghum, and that silicification is enhanced under oxidative stress as a result of increased deposition of the ASZ lignin.

One sentence summaryLignin with carbonyl modifications is patterned by the activity of H2O2 to nucleate silica aggregations in sorghum roots.

sentences: Eye movements during text reading align with the rate of speech production Across languages, the speech signal is characterized by a predominant modulation of the amplitude spectrum between about 4.3-5.5Hz, reflecting the production and processing of linguistic information chunks (syllables, words) every [~]200ms. Interestingly, [~]200ms is also the typical duration of eye fixations during reading. Prompted by this observation, we demonstrate that German readers sample written text at [~]5Hz. A subsequent meta-analysis with 142 studies from 14 languages replicates this result, but also shows that sampling frequencies vary across languages between 3.9Hz and 5.2Hz, and that this variation systematically depends on the complexity of the writing systems (character-based vs. alphabetic systems, orthographic transparency). Finally, we demonstrate empirically a positive correlation between speech spectrum and eye-movement sampling in low-skilled readers. Based on this convergent evidence, we propose that during reading, our brains linguistic processing systems imprint a preferred processing rate, i.e., the rate of spoken language production and perception, onto the oculomotor system.

sentences: Longitudinal examination of perfusion and angiogenesis markers in primary colorectal tumors shows distinct signatures for metronomic and maximum-tolerated dose strategies 1Metronomic chemotherapy (MET) has been developed to address the shortcomings of maximum-tolerated chemotherapy (MTD) in regard to toxicity and development of resistance mechanisms in the tumor. In colorectal cancer (CRC), MET is a promising novel strategy to treat locally advanced malignancies when used as neoadjuvant chemotherapy (NAC). However, so far there are no preclinical studies to assess the impact of MET NAC in CRC to assess the benefits and challenges of this approach. Here, we used a primary model of CRC (via azoxymethane) to analyze longitudinal changes in angiogenesis in primary tumors under MET and MTD NAC using a combination of diffuse reflectance spectroscopy and mRNA expression (via qPCR). Our results show that MET and MTD NAC lead to increased mean tissue oxygen saturation (8% and 5%, respectively) and oxyhemoglobin (15% and 10%) between weeks 2 and 5 of NAC, and that such increases are caused by distinct molecular signatures in the angiogenic program. Specifically, we find that in the MET group there is a sustained increase in Hif-1a, Aldoa, and Pgk1 expression, suggesting upregulated glycolysis, whereas MTD NAC causes a significant reduction in the expression of the aforementioned genes and of Vegf, leading to vascular remodeling in MTD-treated tumors. Taken together, this study demonstrates the ability of combined optical and molecular methodologies to provide a holistic picture of tumor response to therapy in CRC in a minimally invasive manner.

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Complete genomes of clade G6 Saccharibacteria suggest a divergent ecological niche and lifestyle AO_SCPLOWBSTRACTC_SCPLOWSaccharibacteria (formerly TM7) have reduced genomes, a small size, and appear to have a parasitic lifestyle dependent on a bacterial host. Although there are at least 6 major clades of Saccharibacteria inhabiting the human oral cavity, cultured isolates or complete genomes of oral Saccharibacteria have been previously limited to the G1 clade. In this study, nanopore sequencing was used to obtain three complete genome sequences from clade G6. Phylogenetic analysis suggested the presence of at least 3-5 distinct species within G6, with two discrete taxa represented by the 3 complete genomes. G6 Saccharibacteria were highly divergent from the more well-studied clade G1, and had the smallest genomes and lowest GC-content of all Saccharibacteria. Pangenome analysis showed that although 97% of shared pan-Saccharibacteria core genes and 89% of G1-specific Core Genes had putative functions, only 50% of the 244 G6-specific Core Genes had putative functions, highlighting the novelty of this group. Compared to G1, G6 encoded divergent metabolic pathways. G6 genomes lacked an F1F0 ATPase, the pentose phosphate pathway, and several genes involved in nucleotide metabolism, which were all core genes for G1. G6 genomes were also unique compared to G1 in that they encoded lactate dehydrogenase, adenylate cyclase, limited glycerolipid metabolism, a homolog to a lipoarabinomannan biosynthesis enzyme, and the means to degrade starch. These differences at key metabolic steps suggest a distinct lifestyle and ecological niche for clade G6, possibly with alternative hosts and/or host-dependencies, which would have significant ecological, evolutionary, and likely pathogenic, implications.

IO_SCPLOWMPORTANCEC_SCPLOWSaccharibacteria are ultrasmall, parasitic bacteria that are common members of the oral microbiota and have been increasingly linked to disease and inflammation. However, the lifestyle and impact on human health of Saccharibacteria remains poorly understood, especially for the 5 clades (G2-G6) with no complete genomes or cultured isolates. Obtaining complete genomes is of particular importance for Saccharibacteria, because they lack many of the "essential" core genes used for determining draft genome completeness and few references exist outside of clade G1. In this study, complete genomes of 3 G6 strains, representing two candidate species, were obtained and analyzed. The G6 genomes were highly divergent from G1, and enigmatic, with 50% of the G6 core genes having no putative functions. The significant difference in encoded functional pathways is suggestive of a distinct lifestyle and ecological niche, probably with alternative hosts and/or host-dependencies, which would have major implications in ecology, evolution, and pathogenesis.

sentences: Cis-Regulatory Element Hijacking by Structural Variants Overshadows Topological Changes in Primary Prostate Cancer Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However the impact of this instability on the three-dimensional chromatin organization and how this drives progression is unclear. Using primary benign and tumour tissue, we find a high concordance in the higher-order three-dimensional genome organization across normal and prostate cancer cells. This concordance argues for constraints to the topology of prostate tumour genomes. Nonetheless, we identify changes to focal chromatin interactions and show how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events result in opposing differential expression on genes found at antipodes of rearrangements. Collectively, our results argue that cis-regulatory element hijacking from structural variant-induced altered focal chromatin interactions overshadows higher-order topological changes in the development of primary prostate cancer.

sentences: A meta-analysis of Microarray Data is Effective for Identifying Gravity-Sensitive Genes Gravity affects the function and maintenance of organs, such as bones, muscles, and the heart. Several studies have used DNA microarrays to identify genes with altered expressions in response to gravity. However, it is technically challenging to combine the results from various microarray datasets because of their different data structures. We hypothesized it is possible to identify common changes in gene expression from the DNA microarray datasets obtained under various conditions and methods. In this study, we grouped homologous genes to perform a meta-analysis of multiple vascular endothelial cell and skeletal muscle datasets. According to the t-distributed stochastic neighbor embedding (t-SNE) analysis, the changes in the gene expression pattern in vascular endothelial cells formed specific clusters. We also identified candidate genes in endothelial cells that responded to gravity. Further, we exposed human umbilical vein endothelial cells to simulated microgravity using a clinostat and measured the expression levels of the candidate genes. Gene expression analysis using qRT-PCR revealed that the expression level of the prostaglandin transporter gene SLCO2A1 decreased in response to microgravity, consistent with the meta-analysis of microarray datasets. Furthermore, the direction of gravity affected the expression level of SLCO2A1, buttressing the finding that its expression was affected by gravity. These results suggest that a meta-analysis of DNA microarray datasets may help identify new target genes previously overlooked in individual microarray analyses.

sentences: The small GTPase Arf1 regulates ATP synthesis and mitochondria homeostasis by modulating fatty acid metabolism Lipid mobilization through fatty acid {beta}-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where {beta}-oxidation products enter mitochondria and fuel the TCA cycle. Little is known about the physical and metabolic cooperation between these organelles. We found that expression of fatty acid transporters and of the rate-limiting enzyme involved in {beta}-oxidation are decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. As a consequence, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although {beta}-oxidation occurs mainly in mitochondria in mammals, Arf1s role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact-sites.

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Context dependency of nucleotide probabilities and variants in human DNA BackgroundGenomic DNA has been shaped by mutational processes through evolution. The cellular machinery for error correction and repair has left its marks in the nucleotide composition along with structural and functional constraints. Therefore, the probability of observing a base in a certain position in the human genome is highly context-dependent.

ResultsHere we develop context-dependent nucleotide models. We first investigate models of nucleotides conditioned on sequence context. We develop a bidirectional Markov model that use an average of the probability from a Markov model applied to both strands of the sequence and thus depends on up to 14 bases to each side of the nucleotide. We show how the genome predictability varies across different types of genomic regions. Surprisingly, this model can predict a base from its context with an average of more than 50% accuracy. For somatic variants we show a tendency towards higher probability for the variant base than for the reference base. Inspired by DNA substitution models, we develop a model of mutability that estimates a mutation matrix (called the alpha matrix) on top of the nucleotide distribution. The alpha matrix can be estimated from a much smaller context than the nucleotide model, but the final model will still depend on the full context of the nucleotide model. With the bidirectional Markov model of order 14 and an alpha matrix dependent on just one base to each side, we obtain a model that compares well with a model of mutability that estimates mutation probabilities directly conditioned on three nucleotides to each side. For somatic variants in particular, our model fits better than the simpler model. Interestingly, the model is not very sensitive to the size of the context for the alpha matrix.

ConclusionsOur study found strong context dependencies of nucleotides in the human genome. The best model uses a context of 14 nucleotides to each side. Based on these models, a substitution model was constructed that separates into the context model and a matrix dependent on a small context. The model fit somatic variants particularly well.

sentences: A sinusoidal transform of the visual field in cortical area V2 The retinotopic maps of many visual cortical areas are thought to follow the fundamental principles that have been described for primary visual cortex (V1) where nearby points on the retina map to nearby points on the surface of V1, and orthogonal axes of the retinal surface are represented along orthogonal axes of the cortical surface. Here we demonstrate a striking departure from this conventional mapping in the secondary visual area (V2) of the tree shrew. Although local retinotopy is preserved, orthogonal axes of the retina are represented along the same axis of the cortical surface, an unexpected geometry explained by an orderly sinusoidal transform of the retinal surface. This sinusoidal topography is ideally suited for achieving uniform coverage in an elongated area like V2, is predicted by mathematical models designed to achieve wiring minimization, and provides a novel explanation for stripe-like patterns of intra-cortical connections and stimulus response properties in V2. Our findings suggest that cortical circuits flexibly implement solutions to sensory surface representation, with dramatic consequences for the large-scale layout of topographic maps.

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Predicting human decision making in psychological tasks with recurrent neural networks Unlike traditional time series, the action sequences of human decision making usually involve many cognitive processes such as beliefs, desires, intentions and theory of mind, i.e. what others are thinking. This makes predicting human decision making challenging to be treated agnostically to the underlying psychological mechanisms. We propose to use a recurrent neural network architecture based on long short-term memory networks (LSTM) to predict the time series of the actions taken by the human subjects at each step of their decision making, the first application of such methods in this research domain. In this study, we collate the human data from 8 published literature of the Iterated Prisoners Dilemma comprising 168,386 individual decisions and postprocess them into 8,257 behavioral trajectories of 9 actions each for both players. Similarly, we collate 617 trajectories of 95 actions from 10 different published studies of Iowa Gambling Task experiments with healthy human subjects. We train our prediction networks on the behavioral data from these published psychological experiments of human decision making, and demonstrate a clear advantage over the state-of-the-art methods in predicting human decision making trajectories in both single-agent scenarios such as the Iowa Gambling Task and multi-agent scenarios such as the Iterated Prisoners Dilemma. In the prediction, we observe that the weights of the top performers tends to have a wider distribution, and a bigger bias in the LSTM networks, which suggests possible interpretations for the distribution of strategies adopted by each group.

Author summaryThe challenge of predicting human decision is important for many application domains like economy, marketing and artificial intelligence. But the domain where the human behavior modeling is especially crucial is psychology, where the goal is to describe, explain and predict these behaviors. Within this domain, creating agents that effectively mimic human decision making is particularly important. However, despite the many behavioral tasks and analytical methods developed to simulate the process of human decision making in real situations, there have not been a predictive model that can well predict the human decision making trajectories in these psychological tasks. In this predictive modeling problem, we propose to use the long short-term memory networks (LSTM), a popular recurrent neural network architecture and effectively predict the time series of the actions taken by the human subjects at each step of their decision making in both the Iowa Gambling Task, a single-agent game with monetary reward, and the Iterated Prisoners Dilemma, a complex multi-agent social game with non-monetary capital at stake. To the best of our knowledge, this is the first application of such methods in this research domain.

sentences: Genetic and epigenetic driven variation in regulatory regions activitycontribute to adaptation and evolution under endocrine treatment Comprehensive profiling of hormone-dependent breast cancer (HDBC) has identified hundreds of protein-coding alterations contributing to cancer initiation1, 2, but only a handful have been linked to endocrine therapy resistance, potentially contributing to 40% of relapses1, 3-9. If other mechanisms underlie the evolution of HDBC under adjuvant therapy is currently unknown. In this work, we employ integrative functional genomics to dissect the contribution of cis-regulatory elements (CREs) to cancer evolution by focusing on 12 megabases of non-coding DNA, including clonal enhancers10, gene promoters, and boundaries of topologically associating domains11. Massive parallel perturbation in vitro reveals context-dependent roles for many of these CREs, with a specific impact on dormancy entrance12, 13 and endocrine therapy resistance9. Profiling of CRE somatic alterations in a unique, longitudinal cohort of patients treated with endocrine therapies identifies non-coding changes involved in therapy resistance. Overall, our data uncover actionable transient transcriptional programs critical for dormant persister cells and unveil new regulatory nodes driving evolutionary trajectories towards disease progression.

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Hierarchical Timescales in the Neocortex: Mathematical Mechanism and Biological Insights A cardinal feature of the neocortex is the progressive increase of the spatial receptive fields along the cortical hierarchy. Recently, theoretical and experimental findings have shown that the temporal response windows also gradually enlarge, so that early sensory neural circuits operate on short-time scales whereas higher association areas are capable of integrating information over a long period of time. While an increased receptive field is accounted for by spatial summation of inputs from neurons in an upstream area, the emergence of timescale hierarchy cannot be readily explained, especially given the dense inter-areal cortical connectivity known in modern connectome. To uncover the required neurobiological properties, we carried out a rigorous analysis of an anatomically-based large-scale cortex model of macaque monkeys. Using a perturbation method, we show that the segregation of disparate timescales is defined in terms of the localization of eigenvectors of the connectivity matrix, which depends on three circuit properties: (1) a macroscopic gradient of synaptic excitation, (2) distinct electrophysiological properties between excitatory and inhibitory neuronal populations, and (3) a detailed balance between long-range excitatory inputs and local inhibitory inputs for each area-to-area pathway. Our work thus provides a quantitative understanding of the mechanism underlying the emergence of timescale hierarchy in large-scale primate cortical networks.

Significance StatementIn the neocortex, while early sensory areas encode and process external inputs rapidly, higher association areas are endowed with slow dynamics suitable for accumulating information over time. Such a hierarchy of temporal response windows along the cortical hierarchy naturally emerges in a model of multi-areal primate cortex. This finding raises the question of why diverse temporal modes are not mixed in roughly the same way across the whole cortex, despite high connection density and an abundance of feedback loops. We investigate this question by mathematically analyzing the anatomically-based network model of macaque cortex, and show that three general principles of synaptic excitation and inhibition are crucial for timescale segregation in a hierarchy, a functionally important characteristic of the cortex.

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Impaired succinate oxidation prevents growth and influences drug susceptibility in Mycobacterium tuberculosis Succinate is a major focal point in mycobacterial metabolism and respiration, serving as both an intermediate of the TCA cycle and a direct electron donor for the respiratory chain. Mycobacterium tuberculosis encodes multiple enzymes predicted to be capable of catalyzing the oxidation of succinate to fumarate, including two different succinate dehydrogenases (Sdh1 and Sdh2) and a separate fumarate reductase (Frd) with possible bi-directional behavior. Previous attempts to investigate the essentiality of succinate oxidation in M. tuberculosis have relied on the use of single-gene deletion mutants, raising the possibility that the remaining enzymes could catalyze succinate oxidation in the absence of the other. To address this, we report on the use of mycobacterial CRISPR interference (CRISPRi) to construct single, double, and triple transcriptional knockdowns of sdhA1, sdhA2, and frdA in M. tuberculosis. We show that the simultaneous knockdown of sdhA1 + sdhA2 is required to prevent succinate oxidation and overcome the functional redundancy within these enzymes. Succinate oxidation was demonstrated to be essential for the optimal growth of M. tuberculosis, with the combined knockdown of sdhA1 + sdhA2 significantly impairing the activity of the respiratory chain and preventing growth on a range of carbon sources. Moreover, impaired succinate oxidation was shown to influence the activity of several antitubercular drugs against M. tuberculosis, including potentiating the activity of bioenergetic inhibitors and attenuating the activity of cell wall inhibitors. Together, these data provide fundamental insights into mycobacterial physiology, energy metabolism, and antimicrobial susceptibility.

ImportanceNew drugs are urgently required to combat the tuberculosis epidemic that claims 1.5 million lives annually. Inhibitors of mycobacterial energy metabolism have shown significant promise clinically; however, further advancing this nascent target space requires a more fundamental understanding of the respiratory enzymes and pathways used by Mycobacterium tuberculosis. Succinate is a major focal point in mycobacterial metabolism and respiration; yet the essentiality of succinate oxidation, and the consequences of inhibiting this process, are poorly defined. In this study, we demonstrate that impaired succinate oxidation prevents the optimal growth of M. tuberculosis on a range of carbon sources and significantly reduces the activity of the electron transport chain. Moreover, we show that impaired succinate oxidation both positively and negatively influences the activity of a variety of anti-tuberculosis drugs. Combined, these findings provide fundamental insights into mycobacterial physiology and drug susceptibility that will be useful in the continued development of bioenergetic inhibitors.

sentences: Perivascular cells support folliculogenesis in the developing ovary Granulosa cells, supporting cells of the ovary, are essential for ovarian differentiation by providing a nurturing environment for oogenesis. Sufficient numbers of granulosa cells are vital for establishment of follicles and the oocyte reserve; therefore, identifying the cellular source from which granulosa cells are derived is critical for understanding basic ovarian biology. One cell type that has received little attention in this field is the perivascular cell. Here we use lineage tracing and organ culture techniques in mice to identify ovarian Nestin+ perivascular cells as multipotent progenitors that contribute to granulosa, thecal, and pericyte lineages. Maintenance of these progenitors was dependent on vascular-mesenchymal Notch signaling. Ablation of postnatal Nestin+ cells resulted in a disruption of granulosa cell specification and an increased incidence of polyovular ovarian follicles, thus uncovering key roles for vasculature in ovarian differentiation. These findings may provide new insights into the origins of female gonad dysgenesis and infertility.

sentences: Comprehensive PAM prediction for CRISPR-Cas systems reveals evidence for spacer sharing, preferred strand targeting and conserved links with CRISPR repeats The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database. By using vast metagenomic sequence databases, we mapped one third ([~]70,000) of more than 200,000 unique CRISPR spacers from a variety of microbes, and derived a catalog of more than one hundred unique PAM sequences associated with specific CRISPR subtypes. These PAMs were further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. From the curated CRISPR arrays dataset we could also deduce CRISPR subtype specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (e.g. Type I-E and Type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (i.e. Type I-A, I-B and Type III systems) prefer the coding strand and mRNA. In addition, we found large scale evidence that both CRISPR adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA- and RNA targeting of invaders, which may be effective at combating mobile genetic invaders.

sentences: Lower Vibrio spp. abundances in Zostera marina leaf canopies suggest a novel ecosystem function for temperate seagrass beds Seagrasses, a polyphyletic group of about 60 marine angiosperm species, are the foundation of diverse and functionally important marine habitats along sheltered sedimentary coasts. As a novel ecological function with high societal relevance, a role of the leaf canopy for reducing potentially harmful bacteria has recently been hypothesized. Accordingly, we tested whether or not the abundance of general bacteria and more specifically, those belonging to the genus Vibrio were reduced within temperate Zostera marina (eelgrass) meadows compared to adjacent sand flats and sampled 5 sites in the south-western Baltic Sea using SCUBA. Compared to non-vegetated area, we found an average reduction of 39% for all Vibrio and 63% for the potentially harmful V. vulnificus/cholerae subtype based on robust plate counting data on Vibrio selective agar. The underlying mechanism is currently elusive and clearly merits further study. Our results underline the critical importance of seagrasses in maintaining shallow water ecosystem functioning including water quality and provide further motivation for their protection and restoration.

sentences: A New Classification Method Based on Dynamic Ensemble Selection and its Application to Predict Variance Patterns in HIV-1 Env Therapeutics that target the envelope glycoproteins (Envs) of human immunodeficiency virus type 1 (HIV-1) effectively reduce virus levels in patients. However, due to mutations, new Env variants are frequently generated, which may be resistant to the treatments. The appearance of such sequence variance at any Env position is seemingly random. A better understanding of the spatiotemporal patterns of variance across Env may lead to the development of new therapeutic strategies. We hypothesized that, at any time point in a patient, positions with sequence variance are clustered on the three-dimensional structure of Env. To test this hypothesis, we examined whether variance at any Env position can be predicted by the variance measured at adjacent positions. Sequences from 300 HIV-infected patients were applied to a new algorithm we developed. The k-best classifiers (KBC) method is a dynamic ensemble selection technique that identifies the best classifier(s) within the neighborhood of a new observation. It applies bootstrap resampling to generate out-of-bag samples that are used with the resampled set to evaluate each classifier. For many positions of Env, primarily in the CD4-binding site, KBC accurately predicted variance based on the variance at their adjacent positions. KBC improved performance compared to the initial learners, static ensemble, and other baseline models. KBC also outperformed other algorithms for predicting variance at multi-position footprints of therapeutics on Env. These understandings can be applied to refine models that predict future changes in HIV-1 Env. More generally, we propose KBC as a new high-performance dynamic ensemble selection technique.

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Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveal Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation Yarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504, which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing non-preferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica. To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, {beta}-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation and degradation in biomass hydrolysates.

IMPORTANCEYarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates. Our investigation into the genetic diversity of undomesticated Y. lipolytica strains, coupled with detailed strain characterization and proteomic analysis, revealed metabolic processes and regulatory elements conferring desirable phenotypes for growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates by these natural variants. This study provides a better understanding of the robust metabolism of Y. lipolytica and suggests potential metabolic engineering strategies to enhance its performance.

sentences: The 3-D structure of fibrous material is fully restorable from its X-ray diffraction pattern X-ray fiber diffraction is potentially a powerful technique to study the structure of fibrous materials, such as DNA and synthetic polymers. However, only rotationally averaged diffraction patterns can be recorded, and it is difficult to correctly interpret them without the knowledge of esoteric diffraction theories. Here we demonstrate that, in principle, the non-rotationally averaged 3-D structure of the material can be restored from its fiber diffraction pattern. The method is a simple puzzle-solving process, and in ideal cases, it does not require any prior knowledge about the structure, such as helical symmetry. We believe that the proposed method has a potential to transform the fiber diffraction to a 3-D imaging technique, and will be useful for a wide field of life and materials sciences.

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Graph convolutional network for protein sequence sampling and prediction using structural data The increasing integration between protein engineering and machine learning has led to many interesting results. A problem still to solve is to evaluate the likelihood that a sequence will fold into a target structure. This problem can be also viewed as sequence prediction from a known structure.

In the current work, we propose improvements in the recent architecture of Geometric Vector Perceptrons [1] in order to optimize the sampling of sequences from a known backbone structure. The proposed model differs from the original in that there is: (i) no updating in the vectorial embedding, only in the scalar one, (ii) only one layer of decoding. The first aspect improves the accuracy of the model and reduces the use of memory, the second allows for training of the model with several tasks without incurring data leakage.

We treat the trained classifier as an Energy-Based Model and sample sequences by sampling amino acids in a non-autoreggresive manner in the empty positions of the sequence using energy-guided criteria and followed by random mutation optimization. We improve the median identity of samples from 40.2% to 44.7%.

An additional question worth investigating is whether sampled and original sequences fold into similar structures independent of their identity. We chose proteins in our test set whose sampled sequences show low identity (under 30%) but for which our model predicted favorable energies. We used AlphaFold [2, 3] and observed that the predicted structures for sampled sequences highly resemble the predicted structures for original sequences, with an average TM-score of 0.84.

sentences: Thin-Film Freeze-Drying of a Norovirus Vaccine Candidate A bivalent Norovirus vaccine candidate has been developed that contains Norovirus strain GI.1 Norwalk-virus like particles (VLP) and strain GII.4 Consensus VLP adsorbed onto aluminum (oxy)hydroxide. In the present study, we tested the feasibility of converting the vaccine from a liquid suspension into dry powder by thin-film freeze-drying (TFFD). With the proper amount of trehalose and/or sucrose as cryoprotectant, TFFD can be applied to transform the Norovirus vaccine candidate into dry powders without causing antigen loss or particle aggregation, while maintaining the potency of the antigens within a specified acceptable range. In an accelerated stability study, the potency of the antigens was also maintained in the specified acceptable range after the dry powders were stored for eight weeks at 40{degrees}C, 75% relative humidity. The dry powder Norovirus vaccine offers the potential to eliminate the cold chain requirement for transport and/or storage of the vaccine.

sentences: The runaway evolution of SARS-CoV-2 leading to the highly evolved Delta strain In new epidemics after the host shift, the pathogens may experience accelerated evolution driven by novel selective pressures. When the accelerated evolution enters a positive feedback loop with the expanding epidemics, the pathogens runaway evolution may be triggered. To test this possibility in COVID-19, we analyze the extensive databases and identify 5 major waves of strains, one replacing the previous one in 2020 - 2021. The mutations differ entirely between waves and the number of mutations continues to increase, from 3-4 to 21-31. The latest wave is the Delta strain which accrues 31 new mutations to become highly prevalent. Interestingly, these new mutations in Delta strain emerge in multiple stages with each stage driven by 6 - 12 coding mutations that form a fitness group. In short, the evolution of SARS-CoV-2 from the oldest to the youngest wave, and from the earlier to the later stages of the Delta wave, is a process of acceleration with more and more mutations. The global increase in the viral population size (M(t), at time t) and the mutation accumulation (R(t)) may have indeed triggered the runaway evolution in late 2020, leading to the highly evolved Alpha and then Delta strain. To suppress the pandemic, it is crucial to break the positive feedback loop between M(t) and R(t), neither of which has yet to be effectively dampened by late 2021. New waves beyond Delta, hence, should not be surprising.

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Probing the Structural Basis of Citrus Phytochrome B using Computational Modelling and Molecular Dynamics Simulation Approaches Phytochromes (Phys) are known as red/far-red light photoreceptors and are responsible for directing the photosensory responses across the species, majorly from fungal, bacterial and plant kingdoms. Such responses majorly include photosynthetic potential and pigmentation in bacteria, whereas in a plant, they are involved in chloroplast development and photomorphogenesis. Many prokaryotic Phys have been modelled for their structural and functional analysis, but their plant counterparts have not been explored yet. To date, only the crystal structures of the photo-sensing module (PSM) of PhyB isoform from Arabidopsis thaliana and Glycine max have been resolved experimentally. Thus, in this study, we elucidated the complete 3D structure of Citrus PhyB. Initially, the structure and organisation of the Citrus PhyB have been predicted computationally, which were found to have the same domain organisation as A. thaliana and G. max PhyBs, yet their considerable distinct structural difference indicated potential divergence in signaling and functioning. Therefore, to evaluate the structural and functional implications of Citrus PhyB, we compared its structure with A. thaliana and G. max PhyBs using molecular dynamics (MD) simulation approaches. The modeling studies revealed that the region of Citrus PhyB-GAF domain possibly contributes to the variations between Citrus, A. thaliana and G. max PhyBs structures/functions. Hence, structural and molecular insights into Citrus PhyB can help to discover the Phys signaling and thus, an essential framework can be designed for optogenetic reagents and various agricultural/horticulture benefits.

One sentence summaryA complete Citrus PhyB structure together with photo-sensory and out-put modules provides significant information to evaluate its biological activities for agricultural benefits.

sentences: Imaging tools generated by CRISPR/Cas9 tagging reveal cytokinetic diversity in mammalian cells Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This process occurs via the ingression of an actomyosin ring that assembles in anaphase and pulls in the overlying plasma membrane as it constricts. Mechanistic studies have uncovered different pathways that regulate the assembly and position of the ring in mammalian cells, but the majority of these studies were done using HeLa cells with overexpressed transgenes, and the relative requirement for these mechanisms among the majority of cell types is not known. Here, we used CRISPR/Cas9 gene editing to endogenously tag cytokinesis proteins, anillin, Ect2 and RhoA, as well as other cellular components, with fluorescent proteins. These tools enabled the visualization of cytokinesis by live imaging to quantitatively study these proteins at endogenous levels. As a proof-of-concept, tagging anillin in multiple mammalian cell lines revealed cytokinetic diversity, which will be useful for studies of how mechanisms controlling cytokinesis vary among cell types. We also successfully tagged multiple cellular components in the same cell line, demonstrating the versatility of these tagging tools.

sentences: Macroevolutionary dynamics of climatic niche space How and why lineages evolve along niche space as they diversify and adapt to different environments is fundamental to evolution. Progress has been hampered by the difficulties of linking a comprehensive empirical characterization of species niches with flexible evolutionary models that describe their evolution. Consequently, the relative influence of external episodic and biotic factors remains poorly understood. Here we characterize species two-dimensional temperature and precipitation niche space occupied (i.e., species niche envelope) as complex geometries and assess their evolution across a large vertebrate radiation (all Aves) using a model that captures heterogeneous evolutionary rates on time-calibrated phylogenies. We find that extant birds coevolved from warm, mesic climatic niches into colder and drier environments and responded to the K-Pg boundary with a dramatic increase in disparity. Contrary to expectations of subsiding rates of niche evolution as lineages diversify, our results show that overall rates have increased steadily, with some lineages experiencing exceptionally high evolutionary rates, associated with colonization of novel niche spaces, and others showing niche stasis. Both competition- and environmental change-driven niche evolution transpire and result in highly heterogeneous rates near the present. Our findings share the limitations of all work based purely on extant taxa but highlight the growing ecological and conservation insights arising from the model-based integration of increasingly comprehensive and robust environmental and phylogenetic information.

sentences: A variable-rate quantitative trait evolution model using penalized-likelihood In recent years it has become increasingly popular to use phylogenetic comparative methods to investigate heterogeneity in the rate or process of quantitative trait evolution across the branches or clades of a phylogenetic tree. Here, I present a new method for modeling variability in the rate of evolution of a continuously-valued character trait on a reconstructed phylogeny. The underlying model of evolution is stochastic diffusion (Brownian motion), but in which the instantaneous diffusion rate ({sigma}2) also evolves by Brownian motion on a logarithmic scale. Unfortunately, its not possible to simultaneously estimate the rates of evolution along each edge of the tree and the rate of evolution of{sigma} 2 itself using Maximum Likelihood. As such, I propose a penalized-likelihood method in which the penalty term is equal to the log-transformed probability density of the rates under a Brownian model, multiplied by a  smoothing coefficient,{lambda} , selected by the user.{lambda} determines the magnitude of penalty thats applied to rate variation between edges. Lower values of{lambda} penalize rate variation relatively little; whereas larger{lambda} values result in minimal rate variation among edges of the tree in the fitted model, eventually converging on a single value of{sigma} 2 for all of the branches of the tree. In addition to presenting this model here, I have also implemented it as part of my phytools R package in the function multirateBM. Using different values of the penalty coefficient,{lambda} , I fit the model to simulated data with: Brownian rate variation among edges (the model assumption); uncorrelated rate variation; rate changes that occur in discrete places on the tree; and no rate variation at all among the branches of the phylogeny. I then compare the estimated values of{sigma} 2 to their known true values. In addition, I use the method to analyze a simple empirical dataset of body mass evolution in mammals. Finally, I discuss the relationship between the method of this article and other models from the phylogenetic comparative methods and finance literature, as well as some applications and limitations of the approach.

sentences: An intranasal lentiviral booster broadens immune recognition of SARS-CoV-2 variants and reinforces the waning mRNA vaccine-induced immunity that it targets to lung mucosa As the COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines wains and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal humoral and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization because it is non-cytopathic, non-replicative and scarcely inflammatory. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P). mRNA vaccine-primed and -boosted mice, with waning primary humoral immunity at 4 months post-vaccination, were boosted intranasally with LV::SBeta-2P. Strong boost effect was detected on cross-sero-neutralizing activity and systemic T-cell immunity. In addition, mucosal anti-Spike IgG and IgA, lung resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::SBeta-2P vaccine candidate as an intranasal booster against COVID-19.

sentences: Identifying SNP associations and predicting disease risk from Genome-wide association studies using LassoNet In this paper, we show that under certain conditions LassoNet [1] should outperform threshold of significance (P-value) methods for identifying multi-SNP disease associations and predicting disease risk using data from Genome Wide Association Studies. To demonstrate this, we built a genotype-phenotype simulation to comprehensively benchmark each methods performance in variant selection and in predicting disease risk. Our results suggest that LassoNet, with its ease of implementation, should be added to the biomedical informaticians toolkit. We release code to replicate our results.

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Constitutive high expression of NOXA sensitizes human embryonic stem cells for rapid cell death Human embryonic stem (hES) cells are highly sensitive to apoptotic stimuli such as DNA damage, which allows for rapid elimination of mutated cells during development. However, the mechanisms that maintain hES cells in the primed apoptotic state are not completely known. Key activators of apoptosis, the BH3-only proteins, are present at low levels in most cell types. In contrast, hES cells have constitutive high levels of the BH3-only protein, NOXA. We examined the importance of NOXA for enabling apoptosis in hES cells. hES cells deleted for NOXA showed remarkable protection against multiple apoptotic stimuli. NOXA was constitutively localized to the mitochondria, where it interacted with MCL1. Strikingly, inhibition of MCL1 in NOXA knockout cells was sufficient to sensitize these cells to DNA damage, and subsequently, cell death. Our study demonstrates, an essential function of constitutive high levels of NOXA in hES cells is to effectively antagonize MCL1 to permit rapid apoptosis.

Significance statementHuman embryonic stem (hES) cells give rise to the entire organism, hence understanding how these cells regulate their survival and death is important. These cells undergo rapid death in response to DNA damage thereby removing mutated cells from the developing embryo. We focused on identifying the mechanism underlying the sensitivity of these cells to DNA damage. We discovered that the protein NOXA is essential for cell death in hES cells. Further, the crucial function of NOXA is to neutralize high levels of antiapototic protein, MCL1, thus enabling hES cells to respond rapidly to DNA damage.

sentences: Human serum triggers antibiotic tolerance in Staphylococcus aureus Staphylococcus aureus is a frequent cause of bloodstream infections. Treatment can be challenging, even when isolates appear to be drug susceptible, with high rates of persistent and relapsing infection. This is particularly the case with infections caused by methicillin resistant S. aureus (MRSA) strains, which are resistant to frontline antibiotics. To understand how the host environment influences treatment outcomes in MRSA infections, we studied the impact of human serum on staphylococcal susceptibility to daptomycin, an antibiotic of last resort. This revealed that serum triggered a very high degree of tolerance to daptomycin, as well as several other classes of antibiotics and antimicrobial peptides, including gentamicin, nitrofurantoin, vancomycin, nisin and gramicidin. Serum-induced daptomycin tolerance was due to two independent mechanisms. Firstly, the host defence peptide LL-37 present in serum induced tolerance by triggering the staphylococcal GraRS two component system. This led to increased cell wall accumulation that reduced access of daptomycin to its membrane target. Secondly, GraRS-independent changes to the membrane resulted in increased cardiolipin abundance that also contributed to daptomycin tolerance. When both mechanisms were blocked, serum exposed S. aureus cells were as susceptible to daptomycin as bacteria growing in laboratory media. These data demonstrate that host factors can significantly modulate antibiotic susceptibility via diverse mechanisms, which may in turn contribute to treatment failure. The inhibition of serum-induced cell wall accumulation by fosfomycin reduced tolerance, suggesting that this antibiotic may form a useful combination therapy with daptomycin.

sentences: SCARF1-induced efferocytosis plays an immunomodulatory role in humans, and autoantibodies targeting SCARF1 are produced in patients with systemic lupus erythematosus Deficiency in the clearance of cellular debris is a major pathogenic factor in the emergence of autoimmune diseases. We previously demonstrated that mice deficient for scavenger receptor class F member 1 (SCARF1) develop a lupus-like autoimmune disease with symptoms similar to human systemic lupus erythematosus (SLE), including a pronounced accumulation of apoptotic cells (ACs). Therefore, we hypothesized that SCARF1 will be important for clearance of ACs and maintenance of self-tolerance in humans, and that dysregulation of this process could contribute to SLE. Here, we show that SCARF1 is highly expressed on phagocytic cells, where it functions as an efferocytosis receptor. In healthy individuals, we discovered that engagement of SCARF1 by ACs on BDCA1+ dendritic cells (DCs) initiates an interleukin-10 (IL-10) anti-inflammatory response mediated by the phosphorylation of signal transducer and activator of transcription 1 (STAT1). Unexpectedly, there was no significant difference in SCARF1 expression in SLE patient samples compared to healthy donor samples. However, we detected anti-SCARF1 autoantibodies in 26% of SLE patients, which was associated with dsDNA antibody positivity. Furthermore, our data shows a direct correlation of the levels of anti-SCARF1 in the serum and defects in the removal of ACs. Depletion of immunoglobulin restores efferocytosis in SLE serum, suggesting that defects in the removal of ACs is partially mediated by SCARF1 pathogenic autoantibodies. Our data demonstrate that human SCARF1 is an AC receptor in DCs and plays a role in maintaining tolerance and homeostasis.

sentences: Correlation of biomechanics and cancer cell phenotype by combined Brillouin and Raman spectroscopy of U87-MG glioblastoma cells The elucidation of biomechanics furthers understanding of brain tumor biology. Brillouin spectroscopy is a new optical method that addresses viscoelastic properties down to subcellular resolution in contact-free manner. Moreover, it can be combined with Raman spectroscopy to obtain co-localized biochemical information. Here, we applied co-registered Brillouin and Raman spectroscopy to U87-MG human glioblastoma cells in vitro. Using 2D and 3D cultures, we related biomechanical properties with local biochemical composition at subcellular level, as well as cell phenotype. Brillouin and Raman mapping of adherent cells showed that the nucleus and nucleoli are stiffer than the perinuclear region and the cytoplasm. The biomechanics of cell cytoplasm is affected by culturing conditions, i.e. cells grown as spheroids being stiffer than adherent cells. Inside the spheroids, the presence of lipid droplets as assessed by Raman spectroscopy reveals higher Brillouin shifts which is not related to local stiffness increase, but due to a higher refractive index combined with a lower mass density. This highlights the importance of locally defined biochemical reference data for a correct interpretation of the Brillouin shift of cells and tissue in future studies investigating the biomechanics of brain tumor models by Brillouin spectroscopy.

sentences: Cell type-specific interpretation of noncoding variants using deep learning-based methods Interpretation of non-coding genomic variants is one of the most important challenges in human genetics. Machine learning methods have emerged recently as a powerful tool to solve this problem. State-of-the-art approaches allow prediction of transcriptional and epigenetic effects caused by non-coding mutations. However, these approaches require specific experimental data for training and can not generalize across cell types where required features were not experimentally measured. We show here that available epigenetic characteristics of human cell types are extremely sparse, limiting those approaches that rely on specific epigenetic input. We propose a new neural network architecture, DeepCT, which can learn complex interconnections of epigenetic features and infer unmeasured data from any available input. Furthermore, we show that DeepCT can learn cell type-specific properties, build biologically meaningful vector representations of cell types and utilize these representations to generate cell type-specific predictions of the effects of non-coding variations in the human genome.

sentences: Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster Circadian clocks are highly conserved transcriptional regulators that control 24-hour oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcription properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks; we found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. This result suggests that the presumed dominance of the brain clock in regulating peripheral clocks needs to be re-examined. This result further demonstrates that LABL allows rapid, affordable, and direct real-time monitoring of clocks in vivo.

sentences: Biochar alters soil properties, microbial community diversity, and enzyme activities, while decreasing conifer performance Biochars are porous charcoal-like materials that can enhance soil health and plant growth, but its use has not been adequately evaluated in woody cropping systems. We set up an experimental Christmas tree plantation on a Marlette series soil amended with two types of biochar and conducted two studies on the impacts of biochar on the agroecosystem over three years following establishment. The first study investigated the effects of biochar on plant performance, soil physicochemical properties and extracellular enzyme activities, while the second investigated the response of the root-associated fungal community. Both biochars stimulated five extracellular enzyme activities, with increases of between 67% and 446%. Structural equation modelling identified increases to dissolved organic carbon and soil moisture as potential mechanisms of biochars effects on enzyme activities. Tree growth and survival were negatively affected by biochar application, depending on the tree species and biochar applied, which may have been due to induced nitrogen limitation. High-throughput sequencing showed that biochar decreased the diversity of root-associated fungal communities, with the ectomycorrhizal species Wilcoxina mikolae reaching levels of hyper-dominance on balsam fir in response to one of the biochars. Further studies should investigate how biochar can be harnessed to remediate specific soil quality issues or restructure soil ecosystems in ways that improve crop performance.

sentences: Rejuveinix Mitigates Sepsis-Associated Oxidative Stress in the Brain of Mice: Clinical Impact Potential in COVID-19 and Nervous System Disorders Here, we demonstrate that our anti-sepsis and COVID-19 drug candidate Rejuveinix (RJX) substantially improves the survival outcome in the LPS-GalN animal model of sepsis and multi-organ failure. One hundred (100) percent (%) of untreated control mice remained alive throughout the experiment. By comparison, 100% of LPS-GalN injected mice died at a median of 4.6 hours. In contrast to the invariably fatal treatment outcome of vehicle-treated control mice, 40% of mice treated with RJX (n=25) remained alive with a 2.4-fold longer median time survival time of 10.9 hours (Log-rank X2=20.60, P<0.0001). Notably, RJX increased the tissue levels of antioxidant enzymes SOD, CAT, and GSH-Px, and reduced oxidative stress in the brain. These findings demonstrate the clinical impact potential of RJX as a neuroprotective COVID-19 and sepsis drug candidate.

sentences: Effect of Stapling on the Thermodynamics of mdm2-p53 Binding Protein-protein interaction (PPI) is one of the key regulatory features to drive biomolecular processes and hence is targeted for designing therapeutics against diseases. Small peptides are a new and emerging class of therapeutics owing to their high specificity and low toxicity. For achieving efficient targeting of the PPI, amino acid side chains are often stapled together resulting in the rigidification of these peptides. Exploring the scope of these peptides demands a comprehensive understanding of their working principle. In this work, two stapled p53 peptides have been considered to delineate their binding mechanism with mdm2 using computational approaches. Addition of stapling protects the secondary structure of the peptides even in the case of thermal and chemical denaturation. Although the introduction of a stapling agent increases the hydrophobicity of the peptide, the enthalpic stabilization decreases. This is overcome by the lowering of the entropic penalty and the overall binding affinity improves. The mechanistic insights into the benefit of peptide stapling can be adopted for further improvement of peptide therapeutics.

sentences: The transcriptome of Schistosoma mansoni developing eggs reveals key mediators in pathogenesis and life cycle propagation. Schistosomiasis, the most important helminthic disease of humanity, is caused by infection with parasitic flatworms of the genus Schistosoma. The disease is driven by the eggs laid by the parasites and becoming trapped in host tissues, followed by inflammation and granuloma formation. Despite abundant transcriptome data for most developmental stages of the three main human-infective schistosome species, i.e. Schistosoma mansoni, S. japonicum and S. haematobium, the transcriptomic profiles of developing eggs remain largely unexplored. In this study, we performed RNAseq of S. mansoni eggs laid in vitro during early and late embryogenesis (days 1-3 and 3-6 post-oviposition, respectively). Analysis of the transcriptomes identified hundreds of up-regulated genes during the later stage, including venom allergen-like (VAL) proteins, well-established host immunomodulators, and genes involved in organogenesis of the miracidium larva. In addition, the transcriptomes of the in vitro laid eggs were compared with existing publicly available RNA-seq dataset from S. mansoni eggs collected from the livers of murine hosts. Analysis of enriched GO terms and pathway annotations revealed cell division and protein synthesis processes associated with early embryogenesis, whereas cellular metabolic processes, microtubule-based movement, and microtubule cytoskeleton organization were found enriched in the later developmental time point. This is the first transcriptomic analysis of S. mansoni embryonic development, and will facilitate our understanding of infection pathogenesis, miracidia development and life cycle progression of schistosomes.

sentences: Water Pollution and Administrative Division Adjustments: A Quasi-natural Experiment in Chaohu Lake of China The adjustment of administrative divisions introduces a series of uncertain impacts on the social and economic development in the administrative region. Previous studies focused more on the economic effects of the adjustment of administrative divisions, while, in this paper, we also take environmental effects into consideration. The administrative division adjustment for Chaohu Lake is used as a quasi-natural experiment to explore the influence of the adjustment on pollution control. The synthetic control method is used in this study to access the effect of administrative division adjustment on the water quality indicators of Chaohu Lake and its internal mechanism. Some conclusions are as follows. First, after the administrative division adjustment, some water quality indicators, such as ammonia nitrogen, have indeed been alleviated; however, other major pollution indicators, such as chemical oxygen demand and dissolved oxygen, have deteriorated to varying degrees. Second, the results also reveal that improper development ideas, industrial excessive expansion, and the swing of economic growth and environmental goals are problems after the adjustment. Returning to the original intention of adjustment, rationalizing the Chaohu Lake management system and designing a sound and feasible accountability mechanism are fundamental measures to reduce pollution.

sentences: Normal Aging in Mice is Associated with a Global Reduction in Cortical Spectral Power and a Network-Specific Decline in Functional Connectivity Normal aging is associated with a variety of neurologic changes including declines in cognition, memory, and motor activity. These declines correlate with neuronal changes in synaptic structure and function. Degradation of brain network activity and connectivity represents a likely mediator of age-related functional deterioration resulting from these neuronal changes. Human studies have demonstrated both general decreases in spontaneous cortical activity and disruption of cortical networks with aging. Current techniques used to study cerebral network activity are hampered either by limited spatial resolution (e.g. electroencephalography, EEG) or limited temporal resolution (e.g., functional magnetic resonance imaging, fMRI). Here we utilize mesoscale imaging of neuronal activity in Thy1-GCaMP6f mice to characterize neuronal network changes in aging with high spatial resolution across a wide frequency range. We show that while evoked activity is unchanged with aging, spontaneous neuronal activity decreases across a wide frequency range (0.01-4Hz) involving all regions of the cortex. In contrast to this global reduction in cortical power, we found that aging is associated with functional connectivity (FC) deterioration of select networks including somatomotor, cingulate, and retrosplenial nodes. These changes are corroborated by reductions in homotopic FC and node degree within somatomotor and visual cortices. Finally, we found that whole cortex delta power and delta band node degree correlate with exploratory activity in young but not aged animals. Together these data suggest that aging is associated with global declines in spontaneous cortical activity and focal deterioration of network connectivity, and that these reductions may be associated with age-related behavioral declines.

sentences: Slow TCA flux implies low ATP production in tumors The tricarboxylic acid (TCA) cycle oxidizes carbon substrates to carbon dioxide, with the resulting high energy electrons fed into the electron transport chain to produce ATP by oxidative phosphorylation. Healthy tissues derive most of their ATP from oxidative metabolism, and the remainder from glycolysis. The corresponding balance in tumors remains unclear. Tumors upregulate aerobic glycolysis (the Warburg effect), yet they also typically require an intact TCA cycle and electron transport chain1-6. Recent studies have measured which nutrients contribute carbon to the tumor TCA metabolites7,8, but not tumor TCA flux: how fast the cycle turns. Here, we develop and validate an in vivo dynamic isotope tracing-mass spectrometry strategy for TCA flux quantitation, which we apply to all major mouse organs and to five tumor models. We show that, compared to the tissue of origin, tumor TCA flux is markedly suppressed. Complementary glycolytic flux measurements confirm tumor glycolysis acceleration, but the majority of tumor ATP is nevertheless made aerobically, and total tumor ATP production is suppressed compared to healthy tissues. In murine pancreatic cancer, this is accommodated by downregulation of the major energy-using pathway in the healthy exocrine pancreas, protein synthesis. Thus, instead of being hypermetabolic as commonly assumed, tumors apparently make ATP at a lower than normal rate. We propose that, as cells de-differentiate into cancer, they eschew ATP-intensive processes characteristic of the host tissue, and that the resulting suppressed ATP demand contributes to the Warburg effect and facilitates cancer growth in the nutrient-poor tumor microenvironment.

sentences: SARS-CoV-2 variant B.1.617 is resistant to Bamlanivimab and evades antibodies induced by infection and vaccination The emergence of SARS-CoV-2 variants threatens efforts to contain the COVID-19 pandemic. The number of COVID-19 cases and deaths in India has risen steeply in recent weeks and a novel SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding domain, which interacts with the ACE2 receptor and constitutes the main target of neutralizing antibodies. Therefore, we analyzed whether B.1.617 is more adept in entering cells and/or evades antibody responses. B.1.617 entered two out of eight cell lines tested with slightly increased efficiency and was blocked by entry inhibitors. In contrast, B.1.617 was resistant against Bamlanivimab, an antibody used for COVID-19 treatment. Finally, B.1.617 evaded antibodies induced by infection or vaccination, although with moderate efficiency. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to the rapid spread of this variant.

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Trimeric Photosystem I facilitates energy transfer from phycobilisomes in Synechocystis sp. PCC 6803 In cyanobacteria, phycobilisomes serve as peripheral light-harvesting complexes of the two photosystems, extending their antenna size and the wavelength range of photons available for photosynthesis. The abundance of phycobilisomes, the number of phycobiliproteins they contain, and their light-harvesting function are dynamically adjusted in response to the physiological conditions. Phycobilisomes are also thought to be involved in state transitions that maintain the excitation balance between the two photosystems. Unlike its eukaryotic counterpart, PSI is trimeric in many cyanobacterial species and the physiological significance of this is not well understood. Here we compared the composition and light-harvesting function of phycobilisomes in cells of Synechocystis sp. PCC 6803, which has primarily trimeric PSI, and the{Delta} psaL mutant unable to form trimers. We also investigated a mutant additionally lacking the PsaJ and PsaF subunits of PSI. Both strains with monomeric PSI accumulated significantly more allophycocyanin per chlorophyll, indicating higher abundance of phycobilisomes. On the other hand, a higher phycocyanin:allophycocyanin ratio in WT suggests larger phycobilisomes or the presence of APC-less phycobilisomes (CpcL-type), that are not assembled in cells with monomeric PSI. Steady-state and time-resolved fluorescence spectroscopy at room temperature and 77 K revealed that PSII receives more energy from the phycobilisomes at the expense of PSI in cells with monomeric PSI, regardless of the presence of PsaF. Taken together, these results show that the oligomeric state of PSI has an impact on the excitation energy flow in Synechocystis.

One-sentence summaryCyanobacterial mutants with monomeric PSI show changes in the composition and abundance of phycobilisomes and in the excitation energy transfer to PSII and PSI.

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Repression of Hypoxia-Inducible Factor-1 Contributes to Increased Mitochondrial Reactive Oxygen Species Production in Diabetes BackgroundExcessive production of mitochondrial reactive oxygen species (ROS) is a central mechanism for the development of diabetes complications. Recently, hypoxia has been identified to play an additional pathogenic role in diabetes. In this study, we hypothesized that ROS overproduction was secondary to the impaired responses to hypoxia due to the inhibition of hypoxia-inducible factor-1 (HIF-1) by hyperglycemia.

MethodsThe dynamic of ROS levels was analysed in the blood of healthy subjects and individuals with type 1 diabetes after exposure to hypoxia (ClinicalTrials.gov registration no. NCT02629406). The relation between HIF-1, glucose levels, ROS production and its functional consequences were analyzed in renal mIMCD-3 cells and in kidneys of mouse models of diabetes.

ResultsExposure to hypoxia increased circulating ROS in subjects with diabetes, but not in subjects without diabetes. High glucose concentrations repressed HIF-1 both in hypoxic cells and in kidneys of animals with diabetes, through a HIF prolyl-hydroxylase (PHD) - dependent mechanism. The impaired HIF-1 signaling contributed to excess production of mitochondrial ROS through increased mitochondrial respiration that was mediated by Pyruvate dehydrogenase kinase 1 (PDK1) and was followed by functional consequences. The restoration of HIF-1 function attenuated ROS overproduction despite persistent hyperglycemia, and conferred protection against apoptosis and renal injury in diabetes.

ConclusionsWe conclude that the repression of HIF-1 plays a central role in mitochondrial ROS overproduction in diabetes and is a potential therapeutic target for diabetic complications. These findings are highly significant and timely since the first PHD inhibitor that can activate HIF-1 has been newly approved for clinical use.

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The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells BackgroundDNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESC) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss.

ResultsWe determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 {micro}M or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within two days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole genome bisulfite sequencing (WGBS) showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862, similar to the methylation level observed in Dnmt1 deficient mESCs.

ConclusionsGSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.

sentences: Epithelial-mesenchymal plasticity determines estrogen receptor positive (ER+) breast cancer dormancy and reacquisition of an epithelial state drives awakening Estrogen receptor -positive (ER+) breast cancers (BCs) represent more than 70% of all breast cancers and pose a particular clinical challenge because they recur up to decades after initial diagnosis and treatment. The mechanisms governing tumor cell dormancy and latent disease remain elusive due to a lack of adequate models. Here, we compare tumor progression of ER+ and triple-negative (TN) BC subtypes with a clinically relevant mouse intraductal xenografting approach (MIND). Both ER+ and TN BC cells disseminate already during the in situstage. However, TN disseminated tumor cells (DTCs) proliferate at the same rate as cells at the primary site and give rise to macro-metastases. ER+ DTCs have low proliferative indices, form only micro-metastases and lose epithelial characteristics. Expression of CDH1 is decreased whereas the mesenchymal marker VIM and the transcription factors, ZEB1/ZEB2, which control epithelial-mesenchymal plasticity (EMP) are increased. EMP is not detected earlier during ER+ BC development and not required for invasion or metastasis. In vivo, forced transition to the epithelial state through ectopic E-cadherin expression overcomes dormancy with increased growth of lung metastases. We conclude that EMP is essential for the generation of a dormant cell state and the development of latent disease. Targeting exit from EMP is of therapeutic potential.

sentences: Identification of candidate biomarkers and pathways associated with type 1 diabetes mellitus using bioinformatics analysis Type 1 diabetes mellitus (T1DM) is a metabolic disorder for which the underlying molecular mechanisms remain largely unclear. This investigation aimed to elucidate essential candidate genes and pathways in T1DM by integrated bioinformatics analysis. In this study, differentially expressed genes (DEGs) were analyzed using DESeq2 of R package from GSE162689 of the Gene Expression Omnibus (GEO). Gene ontology (GO) enrichment analysis, REACTOME pathway enrichment analysis, and construction and analysis of protein-protein interaction (PPI) network, modules, miRNA-hub gene regulatory network and TF-hub gene regulatory network, and validation of hub genes were then performed. A total of 952 DEGs (477 up regulated and 475 down regulated genes) were identified in T1DM. GO and REACTOME enrichment result results showed that DEGs mainly enriched in multicellular organism development, detection of stimulus, diseases of signal transduction by growth factor receptors and second messengers, and olfactory signaling pathway. The top hub genes such as MYC, EGFR, LNX1, YBX1, HSP90AA1, ESR1, FN1, TK1, ANLN and SMAD9 were screened out as the critical genes among the DEGs from the PPI network, modules, miRNA-hub gene regulatory network and TF-hub gene regulatory network. Receiver operating characteristic curve (ROC) analysis and RT-PCR confirmed that these genes were significantly associated with T1DM. In conclusion, the identified DEGs, particularly the hub genes, strengthen the understanding of the advancement and progression of T1DM, and certain genes might be used as candidate target molecules to diagnose, monitor and treat T1DM.

sentences: Biogenesis of mature piRNAs in Caenorhabditis elegans is dependent on orchestration between multiple ribonucleases and PRG-1 Piwi-interacting RNAs (piRNAs) are an animal-specific class of germline-enriched small non-coding RNAs that shape transcriptome, as well as ensure genomic integrity and fertility by regulating transposons and other selfish genetic elements. In Caenorhabditis elegans mature piRNAs are 21-nucleotides long, begin with a monophosphorylated uridine, and they associate with PRG-1 to form piRISCs that scan the transcriptome for  non-self sequences. However, these piRNAs are born as longer 5-capped transcripts, where PARN-1, a 3-5 exoribonuclease, contributes to the formation of the mature 3-end. But, till date, the 5-processing events remain elusive. We demonstrate that the recently identified endoribonuclease activity of XRN-2 is involved in the processing of the 5-end of precursor piRNAs in worms. Depletion of XRN-2 results in reduced mature piRNA levels, with concomitant increase in levels of the 5-capped precursors. We also reveal that the piRNAs born as longer precursor molecules ([≥]60 nt), prior to 5-end processing, undergo ENDU-1-mediated endoribonucleolytic processing of their 3-ends. Our in vitro RNA-protein interaction studies unravel the mechanistic interactions between XRN-2 and PRG-1 towards the formation of mature 5-ends of piRNAs. In vivo experiments employing prg-1 mutant worms indicate that XRN-2 has the potential to perform clearance of precursors that are not bound and protected by PRG-1. Finally, we also demonstrate that XRN-2 is not only important for the generation of mature piRNAs and piRNA-dependent endo-siRNAs, but through yet unknown pathways, it also affects piRNA-independent endo-siRNAs that shape transcriptome, as well as contribute to genomic integrity via regulation of transposable elements.

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Transendothelial migration induces differential migration dynamics of leukocytes in tissue matrix Leukocyte extravasation into inflamed tissue is a complex process that is difficult to capture as a whole in vitro. We employed a blood-vessel-on-a-chip model in which endothelial cells were cultured in a tube-like lumen in a collagen-1 matrix. The vessels are leak-tight, creating a barrier for molecules and leukocytes. Addition of inflammatory cytokine TNF- caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualisation of all different steps of the leukocyte transmigration cascade including migration into the extracellular matrix. Individual cell tracking over time distinguished striking differences in migratory behaviour between T-cells and neutrophils. Neutrophils cross the endothelial layer more efficiently than T-cells, but upon entering the matrix, neutrophils display high speed but low persistence, whereas T-cells migrate with low speed and rather linear migration. In conclusion, 3D imaging in real-time of leukocyte extravasation in a vessel-on-a-chip enables detailed qualitative and quantitative analysis of different stages of the full leukocyte extravasation process in a single assay.

SummaryA functional hydrogel-based blood-vessel-on-a-chip model is used to study the complete leukocyte transendothelial migration process in real time. T-lymphocytes and neutrophils exhibit distinct migration dynamics in the extravascular matrix after transendothelial migration, which can be altered using a chemotactic gradient.

sentences: Sequence characterization of the complete chloroplast genome of Eupatorium fortunei Eupatorium fortunei has been utilized as herbal medicine to cure various diseases in various regions of the world especially China, Korea, and other Asian countries. The available literature shows Eupatorium fortunei contain anti-metastatic, anti-angiogenic, anti-bacterial, and anti-oxidant, along with compounds to cure human cancer. In present study, Eupatorium fortunei was used as an experimental material, and its genome was sequenced based on high-throughput sequencing technology. We assembled the complete chloroplast genome of Eupatorium fortunei and obtained NCBI accession number (OK545755). We analyzed chloroplast genome structure of Eupatorium fortunei and systematic analysis was conducted, including the size of the entire genome sequence, genome volume, large single-copy (LSC), small single-copy (SSC), inverted repeat region (IR)-LSC/SSC boundaries, etc. The obtained results showed that chloroplast genome of Eupatorium fortunei is a typical tetrad structure with a total length of 152,401 bp and a sum of 133 annotated genes, including eight ribosomal RNA (rRNA)-encoding genes, 37 transfer RNA (tRNA)-encoding genes, 87 protein-encoding genes, and one pseudo-gene. A sum of 29 Single Sequence Repeats (SSR)s were identified for mono-nucleotide, di-nucleotide and tri-nucleotide microsatellites with mono-nucleotides in majority. The phylogenetic analysis revealed that Eupatorium fortunei is more closely related to Litothamnus nitidus and belongs to the same branch. The genome assembly and analysis of the chloroplast genome of Eupatorium fortunei provides more accurate evidence for further taxonomic identification and localization of Asteraceae/Compositae plant.

sentences: EXTENDED TUDOR-DOMAINS of the piRNA BIOGENESIS PATHWAY HAVE RNA-SPECIFIC NUCLEASE ACTIVITY piRNAs are essential for transposon repression and protecting the germline from deleterious mutations. piRNA biogenesis comprises a primary and secondary pathway, and involves PIWI clade argonaute proteins and ancillary factors. Secondary piRNA biogenesis is tightly coupled to transposon repression. It requires processing of the 3 end of pre-piRNA during an amplification loop by an as yet unidentified endonuclease. Here, using crystallography, and biochemical assays, we discover that the Drosophila Qin protein, which is a critical member of the core amplification complex, has endonuclease activity. Qin contains five extended Tudor domains, which had been proposed to recognize methylated ligands. Instead, we show that these domains act as RNA-specific nucleases. This supports a role for Qin in the 3 end processing of Ago3-bound pre-piRNAs. Extended Tudor domains are frequent in piRNA-processing proteins, suggesting that the uncovered nuclease activity of this protein fold may be key to understanding the piRNA biogenesis.

sentences: Corynebacterium matruchotii fitness enhancement of adjacent streptococci by multiple mechanisms Polymicrobial biofilms are present in many environments particularly in the human oral cavity where they can prevent or facilitate the onset of disease. While recent advances have provided a clear picture of both the constituents and their biogeographical arrangement, it is still unclear what mechanisms of interaction occur between individual species in close proximity within these communities. In this study we investigated two mechanisms of interaction between the highly abundant supragingival plaque (SUPP) commensal Corynebacterium matruchotii and Strepto-coccus mitis which are directly adjacent in vivo. We discovered that C. matruchotii enhanced the fitness of streptococci dependent on its ability to detoxify streptococcal-produced hydrogen per-oxide and its ability to oxidize lactate also produced by streptococci. We demonstrate that the fitness of adjacent streptococci was linked to that of C. matruchotii and that these mechanisms support the previously described corncob arrangement between these species but that this is favorable only in aerobic conditions. Further we utilized scanning electrochemical microscopy (SECM) to quantify lactate production and consumption between individual bacterial cells for the 1st time, revealing that lactate oxidation provides a fitness benefit to S. mitis and not pH mitigation. This study describes mechanistic interactions between two highly abundant human com-mensals that can explain their observed in vivo spatial arrangements and suggest a way by which they may help preserve a healthy oral bacterial community.

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LuxT is a Global Regulator of Low-Cell Density Behaviors Including Type III Secretion, Siderophore Production, and Aerolysin Production in Vibrio harveyi Quorum sensing (QS) is a chemical communication process in which bacteria produce, release, and detect extracellular signaling molecules called autoinducers. Via combined transcriptional and post-transcriptional regulatory mechanisms, QS allows bacteria to collectively alter gene expression on a population-wide scale. Recently, the TetR-family transcriptional regulator LuxT was shown to control V. harveyi qrr1, encoding the Qrr1 small RNA that functions at the core of the QS regulatory cascade. Here, we use RNA-Sequencing to reveal that, beyond control of qrr1, LuxT is a global regulator of 414 V. harveyi genes including those involved in type III secretion, siderophore production, and aerolysin toxin biosynthesis. Importantly, LuxT directly represses swrZ, encoding a GntR-family transcriptional regulator, and LuxT control of type III secretion, siderophore, and aerolysin genes occurs by two mechanisms, one that is SwrZ-dependent and one that is SwrZ-independent. All of these target genes specify QS-controlled behaviors that are enacted when V. harveyi is at low cell density. Thus, LuxT and SwrZ function in parallel with QS to drive particular low cell density behaviors. Phylogenetic analyses reveal that luxT is highly conserved among Vibrionaceae, but swrZ is less well conserved. In a test case, we find that in Aliivibrio fischeri, LuxT also represses swrZ. SwrZ is a repressor of A. fischeri siderophore production genes. Thus, LuxT repression of swrZ drives activation of A. fischeri siderophore gene expression. Our results indicate that LuxT is a major regulator among Vibrionaceae, and, in the species that also possess swrZ, LuxT functions with SwrZ to control gene expression.

ImportanceBacteria precisely tune gene expression patterns to successfully react to changes that occur in the environment. Defining the mechanisms that enable bacteria to thrive in diverse and fluctuating habitats, including in host organisms, is crucial for a deep understanding of the microbial world and also for development of effective applications to promote or to combat particular bacteria. In this study, we show that a regulator called LuxT controls over 400 genes in the marine bacterium Vibrio harveyi and, moreover, that LuxT is highly conserved among Vibrionaceae species, ubiquitous marine bacteria that often cause disease. We characterize the mechanisms by which LuxT controls genes involved in virulence and nutrient acquisition. We show that LuxT functions in parallel with a set of regulators of the bacterial cell-to-cell communication process called quorum sensing to promote V. harveyi behaviors at low cell density.

sentences: Chimpanzees communicate to coordinate a cultural practice Human culture is considered to differ from animal culture due to its interactive nature built on shared intentionality and cognitive flexibility. Here, we investigated whether chimpanzees use communication to engage in cultural practices by analyzing grooming handclasp (GHC) interactions - a socio-cultural behavior requiring coordination. Previous accounts attributed GHC initiations to behavioral shaping whereby the initiator physically molds the partners arm into the GHC posture. Using frame-by-frame analysis and matched-control methodology, we find that chimpanzees use gestural communication to initiate GHC, which requires an active and synchronized response from the partner. This showcases a behavioral expression of joint commitment to engage in this shared cultural practice. Moreover, we show that GHC initiators used various initiation strategies, attesting to situation-contingent interactional flexibility. We conclude that chimpanzees can be jointly committed to a cultural practice, which suggests that culture predicated on shared intentionality and flexible communication may not be unique to the human species.

sentences: Fully-automated and ultra-fast cell-type identification using specific marker combinations from single-cell transcriptomic data Single-cell transcriptomics enables systematic charting of cellular composition of complex tissues. Identification of cell populations often relies on unsupervised clustering of cells based on the similarity of their scRNA-seq profiles, followed by manual annotation of cell clusters using established marker genes. However, manual selection of marker genes is a time-consuming process that may lead to sub-optimal annotation results as the selected markers must be informative of both the individual cell clusters and various cell types present in the complex samples. Here, we developed a computational platform, termed ScType, which enables data-driven, fully-automated and ultra-fast cell-type identification based solely on given scRNA-seq data, combined with our comprehensive cell marker database as background information. Using a compendium of six scRNA-seq datasets from various human and mouse tissues, we show how ScType provides an unbiased and accurate cell-type annotation by guaranteeing the specificity of positive and negative marker genes both across cell clusters and cell types. We also demonstrate how ScType enables distinguishing between healthy and malignant cell populations, based on single-cell calling of single-nucleotide variants, making it a versatile tool for exploration and use of single-cell transcriptomic data for anticancer applications. The widely-applicable method is deployed both as an interactive web-tool (https://sctype.app), and as an open-source R-package, connected with a comprehensive ScType database of specific markers.

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Robust Rhythmogenesis via Spike Timing Dependent Plasticity Rhythmic activity has been observed in numerous animal species ranging from insects to humans, and in relation to a wide range of cognitive tasks. Various experimental and theoretical studies have investigated rhythmic activity. The theoretical efforts have mainly been focused on the neuronal dynamics, under the assumption that network connectivity satisfies certain fine-tuning conditions required to generate oscillations. However, it remains unclear how this fine tuning is achieved.

Here we investigated the hypothesis that spike timing dependent plasticity (STDP) can provide the underlying mechanism for tuning synaptic connectivity to generate rhythmic activity. We addressed this question in a modeling study. We examined STDP dynamics in the framework of a network of excitatory and inhibitory neuronal populations that has been suggested to underlie the generation of oscillations in the gamma range. Mean field Fokker Planck equations for the synaptic weights dynamics are derived in the limit of slow learning. We drew on this approximation to determine which types of STDP rules drive the system to exhibit rhythmic activity, and demonstrate how the parameters that characterize the plasticity rule govern the rhythmic activity. Finally, we propose a novel mechanism that can ensure the robustness of self-developing processes, in general and for rhythmogenesis in particular.

sentences: GABA facilitates spike propagation through branch points of sensory axons in the spinal cord Movement and posture depend on sensory feedback that is regulated by specialized GABAergic neurons (GAD2+) that form axo-axonic contacts onto myelinated proprioceptive sensory axons and are thought to be inhibitory. However, we report here that activating GAD2+ neurons, directly with optogenetics or indirectly by cutaneous stimulation, facilitates sensory feedback to motoneurons in awake rodents and humans. GABAA receptors and GAD2+ innervation at or near nodes of Ranvier of sensory axons cause this facilitation, preventing spike propagation failure at the many axon branch points, which is otherwise common without GABA. In contrast, GABAA receptors are generally lacking from axon terminals (unlike GABAB) and do not presynaptically inhibit transmitter release onto motoneurons. GABAergic innervation near nodes and branch points allows individual branches to function autonomously, with GAD2+ neurons regulating which branches conduct, adding a computational layer to the neuronal networks generating movement and likely generalizing to other CNS axons.

sentences: Using ddRAD-seq phylogeography to test for genetic effects of headwater river capture in suckermouth armored catfish (Loricariidae: Hypostomus) from the central Brazilian Shield River capture is a geological process of potentially great importance in shaping the genetic diversity, distributions, and community composition of freshwater taxa. Using phylogeographic analyses of ddRAD-seq data from suckermouth armored catfish (Hypostomus sp. 2) populations, we tested for predicted genetic effects of headwater river capture events in central Brazil, previously supported by geological and community ecological data. We analyzed 227 ddRAD tags (3829 SNP loci) across 42 samples. Molecular results strongly supported six Hypostomus genetic clusters/lineages, with the deepest divergence [~]1.25 million years ago in the early Pleistocene between a clade from the Upper Parana and Upper Sao Francisco river basins versus all other lineages. Consistent with the  Parana Capture Hypothesis, several lines of evidence supported mid-Pleistocene colonization and vicariant isolation of Hypostomus populations from an ancestral Upper Parana population, including: (1) significant phylogeographic structure, with predicted phylogenetic patterns, (2) higher Parana lineage diversity, (3) ancestral geographic locations reconstructed in the Parana basin, and (4) non-random interdrainage dispersal and vicariance events, indicating river captures primarily into the Tocantins and Upper Sao Francisco basins c. [~]220,000-145,500 years ago. Phylogeographic inference was complicated by lack of lineage monophyly across loci and lineages distributed in multiple basins, the latter of which lent support to the non-mutually exclusive  Frequent Interdrainage Dispersal Hypothesis. However, species tree and demographic modeling results suggested these were artefacts of incomplete sorting of alleles in large ancestral populations over a geologically recent timeframe of divergence. Qualitative and quantitative sensitivity analyses demonstrated that our downstream genetic results were robust to effects of varying ddRAD-seq assembly parameters, which heavily influenced the number of output loci. We predict that codistributed freshwater taxa in Central Brazil may not exhibit phylogeographic patterns similar to Hypostomus sp. 2 due to complex patterns of superimposed river capture events, or if smaller ancestral population sizes have allowed more complete lineage sorting in other taxa.

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Environmental differences explain subtle yet detectable genetic structure in a widespread pollinator BackgroundThe environment is a strong driver of genetic structure in many natural populations, yet often neglected in population genetic studies. This may be a particular problem in vagile species, where subtle structure cannot be explained by limitations to dispersal. These species might falsely be considered panmictic and hence potentially mismanaged. Here we analysed the genetic structure in an economically important and widespread pollinator, the buff-tailed bumble bee (Bombus terrestris), which is considered to be quasi-panmictic at mainland continental scales. We first quantified population structure in Romania and Bulgaria with spatially implicit Fst and Bayesian clustering analyses. We then incorporated environmental information to infer the influence of the permeability of the habitat matrix between populations (resistance distances) as well as environmental differences among sites in explaining population divergence.

ResultsGenetic structure of the buff-tailed bumble bee was subtle and not detected by Bayesian clustering. As expected, geographic distance and habitat permeability were not informative in explaining the spatial pattern of genetic divergence. Yet, environmental variables related to temperature, vegetation and topography were highly informative, explaining between 33 and 39% of the genetic variation observed.

ConclusionsWhere in the past spatially implicit approaches had repeatedly failed, incorporating environmental data proved to be highly beneficial in detecting and unravelling the drivers of genetic structure in this vagile and opportunistic species. Indeed, structure followed a pattern of isolation by environment, where the establishment of dispersers is limited by environmental differences among populations, resulting in the disruption of genetic connectivity and the divergence of populations through genetic drift and divergent natural selection. With this work, we highlight the potential of incorporating environmental differences among population locations to complement the more traditional approach of isolation by geographic distance, in order to obtain a holistic understanding of the processes driving structure in natural populations.

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Natural enemies have inconsistent impacts on the coexistence of competing species O_LIThe role of natural enemies in promoting coexistence of competing species has generated substantial debate. Modern coexistence theory provides a detailed framework to investigate this topic, but there have been remarkably few empirical applications to the impact of natural enemies.
C_LIO_LIWe tested experimentally the capacity for a generalist enemy to promote coexistence of competing insect species, and the extent to which any impact can be predicted by trade-offs between reproductive rate and susceptibility to natural enemies.
C_LIO_LIWe used experimental mesocosms to conduct a fully-factorial pairwise competition experiment for six rainforest Drosophila species, with and without a generalist pupal parasitoid. We then parameterised models of competition and examined the coexistence of each pair of Drosophila species within the framework of modern coexistence theory.
C_LIO_LIWe found idiosyncratic impacts of parasitism on pairwise coexistence, mediated through changes in fitness differences, not niche differences. There was no evidence of an overall reproductive rate - susceptibility trade-off. Pairwise reproductive rate - susceptibility relationships were not useful shortcuts for predicting the impact of parasitism on coexistence.
C_LIO_LIOur results exemplify the value of modern coexistence theory in multi-trophic contexts and the importance of contextualising the impact of natural enemies. In the set of species investigated, competition was affected by the higher trophic level, but the overall impact on coexistence cannot be easily predicted just from knowledge of relative susceptibility. Methodologically, our Bayesian approach highlights issues with the separability of model parameters within modern coexistence theory and shows how using the full posterior parameter distribution improves inferences. This method should be widely applicable for understanding species coexistence in a range of systems.
C_LI

sentences: Single nuclei RNAseq stratifies multiple sclerosis patients into three distinct white matter glia responses The lack of understanding as to the cellular and molecular basis of clinical and genetic heterogeneity in progressive multiple sclerosis (MS) has hindered the search for new effective therapies and biomarkers. Here, to address this gap, we analysed 740,000 single nuclei RNAseq profiles of 165 samples of white matter (WM) lesions, normal appearing WM, grey matter (GM) lesions and normal appearing GM from 55 MS patients and 28 controls. We find that gene expression changes in response to MS are highly cell-type specific in WM and GM lesions but are largely shared within an individual cell-type across lesions, following a continuum rather than discrete lesion-specific molecular programs. The major biological determinants of variability in gene expression in MS samples relate to individual patient effects, rather than to lesion types or other metadata. Using multi-omics factor analysis (MOFA+), we identify three subgroups of MS patients with distinct oligodendrocyte composition and WM glial gene expression signatures, suggestive of engagement of different pathological/regenerative processes. The discovery of these three patterns significantly advances our mechanistic understanding of progressive MS, provides a framework to use molecular biomarkers to stratify patients for best therapeutic approaches for progressive MS, and highlights the need for precision-medicine approaches to address heterogeneity among MS patients.

sentences: Rating enrichment items by group-housed laboratory mice in multiple binary choice tests using an RFID-based tracking system There is growing evidence that enrichment of housing conditions of laboratory animals has positive effects on behavior, growth, and health. Laboratory mice spend most of their lives in their housing rather than in experimental apparatus, so improving housing conditions is a first-choice approach to improving their welfare. Despite the increasing popularity of enrichment, little is known about whether it is also perceived as being beneficial from the animals point of view. This is especially true due to the fact that  enrichment has become an umbrella term that encompasses a wide variety of different elements. Therefore, we categorized enrichment items according to their prospective use into the categories  structural,  housing, and  foraging. In multiple binary choice tests we let 12 female C57BL/6J mice chose and rank 5 enrichment items per category. All possible pair combinations of enrichment items within each category were presented counterbalanced for a 46-hour period in a home cage based system consisting of two interconnected cages. A new analyzing method combined the binary decisions and ranked the enrichment items within each category by calculating worth values and consensus errors. Mice ranked the lattice ball (foraging), the rope (structural) and the second plane (structural) in upper positions. No clear preferences were determined for different types of housing enrichment during inactive times (light phase) whereas these objects were actively explored during the dark phase. Here the floorhouse and the paperhouse revealed high worth values. Overall, a high consensus error in ranking positions was observed reflecting strong individual differences in preferences. This highlights the importance of a varied enrichment approach as not all mice prefer the same item at all times. Given the known overall beneficial effects of enrichment, these data will help to provide appropriate enrichment elements to improve animal welfare and refine animal experimentation.

sentences: CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR) components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-associated (Cas) nuclease. Here we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors, derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.

sentences: Day length-dependent thermal COP1 dynamics integrate conflicting seasonal cues in the control of Arabidopsis elongation As the summer approaches, plants experience enhanced light inputs and elevated temperatures, two environmental cues with an opposite morphogenic impact. How plants integrate this conflicting information throughout seasons remains unclear. Key components of the plant response to light and temperature include phytochrome B (phyB), PHYTOCROME INTERACTING FACTOR 4 (PIF4), EARLY FLOWERING 3 (ELF3) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). Here, we used hypocotyl lengths of single and double mutant/over-expression lines to fit a mathematical model incorporating known interactions of these genes. The fitted model recapitulates day length-dependent thermoelongation of all lines studied, and correctly predicts temperature responsiveness of new genotypes. Whilst previous works pointed to a lightindependent thermal function of COP1, simulations of our model suggested that COP1 has a role in temperature-signaling only during daytime. Based on by this prediction, we show that COP1 overexpression increases thermal response in continuous white light, while it has little effect in darkness. Defective thermal response of cop1-4 mutants is epistatic to phyB-9 and elf3-8, indicating that COP1 activity is essential to the transduction of phyB and ELF3 thermosensory function. Our model accurately captures phyB, ELF3 and PIF4 dynamics, providing an excellent toolbox for identification of best allelic combinations towards optimized crops resilience to climate change at different geographical latitudes.

sentences: MPTHub: an open-source software for characterizing the transport of particles in biorelevant media The study of the transport of particles in different environments plays an essential role in understanding interactions with humans and other living organisms. Importantly, obtained data can be directly used for multiple applications in fields such as fundamental biology, toxicology or medicine. Particle movement in biorelevant media can be readily monitored using microscopy and converted into time-resolved trajectories using freely available tracking software. However, translation into tangible and meaningful parameters is time-consuming and not always intuitive. Thus, we developed a new software - MPTHub - as an open-access, stand-alone, user-friendly tool for the rapid and reliable analysis of particle trajectories extracted from video microscopy. The software was programmed using Python and allowed to import and analyze trajectory data, and export relevant data such as individual and ensemble time-averaged mean square displacements and effective diffusivity, and anomalous transport exponent. Data processing was reliable, fast (total processing time of less than 10 sec) and required minimal memory resources (up to a maximum of around 150 MB in RAM). Demonstration of software applicability was conducted by studying the transport of different polystyrene nanoparticles (100-200 nm) in mucus surrogates. Overall, MPTHub represents a freely available software tool that can be used even by unexperienced users for studying the transport of particles in biorelevant media.

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A panel of KSHV mutants in the polycistronic kaposin locus for precise analysis of individual protein products Kaposis sarcoma-associated herpesvirus (KSHV) is the cause of several human cancers including the endothelial cell (EC) malignancy, Kaposis sarcoma. Unique KSHV genes absent from other human herpesvirus genomes, the "K-genes", are important for KSHV replication and pathogenesis. Among these, the kaposin transcript is highly expressed in all phases of infection, but its complex polycistronic nature has hindered functional analysis to date. At least three proteins are produced from the kaposin transcript: Kaposin A (KapA), B (KapB), and C (KapC). To determine the relative contributions of kaposin proteins during KSHV infection, we created a collection of mutant viruses unable to produce kaposin proteins individually or in combination. Kaposin-deficient latent iSLK cell lines displayed reduced viral genome copy number and often exhibited small LANA nuclear bodies; despite this, all were capable of progeny virion production. Primary infection with {Delta}KapB virus revealed decreased LANA expression and viral genome copy number, yet providing KapB protein in trans failed to complement these defects, suggesting a requirement for the kaposin locus in cis. Our previous work showed that KapB was sufficient to recapitulate the elevated proinflammatory cytokine transcripts associated with KS via the disassembly of RNA granules called processing bodies (PBs). We now show that KapB is necessary for PB disassembly during latent KSHV infection. These findings demonstrate that our panel of kaposin-deficient viruses enables precise analysis of the respective contributions of individual kaposin proteins to KSHV replication. Moreover, our mutagenesis approach serves as a guide for the functional analysis of other complex multicistronic viral loci.

ImportanceKaposis sarcoma-associated herpesvirus (KSHV) expresses high levels of the kaposin transcript during both latent and lytic phases of replication. Due to its repetitive, GC-rich nature and polycistronic coding capacity, until now no reagents existed to permit a methodical analysis of the role of individual kaposin proteins in KSHV replication. We report the creation of a panel of recombinant viruses and matched producer cell lines that delete kaposin proteins individually or in combination. We demonstrate the utility of this panel by confirming the requirement of one kaposin translation product to a key KSHV latency phenotype. This study describes a new panel of molecular tools for the KSHV field to enable precise analysis of the roles of individual kaposin proteins during KSHV infection.

sentences: Traditional use of Cissampelos pareira L. for hormone disorder and fever provides molecular links of ESR1 modulation to viral inhibition Bioactive fractions or compounds obtained from medicinal plants have been used for the treatment of multiple diseases. This effect could be due to common pathways underlying these conditions that are targeted by such medicines. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira, used for the treatment of female hormone disorders and fever. Genome-wide expression studies on MCF7 cell lines treated with Cipa extract were carried out using Affymetrix arrays. Transcriptome analysis revealed a downregulation of signatures of estrogen response governed by estrogen receptor  (ER). Molecular docking analysis identified 38 constituent molecules in Cipa that potentially bind ({Delta}G< -7.5) with ER at the same site as estrogen. Cipa transcriptome signatures show high positive connectivity (https://clue.io/) scores with protein translation inhibitors such as emetine (score: 99.61) and knockdown signatures of genes linked to the antiviral response such as ribosomal protein RPL7 (score: 99.92), which is also an ER coactivator. Cipa exhibits antiviral activity in dengue infected MCF7 cells that is decreased upon ESR1 (estrogen receptor 1) gene knockdown. This approach reveals a novel pathway involving ESR1-RPL7 axis that could be a potential target in dengue viral infection.

sentences: Limited selection towards lighter seeds in Danish grasslands over an eight-year period Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes.

sentences: Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defence compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.

sentences: Enhancer RNAs stimulate Pol II pause release by harnessing multivalent interactions to NELF Enhancer RNAs (eRNAs) are long non-coding RNAs that originate from enhancers. Although eRNA transcription is a canonical feature of activated enhancers, the molecular features required for eRNA function and the mechanism of how eRNAs impinge on target gene transcription have not been established. Thus, using eRNA-dependent RNA polymerase II (Pol II) pause release as a model, we examined the requirement of sequence, structure and length of eRNAs for their ability to stimulate Pol II pause release by detaching NELF from paused Pol II. We found eRNA not to exert their function through common structural or sequence motifs. Instead, efficient NELF release requires a single eRNA molecule that must contain unpaired guanosines to make multiple, allosteric contacts with several NELF subunits. By revealing the molecular determinants for eRNA function, our study mechanistically links eRNAs to Pol II pause release and provides new insight into the regulation of metazoan transcription.

sentences: Validation of binary non-targeted approaches: mathematical framework and experimental designs Non-targeted methods (NTMs) are being increasingly developed and adopted to detect food fraud and identify the authenticity of the food. Method validation is a critical step before bringing the NTMs to the routine to be assured of method performance and trust that its outcomes are reliable. However, the paucity of well structured and harmonized validation strategies has been one of the hurdles, withholding the exploitation of NTMs. This report aims to describe a validation framework for methods that involve binary classification, which are prevalent in non-targeted workflows. We foresee this work to contribute to filling the current gap in the provisions for NTM method validation; perhaps also push the dialogue further to collectively resolve existing challenges.

sentences: Modeling Brain Connectivity Dynamics in Functional Magnetic Resonance Imaging via Particle Filtering Interest in the studying of functional connections in the brain has grown considerably in the last decades, as many studies have pointed out that these interactions can play a role as markers of neurological diseases. Most studies in this field treat the brain network as a system of connections stationary in time, but dynamic features of brain connectivity can provide useful information, both on physiology and pathological conditions of the brain. In this paper, we propose the application of a computational methodology, named Particle Filter (PF), to study non-stationarities in brain connectivity in functional Magnetic Resonance Imaging (fMRI). The PF algorithm estimates time-varying hidden parameters of a first-order linear time-varying Vector Autoregressive model (VAR) through a Sequential Monte Carlo strategy. On simulated time series, the PF approach effectively detected and enabled to follow time-varying hidden parameters and it captured causal relationships among signals. The method was also applied to real fMRI data, acquired in presence of periodic tactile or visual stimulations, in different sessions. On these data, the PF estimates were consistent with current knowledge on brain functioning. Most importantly, the approach enabled to detect statistically significant modulations in the cause-effect relationship between brain areas, which correlated with the underlying visual stimulation pattern presented during the acquisition.

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Tracking age differences in neural distinctiveness across representational levels The distinctiveness of neural information representation is crucial for successful memory performance but declines with advancing age. Computational models implicate age-related neural dedifferentiation on the level of item representations, but previous studies mostly focused on age differences of categorical information representation in higher-order visual regions. In an age-comparative fMRI study, we combined univariate analyses and whole-brain searchlight pattern similarity analyses to elucidate age differences in neural distinctiveness at both category and item levels and their relation to memory. Thirty-five younger (18-27 years old) and 32 older (67-75 years old) women and men incidentally encoded images of faces and houses, followed by an old/new recognition memory task. During encoding, age-related neural dedifferentiation was shown as reduced category-selective processing in ventral visual cortex and impoverished item specificity in occipital regions. Importantly, successful subsequent memory performance built upon high item stability, that is, high representational similarity between initial and repeated presentation of an item, which was greater in younger than older adults. Overall, we found that differences in representational distinctiveness coexist across representational levels and contribute to interindividual and intraindividual variability in memory success, with item specificity being the strongest contributor. Our results close an important gap in the literature, showing that older adults' neural representation of item-specific information in addition to categorical information is reduced compared to younger adults.

Significance StatementA long-standing hypothesis links age-related cognitive decline to a loss of neural specificity. While previous evidence supports the notion of age-related neural dedifferentiation of category-level information in ventral visual cortex, whether or not age differences exist at the item level was a matter of debate. Here, we observed age group differences at both levels as well as associations between both categorical distinctiveness and item specificity to memory performance, with item specificity being the strongest contributor. Importantly, age differences in occipital item specificity were largely due to reduced item stability across repetitions in older adults. Our results suggest that age differences in neural representations can be observed across the entire cortical hierarchy and are not limited to category-level information.

sentences: Syntactic representations in the human brain: beyond effort-based metrics While studying semantics in the brain, neuroscientists use two approaches. One is to identify areas that are correlated with semantic processing load. Another is to find areas that are predicted by the semantic representation of the stimulus words. However, in the domain of syntax, most studies have focused only on identifying areas correlated with syntactic processing load. One possible reason for this discrepancy is that representing syntactic structure in an embedding space such that it can be used to model brain activity is a non-trivial computational problem. Another possible reason is that it is unclear if the low signal-to-noise ratio of neuroimaging tools such as functional Magnetic Resonance Imaging (fMRI) can allow us to reveal correlates of complex (and perhaps subtle) syntactic representations. In this study, we propose novel multi-dimensional features that encode information about the syntactic structure of sentences. Using these features and fMRI recordings of participants reading a natural text, we model the brain representation of syntax. First, we find that our syntactic structure-based features explain additional variance in the brain activity of various parts of the language system, even after controlling for complexity metrics that capture processing load. At the same time, we see that regions well-predicted by syntactic features are distributed in the language system and are not distinguishable from those processing semantics.

sentences: Graph-based machine learning reveals rules of spatiotemporal cell interactions in tissues Robustness in developing and homeostatic tissues is supported by various types of spatiotemporal cell-to-cell interactions. Although live imaging and cell tracking are powerful in providing direct evidence of cell coordination rules, extracting and comparing these rules across many tissues with potentially different length and timescales of coordination requires a versatile framework of analysis. Here we demonstrate that graph neural network (GNN) models are suited for this purpose, by showing how they can be applied to predict cell fate in tissues and utilized to infer the cell interactions governing the multicellular dynamics. Analyzing the live mammalian epidermis data, where spatiotemporal graphs constructed from cell tracks and cell contacts are given as inputs, GNN discovers distinct neighbor cell fate coordination rules that depend on the region of the body. This approach demonstrates how the GNN framework is powerful in inferring general cell interaction rules from live data without prior knowledge of the signaling involved.

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Gene flux and acid-imposed selection are the main drivers of antimicrobial resistance in broiler chicks infected with Salmonella enterica serovar Heidelberg Antimicrobial resistance (AR) spread is a worldwide health challenge, stemming in large part, from the ability of microbes to share their genetic material through horizontal gene transfer (HGT). Overuse and misuse of antibiotics in clinical settings and in food production have been linked to this increased prevalence and spread of AR. Consequently, public health and consumer concerns have resulted in a remarkable recent reduction in antibiotics used for food animal production. This is driven by the assumption that removing this selective pressure will favor the recovery of antibiotic susceptible taxa and will limit AR sharing through HGT, allowing the currently available antibiotic arsenal to be effective for a longer period. In this study we used broiler chicks raised antibiotic-free and Salmonella enterica serovar Heidelberg (SH), as a model food pathogen, to test this hypothesis. Our results show that neonatal broiler chicks challenged with an antibiotic susceptible SH strain and raised without antibiotics carried susceptible and multidrug resistance SH strains 14 days after challenge. SH infection perturbed the microbiota of broiler chicks and gavaged chicks acquired antibiotic resistant SH at a higher rate. We determined that the acquisition of a plasmid from commensal Escherichia coli population conferred multidrug resistance phenotype to SH recipients and carriage of this plasmid increased the fitness of SH under acidic selection pressure. These results suggest that HGT of AR shaped the evolution of SH and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella.

ImportanceThe reported increase in antibiotic resistant bacteria in humans have resulted in a major shift away from antibiotics use in food animal production. This has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, and this in turn will allow the currently available antibiotic arsenal to be more effective. This shift in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic susceptible Salmonella Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggests that simply removing antibiotics from food-animal production might not be sufficient to limit the spread of antimicrobial resistance.

sentences: ARID1A loss derepresses human endogenous retrovirus-H to modulate BRD4-dependent transcription The transposable elements (TEs) through evolutionary exaptation have become an integral part of human genome, offering ample regulatory sequences and shaping chromatin 3D architecture. While the functional impacts of TE-derived sequences on early embryogenesis are recognized, their role in malignancy has only started to emerge. Here we show that many TEs, especially the pluripotency-related endogenous retrovirus H (HERVH), are abnormally activated in colorectal cancer (CRC) samples. The transcriptional upregulation of HERVH is associated with mutations of several tumor suppressors including ARID1A. Knockout of ARID1A in CRC cells leads to increased accessibility at HERVH loci and enhanced transcription, which is dependent on ARID1B. Suppression of HERVH in CRC cells and patient-derived organoids impairs tumor growth. Mechanistically, HERVH transcripts colocalize with nuclear BRD4 foci, modulate their dynamics, and co-regulate many target genes. Altogether, we uncover a critical role for ARID1A in restraining HERVH, which can promote tumorigenesis by stimulating BRD4-dependent transcription when ARID1A is mutated.

sentences: Functional and comparative genome analysis reveals clade-specific genome innovations in the killer fungus Candida auris The thermotolerant multidrug-resistant ascomycete Candida auris rapidly emerged since 2009 and simultaneously evolved in different geographical zones worldwide, causing superficial as well as systemic infections. The molecular events that orchestrated this sudden emergence of the killer fungus remain mostly elusive. Here, we identify centromeres in C. auris and related species, using a combined approach of chromatin immunoprecipitation and comparative genomic analyses. We find that C. auris and multiple other species in the Clavispora/Candida clade shared a conserved small regional centromere landscape lacking pericentromeres. Further, a centromere inactivation event led to karyotypic alterations in this species complex. Inter-species genome analysis identified several structural chromosomal changes around centromeres. In addition, centromeres are found to be rapidly evolving loci among the different geographical clades of the same species of C. auris. Finally, we reveal an evolutionary trajectory of the unique karyotype associated with clade 2 that consists of the drug susceptible isolates of C. auris.

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Sucrose transport and metabolism control carbon partitioning between stem and grain in rice The source-sink relationship is key to overall crop performance. Detailed understanding of the factors that determine source-sink dynamics is imperative for the balance of biomass and grain yield in crop plants. We investigated the differences in the source-sink relationship between a cultivated rice Oryza sativa cv. Nipponbare and a wild rice Oryza australiensis that show striking differences in biomass and grain yield. Oryza australiensis, accumulating higher biomass, not only showed higher photosynthesis per unit leaf area but also exported more sucrose from leaves than Nipponbare. However, grain features and sugar levels suggested limited sucrose mobilization to the grains in the wild rice due to vasculature and sucrose transporter functions. Low cell wall invertase activity and high sucrose synthase cleavage activity followed by higher expression of cellulose synthase genes in Oryza australiensis stem utilized photosynthates preferentially for the synthesis of structural carbohydrates, resulting in high biomass. In contrast, the source-sink relationship favored high grain yield in Nipponbare via accumulation of transitory starch in the stem, due to higher expression of starch biosynthetic genes, which is mobilized to panicles at the grain filling stage. Thus, vascular features, sucrose transport, and functions of sugar metabolic enzymes explained the differences in the source-sink relationship between Nipponbare and Oryza australiensis.

HighlightVascular features, sucrose transport, and sugar metabolic enzyme activity contribute to the differential source-sink relationship between the selected cultivated and wild rice that differ in biomass and grain yield.

sentences: The Amyloid Precursor Protein regulates human cortical neurogenesis The approximately 16 billion neurons of the human neocortex are derived from a relatively limited number of developmental neural stem cells (NSCs). During embryogenesis, human cortical NSCs initially generate neurons at a particularly slow rate while preserving their progenitor state for a relatively long time. How this balance between the progenitor state and neurogenic state is regulated, and whether it contributes to species-specific brain patterning, is poorly understood. Here we show that the characteristic potential of human NSCs to remain in a progenitor state as they generate neurons for a prolonged amount of time requires the Amyloid Precursor Protein (APP). In contrast, APP is dispensable in mouse NSCs, which undergo neurogenesis at a much faster rate. Mechanistically, loss of APP cell-autonomously accelerates neurogenesis through activation of the AP1 transcription factor and repression of WNT signaling. We propose that the fine balance between self-renewal and differentiation is homeostatically regulated by APP, which may contribute to human-specific temporal patterns of neurogenesis.

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Ultra-low Input Circulating Tumor DNA Detection by MED-Amp in Early-Stage Pancreatic Cancer PurposeThe clinical utility of circulating tumor DNA (ctDNA) has been shown in advanced pancreatic ductal adenocarcinoma (PDA). However, diagnostic sensitivity of many ctDNA assays is low in resectable and locally advanced disease, where tumor burden is substantially lower. We have previously described Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp), a multiplexed panel for the detection of the most common oncogenic KRAS mutations in PDA. In this study, we aimed to assess the diagnostic sensitivity of MED-Amp for detection of rare mutant alleles present in the plasma of patients with localized PDA.

Experimental DesignWe retrospectively analyzed ninety-eight plasma samples from 51 patients with various stages of localized disease. For comparison, we measured ctDNA levels in 20 additional patients with metastatic PDA. The MED-Amp assay was used to measure the abundance of the four most common KRAS codon 12 mutations (G12C/D/R/V). We correlated the presence and quantity of ctDNA with overall survival (OS) as well as progression-free survival (PFS). Using serial plasma draws, we also assessed the relationship between changes in ctDNA allelic frequency and progression.

ResultsKRAS-positive ctDNA was detected in 52.9% of localized PDA and 75% of metastatic samples tested using DNA inputs as low as 2 ng. As previously reported, the presence of KRAS mutant ctDNA was correlated with worse OS for all disease stages (p = 0.02). In patients with localized PDA high ctDNA levels also correlated with significantly worse median OS (533 days vs 1090 days) and PFS (192 days vs 787 days). We also studied a small cohort of serial plasma draws to observe the relationship between ctDNA fold change and PFS. We found 83% of patients with increased fold change in mutant KRAS experienced disease progression (n=6). In contrast, 75% (n=4) of patients with decreased fold change remained disease-free (p=0.03).

ConclusionsMED-Amp is a flexible and cost-effective approach for measurement of ctDNA in patients with localized cancer. Though this study focused on KRAS mutation detection, this assay could be adapted for a number of common oncogenic alterations.

Statement of translational relevanceOnly 25% of pancreatic ductal adenocarcinoma (PDA) patients with localized disease survive five years post-resection. It is hypothesized PDA undergoes dissemination at the earliest stages of tumor formation, driving formation of occult metastases which go undetected using conventional screening methods. Development of high specificity, high sensitivity biomarkers is critical to improving patient mortality. Circulating tumor DNA (ctDNA) has gained increasing acceptance as a non-invasive prognostic in metastatic disease. However, the sensitivity of most targeted ctDNA assays precludes reliable detection of localized and resected disease. Here, we present a digital droplet PCR assay for multiplexed enrichment and detection of KRAS mutations, the most commonly mutated oncogene in PDA. This assay preserves ctDNA allelic frequency in the original sample, while increasing the molecular signal over 50-fold. This study shows the ctDNA has potential diagnostic value in early-stage PDA, and that digital preenrichment of cell-free DNA increases overall assay sensitivity without sacrificing specificity.

sentences: Leaky wiring of the brain: local cluster of coupled synapses and extracellular signal integration The wiring scheme of neurons is key to the function of the brain. Neurons are structurally wired by synapses and it is a long-held view that most synapses in the CNS are sufficiently isolated to avoid cross-talk to AMPA receptors of neighboring synapses. Here we report in hippocampal brain slices that quantal glutamate release activated optical reporter proteins >1.5 {micro}m distant to the releasing synapse. 2P-glutamate uncaging was used to quantitatively probe glutamate spread in the neuropil. Releasing [~]35000 molecules of glutamate ([~]5 vesicles) at a distance of 500 nm to a spine generated an uncaging EPSC reaching [~]30% of the quantal amplitude at synaptic AMPA-Rs. The same stimulus activated [~]70% of the quantal amplitude at NMDA-Rs and still generated clear current and calcium responses when applied at >= 2 {micro}m remote to the spine. Extracellular spread of glutamate on the sub-micrometer scale appeared cooperative and caused supra-additive activation of AMPA-Rs in a spine. These observations are not predicted by previously used models of glutamate diffusion in the neuropil. An extracellular glutamate scavenger system weakly reduced field potential responses but not the quantal amplitude, indicating that a cross-talk component regularly contributes to synaptic transmission. Our data suggest that slight synaptic crosstalk responses at AMPA receptors of [~]2-4 adjacent synapses may be common (>70 synapses for NMDA receptors). Such broadcasting of synaptic signals to very local neighborhoods could stabilize network learning performance and allow for integration of synaptic activity within the extracellular space.

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Impaired photoprotection in Phaeodactylum tricornutum KEA3 mutants reveals the proton regulatory circuit of diatoms light acclimation. Diatoms are amongst the most successful clades of oceanic phytoplankton, significantly contributing to photosynthesis on Earth. Their ecological success likely stems from their ability to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity of via Non Photochemical Quenching (NPQ). While the molecular effectors of this process, as well as the role of the Proton Motive Force (PMF) in its regulation are known, the putative regulators of the PET/PMF relationship in diatoms remain unidentified. Here, we demonstrate that the H+/K+ antiporter KEA3 is the main regulator of the coupling between PMF and PET in the model diatom Phaeodactylum tricornutum. By controlling the PMF, it modulates NPQ responses at the onset of illumination, during transients and in steady state conditions. Under intermittent light KEA3 absence results in reduced fitness. Using a parsimonious model including only two components, KEA3 and the diadinoxanthin de-epoxidase, we can describe most of the feedback loops observed between PET and NPQ. This two-components regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory Ca2+-binding domain in KEA3 that controls its activity. This circuit is likely finely tuned by the NPQ effector proteins LHCX, providing diatoms with the required flexibility to thrive in different ocean provinces.

One sentence summaryThe author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is Giovanni Finazzi.

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Postdiction: when temporal regularity drives space perception through pre-stimulus alpha oscillations During postdiction, the last stimulus of a sequence changes the perception of the preceding ones: in the rabbit illusion, a three-flash series presented regularly in time, but not in space, is - illusory - perceived as spatially regular. Such a reorganization of (spatial) perception could be driven by internal priors, e.g. favoring slow motion for the rabbit illusion. Although postdiction is a ubiquitous phenomenon, its neural underpinnings remain poorly understood. Here, we focused on the role of priors during postdiction and hypothesized that these could be reflected by alpha oscillations (8-12Hz), previously observed to correlate with idiosyncratic biases. We presented human participants with ambiguous visual stimuli that elicited the rabbit illusion on about half the trials, allowing us to contrast MEG-EEG brain responses to the same physical events causing distinct percepts. Given that a strong prior will increase the overall probability of perceiving the illusion, we used the percentage of perceived illusion as a proxy for an individuals prior. We found that high fronto-parietal alpha power was associated with perceiving the sequence according to individual biases: participants with high susceptibility to the illusion would report the illusion, while participants with low susceptibility would report the veridical sequence. Additionally, we found that pre-stimulus alpha phase in occipital and frontal areas dissociated illusory from non-illusory trials. These results point to a dissociated relation of the power and timing of alpha band activity to illusory perception, with power reflecting prior expectations and phase influencing behavioral performance, potentially due to the modulation of sensory uncertainty.

Significance StatementLate events may sometimes influence how earlier events are perceived, as if the arrow of time was reversed in the brain. This surprising phenomenon, called postdiction, is observed in the rabbit illusion, and highlights a predominant mechanism for perceptual processes. Perception builds up from the combination of prior expectations with incoming sensory evidence, which takes time. We showed that pre-stimulus neural activity, and more specifically alpha oscillations (8-12Hz), play a double role in postdiction. Fronto-parietal alpha power reflects individual prior expectation, while occipital and frontal alpha phase predicts illusory perception. Postdiction might actually be a means of compensating for the neural delays inherent in perceptual processes, so that the arrow of perceptual time matches the arrow of physical time.

sentences: Readers move their eyes mindlessly using midbrain visuo-motor principles Saccadic eye movements rapidly shift our gaze over 100,000 times daily, enabling countless tasks ranging from driving to reading. Long regarded as a window to the mind1 and human information processing2, they are thought to be cortically/cognitively controlled movements aimed at objects/words of interest3-10. Saccades however involve a complex cerebral network11-13 wherein the contribution of phylogenetically older sensory-motor pathways14-15 remains unclear. Here we show using a neuro-computational approach16 that mindless visuo-motor computations, akin to reflexive orienting responses17 in neonates18-19 and vertebrates with little neocortex15,20, guide humans eye movements in a quintessentially cognitive task, reading. These computations occur in the superior colliculus, an ancestral midbrain structure15, that integrates retinal and (sub)cortical afferent signals13 over retinotopically organized, and size-invariant, neuronal populations21. Simply considering retinal and primary-visual-cortex afferents, which convey the distribution of luminance contrast over sentences (visual-saliency map22), we find that collicular population-averaging principles capture readers prototypical word-based oculomotor behavior2, leaving essentially rereading behavior unexplained. These principles reveal that inter-word spacing is unnecessary23-24, explaining metadata across languages and writing systems using only print size as a predictor25-26. Our findings demonstrate that saccades, rather than being a window into cognitive/linguistic processes, primarily reflect rudimentary visuo-motor mechanisms in the midbrain that survived brain-evolution pressure27.

sentences: Synaptotagmin-7 enhances phasic dopamine release Dopamine released from substantia nigra pars compacta (SNc) neurons modulates movement, motivation, and reward. In addition to their tonic firing pattern, dopamine neurons also fire high-frequency bursts that cause superlinear increases in dopamine release. To study this poorly understood form of short-term plasticity, we used the fluorescent dopamine sensor dLight1.3b to examine the role of the calcium-binding protein synaptotagmin-7 (SYT7). We report that SYT7 mediates a hidden component of facilitation, which was unmasked by lowering initial release probability, or by low-frequency stimulation of nerve terminals. In Syt7 KO neurons, there was profound synaptic depression that significantly reduced release during stimulations that mimic in vivo firing patterns of SNc neurons. D2-mediated inhibitory postsynaptic currents in the SNc revealed a similar role for SYT7 in somatodendritic release. Our results indicate that SYT7 drives short-term facilitation of release from dopamine neurons, which likely underlies frequency-dependence of dopamine signaling in vivo.

sentences: Cryo-electron tomography of the apicomplexan invasion machinery in its native state reveals rigid body motion of the conoid and docked secretory machinery The apical complex is a conserved cytoskeletal structure that organizes the secretory and invasion machinery of all apicomplexan parasites, the causative agents of diseases such as malaria, toxoplasmosis, and cryptosporidiosis. The structure of the apical complex and its mechanism of motion, which is thought to be required for host cell invasion, is not well understood. Recent advances in cryo-electron tomography (cryo-ET) have allowed the high-resolution reconstruction of large complexes in their near-native states. Here, we use cryo-focused ion beam milling to generate a thin specimen of the coccidian apical complex, allowing us to visualize and compare its three-dimensional structure in the protruded and retracted states in situ by cryo-ET. Subtomogram averaging of the 8 nm repeats from the conoid fibers (specialized microtubules) revealed their polarity and unusual nine- protofilament arrangement with associated proteins that connect and likely stabilize the fibers. In spite of the open shape of the conoid fibers, neither the structure of the conoid fibers nor the architecture of the spiral-shaped conoid complex as a whole are altered during cycles of protrusion and retraction. Thus the conoid moves as a rigid body, and is not compressible like a spring, as has been previously suggested. Instead, we found that the apical polar rings (APR), previously considered a rigid structure to which the conoid is docked, dilates during conoid protrusion. Furthermore, we identified previously undescribed actin-like filaments connecting the conoid to the APR during protrusion, consistent with a role of actin in driving conoid movements. In addition, our data capture the parasites in the act of secretion during conoid extension, allowing us to identify a network of interactions between the apical complex cytoskeleton and the parasites secretory machinery.

sentences: Predicting T cell receptor antigen specificity from structural features derived from homology models of receptor-peptide-major histocompatibility complexes. 1The physical interaction between the T cell receptor (TCR) and its cognate antigen causes T cells to activate and participate in the immune response. Understanding this physical interaction is important in predicting TCR binding to a target epitope, as well as potential cross-reactivity. Here, we propose a way of collecting informative features of the binding interface from homology models of T cell receptor-peptide-major histocompatibility complex (TCR-pMHC) complexes. The information collected from these structures is sufficient to discriminate binding from non-binding TCR-pMHC pairs in multiple independent datasets. The classifier is limited by the number of crystal structures available for the homology modelling and by the size of the training set. However, the classifier shows comparable performance to sequence-based classifiers requiring much larger training sets.

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Striatal dopamine explains novelty-induced behavioral dynamics and individual variability in threat prediction Animals exhibit diverse behavioral responses, such as exploration and avoidance, to novel cues in the environment. However, it remains unclear how dopamine neuron-related novelty responses influence behavior. Here, we characterized dynamics of novelty exploration using multi-point tracking (DeepLabCut) and behavioral segmentation (MoSeq). Novelty elicits a characteristic sequence of behavior, starting with investigatory approach and culminating in object engagement or avoidance. Dopamine in the tail of striatum (TS) suppresses engagement, and dopamine responses were predictive of individual variability in behavior. Behavioral dynamics and individual variability were explained by a novel reinforcement learning (RL) model of threat prediction, in which behavior arises from a novelty-induced initial threat prediction (akin to "shaping bonus"), and a threat prediction that is learned through dopamine-mediated threat prediction errors. These results uncover an algorithmic similarity between reward- and threat-related dopamine sub-systems.

HighlightsO_LINovelty-induced behaviors are analyzed using modern machine-learning methods
C_LIO_LINovelty induces risk assessment which develops into engagement or avoidance
C_LIO_LIDopamine in the tail of striatum correlates with individual behavioral variability
C_LIO_LIReinforcement learning with shaping bonus and uncertainty explains the data
C_LI

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The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host and tissue tropism and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by viral attachment protein {sigma}1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule-A (JAM-A) and Nogo 66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease following inoculation of wild-type (WT) and NgR1-/- mice. Genetic ablation of NgR1 did not alter replication of neurotropic reovirus strain T3SA- in the intestine and transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1-/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of both NgR1 and JAM-A also did not alter replication, neural tropism, and virulence of T3SA- following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro. These results eliminate functions for JAM-A and NgR1 in shaping CNS tropism in mice and suggest that other receptors, yet to be identified, support this function.

IMPORTANCEThe CNS presents a range of barriers to pathogen invasion. Yet neurotropic viruses have evolved strategies to breach these barriers and establish infection by engagement of host factors that allow navigation to the CNS and neural cell entry. Human NgR1 was identified as a reovirus receptor in an RNA interference screen and is expressed in CNS neurons in a pattern overlapping with reovirus tropism. Using mice genetically lacking NgR1 expression, and following different routes of inoculation, we discovered that murine NgR1 is dispensable for reovirus dissemination to the CNS, tropism and replication in the brain, and resultant disease. Concordant with these results, expression of human but not murine NgR1 confers reovirus binding and infection of nonsusceptible cells in vitro. These results point to species-specific use of alternate receptors by reovirus. A detailed understanding of species- and tissue-specific factors that dictate viral tropism will inform development of interventions and targeted gene delivery and therapeutic viral vectors.

sentences: Comparison of breeding strategies for the creation of a synthetic pig line Creating a new synthetic line by crossbreeding means complementary traits from pure breeds can be combined in the new population. Although diversity is generated during the crossbreeding stage, in this study, we analyze diversity management before selection starts. Using genomic and phenotypic data from animals belonging to the first generation (G0) of a new line, different simulations were run to evaluate diversity management during the first generations of a new line and to test the effects of starting selection at two alternative times, G3 and G4. Genetic diversity was characterized by allele frequency, inbreeding coefficients based on genomic and pedigree data, and expected heterozygosity. Breeding values were extracted at each generation to evaluate differences in starting selection at G3 or G4. All simulations were run for ten generations. A scenario with genomic data to manage diversity during the first generations of a new line was compared with a random and a selection scenario. As expected, loss of diversity was higher in the selection scenario, while the scenario with diversity control preserved diversity. We also combined the diversity management strategy with different selection scenarios involving different degrees of diversity control. Our simulation results show that a diversity management strategy combining genomic data with selection starting at G4 and a moderate degree of diversity control generates genetic progress and preserves diversity.

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Systematic analysis of Epstein-Barr virus genes and their individual contribution to virus production and composition Epstein-Barr virus (EBV), a member of the large herpesvirus family is a very complex human{gamma} -herpesvirus. Its complexity with respect to viral gene regulation, its preferred latent life style and technical difficulties are major obstacles, which hinder efficient virus generation in vitro to mass-produce or establish recombinant wild-type EBV or mutant derivatives. To explore conditions optimizing and improving virus production, we established and tested an EBV gene library with 77 expression plasmids and a set of designed shRNAs. With this tool set we investigated the contributions of individual viral genes in the context of virus synthesis and virion functions. Engineered virus stocks were systematically analyzed with respect to physical and bioparticle concentration, virus titer and virus uptake by primary human B cells, EBVs target cells in vivo. To quantitate virus uptake by these cells, we developed a novel {beta}-lactamase-based assay that can monitor fusion events of the viral envelope with membranes of recipient cells at the level of single cells. Based on our findings, EBV does not encode a dominant regulator that governs virus production, but our results identified several EBV genes such as BALF4, BVLF1 and BKRF4, encoding a viral glycoprotein, a transcriptional regulator of late viral genes, and a possible tegument protein, respectively, that improve virus production regarding virus yield, virus composition and quality, and virus uptake by primary human B cells.

ImportanceFor more than 20 years HEK293 cells have been instrumental to produce virus stocks of recombinant EBV. The identification of this cell line as a source of infectious EB virions was unexpected because HEK293 cells are very distant from B cells and in particular plasma cells which produce EBV progeny in vivo. To our knowledge, no systematic analysis has addressed the fundamental question whether virus yield with respect to virus concentration, virion composition and functionality can be improved to produce recombinant EBV stocks in HEK293 cells. We tackled this question and analyzed 77 individual EBV genes for their possible contribution to virus yield. To analyze and compare different virus stocks we used established and developed novel assays to characterize important parameters of EB virions and their functions.

sentences: Single-cell profiling of the human primary motor cortex in ALS and FTLD Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two devastating and fatal neurodegenerative conditions. While distinct, they share many clinical, genetic, and pathological characteristics1, and both show selective vulnerability of layer 5b extratelencephalic-projecting cortical populations, including Betz cells in ALS2,3 and von Economo neurons (VENs) in FTLD4,5. Here, we report the first high resolution single-cell atlas of the human primary motor cortex (MCX) and its transcriptional alterations in ALS and FTLD across ~380,000 nuclei from 64 individuals, including 17 control samples and 47 sporadic and C9orf72-associated ALS and FTLD patient samples. We identify 46 transcriptionally distinct cellular subtypes including two Betz-cell subtypes, and we observe a previously unappreciated molecular similarity between Betz cells and VENs of the prefrontal cortex (PFC) and frontal insula. Many of the dysregulated genes and pathways are shared across excitatory neurons, including stress response, ribosome function, oxidative phosphorylation, synaptic vesicle cycle, endoplasmic reticulum protein processing, and autophagy. Betz cells and SCN4B+ long-range projecting L3/L5 cells are the most transcriptionally affected in both ALS and FTLD. Lastly, we find that the VEN/Betz cell-enriched transcription factor, POU3F1, has altered subcellular localization, co-localizes with TDP-43 aggregates, and may represent a cell type-specific vulnerability factor in the Betz cells of ALS and FTLD patient tissues.

sentences: Bacteroidales species are a reservoir of phase-variable antibiotic resistance genes in the human gut microbiome Phase-variable antibiotic resistance genes (ARGs) can mitigate the fitness cost of maintaining ARGs in the absence of antibiotics and could potentially prolong the persistence of ARGs in bacterial populations. However, the origin, prevalence, and distribution of phase-variable ARGs remains poorly understood. Here, we sought to assess the threat posed by phase-variable ARGs by systematically searching for phase-variable ARGs in the human gut microbiome and examining their origin, prevalence, and distribution. Through metagenomic assembly of 2227 human gut metagenomes and genomic analysis of the Unified Human Gastrointestinal Genome (UHGG) collection, we identified phase-variable ARGs and categorized them into three classes based on the invertase regulating phase variation. In the human gut microbiome, phase-variable ARGs are commonly and exclusively distributed in Bacteroidales species. Through genomic analysis, we observed that phase-variable ARGs have convergently originated from ARG insertions into phase-variable capsule polysaccharide biosynthesis (CPS) loci at least three times. Moreover, all identified phase-variable ARGs are located within integrative conjugative elements (ICEs). Therefore, horizontal transfer via ICEs could explain the wide taxonomic distribution of phase-variable ARGs. Overall, these findings reveal that phase-variable CPS loci in Bacteroidales species are an important hotspot for the emergence of clinically-relevant phase-variable ARGs.

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Microglial ferroptotic stress causes non-cell autonomous neuronal death BackgroundFerroptosis is a form of regulated cell death characterised by lipid peroxidation as the terminal endpoint and a requirement for iron. Although it protects against cancer and infection, ferroptosis is also implicated in causing neuronal death in degenerative diseases of the central nervous system (CNS). The precise role for ferroptosis in causing neuronal death is yet to be fully resolved.

MethodsTo elucidate the role of ferroptosis in neuronal death we utilised co-culture and conditioned medium transfer experiments involving microglia, astrocytes and neurones. We ratified clinical significance of our cell culture findings via assessment of human CNS tissue from cases of the fatal, paralysing neurodegenerative condition of amyotrophic lateral sclerosis (ALS). Finally, we utilised the SOD1G37R mouse model of ALS and a novel CNS-permeant ferroptosis inhibitor to verify pharmacological significance in vivo.

ResultsWe found that sublethal ferroptotic stress selectively affecting microglia triggers an inflammatory cascade that results in non-cell autonomous neuronal death. Central to this cascade is the conversion of astrocytes to a neurotoxic state. We show that spinal cord tissue from cases of ALS exhibits a signature of ferroptosis that encompasses atomic, molecular and biochemical features. Moreover, a molecular correlation between ferroptosis and neurotoxic astrocytes evident in ALS-affected spinal cord is recapitulated in the SOD1G37R mouse model where treatment with the novel, CNS-permeant ferroptosis inhibitor, CuII(atsm), ameliorated these markers and was neuroprotective.

ConclusionsBy showing that microglia responding to sublethal ferroptotic stress culminates in non-cell autonomous neuronal death, our results implicate microglial ferroptotic stress as a rectifiable cause of neuronal death in neurodegenerative disease. As ferroptosis is currently primarily regarded as an intrinsic cell death phenomenon, these results introduce an entirely new pathophysiological role for ferroptosis in disease.

sentences: PharmaNet: Pharmaceutical discovery with deep recurrent neural networks. The discovery and development of novel pharmaceuticals is an area of active research mainly due to the large investments required and long payback times. As of 2016, the development of a novel drug candidate required up to $ USD 2.6 billion in investment for only 10% rate of approval by the FDA. To help decreasing the costs associated with the process, a number of in silico approaches have been developed with relatively low success due to limited predicting performance. Here, we introduced a machine learning-based algorithm as an alternative for a more accurate search of new pharmacological candidates, which takes advantage of Recurrent Neural Networks (RNN) for active molecule prediction within large databases. Our approach, termed PharmaNet was implemented here to search for ligands against specific cell receptors within 102 targets of the DUD-E database, which contains 22886 active molecules. PharmaNet comprises three main phases. First, a SMILES representation of the molecule is converted into a raw molecular image. Second, a convolutional encoder processes the data to obtain a fingerprint molecular image that is finally analyzed by a Recurrent Neural Network (RNN). This approach enables precise predictions of the molecules target on the basis of the feature extraction, the sequence analysis and the relevant information filtered out throughout the process. Molecule Target prediction is a highly unbalanced detection problem and therefore, we propose that an adequate evaluation metric of performance is the area under the Normalized Average Precision (NAP) curve. PharmaNet largely surpasses the previous state-of-the-art method with 97.7% in the Receiver Operating Characteristic curve (ROC-AUC) and 65.5% in the NAP curve. We obtained a perfect performance for human farnesyl pyrophosphate synthase (FPPS), which is a potential target for antimicrobial and anticancer treatments. We decided to test PharmaNet for activity prediction against FPPS by searching in the CHEMBL data set. We obtained three (3) potential inhibitors that were further validated through both molecular docking and in silico toxicity prediction. Most importantly, one of this candidates, CHEMBL2007613, was predicted as a potential antiviral due to its involvement on the PCDH17 pathway, which has been reported to be related to viral infections.

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Monomodular Pseudomonas aeruginosa phage JG004 lysozyme (Pae87) contains a bacterial surface-active antimicrobial peptide-like region and a possible substrate-binding subdomain Phage lysins are a source of novel antimicrobials to tackle the bacterial antibiotic resistance crisis. The engineering of phage lysins is being explored as a game-changing technological strategy for introducing a more precise approach in the way we apply antimicrobial therapy. Such engineering efforts will benefit from a better understanding of lysin structure and function. In this work, the antimicrobial activity of the endolysin from Pseudomonas aeruginosa phage JG004, termed Pae87, has been characterized. This lysin had been previously identified as an antimicrobial agent candidate, able to interact with the Gram-negative surface and disrupt it. Further evidence is hereby provided on this matter, based on a structural and biochemical study. A high-resolution crystal structure of Pae87 complexed with a peptidoglycan fragment showed a separate substrate-binding region within the catalytic domain, 18 [A] away from the catalytic site and located at the opposite side of the lysin molecule. This substrate binding region was conserved among phylogenetically related lysins lacking an additional cell wall binding domain, but not among those containing such a module. Two glutamic acids were identified as relevant for the peptidoglycan degradation activity, although Pae87 antimicrobial activity was seemingly unrelated to it. In contrast, an antimicrobial peptide-like region within Pae87 C-terminus, named P87, was found to be able to actively disturb the outer membrane and have antibacterial activity by itself. Therefore, we propose an antimicrobial mechanism for Pae87 in which the P87 peptide plays the role of binding to the outer membrane and disrupting the cell wall function, either with or without the participation of Pae87 catalytic activity.

SynopsisThe structure of the monomodular Pseudomonas aeruginosa bacteriophage JG004 lysin Pae87 is presented and investigated in relationship with its function repurposed as an antimicrobial agent. The structure with its peptidoglycan ligand revealed a possible cell wall binding region. A C-terminal antimicrobial peptide-like region is shown to be important for disrupting the bacterial cell wall.

sentences: Z-RNA and the flipside of the SARS Nsp13 helicase We present evidence that the severe acute respiratory syndrome coronavirus (SARS) non-structural protein 13 (Nsp13) modulates the Z-RNA dependent regulated cell death pathways [1]. We show that Z-prone sequences (called flipons [2]) exist in coronavirus and provide a signature (Z-sig) that enables identification of the animal viruses from which the human pathogens arose. We also identify a potential RIP Homology Interaction Motif (RHIM) in the helicase Nsp13 that resembles those present in proteins that initiate Z-RNA-dependent cell death through interactions with the Z-RNA sensor protein ZBP1. These two observations allow us to suggest a model in which Nsp13 down regulates Z-RNA activated innate immunity by two distinct mechanisms. The first involves a novel ATP-independent Z-flipon helicase (flipase) activity in Nsp13 that differs from that of canonical A-RNA helicases. This flipase prevents formation of Z-RNAs that would otherwise activate cell death pathways. The second mechanism likely inhibits the interactions between ZBP1 and the Receptor Interacting Proteins Kinases RIPK1 and RIPK3 by targeting their RHIM domains. Together the described Nsp13 RHIM and flipase activities have the potential to alter the host response to coronaviruses and impact the design of drugs targeting the Nsp13 protein. The Z-sig and RHIM domains may provide a way of identifying previously uncharacterized viruses that are potentially pathogenic for humans.

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Genomic landscape of drug response reveals novel mediators of anthelmintic resistance Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to improving the efficacy and sustainability of parasite control. Here, we use a genetic cross in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies novel alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor, cky-1, in ivermectin resistance. This gene is within a locus under selection in ivermectin resistant populations worldwide; functional validation using knockout experiments supports a role for cky-1 overexpression in ivermectin resistance. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode, and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

One-Sentence SummaryGenetic mapping of known and novel anthelmintic resistance-associated alleles in a multi-drug resistant parasitic nematode

sentences: Optimal strategies to protect a sub-population at risk due to an established epidemic Epidemics can particularly threaten certain sub-populations. For example, for SARS-CoV-2, the elderly are often preferentially protected. For diseases of plants and animals, certain sub-populations can drive mitigation because they are intrinsically more valuable for ecological, economic, socio-cultural or political reasons. Here we use optimal control theory to identify strategies to optimally protect a "high value" sub-population when there is a limited budget and epidemiological uncertainty. We use protection of the Redwood National Park in California in the face of the large ongoing state-wide epidemic of sudden oak death (caused by Phytophthora ramorum) as a case study. We concentrate on whether control should be focused entirely within the National Park itself, or whether treatment of the growing epidemic in the surrounding "buffer region" can instead be more profitable. We find that, depending on rates of infection and the size of the ongoing epidemic, focusing control on the high value region is often optimal. However, priority should sometimes switch from the buffer region to the high value region only as the local outbreak grows. We characterise how the timing of any switch depends on epidemiological and logistic parameters, and test robustness to systematic misspecification of these factors due to imperfect prior knowledge.

sentences: The solution structure of Dead End bound to AU-rich RNA reveals an unprecedented mode of tandem RRM-RNA recognition required for mRNA regulation Dead End (DND1) is an RNA-binding protein essential for germline development through its role in post-transcriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1s tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA. The structure reveals how an NYAYUNN element is specifically recognized, reconciling seemingly contradictory sequence motifs discovered in recent genome-wide studies. RRM1 acts as a main binding platform, including atypical extensions to the canonical RRM fold. RRM2 acts cooperatively with RRM1, capping the RNA using an unusual binding pocket, leading to an unprecedented mode of tandem RRM-RNA recognition. We show that the consensus motif is sufficient to mediate upregulation of a reporter gene in human cells and that this process depends not only on RNA binding by the RRMs, but also on DND1s double-stranded RNA binding domain (dsRBD), which is dispensable for target binding in cellulo. Our results point to a model where DND1 target selection is mediated by a non-canonical mode of AU-rich RNA recognition by the tandem RRMs and a role for the dsRBD in the recruitment of effector complexes responsible for target regulation.

sentences: Durotaxis of passive nanoparticles on elastic membranes The transport of macromolecules and nanoscopic particles to a target cellular site is a crucial aspect in many physiological processes. This directional motion is generally controlled via active mechanical and chemical processes. Here we show, by means of molecular dynamics simulations and an analytical theory, that completely passive nanoparticles can exhibit directional motion when embedded in non-uniform mechanical environments. Specifically, we study the motion of a passive nanoparticle adhering to a mechanically non-uniform elastic membrane. We observe a non-monotonic affinity of the particle to the membrane as a function of the membranes rigidity, which results in the particle transport. This transport can be both up or down the rigidity gradient, depending on the absolute values of the rigidities that the gradient spans across. We conclude that rigidity gradients can be used to direct average motion of passive macromolecules and nanoparticles on deformable membranes, resulting in the preferential accumulation of the macromolecules in regions of certain mechanical properties.

sentences: High-fidelity dendritic sodium spike generation in human layer 2/3 neocortical pyramidal neurons Dendritic computations have a central role in rodent neocortical circuit function, which are signaled into neuronal output by the initiation and propagation of regenerative dendritic spikes. However, it remains poorly explored whether these mechanisms are operational in the human neocortex. To directly examine the dendritic computational properties of the most numerous class of pyramidal neuron in the human and rat neocortex we made simultaneous electrical recordings from the soma and apical dendrites of layer 2/3 pyramidal neurons maintained in acute brain slices of analogous cortical areas under identical experimental conditions. In both species correlated dendritic excitatory input led to the initiation of sodium channel-mediated dendritic spikes with conserved biophysical properties. Dendritic sodium spikes in human and rat layer 2/3 pyramidal neurons could be generated across a similar wide input range, exhibited a similar frequency range of activation, and forward propagated with high fidelity to drive neuronal output to implement stereotyped computations. Our findings therefore reveal that the dendritic computational properties of human layer 2/3 pyramidal neurons are phylogenetically conserved.

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Simulated docking reveals putative channels for the transport of long-chain fatty acids in Vibrio cholerae Fatty acids (FA) play an important role in biological functions, such as membrane homeostasis, metabolism, and as signaling molecules. FadL is the only known protein that uptakes long-chain fatty acids in Gram-negative bacteria, and this uptake has traditionally been thought to be limited to fatty acids up to 18 carbon atoms in length. Recently however, it was found Vibrio cholerae has the ability to uptake fatty acids greater than 18 carbon atoms and this uptake corresponds to higher bacterial survivability. Using E. colis FadL as a template, V. cholerae FadL homologs vc1042, vc1043, and vca0862 have been folded, simulated on an atomistic level using molecular dynamics, and analyzed revealing the FadL transport channels. For vc1042 and vc1043 these transport channels have more structural accommodations for the many rigid unsaturated bonds of long-chain polyunsaturated fatty acids, while vca0862 was found to lack transport channels within the signature beta barrel of FadL proteins.

IMPORTANCEFatty acids are important precursors for membrane phospholipids as well as energy sources for bacteria. V. choleraes uptake of long-chain fatty acids has not been studied to the atomistic level at this point, and by doing so, the resulting putative pathways show important structural details of the transport protein. Not only do the transport channels have unique differences between one another, the predicted transport mechanics of V. choleraes protein homologs differ substantially from the E. coli FadLs pathway found from X-ray crystallography studies.

sentences: Phagocytic Uptake of Polymeric Particles by Immune cells Under Flow Conditions Particles injected intravenously are thought to be cleared by macrophages residing in the liver and spleen, but they also encounter circulating immune cells. It remains to be established if the circulating cells can take up particles while flowing, and if the uptake capacity is similar under static and flow conditions. Here, we use an in vitro peristaltic pump setup that mimics pulsatile blood flow to determine if immune cells take up particles under constant fluidic flow. We use polystyrene particles of varying sizes as the model of a polymeric particle for these studies. Our results show that the immune cells do phagocytose under flow conditions. We demonstrate that cell lines representing myeloid cells, primary human neutrophils and monocytes take up sub-micrometer-sized particles at similar or better rates under flow compared to static conditions. Experiments with whole human blood show that even under the crowding effects of red blood cells, neutrophils and monocytes take up particles while flowing. Together, these data suggest that circulating immune cells are likely to phagocytose intravenously injected particulates, which has implications for design of particles to evade or target these cells.

sentences: A novel 5 prime DNA binding site in RFC facilitates PCNA loading for gap DNA repair RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases {delta} and {gamma}. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures revealing a novel 2nd DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located in the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. It appears 5' DNA binds after 3' DNA binding to RFC. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. We propose that the 5' site facilitates RFCs PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5' DNA site in longer gaps including during lagging strand DNA synthesis.

sentences: The structured flow on the brain's resting state manifold AO_SCPLOWBSTRACTC_SCPLOWSpontaneously fluctuating brain activity patterns emerge at rest and relate to brain functional networks involved in task conditions. Despite detailed descriptions of the spatio-temporal brain patterns, our understanding of their generative mechanism is still incomplete. Using a combination of computational modeling and dynamical systems analysis we provide a complete mechanistic description in terms of the constituent entities and the productive relation of their causal activities leading to the formation of a resting state manifold via the network connectivity. We demonstrate that the symmetry breaking by the connectivity creates a characteristic flow on the manifold, which produces the major empirical data features including spontaneous high amplitude co-activations, neuronal cascades, spectral cortical gradients, multistability and characteristic functional connectivity dynamics. The understanding of the brains resting state manifold is fundamental for the construction of task-specific flows and manifolds used in theories of brain function such as predictive coding.

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Local administration of bicuculline into the ventrolateral and medial preoptic nuclei modifies sleep and maternal behavior in lactating rats. The preoptic area (POA) is a brain structure classically involved in a wide variety of animal behavior including sleep and maternal care. In the current study, we evaluate the specific effect of disinhibition of two specific regions of the POA, the medial POA nucleus (mPOA) and the ventrolateral POA area (VLPO) on sleep and maternal behavior in lactating rats. For this purpose, mother rats on postpartum day 1 (PPD1) were implanted for polysomnographic recordings and with bilateral cannulae either in the mPOA or in the VLPO. The rats were tested for sleep and maternal behavior on PPD4-8 after the infusion of the GABA-A antagonist, bicuculline (0, 10 or 30 ng/0.2 {micro}l/side). Infusion of bicuculline into the mPOA augmented retrieving and nest building behaviors and reduced both nursing and milk ejections but had almost no effect on sleep. When bicuculine was microinjected into the VLPO, the rats significantly increase the number of retrievings and mouthings and reduced the nursing time without changes in milk ejections, which was associated with an increase in wakefulness and a reduction in light sleep.

Our results show that disinhibition of the mPOA, a key area in the control of maternal behavior, increased active maternal behaviors and reduced nursing without affecting wakefulness or sleep time. In contrast, the enhancement of some active maternal behaviors when the drug was infused into the VLPO, a sleep-promoting area, with a concomitant increase in wakefulness suggests that mother rats devote this additional waking time in the active maternal care of the pups. We hypothesize that maternal behavior changes after bicuculine microinjection into the VLPO is caused by a reduction in the sleep drive, rather than a direct effect on maternal behavior.

sentences: Structures of topoisomerase V in complex with DNA reveal unusual DNA binding mode and novel relaxation mechanism Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include three AP lyase repair domains. The unusual architecture of this enzyme bears no resemblance to any other known topoisomerase. Here we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA binding mode not observed before and provide information on the way this unusual topoisomerase relaxes DNA.

sentences: Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion Helicases are essential for nearly all nucleic acid processes across the tree of life. Using Nanopore Tweezers we observed the small, fast steps taken by single RecQ helicases as they step along and unwind DNA at ultrahigh spatiotemporal resolution. By directly measuring conformational substates of RecQ we determine the coupling between helicase domain motions and chemical reactions that together produce forward motion along the DNA. Application of assisting and opposing forces shows that RecQ has a highly asymmetric energy landscape that reduces its sensitivity to opposing mechanical forces that could be encountered in vivo by molecular roadblocks such as DNA bound proteins. This energy landscape enables RecQ to maintain speed against an opposing load.

sentences: Osteomacs support osteoclast-mediated resorption and contribute to bone pathology in a postmenopausal osteoporosis mouse model Osteal macrophages (osteomacs) support osteoblast function and promote bone anabolism, but their contribution to osteoporosis has not been explored. While mouse ovariectomy models have been repeatedly used, variation in strain, experimental design and assessment modalities, have contributed to no single model being confirmed as comprehensively replicating the full gamut of osteoporosis pathological manifestations. We validated an ovariectomy model in adult C3H/HeJ mice and demonstrated that it presents with human post-menopausal osteoporosis features, including reduced bone volume in axial and appendicular bone and bone loss in both trabecular and cortical bone including increased cortical porosity. Bone loss was associated with increased osteoclasts on trabecular and endocortical bone and decreased osteoblasts on trabecular bone. Importantly, this OVX model was characterised by delayed fracture healing. Using this validated model, we demonstrated that osteomacs are increased post-ovariectomy on both trabecular and endocortical bone. Dual F4/80 (pan-macrophage marker) and TRAP staining revealed osteomacs frequently located near TRAP+ osteoclasts and containing TRAP+ intracellular vesicles. Using an in vivo inducible macrophage depletion model that does not simultaneously deplete osteoclasts, we observed that osteomac loss was associated with elevated extracellular TRAP in bone marrow interstitium and increased serum TRAP. Using in vitro high-resolution confocal imaging of mixed osteoclast-macrophage cultures on bone substrate, we observed macrophages juxtaposed to osteoclast basolateral functional secretory domains scavenging degraded bone by-products. These data demonstrate a role for osteomacs in supporting osteoclastic bone resorption through phagocytosis and sequestration of resorption by-products. Finally, using Siglec1 knockout mice, we demonstrated that loss of the macrophage-restricted molecule Siglec-1/CD169 is sufficient to cause age-associated low bone mass, emphasizing the macrophages, independent of osteoclasts, contribute to optimal skeletal health. Overall, our data expose a novel role for osteomacs in supporting osteoclast function and provide the first evidence of their involvement in osteoporosis pathogenesis.

sentences: Molecular Mechanism of Hyperactivation Conferred by a Truncated TRPA1 Disease Mutant Suggests New Gating Insights The wasabi receptor, TRPA1, is a non-selective homotetrameric cation channel expressed in primary sensory neurons of the pain pathway, where it is activated by diverse chemical irritants. A direct role for TRPA1 in human health has been highlighted by the discovery of genetic variants associated with severe pain disorders. One such TRPA1 mutant was identified in a father-son pair with cramp fasciculation syndrome (CFS) and neuronal hyperexcitability-hypersensitivity symptoms that may be caused by aberrant channel activity, though the mechanism of action for this mutant is unknown. Here, we show the CFS-associated R919* TRPA1 mutant is functionally inactive when expressed alone in heterologous cells, which is not surprising since it lacks the 201 C-terminal amino acids that house critical channel gating machinery including the pore-lining transmembrane helix. Interestingly, the R919* mutant confers enhanced agonist sensitivity when co-expressed with wild type (WT) TRPA1. This channel hyperactivation mechanism is conserved in distant TRPA1 species orthologues and can be recapitulated in the capsaicin receptor, TRPV1. Using a combination of ratiometric calcium imaging, immunostaining, surface biotinylation, pulldown assays, fluorescence size exclusion chromatography, and proximity biotinylation assays, we show that the R919* mutant co-assembles with WT subunits into heteromeric channels. Within these heteromers, we postulate that R919* TRPA1 subunits contribute to hyperactivation by lowering energetic barriers to channel activation contributed by the missing regions. Additionally, we show heteromer activation can originate from the R919* TRPA1 subunits, which suggests an unexpected role for the ankyrin repeat and coiled coil domains in concerted channel gating. Our results demonstrate the R919* TRPA1 mutant confers gain-of-function thereby expanding the physiological impact of nonsense mutations, reveals a novel and genetically tractable mechanism for selective channel sensitization that may be broadly applicable to other receptors, and uncovers new gating insights that may explain the molecular mechanism of temperature sensing by some TRPA1 orthologues.

sentences: qc3C: reference-free quality control for Hi-C sequencing data Hi-C is a sample preparation method that enables high-throughput sequencing to capture genome-wide spatial interactions between DNA molecules. The technique has been successfully applied to solve challenging problems such as 3D structural analysis of chromatin, scaffolding of large genome assemblies and more recently the accurate resolution of metagenome-assembled genomes (MAGs). Despite continued refinements, however, Hi-C library preparation remains a complex laboratory protocol and diligent quality management is recommended to avoid costly failure. Current wet-lab protocols for Hi-C library QC provide only a crude assay, while commonly used sequence-based QC methods demand a reference genome; the quality of which can skew results. We propose a new, reference-free approach for Hi-C library quality assessment that requires only a modest amount of sequencing data. The algorithm builds upon the observation that proximity ligation events are likely to create k -mers that would not naturally occur in the sample. Our software tool (qc3C) is to our knowledge the first to implement a reference-free Hi-C QC tool, and also provides reference-based QC, enabling Hi-C to be more easily applied to non-model organisms and environmental samples. We characterise the accuracy of the new algorithm on simulated and real datasets and compare it to reference-based methods.

sentences: Resilience in soil bacterial communities of the boreal forest from one to five years after wildfire across a severity gradient Wildfires can represent a major disturbance to ecosystems, including soil microbial communities belowground. Furthermore, fire regimes are changing in many parts of the world, altering and often increasing fire severity, frequency, and size. The boreal forest and taiga plains ecoregions of northern Canada are characterized by naturally-occurring stand-replacing wildfires on a 40-350 year basis. We previously studied the effects of wildfire on soil microbial communities one year post-fire across 40 sites, spanning a range of burn severity. Here, we return to the same sites five years post-fire to test a series of hypotheses about the effects of fire on bacterial community composition. We ask the following questions: (1a) Do the fundamental factors structuring bacterial community composition remain the same five years post-fire? (1b) Do the effects of fire on bacterial community composition decrease between one and five years post-fire? (1c) Do shifts in bacterial community composition between one and five years post-fire suggest resilience? (2a) Does the importance of fast growth diminish between one and five years post-fire? (2b) Do short-term post-fire responders continue to dominate the community five years post-fire? We find the following: (1a) Five years post-fire, vegetation community, moisture regime, pH, total carbon, texture, and burned/unburned all remained significant predictors of bacterial community composition with similar predictive value (R2). (1b and 1c) Bacterial communities became more similar to unburned sites five years post-fire, across the range of severity, suggesting resilience, while general structure of co-occurrence networks remained similar one and five years post-fire. (2a) Fast growth potential, as estimated using predicted 16S rRNA copy numbers, was no longer significantly correlated with burn severity five years post-fire, indicating the importance of this trait for structuring bacterial community composition may be limited to relatively short timescales. (2b) Many taxa that were enriched in burned sites one year post-fire remained enriched five years post-fire, although the degree to which they were enriched generally decreased. Specific taxa of interest from the genera Massilia, Blastococcus, and Arthrobacter all remained significantly enriched, suggesting that they may have traits that allow them to continue to flourish in the post-fire environment, such as tolerance to increased pH or ability to degrade pyrogenic organic matter. This hypothesis-based work expands our understanding of the post-fire recovery of soil bacterial communities and raises new hypotheses to test in future studies.

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Transposable elements in the genome of the lichen-forming fungus Umbilicaria pustulata, and their distribution in different climate zones along elevation BackgroundTransposable elements (TEs) are an important source of genome plasticity across the tree of life. Accumulating evidence suggests that TEs may not be randomly distributed in the genome. Drift and natural selection are important forces shaping TE distribution and accumulation, acting directly on the TE element or indirectly on the host species. Fungi, with their multifaceted phenotypic diversity and relatively small genome size, are ideal models to study the role of TEs in genome evolution and their impact on the hosts ecological and life history traits. Here we present an account of all TEs found in a high-quality reference genome of the lichen-forming fungus Umbilicaria pustulata, a macrolichen species comprising two climatic ecotypes: Mediterranean and cold-temperate. We trace the occurrence of the newly identified TEs in populations along three replicated elevation gradients using a Pool-Seq approach, to identify TE insertions of potential adaptive significance.

ResultsWe found that TEs cover 21.26 % of the 32.9 Mbp genome, with LTR Gypsy and Copia clades being the most common TEs. Out of a total of 182 TE copies we identified 28 insertions displaying consistent insertion frequency differences between the two host ecotypes across the elevation gradients. Most of the highly differentiated insertions were located near genes, indicating a putative function.

ConclusionsThis pioneering study into the content and climate niche-specific distribution of TEs in a lichen-forming fungus contributes to understanding the roles of TEs in fungal evolution. Particularly, it may serve as a foundation for assessing the impact of TE dynamics on fungal adaptation to the abiotic environment, and the impact of TE activity on the evolution and maintenance of a symbiotic lifestyle.

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The view of microbes as energy converters illustrates the trade-off between growth rate and yield The application of thermodynamics to microbial growth has a long tradition that originated in the middle of the 20th century. This approach reflects the view that self-replication is a thermodynamic process that is not fundamentally different from mechanical thermodynamics. The key distinction is that a free energy gradient is not converted into mechanical (or any other form of) energy, but rather into new biomass. As such, microbes can be viewed as energy converters that convert a part of the energy contained in environmental nutrients into chemical energy that drives self-replication. Before the advent of high-throughput sequencing technologies, only the most central metabolic pathways were known. However, precise measurement techniques allowed for the quantification of exchanged extracellular nutrients and heat of growing microbes with their environment. These data, together with the absence of knowledge of metabolic details, drove the development of so-called black box models, which only consider the observable interactions of a cell with its environment and neglect all details of how exactly inputs are converted into outputs. Now, genome sequencing and genome-scale metabolic models provide us with unprecedented detail about metabolic processes inside the cell. However, the derived modelling approaches make surprisingly little use of thermodynamic concepts. Here, we review classical black box models and modern approaches that integrate thermodynamics into genome-scale metabolic models. We also illustrate how the description of microbial growth as an energy converter can help to understand and quantify the trade-off between microbial growth rate and yield.

PerspectiveO_LIMicrobial growth is the foundation of many biotechnological applications. The key to optimizing microbial growth lies in thermodynamics, similar to how classical thermodynamics helped optimize steam engines in the 19th century.
C_LIO_LIGenome-scale metabolic models have become widely available, and are used to predict microbial growth. These predictions often fail because these models do not distinguish between growth rate and yield.
C_LIO_LIClassical black box models present a sound thermodynamic theory, by viewing microbes as energy converters. Incorporating such concepts into genome-scale metabolic models has the promise to advance our fundamental understanding of microbial growth, and thus to improve the predictive power of these models.
C_LI

sentences: Spatial expression programs of the intestinal follicle-associated epithelium The intestine is lined with isolated lymphoid follicles (ILFs) that facilitate sampling of luminal antigens to elicit immune responses. Technical challenges related to the scarcity and small sizes of ILFs and their follicle-associated epithelium (FAE) impeded the characterization of their spatial gene expression programs. Here, we combined RNA sequencing of laser capture microdissected tissues with single molecule transcript imaging to obtain a spatial gene expression map of the ILF and its associated FAE in the mouse small intestine. We identified zonated expression programs in both follicles and FAEs, with a decrease in enterocyte anti-microbial and absorption programs and a partial induction of expression programs normally observed at the villus tip. We further identified Lepr+ sub-epithelial telocytes at the FAE top, which are distinct from villus-tip Lgr5+ telocytes. Our analysis exposes the epithelial and mesenchymal cell states associated with ILFs.

sentences: Enabling high throughput target-based drug discovery in 3D cell cultures through novel bioprinting workflow Advanced three-dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter Rastrum with the high throughput screening assay AlphaLISA, we demonstrate the utility of the workflow in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3{beta} phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.

sentences: Decoupling SARS-CoV-2 ORF6 localization and interferon antagonism Like many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds the nuclear pore components Nup98-Rae1 to inhibit nuclear translocation of activated STAT1/IRF3 transcription factors. However, a direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants can still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6/Nup98 interaction domain robustly inhibited nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral-membrane protein, opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication.

sentences: Mapping yeast mitotic 5' resection at base resolution reveals the sequence and positional dependence of nucleases in vivo Resection of the 5-terminated strand at DNA double strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Biochemical and genetic studies have led to a multi-step model of DSB resection in which endonucleolytic cleavage mediated by Mre11 in partnership with Sae2 is coupled with exonucleolytic cleavage mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing in which molecular identifiers allow exact counting of cleaved 5 ends at base pair resolution. Mutant strains, including exo1{Delta}, mre11-H125N, exo1{Delta} and exo1{Delta} sgs1{Delta}, revealed a major Mre11-dependent cleavage position 60 to 70 bp from the DSB end whose exact position depended on local sequence and tracked a possible motif. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1{Delta} sgs1{Delta} yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo support of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

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Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate NMR-assisted crystallography - the synergistic combination of solid-state NMR, X-ray crystallography, and first-principles computational chemistry - holds remarkable promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in the enzyme active site - including hydrogen atom locations and tautomeric equilibria - it offers insight into structure, dynamics, and function. Here, we make use of this combined approach to characterize the -aminoacrylate intermediate in tryptophan synthase, a defining species for pyridoxal-5'-phosphate-dependent enzymes on the {beta}-elimination and replacement pathway. By uniquely identifying the protonation states of ionizable sites on the cofactor, substrates, and catalytic side chains, as well as the location and orientation of structural waters in the active site, a remarkably clear picture of structure and reactivity emerges. Most incredibly, this intermediate appears to be mere tenths of angstroms away from the preceding transition state in which the {beta}-hydroxyl of the serine substrate is lost. The position and orientation of the structural water immediately adjacent to the substrate {beta}-carbon suggests not only the fate of the hydroxyl group, but also the pathway back to the transition state and the identity of the active site acid-base catalytic residue. Reaction of this intermediate with benzimidazole (BZI), an isostere of the natural substrate, indole, shows BZI bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the BZI position, indole is positioned with C3 in contact with the -aminoacrylate C{beta} and aligned for nucleophilic attack.

Significance StatementThe determination of active site protonation states is critical to gaining a full mechanistic understanding of enzymatic transformations; yet hydrogen positions are challenging to extract using the standard tools of structural biology. Here we make use of a joint solid-state NMR, X-ray crystallography, and first-principles computational approach that unlocks the investigation of enzyme catalytic mechanism at this fine level of chemical detail. For tryptophan synthase, this allows us to peer along the reaction coordinates into and out of the -aminoacrylate intermediate. Through this process, we are developing a high-resolution probe for structural biology that is keenly sensitive to proton positions - rivaling that of neutron diffraction, yet able to be applied under conditions of active catalysis to microcrystalline and non-crystalline materials.

sentences: Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum Experience-dependent plasticity is a key feature of brain synapses for which neuronal N-Methyl-D-Aspartate receptors (NMDARs) play a major role, from developmental circuit refinement to learning and memory. Astrocytes also express NMDARs although their exact function has remained controversial. Here we identify a circuit function for GluN2C NMDAR, a subtype highly expressed in astrocytes, in layer-specific tuning of synaptic strengths in mouse hippocampal CA1 pyramidal neurons. Interfering with astrocyte NMDAR or GluN2C NMDAR activity reduces the range of presynaptic strength distribution specifically in the stratum radiatum inputs without an appreciable change in the mean presynaptic strength. Mathematical modeling shows that narrowing of the width of presynaptic release probability distribution compromises the expression of long-term synaptic plasticity. Our findings suggest a novel feedback signaling system that uses astrocyte GluN2C NMDARs to adjust basal synaptic weight distribution of Schaffer collateral inputs, which in turn impacts computations performed by the CA1 pyramidal neuron.

sentences: Unfolding of RNA via Translocation Through a Nanopore RNA unfolding and refolding are important biological phenomena, which occur during the transfer of genetic information from DNA to RNA to proteins. During these processes, RNA is found in single stranded, secondary and tertiary structures, including secondary conformations like hairpins and pseudoknots. Understanding the diverse conformations of RNA and how these influence the dynamics of unfolding and refolding is crucial to gain insight to fundamental biological processes. In this work, we employ coarse-grained Langevin dynamics simulations of a simple model of different RNA hairpins passing through a geometric nanopore to find the influence of structural changes on the translocation dynamics. The threshold voltage of unfolding depends on the length of the hairpin attached to the tail. The lag time to unfold is longer for smaller applied voltages and for the architectures containing a longer hairpin attached to the tail. Chain translocation dynamics for different architectures are largely collapsed by the threshold. A distinct signature for the base unfolding time was observed for the bases around the unpaired bases in all the RNA hairpin models. These results can motivate future technologies or experiments that use translocation to predict secondary structures of polynucleotides.

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Agonist-independent Prostaglandin EP3 Receptor signaling is required to prevent insulin hypersecretion and metabolic dysfunction in a non-obese mouse model of insulin resistance When homozygous for the LeptinOb mutation (Ob), Black-and-Tan Brachyury (BTBR) mice become morbidly obese and severely insulin resistant, and by 10 weeks of age, frankly diabetic. Previous work has shown Prostaglandin EP3 Receptor (EP3) expression and activity is up-regulated in islets from BTBR-Ob mice as compared to lean controls, actively contributing to their beta-cell dysfunction. In this work, we aimed to test the impact of beta-cell-specific EP3 loss on the BTBR-Ob phenotype by crossing Ptger3 floxed mice with the Rat insulin promoter (RIP)-CreHerr driver strain. Instead, germline recombination of the floxed allele in the founder mouse - an event whose prevalence we identified as directly associated with underlying insulin resistance of the background strain - generated a full-body knockout. Full-body EP3 loss provided no diabetes protection to BTBR-Ob mice, but, unexpectedly, significantly worsened BTBR-lean insulin resistance and glucose tolerance. This in vivo phenotype was not associated with changes in beta-cell fractional area or markers of beta-cell replication ex vivo. Instead, EP3-null BTBR-lean islets had essentially uncontrolled insulin hypersecretion. The selective up-regulation of constitutively-active EP3 splice variants in islets from young, lean BTBR mice as compared to C57BL/6J, where no phenotype of EP3 loss has been observed, provides a potential explanation for the hypersecretion phenotype. In support of this, high islet EP3 expression in Balb/c females vs. Balb/c males was fully consistent with their sexually-dimorphic metabolic phenotype after loss of EP3-coupled Gz protein. Taken together, our findings provide a new dimension to the understanding of EP3 as a critical brake on insulin secretion.

New and NoteworthyIslet Prostaglandin EP3 receptor (EP3) signaling is well-known as up-regulated in the pathophysiological conditions of type 2 diabetes, contributing to beta-cell dysfunction. Unexpected findings in mouse models of non-obese insulin sensitivity and resistance provide a new dimension to our understanding of EP3 as a key modulator of insulin secretion. A previously-unknown relationship between mouse insulin resistance and the penetrance of Rat insulin promoter-driven germline floxed allele recombination is critical to consider when creating beta-cell-specific knockouts.

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sentences: Plant responses to multiple antagonists are mediated by order of attack and phytohormone crosstalk Plants are often attacked by multiple antagonists, and traits of the attacking organisms, and their order of arrival onto hosts, may affect plant defenses. However, few studies have assessed how multiple antagonists, and varying attack order, affect plant defense or nutrition. To address this, we assessed defensive and nutritional responses of Pisum sativum plants after attack by a vector herbivore (Acrythosiphon pisum), a non-vector herbivore (Sitona lineatus), and a pathogen (Pea enation mosaic virus, PEMV). We show PEMV-infectious A. pisum induced several pathogen-specific plant defense signals, but these defenses were inhibited when S. lineatus was present in peas infected with PEMV. In contrast, feeding by S. lineatus induced anti-herbivore defense signals, but these defenses were enhanced by PEMV. Sitona lineatus also increased abundance of plant amino acids, but only when they attacked after PEMV-infectious A. pisum. Our results suggest that diverse communities of biotic antagonists alter defense and nutritional traits of plants through complex pathways that depend on the identity of attackers and their order of arrival onto hosts. Moreover, we show interactions among a group of biotic stressors can vary along a spectrum from antagonism to enhancement/synergism based on the identity and order of attackers, and these interactions are mediated by a multitude of phytohormone pathways.

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Still no evidence for disruption of global patterns of nest predation in shorebirds Many shorebird species are rapidly declining (Piersma et al. 2016; Munro 2017; Studds et al. 2017), but it is not always clear why. Deteriorating and disappearing habitat, e.g. due to intensive agriculture (Donal et al. 2001; Kentie et al. 2013; Kentie et al. 2018), river regulation (Nebel et al. 2008) or mudflat reclamation (Ma et al. 2014; Larson 2017), and hunting (Reed et al. 2018; Gallo-Cajiao et al. 2020) are some of the documented causes. A recent study suggests yet another possible cause of shorebird decline: a global increase in nest predation (Kubelka et al. 2018). The authors compiled an impressive dataset on patterns of nest predation in shorebirds and their analyses suggest that global patterns of nest predation have been disrupted by climate change, particularly in the Arctic. They go as far as to conclude that the Arctic might have become an ecological trap (Kubelka et al. 2018). Because these findings might have far-reaching consequences for conservation and related political decisions, we scrutinized the study and concluded that the main conclusions of Kubelka et al. (2018) are invalid (Bulla et al. 2019a). The authors then responded by reaffirming their conclusions (Kubelka et al. 2019b).

Here, we evaluate some of Kubelka et al.s (2019b) responses, including their recent erratum (2020), and show that the main concerns about the original study still hold. Specifically, (1) we reaffirm that Kubelka et al.s (2018) original findings are confounded by study site. Hence, their conclusions are over-confident because of pseudo-replication. (2) We reiterate that there is no statistical support for the assertion that predation rate has changed in a different way in the Arctic compared to other regions. The relevant test is an interaction between a measure of time (year or period) and a measure of geography (e.g., Arctic vs the rest of the world). The effect of such an interaction is weak, uncertain and statistically non-significant, which undermines Kubelka et al.s (2018) key conclusion. (3) We further confirm that the suggested general increase in predation rates over time is at best a weak and uncertain trend. The most parsimonious hypothesis for the described results is that the temporal changes in predation rate are an artefact of temporal changes in methodology and data quality. Using only high-quality data, i.e. directly calculated predation rates, reveals no overall temporal trend in predation rate. Below we elaborate in detail on each of these points.

We conclude that (i) there is no evidence whatsoever that the pattern in the Arctic is different from that in the rest of the world and (ii) there is no solid evidence for an increase in predation rate over time. While we commend Kubelka et al. for compiling and exploring the data, we posit that the data underlying their study, and perhaps all currently available data, are not sufficient (or of sufficient quality) to test their main hypotheses. We call for standardized and consistent data collection protocols and experimental validation of current methods for estimating nesting success.

sentences: Low Frequency Oscillations drive EEG's complexity changes during wakefulness and sleep Recently, the sleep-wake states have been analysed using novel complexity measures, complementing the classical analysis of EEGs by frequency bands. This new approach consistently shows a decrease in EEGs complexity during slow-wave sleep, yet it is unclear how cortical oscillations shape these complexity variations. In this work, we analyse how the frequency content of brain signals affects the complexity estimates in freely moving rats. We find that the low-frequency spectrum - including the Delta, Theta, and Sigma frequency bands - drives the complexity changes during the sleep-wake states. This happens because low-frequency oscillations emerge from neuronal population patterns, as we show by recovering the complexity variations during the sleep-wake cycle from micro, meso, and macroscopic recordings. Moreover, we find that the lower frequencies reveal synchronisation patterns across the neocortex, such as a sensory-motor decoupling that happens during REM sleep. Overall, our works shows that EEGs low frequencies are critical in shaping the sleep-wake states complexity across cortical scales.

sentences: One mucosal administration of a live attenuated recombinant COVID-19 vaccine protects non-human primates from SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein, spike, for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live attenuated, recombinant human respiratory syncytial virus (RSV) expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200- fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunity in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against two virus variants of concern. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in phase 1 clinical trials as a single-dose intranasal COVID-19 vaccine.

sentences: Genome-wide Association Study Reveals that PvGUX1_1 is Associated with Pod Stringlessness in Snap Bean (Phaseolus vulgaris L.) The suture strings is a particularly important pod trait that determines the quality and texture of snap bean (Phaseolus vulgaris L.). The St locus on chromosome 2 has been described as a major locus associated with suture strings. However, the gene and genetic basis underlying this locus remain unknown. Here, we investigated the suture strings of 138 snap bean accessions across two years. A total of 3.66 million single-nucleotide polymorphisms (SNPs) were obtained by deep resequencing. Based on these SNPs, we identified a strong association signal on Chr02 and a promising candidate gene, PvGUX1_1. Further analysis revealed that the 2-bp deletion in exon of PvGUX1_1 was significantly associated with stringlessness. Comparative mapping indicated that PvGUX1_1 was a domesticated locus and diverged from PvGUX1_2 during an early stage. Our study provides important insights into the genetic mechanism of suture string formation and useful information for snap bean improvement.

sentences: Transverse and axial resolution of femtosecond laser ablation Femtosecond lasers are capable of precise ablation that produce surgical dissections in vivo. The transverse and axial resolution of the laser damage inside the bulk are important parameters of ablation. The transverse resolution is routinely quantified, but the axial resolution is more difficult to measure and is less commonly performed. In some in vivo samples, fine dissections can also be difficult to visualize, but in vitro samples may allow clear imaging. Using a 1040-nm, 400-fs pulsed laser, we performed ablation inside agarose and glass, producing clear and persistent damage spots. Near the ablation threshold of both media, we found that the axial resolution is similar to the transverse resolution. We also ablated neuron cell bodies and fibers in C. elegans and demonstrate submicrometer resolution in both the transverse and axial directions, consistent with our results in agarose and glass. Using simple yet rigorous methods, we define the resolution of laser ablation in transparent media along all directions.

sentences: Inferring cell-cell interactions from pseudotime ordering of scRNA-Seq data A major advantage of single cell RNA-Sequencing (scRNA-Seq) data is the ability to reconstruct continuous ordering and trajectories for cells. To date, such ordering was mainly used to group cells and to infer interactions within cells. Here we present TraSig, a computational method for improving the inference of cell-cell interactions in scRNA-Seq studies. Unlike prior methods that only focus on the average expression levels of genes in clusters or cell types, TraSig fully utilizes the dynamic information to identify significant ligand-receptor pairs with similar trajectories, which in turn are used to score interacting cell clusters. We applied TraSig to several scRNA-Seq datasets. As we show, using the ordering information allows TraSig to obtain unique predictions that improve upon those identified by prior methods. Functional experiments validate the ability of TraSig to identify novel signaling interactions that impact vascular development in liver organoid.

sentences: Fast and accurate bootstrap confidence limits on genome-scale phylogenies using little bootstraps Felsensteins bootstrap resampling approach, applied in thousands of research articles, imposes a high computational burden for very long sequence alignments. We show that the bootstrapping of a collection of little subsamples, coupled with median bagging of subsample confidence limits, produces accurate bootstrap confidence for phylogenetic relationships in a fraction of time and memory. The little bootstraps approach will enhance rigor, efficiency, and parallelization of big data phylogenomic analyses.

sentences: Closed-loop recruitment of striatal parvalbumin interneurons prevents the onset of compulsive behaviors A prominent electrophysiological feature of compulsive behaviours is striatal hyperactivity; but its underlying dysfunctional cellular mechanisms still need to be characterized. Within the striatum, parvalbumin-positive interneurons (PVI) exert a powerful feedforward inhibition essential for the regulation of striatal activity. To investigate the potential implication of striatal PVI in aberrant repetitive behaviors, we used the Sapap3 mutant mice which exhibit compulsive-like behaviours characterized by excessive self-grooming. When striatal PVI in the centromedial striatum of Sapap3 mice were we optogenetically activated, we first showed that the number of compulsive-like events were greatly reduced. To investigate further the critical time-window when striatal PVI needed to be recruited for the behavioural regulation of compulsive-like grooming, we then designed a novel closed-loop stimulation pipeline. We identified a transient 1-4 Hz oscillations in the orbitofrontal cortex that temporally predicted grooming onsets. Exploiting this delta band signal as a biomarker, we were able to provide on-demand stimulation of striatal PVI shortly before predicted grooming events. This targeted closed-loop optogenetics approach greatly reduced grooming events and demonstrated that the recruitment of striatal PVI regulated the initiations of compulsive-like behaviours.

sentences: Butterfly eyespots evolved via co-option of the antennal gene-regulatory network Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via co-option of the antennal gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster with antennae relative to multiple other tissues. Furthermore, three genes essential for eyespot development (Distal-less (Dll), spalt (sal), and Antennapedia (Antp)) share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots and antennae, and also legs and wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely re-used the ancient antennal GRN, a network previously implicated also in the development of legs and wings.

sentences: Cellular genome wide association study identifies common genetic variation influencing lithium induced neural progenitor proliferation Lithium is used in the treatment of bipolar disorder (BD) and is known to increase neural progenitor cell (NPC) proliferation. Though the mechanism of lithiums therapeutic effect is not understood, evidence suggests that genetic variation influences response to treatment. Here, we used a library of genetically diverse human NPCs to identify common genetic variants that modulate lithium induced proliferation. We identified a locus on chr3p21.1 associated with lithium induced proliferation that colocalizes with BD risk. One lithium responsive gene, GNL3, was detected within the locus. The allele associated with increased baseline and lithium-induced GNL3 expression was also associated with increased lithium-induced NPC proliferation. Experimental manipulation of GNL3 expression using CRISPRa/i in NPCs showed that GNL3 was necessary for lithiums full proliferative effects, and sufficient to induce proliferation without lithium treatment. In all, our data suggest that GNL3 expression sensitizes NPCs for a stronger proliferative response to lithium.

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Functional dynamics of brain networks associated with carry-over effects of negative events on subsequent resting state. Affective inertia represents the lasting impact of transient emotions at one time point on affective state at a subsequent time point. Here we describe the neural underpinnings of inertia following negative emotions elicited by sad events in movies. Using a co-activation pattern analysis of dynamic functional connectivity, we examined the temporal expression and reciprocal interactions among brain-wide networks during movies and subsequent resting periods. Our findings revealed distinctive spatiotemporal expression of visual (VIS), default mode (DMN), central executive (CEN), and frontoparietal control (FPCN) networks both in negative movies and in rest periods following these movies. We also identified different reciprocal relationships among these networks, in transitions from movie to rest. While FPCN and DMN expression increased during and after negative movies, respectively, FPCN occurrences during the movie predicted lower DMN and higher CEN expression during subsequent rest after neutral movies, but this relationship was reversed after the elicitation of negative emotions. Changes in FPCN and DMN activity correlated with more negative subjective affect. These findings provide new insights into the transient interactions of intrinsic brain networks underpinning the inertia of negative emotions. More specifically, they describe a major role of FPCN in emotion elicitation processes, with prolonged impact on DMN activity in subsequent rest, presumably involved in emotion regulation and restoration of homeostatic balance after negative events.

HighlightsO_LIModulations of dynamic functional brain connectivity are associated to the temporal inertia of negative emotions.
C_LIO_LIFunctional co-activations patterns (CAPs) during emotional episodes predict changes in spontaneous brain dynamics during subsequent resting state.
C_LIO_LIClassical "task-rest" anticorrelations in network activity are reversed by negative emotions.
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sentences: Morpho-physiological and gene expression responses of wheat by Aegilops cylindrica amphidiploids to salt stress Aegilops cylindrica Host is one of the most salt-tolerant species in the Triticeae tribe. Amphidiploid plants derived from hybridization of  Roshan x Aegilops cylindrica and  Chinese Spring x Ae. cylindrica genotypes contrasting in salt tolerance were assessed for their morpho-physiological responses and the expression patterns of three genes related to ion homeostasis under 250 mM NaCl. Results showed that salt stress caused significant declines in both their morphological and phenological traits. Moreover, salt stress reduced not only their chlorophyll content but also their root and shoot K contents and K/Na ratios, while it led to significant enhancements in the remaining traits. Similar to Ae. cylindrica, the amphidiploids subjected to salt stress exhibited significantly higher H2O2 levels, root and shoot K contents, and root and shoot K/Na ratios accompanied by lower root and shoot Na contents and MDA concentrations when compared with the same traits in the wheat parents. Quantitative Real-Time PCR showed significant differential expression patterns of the HKT1;5, NHX1, and SOS1 genes between the amphidiploids and their parents. The transcript level of HKT1;5 was found to be higher in the roots than in the shoots of both the amphidiploids and Ae. cylindrica while NHX1 exhibited a higher expression in the shoot tissues. The consistency of these data provides compelling support for the hypothesis that active exclusion of Na from the roots and elevated vacuolar sequestration of Na in the leaves might explain the declining Na levels in the shoots and roots of both the amphidiploids and Ae. cylindrica relative to those measured in wheat parents. It is concluded that the hybridized amphiploids are potentially valuable resources for salt improvement in bread wheat through the backcrossing approach.

sentences: Interaural time difference sensitivity under binaural cochlear implant stimulation even at high pulse rates up to 900 pps Spatial hearing remains one of the major challenges for bilateral cochlear implant (biCI) users, and early deaf patients in particular are often completely insensitive to interaural time differences (ITDs) delivered through biCIs. One popular theory held is that this may be due to a lack of early binaural experience. However, previously we have shown that neonatally deafened rats fitted with biCIs in adulthood quickly learn to discriminate ITDs as well as their normal hearing litter mates (Rosskothen-Kuhl et al. 2021), and perform orders of magnitude better than human biCI users. Our unique behaving biCI rat model allows us to investigate other possible limiting factors of prosthetic binaural hearing, such as the effect of stimulus pulse rate and envelope shape. Previous work has indicated that ITD sensitivity may decline substantially at the high pulse rates often used in clinical practice. We therefore measured behavioral ITD thresholds in neonatally deafened, adult implanted biCI rats to pulse trains of 50, 300, 900 and 1800 pulses per second (pps), with either rectangular or Hanning window envelopes. Our rats exhibited very high sensitivity to ITDs at pulse rates up to 900 pps for both envelope shapes, a pulse rate commonly used in clinical practice. However, ITD sensitivity declined to near zero at 1800 pps, for both Hanning and rectangular windowed pulse trains. Current clinical cochlear implant (CI) processors are often set to pulse rates [≥]900 pps, but ITD sensitivity in human CI listeners is often reported to decline sharply above [~]300 pps. Our results suggest that the relatively poor ITD sensitivity seen at >300 pps in human CI users may not reflect the hard upper limit of biCI ITD performance in the mammalian auditory pathway. Perhaps with training or better CI strategies good binaural hearing may be achievable at pulse rates high enough to allow good sampling of speech envelopes while delivering usable ITDs.

sentences: Closed-Loop Control of Anesthetic State in Non-Human Primates Continuous monitoring of electroencephalogram (EEG) recordings in humans under general anesthesia (GA) has demonstrated that changes in EEG dynamics induced by an anesthetic drug are reliably associated with the altered arousal states caused by the drug. This observation suggests that an intelligent, closed-loop anesthesia delivery (CLAD) system operating in real-time could track EEG dynamics and control the infusion rate of a programmable pump to precisely maintain unconsciousness. The United States FDA acknowledges the potential benefits of such automatic physiological closed-loop control devices for patient care. Bringing these devices into clinical practice requires establishing their feasibility in suitable animal models. Therefore, given the close neurophysiological proximity between human and non-human primates (NHPs), we address this problem by developing and validating a propofol CLAD system in rhesus macaques. Our CLAD system has three key components: (1) a data acquisition system that records cortical local field potentials (LFPs) from an NHP in real-time; (2) a computer executing our CLAD algorithm that takes in the LFP signals as input and outputs infusion rates; and (3) a computer-controlled infusion pump that administers intravenous propofol. Our CLAD system controls an empirically-determined LFP marker of unconsciousness (MOU) at a user-prescribed target value by updating every 20 seconds the propofol infusion rate based on real-time processing of the LFP signal. The MOU is the instantaneous power in the 20 to 30 Hz band of the LFP spectrogram. Every cycle (duration{approx} 20 sec), our CLAD algorithm updates the MOU estimate and uses a robust optimal control strategy to adjust the propofol infusion rate based on the instantaneous error. This error is computed as the difference between the current and the user-prescribed target MOU values. Using neural recordings from multiple NHP anesthesia sessions, we first established that our chosen MOU signal was strongly correlated with propofol-induced decreased spiking activity which itself has been shown earlier to be associated with the level of unconsciousness in NHPs. Then we designed robust optimal control strategies that used subject-specific pharmacokinetic-pharmacodynamic models describing the MOU dynamics due to propofol infusion rate changes. Finally, we achieved safe and efficient closed-loop control of level of unconsciousness in 9 CLAD experiments involving 2 NHPs and 2 different 125 min long target MOU profiles with three target MOU changes within a given experiment. Our CLAD system performs stably, accurately and robustly across a total of 1125 min of closed-loop control. The CLAD performance measures, represented as median (25th percentile, 75th percentile), are 3.13 % (2.62%, 3.53%) for inaccuracy, 0.54 %(-0.31%, 0.89%) for bias, -0.02%/min (-0.06%/min, 0.00%/min) for divergence, and 3% (2.49%, 3.59%) for wobble. These performance measures were comparable or superior to previously reported CLAD performance measures from clinical studies (conducted outside USA) as well as rodent-based studies. The key innovations here are: (1) a pre-clinical NHP model for CLAD development and testing, (2) a neuroscience-informed LFP-based MOU for CLAD, (3) parsimonious, pharmacology-informed models to describe MOU dynamics under propofol infusion in rhesus macaques, (4) a novel numerical testing framework for propofol CLAD that incorporates a principled optimal robust control strategy for titrating propofol, and finally (5) experimental findings demonstrating the feasibility of stable, accurate and robust CLAD in the NHP model. Our NHP-based CLAD framework provides a principled pre-clinical research platform that can form the foundation for future clinical studies.

sentences: Prior aerosol infection with lineage A SARS-CoV-2 variant protects hamsters from disease, but not reinfection with B.1.351 SARS-CoV-2 variant The circulation of SARS-CoV-2 has resulted in the emergence of variants of concern (VOCs). It is currently unclear whether previous infection with SARS-CoV-2 provides protection against reinfection with VOCs. Here, we show that low dose aerosol exposure to hCoV-19/human/USA/WA-CDC-WA1/2020 (WA1, lineage A), resulted in a productive mild infection. In contrast, low dose of SARS-CoV-2 via fomites did not result in productive infection in the majority of exposed hamsters and these animals remained non-seroconverted. After recovery, hamsters were re-exposed to hCoV-19/South African/KRISP-K005325/2020 (VOC B.1.351) via an intranasal challenge. Seroconverted rechallenged animals did not lose weight and shed virus for 3 days. They had little infectious virus and no pathology in the lungs. In contrast, shedding, weight loss and extensive pulmonary pathology caused by B.1.351 replication was observed in the non-seroconverted animals. The rechallenged seroconverted animals did not transmit virus to naive sentinels via direct contact transmission, in contrast to the non-seroconverted animals. Reinfection with B.1.351 triggered an anamnestic response that boosted not only neutralizing titers against lineage A, but also titers against B.1.351. Our results confirm that aerosol exposure is a more efficient infection route than fomite exposure. Furthermore, initial infection with SARS-CoV-2 lineage A does not prevent heterologous reinfection with B.1.351 but prevents disease and onward transmission. These data suggest that previous SARS-CoV-2 exposure induces partial protective immunity. The reinfection generated a broadly neutralizing humoral response capable of effectively neutralizing B.1.351 while maintaining its ability to neutralize the virus to which the initial response was directed against.

sentences: Beyond category-supervision: Computational support for domain-general pressures guiding human visual system representation Anterior regions of the ventral visual stream encode substantial information about object categories. Are top-down category-level forces critical for arriving at this representation, or can this representation be formed purely through domain-general learning of natural image structure? Here we present a fully self-supervised model which learns to represent individual images, rather than categories, such that views of the same image are embedded nearby in a low-dimensional feature space, distinctly from other recently encountered views. We find (i) category information implicitly emerges in the local similarity structure of this feature space, and (ii) these models learn hierarchical features which capture the structure of brain responses across the human ventral visual stream, on par with category-supervised models. These results provide computational support for a domain-general framework guiding the formation of visual representation, where the proximate goal is not explicitly about category information, but is instead to learn unique, compressed descriptions of the visual world.

sentences: Multiple modes of regulation control dynamic transcription patterns during the mitosis-G1 transition Following cell division, genomes must reactivate gene expression patterns that reflect the identity of the cell. Here, we use PRO-seq to examine the mechanisms that reestablish transcription patterns after mitosis. We uncover regulation of the transcription cycle at multiple steps including initiation, promoter-proximal pause positioning and escape, poly-A site cleavage and termination during the mitotic-G1 transition. During mitosis, RNA polymerase activity is retained at initiation sites, albeit shifted in position relative to non-mitotic cells. This activity is strongly linked to maintenance of local chromatin architecture during mitosis and is more predictive of rapid gene reactivation than histone modifications previously associated with bookmarking. These molecular bookmarks, combined with sequence-specific transcription factors, direct expression of select cell growth and cell specific genes during mitosis followed by reactivation of functional gene groups with distinct kinetics after mitosis. This study details how dynamic regulation of transcription at multiple steps contributes to gene expression during the cell cycle.

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Immunity to SARS-CoV-2 up to 15 months after infection BackgroundInformation concerning the longevity of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in convalescent coronavirus disease-2019 (COVID-19) patients up to 15 months after symptoms onset.

MethodsThe levels of anti-spike and anti-receptor binding domain antibodies and neutralizing activities were tested in a total of 188 samples from 136 convalescent patients who experience mild to critical COVID-19. Specific memory B and T cell responses were measured in 76 peripheral blood mononuclear cell samples collected from 54 patients. Twenty-three vaccinated individuals were included for comparison.

FindingsFollowing a peak at day 15-28 post-infection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Plasma neutralizing activity against G614 was still detected in 87% of the patients at 6-15 months. Compared to G614, the median neutralizing titers against Beta, Gamma and Delta variants in plasma collected at early (15-103 days) and late (9-15 month) convalescence were 16- and 8-fold lower, respectively. SARS-CoV-2-specific memory B and T cells reached a peak at 3-6 months and persisted in the majority of patients up to 15 months although a significant decrease in specific T cells was observed between 6 and 15 months.

ConclusionThe data suggest that antiviral specific immunity especially memory B cells in COVID-19 convalescent patients is long-lasting, but some variants of concern, including the fast-spreading Delta variant, may at least partially escape the neutralizing activity of plasma antibodies.

FundingEU-ATAC consortium, the Italian Ministry of Health, the Swedish Research Council, SciLifeLab, and KAW.

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Discovery, diversity and functional associations of crAss-like phages in human gut metagenomes from four Dutch cohorts The crAss-like phages are a diverse group of related viruses that includes one of the most abundant viruses of the human gut. To explore their diversity and functional role in human population and clinical cohorts, we analyzed gut metagenomic data collected from more than 2000 individuals from the Netherlands. We discovered 125 novel species-level and 32 novel genus-level clusters of crAss-like phages, all belonging to five previously recognized groups associated with the human gut. Analysis of their genomic features revealed that closely related crAss-like phages can possess strikingly divergent regions responsible for transcription, presumably acquired through recombination. Prediction of crAss-like phage hosts pointed primarily to bacteria of the phylum Bacteroidetes, consistent with previous reports. Finally, we explored the temporal stability of crAss-like phages over a 4-year period and identified associations between the abundance of crAss-like phages and several human phenotypes, including depletion of crAss-like phages in inflammatory bowel disease patients.

HighlightsO_LI125 tentative new species of crAss-like phages were discovered
C_LIO_LIClosely related crAss-like phages often possess highly divergent transcription gene modules possibly acquired via recombination
C_LIO_LICrAss-like fraction of the human gut virome remains relatively stable over the period of 4 years
C_LIO_LIPrevalence of crAss-like phages in the human gut is associated with several metabolic, dietary and health phenotypes
C_LIO_LIGut crAss-like phages are depleted in inflammatory bowel disease patients
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sentences:
Emerging Accessibility Patterns in Long Telomeric Overhangs We present single molecule experimental and computational modeling studies investigating the accessibility of human telomeric overhangs of physiologically relevant lengths. We studied 25 different overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate 1-7 tandem G-quadruplex (GQ) structures. Using FRET-PAINT, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to available sites. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of the underlying folding landscape and folding frustration. Overhangs that have [4n]G-Tracts, (12, 16, 20...), demonstrate the broadest accessibility patterns where the PNA probe accesses G-Tracts throughout the overhang. On the other hand, constructs with [4n+2]G-Tracts, (14, 18, 22...), have narrower patterns where the neighborhood of the junction between single and double stranded telomere is most accessible. We interpret these results as the folding frustration being higher in [4n]G-Tract constructs compared to [4n+2] G-Tract constructs. We also developed a computational model that tests the consistency of different folding stabilities and cooperativities between neighboring GQs with the observed accessibility patterns. Our experimental and computational studies suggest the neighborhood of duplex junction is least stable and most accessible, which has implications for Shelterin complex formation that require access to this site.

SIGNIFICANCE STATEMENTWe report single molecule FRET-PAINT measurements and computational modeling studies probing the accessibility of 25 different human telomeric overhangs which, to our knowledge, is the most comprehensive range studied to date and covers a significant portion of the physiologically relevant length scale. Our measurements demonstrate novel repeating accessibility patterns with a well-defined periodicity, with certain regions having significantly higher accessibility than others. These accessibility patterns also suggest the underlying folding frustration depends on telomere length and constructs with a certain number of telomeric repeats demonstrate elevated levels of frustration compared to others. These patterns have significant implications for telomere organization, protection of free 3-end against exonuclease activity and telomerase-catalyzed extension, and folding cooperativity between neighboring G-quadruplex structures.

sentences: Inter-brain coupling reflects disciplinary differences in real-world classroom learning Classroom learning is one of the most critical scenarios in educational practices worldwide. Classroom learning was always organized into different disciplines (such as Math or language courses) in educational practice. However, we know little about how disciplinary differences are manifested in students neural dynamics supporting successful classroom learning. In order to address this question, we investigated disciplinary differences during the successful classroom learning from the framework of hard and soft disciplines by recording thirty-four high-school students EEGs simultaneously during their regular Chinese (a soft discipline) and Math (a hard discipline) courses across a whole semester. Ones theta-band inter-brain coupling to all other classmates during both the Chinese and Math courses was found to be positively correlated with their learning outcomes in Math. In contrast, ones alpha-band inter-brain coupling to their excellent peers was positively correlated with their learning outcomes in Chinese. These results demonstrated the value of inter-brain coupling as neural correlates of successful classroom learning and highlighted excellent peers as a candidate to represent successful learning for soft disciplines. Our findings provide real-world neuroscience evidence for understanding disciplinary differences and suggest possibly different neurocognitive mechanisms to support successful learning in different disciplines.

sentences: Conjugation of palbociclib with MHI-148 has an increased cytotoxic effect for breast cancer cells and an altered mechanism of action The CDK4/6 inhibitor palbociclib, combined with endocrine therapy, has been shown to be effective in postmenopausal women with estrogen receptor positive, HER2-negative advanced or metastatic breast cancer. However, palbociclib is not as effective in the highly aggressive triple-negative breast cancer that lacks sensitivity to chemotherapy or endocrine therapy. We hypothesized that conjugation of the near-infrared dye MHI-148 with palbociclib can produce a potential theranostic in triple-negative as well as estrogen receptor positive breast cancer cells. In our study, the conjugate was found to have enhanced activity in all mammalian cell lines tested in vitro. However, the conjugate was cytotoxic and did not induce G1 cell cycle arrest in breast cancer cells suggesting the mechanism of action differed from the parent compound palbociclib. The study highlights the importance of investigating the mechanism of conjugates of near-infrared dyes to therapeutic compounds as conjugation can potentially result in a change of mechanism or target, with an enhanced cytotoxic effect in this case.

sentences: Regular aerobic exercise is positively associated with hippocampal structure and function in young and middle-aged adults Regular exercise has numerous benefits for brain health, including the structure and function of the hippocampus. The hippocampus plays a critical role in memory function, and is altered in a number of psychiatric disorders associated with memory impairments (e.g. depression and schizophrenia), as well as healthy ageing. While many studies have focussed on how regular exercise may improve hippocampal integrity in older individuals, less is known about these effects in young to middle-aged adults. Therefore, we assessed the associations of regular exercise and cardiorespiratory fitness with hippocampal structure and function in these age groups. We recruited 40 healthy young to middle-aged adults, comprised of two groups (n = 20) who self-reported either high or low levels of exercise, according to World Health Organisation guidelines. We assessed cardiorespiratory fitness using a graded exercise test (VO2max) and hippocampal structure via manual tracing of T1-weighted magnetic resonance images. We also assessed hippocampal function using magnetic resonance spectroscopy to derive estimates of NAA concentration and hippocampal-dependent associative memory and pattern separation tasks. We observed evidence of increased N-acetyl-aspartate concentration and associative memory performance in individuals engaging in high levels of exercise. However, no differences in hippocampal volume or pattern separation capacity were observed between groups. Cardiorespiratory fitness was positively associated with hippocampal volume (left, right, and bilateral), N-acetyl-aspartate concentration, and pattern separation. However, no association was observed between cardiorespiratory fitness and associative memory. Therefore, we provide evidence that higher levels of exercise and cardiorespiratory fitness are associated with improved hippocampal structure and function. Exercise may provide a low-risk, effective method of improving hippocampal integrity in an early-to-mid-life stage.

sentences: A Structure-based B-cell Epitope Prediction Model Through Combing Local and Global Features B-cell epitopes (BCEs) are a set of specific sites on the surface of an antigen that binds to an antibody produced by B-cell. The recognition of BCEs is a major challenge for drug design and vaccines development. Compared with experimental methods, computational approaches have strong potential for BCEs prediction at much lower cost. Moreover, most of the currently methods focus on using local information around target residue without taking the global information of the whole antigen sequence into consideration. We propose a novel deep leaning method through combing local features and global features for BCEs prediction. In our model, two parallel modules are built to extract local and global features from the antigen separately. For local features, we use Graph Convolutional Networks(GCNs) to capture information of spatial neighbors of a target residue. For global features, Attention-Based Bidirectional Long Short-Term Memory(Att-BLSTM) networks are applied to extract information from the whole antigen sequence. Then the local and global features are combined to predict BCEs. The experiments show that the proposed method achieves superior performance over the state-of-the-art BCEs prediction methods on benchmark datasets. Also, we compare the performance differences between data with or without global features. The experimental results show that global features play an important role in BCEs prediction. Our detailed case study on the BCEs prediction for SARS-Cov-2 receptor binding domain confirms that our method is effective for predicting and clustering true BCEs.

sentences: Regulating bacterial behavior within hydrogels of tunable viscoelasticity Engineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in materials composition and function. Understanding how the spatial confinement in 3D affects the behavior of the embedded cells is crucial to design and predict ELMs function, regulate and minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and decreasing plasticity of the matrix, a decrease in colony volumes and an increase in their sphericity is observed. Protein production surprisingly follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that, bacterial mechanosensitivity can be used to control and regulate the composition and function of ELMs by thoughtful design of the encapsulating matrix, and by following design criteria with interesting similarities to those developed for 3D culture of mammalian cells.

sentences: The NADPH oxidase NOX2 is a marker of adverse prognosis involved in chemoresistance of Acute Myeloid Leukemias Resistance to chemotherapeutic drugs is a major cause of treatment failure in Acute Myeloid Leukemias (AML). To better characterize the mechanisms of chemoresistance, we first identified genes whose expression is dysregulated in AML cells resistant to daunorubicin (DNR) or cytarabine (Ara-C), the main drugs used for the induction therapy. The genes found activated are mostly linked to immune signaling and inflammation. Among them, we identified a strong up-regulation of the NOX2 NAPDH oxidase subunit genes (CYBB, CYBA, NCF1, NCF2, NCF4 and RAC2). The ensuing increase in NADPH oxidase activity, which is particularly strong in DNR-resistant cells, participates in the acquisition and/or maintenance of resistance to DNR. In addition, analyzing gp91phox (CYBB-encoded Nox2 catalytic sub-unit) expression at the surface of leukemic blasts from 74 patients at diagnosis showed that NOX2 is generally more expressed and active in leukemic cells from the FAB M4/M5 subtypes compared to FAB M0-M2 ones. Using a gene expression-based score we demonstrate that high NOX2 subunit genes expression is a marker of adverse prognosis, independent of the cytogenetic-based risk classification, in AML patients.

sentences: A physical theory of movement in small animals All animal behaviour must ultimately be governed by physical laws. As a basis for understanding the physics of behaviour in a simple system, we here develop an effective theory for the motion of the larval form of the fruitfly Drosophila melanogaster, and compare it against a quantitative analysis of the real animals behaviour. We first define a set of fields which quantify stretching, bending, and twisting along the larvas antero- posterior axis, and then perform a search in the space of possible theories that could govern the long-wavelength physics of these fields, using a simplified approach inspired by the renormalisation group. Guided by symmetry considerations and stability requirements, we arrive at a unique, analytically tractable free-field theory with a minimum of free parameters. Unexpectedly, we are able to explain a wide-spectrum of features of Drosophila larval behaviour by applying equilibrium statistical mechanics: our theory closely predicts the animals postural modes (eigenmaggots), as well as distributions and trajectories in the postural mode space across several behaviours, including peristaltic crawling, rolling, self-righting and unbiased substrate exploration. We explain the low-dimensionality of postural dynamics via Boltzmann suppression of high frequency modes, and also propose and experimentally test, novel predictions on the relationships between different forms of body deformation and animal behaviour. We show that crawling and rolling are dominated by similar symmetry properties, leading to identical dynamics/statistics in mode space, while rolling and unbiased exploration have a common dominant timescale. Furthermore, we are able to demonstrate that self-righting behaviour occurs continuously throughout substrate exploration, owing to the decoupling of stretching, bending, and twisting at low energies. Together, our results demonstrate that relatively simple effective physics can be used to explain and predict a wide range of animal behaviours.

sentences: Generalizability in White Blood Cells' Classification Problem Counting and classifying white blood cells (WBCs) in blood samples helps the early diagnosis of the disease. Many works have been done to develop machine learning-based methods to count WBCs. However, most of these works have low generalizability, and their accuracy decreases sharply as the dataset changes. In this paper, a new method is presented that helps to increase the generalization power. In this method, first, the WBCs nucleus is segmented, and then its convex hull is obtained. By subtracting the nucleus from the convex hull, a new image is created called the representative of the convex hull (ROC). Then, by Training a convolutional neural network (CNN) with the cells RGB image as well as the binary images of the nucleus and ROC, the generalization power is increased. The proposed method was first trained on the Raabin-WBC dataset, then its performance was evaluated on the LISC dataset without retraining. The proposed methods accuracy on the Raabin-WBC and LISC datasets is 93.97% and 51.57 %, respectively. Besides, the generalization power of four well-known CNNs named VGG16, ResNext50, MobileNet-V2, and MnasNet1 was investigated. It was found that VGG16 has more generalization power among these models.

sentences: Combined Pan-, Population-, and Phylo-Genomic Analysis of Aspergillus fumigatus Reveals Population Structure and Lineage-Specific Diversity Aspergillus fumigatus is a deadly agent of human fungal disease, where virulence heterogeneity is thought to be at least partially structured by genetic variation between strains. While population genomic analyses based on reference genome alignments offer valuable insights into how gene variants are distributed across populations, these approaches fail to capture intraspecific variation in genes absent from the reference genome. Pan-genomic analyses based on de novo assemblies offer a promising alternative to reference-based genomics, with the potential to address the full genetic repertoire of a species. Here, we use a combination of population genomics, phylogenomics, and pan-genomics to assess population structure and recombination frequency, phylogenetically structured gene presence-absence variation, evidence for metabolic specificity, and the distribution of putative antifungal resistance genes in A. fumigatus. We provide evidence for three primary populations of A. fumigatus, structured by both gene variation (SNPs and indels) and distinct gene presence-absence variation with unique suites of accessory genes present exclusively in each clade. Accessory genes displayed functional enrichment for nitrogen and carbohydrate metabolism, suggesting that populations may be stratified by environmental niche specialization. Similarly, the distribution of antifungal resistance genes and resistance alleles were often structured by phylogeny. A. fumigatus demonstrated exceptionally high levels of recombination and one of the largest fungal pan-genomes reported to date including many genes unrepresented in the Af293 reference genome. These results highlight the inadequacy of relying on a single-reference genome-based approach for evaluating intraspecific variation, and the power of combined genomic approaches to elucidate population structure, genetic diversity, and putative ecological drivers of clinically relevant fungi.

sentences: Face processing of social cognition in patients with first episode psychosis: Its deficits and association with the right subcallosal anterior cingulate cortex The clinical importance of social cognition is well acknowledged in patients with psychosis, in particular those with first episode psychosis (FEP). Nevertheless, its brain substrates and circuitries remain elusive, lacking precise analysis between multimodal brain characteristics and behavioral sub-dimensions within social cognition. In the present study, we examined face processing of social cognition in 79 FEP patients and 80 healthy controls (HCs). We looked for a possible correlation between face processing and multimodal MRI characteristics such as resting-state functional connectivity (rsFC) and brain volume. We observed worse recognition accuracy, longer recognition response time, and longer memory response time in FEP patients when compared with HCs. Of these, memory response time was selectively correlated with specific rsFCs, which included the right subcallosal sub-region of BA24 in the ACC (scACC), only in FEP patients. The volume of this region was also correlated with memory response time in FEP patients. The scACC is functionally and structurally important in FEP-associated abnormalities of face processing measures in social cognition.

sentences: Boundary convection during velocity sedimentation in the Optima analytical ultracentrifuge Analytical ultracentrifugation (AUC) provides the most widely applicable, precise and accurate means for characterizing solution hydrodynamic and thermodynamic properties. In recent times AUC has found broad application in the biopharmaceutical industry as a first-principle means for quantitatively characterizing biopharmaceuticals. Boundary sedimentation velocity AUC (SV-AUC) analysis is widely used to assess protein aggregation, fragmentation and conformational variants in the same solvents used during drug development and production. SV-AUC is especially useful for the analysis of drug substance, drug product and dosing solution, where other techniques may exhibit solvent matrix issues or concentration limitations. Recently, the only manufacturer of the analytical ultracentrifuge, released its newest (third generation) analytical ultracentrifuge, the Optima, in early 2017 to replace its aging 2nd generation XL series ultracentrifuges. However, SV-AUC data from four Optima units used in conducting characterization work on adeno-associated virus (AAV) has shown evidence of sample convection. Further investigation reveals that this problem arises from the temperature control system design, which is prone to producing destabilizing temperature induced density gradients that can lead to density inversions. The observed convection impacts both the qualitative and quantitative data generated by the Optima. The problem is intermittent and variable in severity within a given Optima unit and between Optima units. This convection appears to be mainly associated with low rotor speeds and dilute samples in dilute solvents, such as AAV samples in formulation buffers containing relatively low concentrations of salts, sugars, etc. Under these conditions it is found that a sufficiently robust stabilizing density gradient is not always present during sedimentation, making the sample susceptible to convection by localized density inversions. Because SV-AUC is used as an analytical tool in making critical decisions in the development and quality control of biotherapeutics, it is imperative to alert users about this potential problem. In general special attention to data quality needs to be made by those researchers working with very large biopharmaceutical particles (e.g. gene therapy products that involve viral vectors or nanoparticles), where the conditions leading to convection are most likely to occur. It is important to note that the XL series analytical ultracentrifuges do not suffer from this problem, indicating that this problem is unique to the Optima. Attributes that reveal the presence of this problem and strategies for its elimination or minimization are provided.

sentences: Uncovering the Genetic Profiles Underlying the Intrinsic Organization of the Human Cerebellum The functional diversity of the human cerebellum is largely believed to be derived more from its extensive connections rather than being limited to its mostly invariant architecture. However, whether and how the determination of cerebellar connections in its intrinsic organization interact with microscale gene expression is still unknown. Here we decode the genetic profiles of the cerebellar functional organization by investigating the genetic substrates simultaneously linking cerebellar functional heterogeneity and its drivers, i.e., the connections. We not only identified 443 network-specific genes but also discovered that their co-expression pattern correlated strongly with intra-cerebellar functional connectivity (FC). Ninety of these genes were also linked to the FC of cortico-cerebellar cognitive-limbic networks. To further discover the biological functions of these genes, we performed a "virtual gene knock-out" by observing the change in the coupling between gene co-expression and FC and divided the genes into two subsets, i.e., a positive gene contribution indicator (GCI+) involved in cerebellar neurodevelopment and a negative gene set (GCI-) related to neurotransmission. A more interesting finding is that GCI- is significantly linked with the cerebellar connectivity-behavior association and many recognized brain diseases that are closely linked with the cerebellar functional abnormalities. Our results could collectively help to rethink the genetic substrates underlying the cerebellar functional organization and offer possible micro-macro interacted mechanistic interpretations of the cerebellum-involved high order functions and dysfunctions in neuropsychiatric disorders.

sentences: Complex response of beta diversity to dispersal in meta-community models Dispersal is one of the most important drivers of community assembly. The conventional belief that dispersal leads to biotic homogenization (lower beta diversity) has been recently challenged by an experiment conducted in nectar microbes (Vannette & Fukami, 2017), showing that dispersal could lead to community divergence. In this paper, I re-examined the relationship between beta diversity and local dispersal in a range of theoretical models: from the classic island biogeography model and meta-population model to a meta-community model that incorporates biotic interactions. I find that the emergence of hump-shaped beta diversity-dispersal relationship is closely related to local dispersal (rather than global dispersal), non-neutrality and biotic interactions. The results reveal rich metacommunity dynamics in relation to dispersal types and biotic interactions which might be overlooked in previous theoretical and empirical studies. The findings call for more realistic experimental manipulations on dispersals in future community assembly studies.

sentences: Functional single cell selection and annotated profiling of dynamically changing cancer cells A method connecting single cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, would be transformative for single cell and cancer biology. Here, we present fSCS: functional single cell selection. This technology combines a custom-built ultrawide field-of-view optical screening microscope, fast automated image analysis and a new photolabeling method, phototagging, using a newly synthesized visible-light-photoactivatable dye. Using fSCS, we screen, selectively photolabel and isolate cells of interest from large heterogeneous populations based on functional dynamics like fast migration, morphological variation, small molecule uptake or cell division. We combined fSCS with single cell RNA sequencing for functionally annotated transcriptomic profiling of fast migrating and spindle-shaped MCF10A cells with or without TGF{beta} induction. We identified critical genes and pathways driving aggressive migration as well as mesenchymal-like morphology that could not be detected with state-of-the-art single cell transcriptomic analysis. fSCS provides a crucial upstream selection paradigm for single cell sequencing independent of biomarkers, allows enrichment of rare cells and can facilitate the identification and understanding of molecular mechanisms underlying functional phenotypes.

sentences: Confusions regarding stochastic fluctuations and accumulators in spontaneous movements Experiments on choice-predictive brain signals have played an important role in the debate on free will. In a seminal study, Benjamin Libet and colleagues found that a negative-going EEG signal, the readiness potential (RP), can be observed over motor-related brain regions already a few hundred ms before a participant makes a conscious decision to move. If the onset of the readiness potential is taken as an indicator of the "brains decision to move" this could mean that this decision to move is made early, by unconscious brain activity, rather than later, at the time when the subject believes to be deciding. However, an alternative interpretation has recently been discussed, the stochastic decision model (SDM), that takes its inspiration from models of perceptual decision making. It suggests that the RP originates from an accumulation of stochastic internal fluctuations. In this view the decision happens only at a much later stage when an accumulated noise signal reaches a threshold. Here we address a number of confusions regarding both the evidence for the stochastic decision model as well as its interpretation. We will show: (a) that the evidence for the role of stochastic fluctuations is highly indirect; (b) that there is little direct support for the SDM from animal studies; (c) that deterministic (non-random) processes can explain the data in a similar way; (d) that the relative components of the model have been mischaracterized leading to an over-emphasis on the role of random fluctuations and an under-emphasis of deterministic aspects of the model; (e) that there is confusion regarding the role of "urgency" and "evidence" in the SDM and its link to perceptual decision making; (f) that the question whether the decision happens early or late depends on the nature of the noise fluctuations, specifically, whether they reflect "absolute" or "epistemic" randomness; (g) finally, that the model does not explain the temporal relationship between conscious decision and neural decision. Our aim is not to rehabilitate the role of RPs in the free will debate. Rather we aim to address some confusions and premature conclusions regarding the evidence for accumulators playing a role in these preparatory brain processes.

sentences: VeloViz: RNA-velocity informed embeddings for visualizing cellular trajectories 0Single cell transcriptomic technologies enable genome-wide gene expression measurements in individual cells but can only provide a static snapshot of cell states. RNA velocity analysis can infer cell state changes from single cell transcriptomics data. To interpret these cell state changes as part of underlying cellular trajectories, current approaches rely on visualization with principal components, t-distributed stochastic neighbor embedding, and other 2D embeddings derived from the observed single cell transcriptional states. However, these 2D embeddings can yield different representations of the underlying cellular trajectories, hindering the interpretation of cell state changes. We developed VeloViz to create RNA-velocity-informed 2D and 3D embeddings from single cell transcriptomics data. Using both real and simulated data, we demonstrate that VeloViz embeddings are able to consistently capture underlying cellular trajectories across diverse trajectory topologies, even when intermediate cell states may be missing. By taking into consideration the predicted future transcriptional states from RNA velocity analysis, VeloViz can help visualize a more reliable representation of underlying cellular trajectories. VeloViz is available as an R package on GitHub (https://github.com/JEFworks-Lab/veloviz) with additional tutorials at https://JEF.works/veloviz/.

sentences: Novel Murine models of Mania and Depression Neuropathological mechanisms triggering manic syndrome or manic episodes in bipolar disorder remain poorly characterised, as the research progress is severely limited by the paucity of appropriate animal models. Here we developed a novel manic mice model by combining a series of chronic unpredictable rhythm disturbances (CURD), which include disruption of circadian rhythm, sleep deprivation, exposure to cone light, with subsequent interference of followed spotlight, stroboscopic illumination, high temperature stress, noise disturbance and foot shock. To validate this novel manic model, we used multiple behavioural and cell biology approaches comparing the CURD-model with healthy controls and depressed mice. The depression model was created by an exposure to an improved chronic unpredictable mild stress, which we defined as chronic unpredictable mild restraint (CUMR). A novel manic mice model induced by environmental stressors and free from genetic or pharmacological interventions will benefit research into pathological mechanisms of mania.

sentences: Genetic isolation by distance underlies color pattern divergence in red-eyed treefrogs (Agalychnis callidryas) Investigating the spatial distribution of genetic and phenotypic variation can provide insights into the evolutionary processes that shape diversity in natural systems. We characterized patterns of genetic and phenotypic diversity to learn about drivers of color-pattern diversification in red-eyed treefrogs (Agalychnis callidryas) in Costa Rica. Along the Pacific coast, red-eyed treefrogs have conspicuous leg color patterning that transitions from orange in the north to purple in the south. We measured phenotypic variation of frogs, with increased sampling at sites where the orange-to-purple transition occurs. At the transition zone, we discovered the co-occurrence of multiple color-pattern morphs. To explore possible causes of this variation, we generated a SNP dataset to analyze population genetic structure, measure genetic diversity, and infer the processes that mediate genotype-phenotype dynamics. We investigated how patterns of genetic relatedness correspond with individual measures of color pattern along the coast, including testing for the role of hybridization in geographic regions where orange and purple phenotypic groups co-occur. We found no evidence that color-pattern polymorphism in the transition zone arose through recent hybridization. Instead, a strong pattern of genetic isolation by distance (IBD) indicates that color-pattern variation was either retained through other processes such as ancestral color polymorphisms or ancient secondary contact, or else it was generated by novel mutations. We found that phenotype changes along the Pacific coast more than would be expected based on genetic divergence and geographic distance alone. Combined, our results suggest the possibility of selective pressures acting on color pattern at a small geographic scale.

sentences: Genetic dissection of glutamatergic neuron subpopulations and developmental trajectories in the cerebral cortex Diverse types of glutamatergic pyramidal neurons (PyNs) mediate the myriad processing streams and output channels of the cerebral cortex, yet all derive from neural progenitors of the embryonic dorsal telencephalon. Here, we establish genetic strategies and tools for dissecting and fate mapping PyN subpopulations based on their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target the temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable combinatorial targeting of major progenitor types and projection classes. Intersectional converter lines confer viral access to specific subsets defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of PyN subpopulations that assemble cortical processing networks and output channels.

sentences: The Role of Notch Signaling in Endometrial Mesenchymal Stromal/Stem-like Cells Maintenance Human endometrium undergoes cycles of regeneration in reproductive women. The endometrial mesenchymal stromal/stem cells (eMSC) contribute to this process. Notch signaling is essential for the homeostasis of somatic stem cells. However, its role in eMSC remains unclear. The gain and loss function shows that activation of Notch signaling promotes eMSC maintenance, while inhibition displays opposite effect. Activation of Notch pathway better maintains eMSC in a quiescent state. However, these quiescent eMSC can re-enter into the cell cycle depending on Notch and Wnt activity in the microenvironment, suggesting a crosstalk between two signaling pathways. In a mouse menstrual-like model, we observe that Notch signaling is highly involved in the dynamic endometrial remodeling event. Suppression of Notch signaling significantly reduces the proliferation of Notch1+ label-retaining stromal cells and consequently delays the endometrial repair. Our data demonstrate the importance of Notch signaling in regulating the endometrial stem/progenitor cells in vitro and in vivo.

sentences: Oxidized thioredoxin-1 restrains the NLRP1 inflammasome At least six human proteins detect danger-associated signals, assemble into complexes called inflammasomes, and trigger pyroptotic cell death. NLRP1 was the first protein discovered to form an inflammasome, but the danger signals and molecular mechanisms that control its activation have not yet been fully established. Here, we report that the NACHT-LRR region of NLRP1 directly binds to oxidized form of thioredoxin-1 (TRX1). We found that NLRP1 requires the ATPase activity of its NACHT domain to associate with TRX1, and that this interaction represses inflammasome activation. Moreover, we discovered that several patient-derived missense mutations in the NACHT-LRR region of NLRP1 weaken TRX1 binding, leading to inflammasome hyperactivation and autoinflammatory disease. Overall, our results establish that oxidized TRX1 binds to and restrains the NLRP1 inflammasome, thereby revealing a link between the cellular redox environment and innate immunity.

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Lytic infection with murine gammaherpesvirus 68 activates host and viral RNA polymerase III-dependent promoters to enhance non-coding RNA expression RNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including the gammaherpesviruses ({gamma}HVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during {gamma}HV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here, we sought to investigate how pol III promoters and transcripts are regulated during gammaherpesvirus infection using the murine gammaherpesvirus 68 ({gamma}HV68) system. To compare the transcription of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III promoters for host and viral ncRNAs using a luciferase reporter optimized to measure pol III activity. We measured promoter activity from the reporter gene at the translation level via luciferase activity and at the transcription level via RT-qPCR. We further measured endogenous ncRNA expression at single cell-resolution by flow cytometry. These studies demonstrated that lytic infection with {gamma}HV68 increased the transcription from multiple host and viral pol III promoters, and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. RNA flow cytometry revealed the induction of endogenous pol III-derived ncRNAs that tightly correlated with viral gene expression. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived RNAs, a process that may further modify cellular function and enhance viral gene expression and pathogenesis.

IMPORTANCEGammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small non-coding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III dependent transcription are complicated by multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we characterized a reporter system to directly study RNA polymerase III-dependent responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral non-coding RNA expression within the infected cell.

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When killers become thieves: trogocytosed PD-1 inhibits NK cells in cancer Leucocytes often perform trogocytosis, the process by which cells acquire parts of the plasma membrane from interacting cells. Accumulating evidence indicates that trogocytosis modulates immune responses, but the underlying molecular mechanisms are unclear. Here, using two mouse models of leukemia, we found that cytotoxic lymphocytes perform trogocytosis at high rates with tumor cells. While performing trogocytosis, both Natural Killer and CD8+ T cells acquire the checkpoint receptor PD-1 from leukemia cells. In vitro and in vivo investigation revealed that PD-1 protein found on the surface of Natural Killer cells, rather than being endogenously expressed, was derived entirely from leukemia cells. Mechanistically, SLAM receptors were essential for PD-1 trogocytosis. PD-1 acquired via trogocytosis actively suppressed anti-tumor immunity, as revealed by the positive outcome of PD-1 blockade in PD-1-deficient mice. PD-1 trogocytosis was corroborated in patients with clonal plasma cell disorders, where Natural Killer cells that stained for PD-1 also stained for tumor cell markers. Our results, in addition to shedding light on a previously unappreciated mechanism underlying the presence of PD-1 on Natural Killer and cytotoxic T cells, reveal the immune-regulatory effect of membrane transfer occurring when immune cells contact tumor cells.

Once sentence summaryNatural Killer cells are inhibited by PD-1 acquired from the surface of tumor cells via trogocytosis.

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Spatially distributed computation in cortical circuits The traditional view of neural computation in the cerebral cortex holds that sensory neurons are specialized, i.e., selective for certain dimensions of sensory stimuli. This view was challenged by evidence of contextual interactions between stimulus dimensions in which a neurons response to one dimension strongly depends on other dimensions. Here we use methods of mathematical modeling, psychophysics, and electrophysiology to address shortcomings of the traditional view. Using a model of a generic cortical circuit, we begin with the simple demonstration that cortical responses are always distributed among neurons, forming characteristic waveforms, which we call neural waves. When stimulated by patterned stimuli, circuit responses arise by interference of neural waves. Resulting patterns of interference depend on interaction between stimulus dimensions. Comparison of these modeled responses with responses of biological vision makes it clear that the framework of neural wave interference provides a useful alternative to the standard concept of neural computation.

TeaserInvestigating interference of neural waves helps to overcome limitations of the traditional view of cortical computation.

sentences: The transcription factor network of E. coli steers global responses to shifts in RNAP concentration The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the genes numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.

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A Deep Learning Framework for Prediction of Clinical Drug Response of Cancer Patients and Identification of Drug Sensitivity Biomarkers using Preclinical Samples BackgroundPrediction of the response of cancer patients to different treatments and identification of biomarkers of drug sensitivity are two major goals of individualized medicine. In this study, we developed a deep learning framework called TINDL, completely trained on preclinical cancer cell lines, to predict the response of cancer patients to different treatments. TINDL utilizes a tissue-informed normalization to account for the tissue and cancer type of the tumours and to reduce the statistical discrepancies between cell lines and patient tumours. In addition, this model identifies a small set of genes whose mRNA expression are predictive of drug response in the trained model, enabling identification of biomarkers of drug sensitivity.

ResultsUsing data from two large databases of cancer cell lines and cancer tumours, we showed that this model can distinguish between sensitive and resistant tumours for 10 (out of 14) drugs, outperforming various other machine learning models. In addition, our siRNA knockdown experiments on 10 genes identified by this model for one of the drugs (tamoxifen) confirmed that all of these genes significantly influence the drug sensitivity of the MCF7 cell line to this drug. In addition, genes implicated for multiple drugs pointed to shared mechanism of action among drugs and suggested several important signaling pathways.

ConclusionsIn summary, this study provides a powerful deep learning framework for prediction of drug response and for identification of biomarkers of drug sensitivity in cancer.

sentences: High-Plex Multiomic Analysis in FFPE Tissue at Single-Cellular and Subcellular Resolution by Spatial Molecular Imaging The Spatial Molecular Imaging platform (CosMxTM SMI, NanoString, Seattle, WA) utilizes a high-plex in-situ imaging chemistry for both RNA and protein detection. This automated instrument provides 1000s of plex, at high sensitivity (1 to 2 copies/cell), very low error rate (0.0093 false calls/cell), and background ([~]0.04 counts/cell). The imaging system generates three-dimensional super-resolution localization of analytes (< 50 nm in the X-Y plane), at [~] 1 million cells per sample, four samples per run. Cell segmentation is antibody-based, compatible with FFPE samples. Multiomic data (980 RNAs, 108 proteins) were measured at subcellular resolution using FFPE tissues (non-small cell lung (NSCLC) and breast cancer) and allowed identification of over 18 distinct cell types, 10 unique tumor microenvironments, and 100 pairwise ligand-receptor interactions. Over 800,000 single cells and [~]260 million transcripts data are released into the public domain allowing extended data analysis by the entire spatial biology research community.

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SALARECON connects the Atlantic salmon genome to growth and feed efficiency Atlantic salmon (Salmo salar) is the most valuable farmed fish globally and there is much interest in optimizing its genetics and rearing conditions for growth and feed efficiency. Marine feed ingredients must be replaced to meet global demand, with challenges for fish health and sustainability. Metabolic models can address this by connecting genomes to metabolism, which converts nutrients in the feed to energy and biomass, but such models are currently not available for major aquaculture species such as salmon. We present SALARECON, a model focusing on energy, amino acid, and nucleotide metabolism that links the Atlantic salmon genome to metabolic fluxes and growth. It performs well in standardized tests and captures expected metabolic (in)capabilities. We show that it can explain observed hypoxic growth in terms of metabolic fluxes and apply it to aquaculture by simulating growth with commercial feed ingredients. Predicted limiting amino acids and feed efficiencies agree with data, and the model suggests that marine feed efficiency can be achieved by supplementing a few amino acids to plant- and insect-based feeds. SALARECON is a high-quality model that makes it possible to simulate Atlantic salmon metabolism and growth. It can be used to explain Atlantic salmon physiology and address key challenges in aquaculture such as development of sustainable feeds.

Author summaryAtlantic salmon aquaculture generates billions of euros annually, but faces challenges of sustainability. Salmon are carnivores by nature, and fish oil and fish meal have become scarce resources in fish feed production. Novel, sustainable feedstuffs are being trialed hand in hand with studies of the genetics of growth and feed efficiency. This calls for a mathematical-biological framework to integrate data with understanding of the effects of novel feeds on salmon physiology and its interplay with genetics. We have developed the SALARECON model of the core salmon metabolic reaction network, linking its genome to metabolic fluxes and growth. Computational analyses show good agreement with observed growth, amino acid limitations, and feed efficiencies, illustrating the potential for in silico studies of potential feed mixtures. In particular, in silico screening of possible diets will enable more efficient animal experiments with improved knowledge gain. We have adopted best practices for test-driven development, virtual experiments to assay metabolic capabilities, revision control, and FAIR data and model management. This facilitates fast, collaborative, reliable development of the model for future applications in sustainable production biology.

sentences: Reversible and gain modulation of neuronal responses and sensorimotor behavior by mid-infrared stimulation Neuromodulation serves as a cornerstone for brain sciences and clinical applications. Mid-infrared stimulation (MIRS) has been recently reported to cause non-thermal modulation of brain functions. However little knowledge of mechanisms hampers its application. Here we bridge across ion channels, neuronal signals, and behavioral performances associated with sensorimotor transformation to provide evidence of how the alternation of neuronal activity by MIRS guides the change of behavioral performance in awake-behaving pigeons. We compared effects on visually-guided eye movements by applying MIRS and electrical stimulation (ES) in the pretectal nucleus lentiformis mesencephali (nLM). Distinct from ES, we found a specific gain modulation of MIRS to alter behavior in a manner of the strength of visual inputs. Our simultaneous extracellular recordings showed that MIRS can excite and inhibit the neuronal activity in the same pretectal neuron based on its ongoing sensory responsiveness levels in awake-behaving animals. We further applied computational simulations and found that MIRS can modulate the carbonyl group (-C=O) enriched on the potassium channel to resonate, and could affect action potential generation, alter neuronal responses to sensory inputs and then guide behavior. Our findings suggest that MIRS could be a promising approach for brain researches and neurological diseases, with gene free manipulation.

sentences: Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II Substoichiometric binding of certain actin-binding proteins induces conformational changes in a disproportionally large number of actin protomers in actin filaments. Here, we report a case in which such conformational changes in actin filaments have profound functional consequences. Rng2 is an IQGAP protein implicated in the assembly and contraction of contractile rings in Schizosaccharomyces pombe. We found that the calponin-homology actin-binding domain of Rng2 (Rng2CHD) strongly inhibits the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II-coated surfaces, Rng2CHD halved the sliding speed of actin filaments at a binding ratio of 1.3% (=1/77), and virtually stopped movement at a binding ratio of 11% (=1/9). Rng2CHD also inhibited actin movements on Dictyostelium myosin II, but in this case by inducing the detachment of actin filaments from myosin II-coated surfaces. Rng2CHD induced cooperative structural changes of actin filaments accompanied by shortening of the filament helical pitch, and reduced the affinity between actin filaments and subfragment 1 (S1) of muscle myosin II in the presence of ADP. Intriguingly, actin-activated ATPase of S1 was hardly inhibited by Rng2CHD. We suggest that sparsely bound Rng2CHD induces global structural changes of actin filaments and interferes with the force generation by actin-myosin II.

sentences: A covariation analysis reveals elements of selectivity in quorum sensing systems Many bacteria communicate with kin and coordinate group behaviors through a form of cell-cell signaling called acyl-homoserine lactone (AHL) quorum sensing (QS). In these systems, a signal synthase produces an AHL to which its paired receptor selectively responds. Selectivity is fundamental to cell signaling. Despite its importance, it has been challenging to determine how this selectivity is achieved and how AHL QS systems evolve and diversify. We hypothesized that we could use covariation within the protein sequences of AHL synthases and receptors to identify selectivity residues. We began by identifying about 6,000 unique synthase-receptor pairs. We then used the protein sequences of these pairs to identify covariation patterns and mapped the patterns onto the LasI/R system from Pseudomonas aeruginosa PAO1. The covarying residues in both proteins cluster around the ligand binding sites. We demonstrate that these residues are involved in system selectivity toward the cognate signal and go on to engineer the Las system to both produce and respond to an alternate AHL signal. We have thus demonstrated a new application for covariation methods and have deepened our understanding of how communication systems evolve and diversify.

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Utility of SARS-CoV-2 infection models using in vitro and in vivo ACE2-lentivirus transduction SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intranasal hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lungs. RNA-Seq analyses illustrated that the model involves an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. Intranasal hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mice lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

AUTHOR SUMMARYSARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research.

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BRPF3 knockdown or inhibition moderately reverses olaparib resistance in high grade serous ovarian carcinoma, but depletion of H3K14 acetylation has no effect BackgroundPARP inhibitors (PARPi) kill cancer cells by stalling DNA replication and preventing DNA repair, resulting in a critical accumulation of DNA damage. Resistance to PARPi is a growing clinical problem in the treatment of high grade serous ovarian carcinoma (HGSOC). Acetylation of histone H3 lysine 14 (H3K14ac) and associated histone acetyltransferases (HATs) have known functions in DNA repair and replication, but their expression and activities have not been examined in the context of PARPi-resistant HGSOC.

ResultsUsing mass spectrometry profiling of histone modifications, we observed altered H3K14ac enrichment in PARPi-resistant HGSOC cells relative to isogenic PARPi-sensitive lines. By RT-qPCR and RNA-Seq, we also observed altered expression of numerous HATs in PARPi-resistant HGSOC cells and a PARPi-resistant PDX model. Knockdown of HATs only modestly altered PARPi response, although knockdown and inhibition of PCAF significantly increased resistance. Pharmacologic inhibition of HBO1 severely depleted H3K14ac but did not affect PARPi response. However, knockdown and inhibition of BRPF3, which is known to interact in a complex with HBO1, did reduce PARPi resistance.

ConclusionsThis study demonstrates that severe depletion of H3K14ac does not affect PARPi response in HGSOC. Our data suggest that bromodomain functions of HAT proteins such as PCAF, or accessory proteins such as BRPF3, may play a greater role in PARPi response than acetyltransferase functions.

sentences: A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

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Imputation of Low-density Marker Chip Data in Plant Breeding: Evaluation of Methods Based on Sugar Beet Low-density genotyping followed by imputation reduces genotyping costs while still providing high-density marker information. An increased marker density has the potential to improve the outcome of all applications that are based on genomic data. This study investigates techniques for 1k to 20k genomic marker imputation for plant breeding programs with sugar beet as an example crop, where these are realistic marker numbers for modern breeding applications.

The generally accepted  gold standard for imputation, Beagle 5.1, was compared to the recently developed software AlphaPlantImpute2 which is designed specifically for plant breeding. For Beagle 5.1 and AlphaPlantImpute2, the imputation strategy as well as the imputation parameters were optimized in this study. We found that the imputation accuracy of Beagle could be tremendously improved (0.22 to 0.67) by tuning parameters, mainly by lowering the values for the parameter for the effective population size and increasing the number of iterations performed. Separating the phasing and imputation steps also improved accuracies when optimized parameters were used (0.67 to 0.82). We also found that the imputation accuracy of Beagle decreased when more low-density lines were included for imputation. AlphaPlantImpute2 produced very high accuracies without optimization (0.89) and was generally less responsive to optimization. Overall, AlphaPlantImpute2 performed relatively better for imputation while Beagle was better for phasing. Combining both tools yielded the highest accuracies.

SummaryGenotype marker information allows the prediction of an individuals breeding value without the need to observe its actual phenotype which can accelerate the breeding progress. The more markers are genotyped, the better the genomic prediction may be. However, analyzing many markers is costly, particularly in commercial breeding programs where thousands of new individuals are genotyped. A solution to obtain information for all markers, while spending comparatively little on genotyping, is to genotype only a small fraction of markers in most individuals. Together with high-density information on other individuals, the low-density individuals can be imputed to high-density. High-density individuals are typically parents or highly influential individuals.

In this study, we compare the widely used software Beagle with the recently developed software AlphaPlantImpute2 on plant breeding data. To allow a fair comparison, we first optimized existing methods and developed new approaches. This was done to avoid comparing results of a less ideal version of one software to optimized settings of another software. After optimization, the software were evaluated in different scenarios with regards to genotyping errors, population types and number of markers based on simulated data. Simulated data were based on real marker data from a sugar beet population as input to mimic the population history of a commercial breeding population.

AlphaPlantImpute2 performs well with default parameters, while much optimization with regards to parameters and strategy was needed to boost accuracies of Beagle. A pipeline is presented which uses Beagle for phasing and AlphaPlantImpute2 for imputation. This pipeline yielded the highest accuracies and shortest run time.

Core IdeasBeagle is sensitive to parameter tuning

Best imputation accuracies could be achieved by using a combination of Beagle and AlphaPlantImpute2

The population structure influence imputation accuracy

sentences: HOPX governs a molecular and physiological switch between cardiomyocyte progenitor and maturation gene programs This study establishes the homeodomain only protein, HOPX, as a determinant controlling the molecular switch between cardiomyocyte progenitor and maturation gene programs. Time-course single-cell gene expression with genome-wide footprinting reveal that HOPX interacts with and controls core cardiac networks by regulating the activity of mutually exclusive developmental gene programs. Upstream hypertrophy and proliferation pathways compete to regulate HOPX transcription. Mitogenic signals override hypertrophic growth signals to suppress HOPX and maintain cardiomyocyte progenitor gene programs. Physiological studies show HOPX directly governs genetic control of cardiomyocyte cell stress responses, electro-mechanical coupling, proliferation, and contractility. We use human genome-wide association studies (GWAS) to show that genetic variation in the HOPX-regulome is significantly associated with complex traits affecting cardiac structure and function. Collectively, this study provides a mechanistic link situating HOPX between competing upstream pathways where HOPX acts as a molecular switch controlling gene regulatory programs underpinning metabolic, signaling, and functional maturation of cardiomyocytes.

sentences: Blind cavefish retain functional connectivity in the tectum despite loss of retinal input Sensory systems display remarkable plasticity and are under strong evolutionary selection. The Mexican cavefish, Astyanax mexicanus, consists of eyed river-dwelling surface populations, and multiple independent cave populations which have converged on eye loss, providing the opportunity to examine the evolution of sensory circuits in response to environmental perturbation. Functional analysis across multiple transgenic populations expressing GCaMP6s showed that functional connectivity of the optic tectum largely did not differ between populations, except for the selective loss of negatively correlated activity within the cavefish tectum, suggesting positively correlated neural activity is resistant to an evolved loss of input from the retina. Further, analysis of surface-cave hybrid fish reveals that changes in the tectum are genetically distinct from those encoding eye-loss. Together, these findings uncover the independent evolution of multiple components of the visual system and establish the use of functional imaging in A. mexicanus to study neural circuit evolution.

sentences: The effect of different exercise training modes on dentate gyrus neurodegeneration and synaptic plasticity in morphine-dependent rats Various impacts of exercise on brain performance have been documented following morphine dependence induction; however, the underlying neuronal mechanisms remain unclear. The present research was done to investigate the impact of different exercise training modes on neuronal maturation, and synaptic plasticity in the perforant pathway (PP)-dentate gyrus (DG) synapse in the morphine-dependent rats. Five groups, including a control group (Con, ten healthy rats) and forty morphine-dependent rats were considered as follows (n=10/group): 1) sedentary-dependent (Sed-D); 2) endurance exercise-dependent (En-D); 3) strength exercise-dependent (St-D); and 4) concurrent exercise-dependent (Co-D). The exercise training groups were subjected to endurance, strength, and concurrent training 5 days per week for 10 weeks. After training sessions, the field excitatory postsynaptic potential (fEPSP) slope and population spike (PS) amplitude in DG were determined in response to high-frequency stimulation (HFS) of the PP. For assessing neurogenesis NeuroD level was evaluated after performing all experiments. Concurrent training increased PS amplitude and EPSP than the control group. NeuroD in the morphine-dependent rats significantly decreased, but concurrent training returned the NeuroD to the healthy rat level. Concurrent training can ameliorate synaptic plasticity impairment in morphine-dependent rats through neurogenesis promotion. According to the results, concurrent training can be an appropriate novel candidate for treating opioid addiction.

sentences: Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells Many organisms can survive extreme conditions and successfully recover to normal life. This extremotolerant behavior has been attributed in part to repetitive, amphipathic, and intrinsically disordered proteins that are upregulated in the protected state. Here, we assemble a library of approximately 300 naturally-occurring and designed extremotolerance-associated proteins to assess their ability to protect human cells from chemically-induced apoptosis. We show that several proteins from tardigrades, nematodes, and the Chinese giant salamander are apoptosis protective. Notably, we identify a region of the human ApoE protein with similarity to extremotolerance-associated proteins that also protects against apoptosis. This region mirrors the phase separation behavior seen with such proteins, like the tardigrade protein CAHS2. Moreover, we identify a synthetic protein, DHR81, that shares this combination of elevated phase separation propensity and apoptosis protection. Finally, we demonstrate that driving protective proteins into the condensate state increases apoptosis protection, and highlight the ability for DHR81 condensates to sequester caspase-7. Taken together, this work draws a link between extremotolerance-associated proteins, condensate formation, and designing human cellular protection.

sentences: Beeporter: a high-throughput tool for non-invasive analysis of honey bee virus infection Honey bees face numerous pests and pathogens but arguably none are as devastating as Deformed wing virus (DWV). Development of antiviral therapeutics and virus-resistant honey bee lines to control DWV in honey bees is slowed by the lack of a cost-effective high-throughput screening of DWV infection. Currently, analysis of virus infection and screening for antiviral treatments in bees and their colonies is tedious, requiring a well-equipped molecular biology laboratory and the use of hazardous chemicals. Here we utilize a cDNA clone of DWV tagged with green fluorescent protein (GFP) to develop the Beeporter assay, a method for detection and quantification of DWV infection in live honey bees. The assay involves infection of honey bee pupae by injecting a standardized DWV-GFP inoculum, followed by incubation for up to 44 hours. GFP fluorescence is recorded at intervals via commonly available long-wave UV light sources and a smartphone camera or a standard ultraviolet transilluminator gel imaging system. Nonlethal DWV monitoring allows high-throughput screening of antiviral candidates and a direct breeding tool for identifying honey bee parents with increased antivirus resistance. For even more rapid drug screening, we also describe a method for screening bees using 96-well trays and a spectrophotometer.

sentences: {Pi}-{Pi} Interactions Stabilize PeptoMicelle-Based Formulations of Pretomanid Derivatives Leading to Promising Therapy Against Tuberculosis in Zebrafish and Mouse Models Tuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and impede the development of drug resistance are desperately needed. Here, we used polymeric micelles to encapsulate four second-generation derivatives of the antitubercular drug pretomanid that had previously displayed much better in vivo activity against Mycobacterium tuberculosis than pretomanid itself. Because these compounds were relatively hydrophobic, we expected that such micellar formulations would increase drug bioavailability, reduce toxicities, and improve therapeutic outcomes. The polymeric micelles were based on polypept(o)ides (PeptoMicelles) and were stabilized in their hydrophobic core by {pi}-{pi} interactions, allowing the efficient encapsulation of aromatic pretomanid derivatives. The stability of these {pi}-{pi}-stabilized PeptoMicelles was demonstrated in water, blood plasma, and lung surfactant by fluorescence cross-correlation spectroscopy and was further supported by prolonged circulation times of several days in the vasculature of zebrafish larvae. The pretomanid derivative with the best in vitro potency against Mycobacterium marinum ("drug D") was also the most efficacious PeptoMicelle formulation tested in the zebrafish larvae infection model, almost completely eradicating the bacteria at non-toxic doses. This lead formulation was further assessed against Mycobacterium tuberculosis in the susceptible C3HeB/FeJ mouse model, which develops human-like necrotic granulomas. Following intravenous administration, the drug D micellar formulation significantly reduced bacterial burden and inflammatory responses in the lungs and spleens of infected mice.

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Graph Transformer for drug response prediction BackgroundPrevious models have shown that learning drug features from their graph representation is more efficient than learning from their strings or numeric representations. Furthermore, integrating multi-omics data of cell lines increases the performance of drug response prediction. However, these models showed drawbacks in extracting drug features from graph representation and incorporating redundancy information from multi-omics data. This paper proposes a deep learning model, GraTransDRP, to better drug representation and reduce information redundancy. First, the Graph transformer was utilized to extract the drug representation more efficiently. Next, Convolutional neural networks were used to learn the mutation, meth, and transcriptomics features. However, the dimension of transcriptomics features is up to 17737. Therefore, KernelPCA was applied to transcriptomics features to reduce the dimension and transform them into a dense presentation before putting them through the CNN model. Finally, drug and omics features were combined to predict a response value by a fully connected network. Experimental results show that our model outperforms some state-of-the-art methods, including GraphDRP, GraOmicDRP.

Availability of data and materialshttps://github.com/chuducthang77/GraTransDRP.

sentences: Organism-wide mapping of MHC class I and II expression in mouse lemur cells and tissues The major histocompatibility complex (MHC) class I and II glycoproteins have been associated with numerous disease phenotypes, mechanisms and outcomes. These associations can be due to allotypic polymorphism or to altered levels of allotype expression. Although well studied in a range of cell types and microenvironments, no study has encompassed all cell-types present in an individual mouse lemur. In this study the droplet-based single cell RNA sequence data from the mouse lemur cell atlas, Tabula Microcebus, were used to examine the patterns of MHC class I and II expression. The cell atlas comprises data obtained from 27 organs from four mouse lemurs, enabling comparison of expression pattern between both cell types and the individual lemurs. Patterns of gene expression showed a good concordance among the four mouse lemurs. Three primary patterns of expression were identified and associated with different cell-types. Mapping MHC expression onto existing cell trajectories of cell development and spatial gradients revealed fine scale differences in expression level and pattern in single tissues. The bioinformatic pipeline developed here is applicable to other cell atlas projects.

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Daily rhythms in the transcriptomes of the human parasite Schistosoma mansoni BackgroundThe consequences of the earths daily rotation have led to 24-hour biological rhythms in most organisms. Even parasites have daily rhythms, which, when in synchrony with host rhythms, optimize their fitness. Understanding these rhythms may enable development of novel control strategies that take advantage of rhythmic vulnerabilities. Recent work on blood-dwelling protozoan parasites has revealed daily rhythms in gene expression, physiology, drug sensitivity and the presence of an intrinsic circadian clock. However, similar studies on metazoan parasites are lacking. The aims of this study were to investigate if a metazoan parasite has daily molecular oscillations, whether they give us insight into how these longer-lived organisms can survive host daily cycles over a life-span of many years and to determine whether canonical metazoan circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni, that lives in the vasculature for decades and causes the serious neglected tropical disease schistosomiasis.

ResultsUsing round-the-clock transcriptomics of male and female adult worms we discovered that [~]2% of its genes followed a daily pattern of expression. Rhythmic processes included a night-time stress response and a day-time metabolic  rush hour. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include drug targets and a vaccine candidate. These 24hr rhythms may be driven by host rhythms and/or generated by a circadian clock. However, core clock genes are missing and orthologues of secondary clock genes show no 24hr rhythmicity in transcript abundance.

ConclusionsThe daily rhythms identified here reveal temporally compartmentalised internal processes and host interactions over the daily cycle, including processes relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other Metazoa. Most importantly, knowing which gene transcripts oscillate at this temporal scale is relevant to functional genomic studies that will lead to the development and delivery of therapeutics against schistosomiasis.

sentences: Specific phenotypic, genomic, and fitness evolutionary trajectories toward antibiotic resistance induced by pesticide co-stressors in Escherichia coli To explore how co-occurring non-antibiotic environmental stressors affect evolutionary trajectories toward antibiotic resistance, we exposed susceptible Escherichia coli K-12 populations to environmentally relevant levels of pesticides and streptomycin for 500 generations. The coexposure substantially changed the phenotypic, genotypic, and fitness evolution trajectories, resulting in much stronger streptomycin resistance (>15-fold increase) of the populations. Antibiotic target modification mutations in rpsL and rsmG, which emerged and dominated at late stages of evolution, conferred the strong resistance even with less than 1% abundance, while the off-target mutations in nuoG, nuoL, glnE, and yaiW dominated at early stages only led to mild resistance (2.5 - 6-fold increase). Moreover, the strongly resistant mutants exhibited lower fitness costs even without the selective pressure and had lower minimal selection concentrations than the mildly resistant ones. Removal of the selective pressure did not reverse the strong resistance of coexposed populations at a later evolutionary stage. The findings suggest higher risks of the selection and propagation of strong antibiotic resistance in environments potentially impacted by antibiotics and pesticides.

sentences: Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

sentences: Prolonged Pain Reliably Slows Peak Alpha Frequency by Reducing Fast Alpha Power The relationship between the 8-12 Hz "alpha: rhythm, the predominant oscillatory activity of the brain, and pain remains unclear. In healthy individuals, acute, noxious stimuli suppress alpha power while patients with chronic pain demonstrate both enhanced alpha power and slowing of the peak alpha frequency (PAF). To investigate these apparent differences, EEG was recorded from healthy individuals while they completed two models of prolonged pain, Phasic Heat Pain and Capsaicin Heat Pain, at two testing visits occurring roughly 8 weeks apart. We report that PAF is reliably slowed and that alpha power is reliably decreased in response to prolonged pain. Furthermore, we show that alpha power changes, but not PAF changes, are fully reversed with stimulus removal suggesting that PAF slowing reflects pain associated states such as sensitization rather than the presence of ongoing pain. Finally, we provide evidence that changes to alpha power and PAF are due to power decreases in the "fast" (10-12 Hz) range of the alpha rhythm. This frequency dependent pain response aligns with the hypothesis that the alpha rhythm is composed of multiple, independent oscillators, and suggest that modulation of a putative "fast" oscillator may represent a promising therapeutic target for treating ongoing pain. In sum, we provide strong evidence that PAF is reliably slowed during prolonged pain and additionally identify a mechanism, "fast" alpha Power, which is responsible for these PAF changes.

sentences: The physiological basis for the computation of direction selectivity in the Drosophila OFF pathway In Drosophila, direction-selective neurons implement a mechanism of motion computation similar to cortical neurons, using contrast-opponent receptive fields with ON and OFF subunits. It is not clear how the presynaptic circuitry of direction-selective neurons in the OFF pathway supports this computation, because all major inputs are OFF-rectified neurons. Here, we reveal the biological substrate for motion computation in the OFF pathway. Three interneurons, Tm2, Tm9 and CT1, also provide information about ON stimuli to the OFF direction-selective neuron T5 across its receptive field, supporting a contrast-opponent receptive field organization. Consistent with its prominent role in motion detection, variability in Tm9 receptive field properties is passed on to T5, and calcium decrements in Tm9 in response to ON stimuli are maintained across behavioral states, while spatial tuning is sharpened by active behavior. Together, our work shows how a key neuronal computation is implemented by its constituent neuronal circuit elements to ensure direction selectivity.

sentences: Dynamics of sex-biased gene expression over development in the stick insect Timema californicum AO_SCPLOWBSTRACTC_SCPLOWSexually dimorphic phenotypes are thought to arise primarily from sex-biased gene expression during development. Major changes in developmental strategies, such as the shift from hemimetabolous to holometabolous development, are therefore expected to have profound consequences for the dynamics of sex-biased gene expression. However, no studies have previously examined sex-biased gene expression during development in hemimetabolous insects, precluding comparisons between developmental strategies. Here we characterized sex-biased gene expression at three developmental stages in a hemimetabolous stick insect (Timema californicum): hatchlings, juveniles, and adults. As expected, the proportion of sex-biased genes gradually increased during development, mirroring the gradual increase of phenotypic sexual dimorphism. Sex-biased genes identified at early developmental stages were generally consistently male- or female-biased at later stages, suggesting their importance in sexual differentiation. Additionally, we compared the dynamics of sex-biased gene expression during development in T. californicum to those of the holometabolous fly Drosophila melanogaster by reanalyzing publicly available RNA-seq data from third instar larval, pupal and adult stages. In D. melanogaster, 84% of genes were sex-biased at the adult stage (compared to only 20% in T. californicum), and sex-biased gene expression increased abruptly at the adult stage when morphological sexual dimorphism is manifested. Our findings are consistent with the prediction that the dynamics of sex-biased gene expression during development differ extensively between holometabolous and hemimetabolous insect species.

sentences: Genomic characterization of four novel bacteriophages infecting the clinical pathogen Klebsiella pneumoniae Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0-13%) identity to genomic phage sequences deposited in the Genbank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections.

sentences: Conservation and convergence of genetic architecture in the adaptive radiation of Anolis lizards The G matrix, which quantifies the genetic architecture of traits, is often viewed as an evolutionary constraint. However, G can evolve in response to selection and may also be viewed as a product of adaptive evolution. Convergent evolution of G in similar environments would suggest that G evolves adaptively, but it is difficult to disentangle such effects from phylogeny. Here, we use the adaptive radiation of Anolis lizards to ask whether convergence of G accompanies the repeated evolution of habitat specialists, or ecomorphs, across the Greater Antilles. We measured G in seven species representing three ecomorphs (trunk-crown, trunk- ground, and grass-bush). We found that the overall structure of G does not converge. Instead, the structure of G is well conserved and displays a phylogenetic signal consistent with Brownian motion. However, several elements of G showed signatures of convergence, indicating that some aspects of genetic architecture have been shaped by selection. Most notably, genetic correlations between limb traits and body traits were weaker in long-legged trunk-ground species, suggesting effects of recurrent selection on limb length. Our results demonstrate that common selection pressures may have subtle but consistent effects on the evolution of G, even as its overall structure remains conserved.

sentences: Genomic features underlie the co-option of SVA transposons as cis-regulatory elements in human pluripotent stem cells Domestication of transposable elements (TEs) into functional cis-regulatory elements is a widespread phenomenon. However, the mechanisms behind why some TEs are co-opted as functional enhancers while others are not are underappreciated. SINE-VNTR-Alus (SVAs) are the youngest group of transposons in the human genome, where [~]3,700 copies are annotated, nearly half of which are human-specific. Many studies indicate that SVAs are among the most frequently co-opted TEs in human gene regulation, but the mechanisms underlying such processes have not yet been thoroughly investigated. Here, we leveraged CRISPR-interference (CRISPRi), computational and functional genomics to elucidate the genomic features that underlie SVA domestication into human stem-cell gene regulation. We found that [~]750 SVAs are co-opted as functional cis-regulatory elements in human induced pluripotent stem cells. These SVAs are significantly closer to genes and harbor more transcription factor binding sites than non-co-opted SVAs. We show that a long DNA motif composed of flanking YY1/2 and OCT4 binding sites is enriched in the co-opted SVAs and that these two transcription factors bind consecutively on the TE sequence. We used CRISPRi to epigenetically repress active SVAs in stem cell-like NCCIT cells. Epigenetic perturbation of active SVAs strongly attenuated YY1/OCT4 binding and influenced neighboring gene expression. Ultimately, SVA repression resulted in [~]3,000 differentially expressed genes, 131 of which were the nearest gene to an annotated SVA. In summary, we demonstrated that SVAs modulate human gene expression, and uncovered that location and sequence composition contribute to SVA domestication into gene regulatory networks.

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Design and execution of a Verification, Validation, and Uncertainty Quantification plan for a numerical model of left ventricular flow after LVAD implantation Backgroundleft ventricular assist devices (LVADs) are implantable pumps that act as a life support therapy for patients with severe heart failure. Despite improving the survival rate, LVAD therapy can carry major complications. Particularly, the flow distortion introduced by the LVAD in the left ventricle (LV) may induce thrombus formation. While previous works have used numerical models to study the impact of multiple variables in the intra-LV stagnation regions, a comprehensive validation analysis has never been executed. The main goal of this work is to present a model of the LV-LVAD system and to design and follow a verification, validation and uncertainty quantification (VVUQ) plan based on the ASME V&V40 and V&V20 standards to ensure credible predictions.

MethodsThe experiment used to validate the simulation is the SDSU cardiac simulator, a bench mock-up of the cardiovascular system that allows mimicking multiple operation conditions for the heart-LVAD system. The numerical model is based on Alya, the BSCs in-house platform for numerical modelling. Alya solves the Navier-Stokes equation with an Arbitrarian Lagrangian-Eulerian (ALE) formulation in a deformable ventricle and includes pressure-driven valves, a 0D Windkessel model for the arterial output and a LVAD boundary condition modeled through a dynamic pressure-flow performance curve. The designed VVUQ plan involves: (a) a risk analysis and the associated credibility goals; (b) a verification stage to ensure correctness in the numerical solution procedure; (c) a sensitivity analysis to quantify the impact of the inputs on the four quantities of interest (QoIs) (average aortic root flow [Formula], maximum aortic root flow [Formula], average LVAD flow [Formula], and maximum LVAD flow [Formula]); (d) an uncertainty quantification using six validation experiments that include extreme operating conditions.

ResultsNumerical code verification tests ensured correctness of the solution procedure and numerical calculation verification showed small numerical errors. The total Sobol indices obtained during the sensitivity analysis demonstrated that the ejection fraction, the heart rate, and the pump performance curve coefficients are the most impactful inputs for the analysed QoIs.

The Minkowski norm is used as validation metric for the uncertainty quantification. It shows that the midpoint cases have more accurate results when compared to the extreme cases. The total computational cost of the simulations was above 100 [core-years] executed in around three weeks time span in Marenostrum IV supercomputer.

ConclusionsThis work details a novel numerical model for the LV-LVAD system, that is supported by the design and execution of a VVUQ plan created following recognised international standards. We present a methodology demonstrating that stringent VVUQ according to ASME standards is feasible but computationally expensive.

sentences: Shared and disease-specific host gene-microbiome interactions across human diseases While the gut microbiome and host gene regulation separately contribute to gastrointestinal disorders, it is unclear how the two may interact to influence host pathophysiology. Here, we developed a machine learning-based framework to jointly analyze host transcriptomic and microbiome profiles from 416 colonic mucosal samples of patients with colorectal cancer, inflammatory bowel disease, and irritable bowel syndrome. We identified potential interactions between gut microbes and host genes that are disease-specific, as well as interactions that are shared across the three diseases, involving host genes and gut microbes previously implicated in gastrointestinal inflammation, gut barrier protection, energy metabolism, and tumorigenesis. In addition, we found that mucosal gut microbes that have been associated with all three diseases, such as Streptococcus, interact with different host pathways in each disease, suggesting that similar microbes can affect host pathophysiology in a disease-specific manner through regulation of different host genes.

sentences: The Drosophila Baramicin polypeptide gene protects against fungal infection The fruit fly Drososphila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be characterized and exactly how each of these effectors contributes to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in the nervous system. Importantly, we show that flies lacking BaraA are viable but susceptible to the enomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA males but not females display an erect wing phenotype upon infection. Collectively, we identify a new antifungal immune effector downstream of Toll signalling, improving our knowledge of the Drosophila antimicrobial response.

sentences: Unifying turbulent dynamics framework distinguishes different brain states Recently, significant advances have been made by identifying the levels of synchronicity of the underlying dynamics of a given brain state. This research has demonstrated that unconscious dynamics tend to be more synchronous than those found in conscious states, which are more asynchronous. Here we go beyond this dichotomy to demonstrate that the different brain states are always underpinned by spatiotemporal chaos but with dissociable turbulent dynamics. We investigated human neuroimaging data from different brain states (resting state, meditation, deep sleep, and disorders of consciousness after coma) and were able to distinguish between them using complementary model-free and model-based measures of turbulent information transmission. Our model-free approach used recent advances describing a measure of information cascade across spatial scales using tools from turbulence theory. Complementarily, our model-based approach used exhaustive in silico perturbations of whole-brain models fitted to the empirical neuroimaging data, which allowed us to study the information encoding capabilities of the brain states. Overall, the current framework demonstrates that different levels of turbulent dynamics are fundamental for describing and differentiating between brain states.

sentences: Distinct allosteric mechanisms of first-generation MsbA inhibitors Present in all kingdoms of life, ATP-binding cassette (ABC) transporters couple ATP hydrolysis to mechanical force and facilitate trafficking of diverse substrates across biological membranes. Although many ABC transporters are promising drug targets, their mechanisms of regulation by small molecule inhibitors remain largely unknown. Herein, we used the lipopolysaccharide (LPS) flippase MsbA, a prototypical ABC exporter, as a model system to probe mechanisms of allosteric modulation by compounds binding to the transmembrane domains (TMDs). Recent chemical screens have identified intriguing LPS transport inhibitors targeting MsbA: the ATPase stimulator TBT1 and the ATPase inhibitor G247. Despite preliminary biochemical and structural data, it is unclear how TBT1 and G247 bind to the MsbA TMDs yet induce opposite allosteric effect in the nucleotide-binding domains (NBDs). Through single-particle EM, mutagenesis and activity assay, we show that TBT1 and G247 bind adjacent yet separate locations in the TMDs, inducing drastic changes in TMD conformation and NBD positioning. Two TBT1 molecules asymmetrically occupy the LPS binding site to break the symmetry of MsbA, resulting in disordered transmembrane helices and decreased NBD distance. In this novel inhibited ABC transporter state, decreased distance between the NBDs causes stimulation of ATP hydrolysis yet LPS transport blockage. In contrast, G247 acts as a TMDs wedge, symmetrically increasing NBD separation and preventing conformational transition of MsbA. Our study uncovers the distinct mechanisms of the first-generation MsbA-specific inhibitors and demonstrates that rational design of substrate-mimicking compounds can be exploited to develop useful ABC transporter modulators.

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Animal soundscapes reveal key markers of Amazon forest degradation from fire and logging Safeguarding tropical forest biodiversity requires solutions for monitoring ecosystem composition over time. In the Amazon, logging and fire reduce forest carbon stocks and alter tree species diversity, but the long-term consequences for wildlife remain unclear, especially for lesser-known taxa. Here, we combined data from multi-day acoustic surveys, airborne lidar, and satellite timeseries covering logged and burned forests (n=39) in the southern Brazilian Amazon to identify acoustic markers of degradation. Our findings contradict theoretical expectations from the Acoustic Niche Hypothesis that animal communities in more degraded habitats occupy fewer  acoustic niches. Instead, we found that habitat structure (e.g., aboveground biomass) was not a consistent proxy for biodiversity based on divergent patterns of acoustic space occupancy (ASO) in logged and burned forests. Full 24-hr soundscapes highlighted a stark and sustained reorganization in community structure after multiple fires; animal communication networks were quieter, more homogenous, and less acoustically integrated in forests burned multiple times than in logged or once-burned forests. These findings demonstrate strong biodiversity co-benefits from protecting Amazon forests from recurrent fire activity. By contrast, soundscape changes after logging were subtle and more consistent with community recovery than reassembly. In both logged and burned forests, insects were the dominant acoustic markers of degradation, particularly during midday and nighttime hours that are not typically sampled by traditional field surveys of biodiversity. The acoustic fingerprints of degradation history were conserved across replicate recording locations at each site, indicating that soundscapes offer a robust, taxonomically inclusive solution for tracking changes in community composition over time.

Significance StatementFire and logging reduce the carbon stored in Amazon forests, but little is known about how human degradation alters animal communities. We recorded thousands of hours of ecosystem sounds to investigate animal community assembly and the associations between biodiversity and biomass following Amazon forest degradation over time. 24-hr patterns of acoustic activity differed between logged and burned forests, and we observed large and sustained breakpoints in community structure after multiple burns. Soundscape differences among degraded forests were clearest during insect-dominated hours rarely sampled in field studies of biodiversity. These findings demonstrate that acoustic monitoring holds promise for routine biodiversity accounting, even by non-experts, to capture a holistic measure of animal communities in degraded tropical forests and benchmark change over time.

sentences: A novel RyR1 inhibitor prevents and rescues sudden death in a mouse model of malignant hyperthermia and heat stroke Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that a novel RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduced resting intracellular Ca2+, inhibited halothane- and isoflurane-induced Ca2+ release, suppressed caffeine-induced contracture in skeletal muscle, reduced sarcolemmal cation influx, and prevented or reversed the fulminant MH crisis induced by isoflurane anesthesia and rescued animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising new candidate for effective treatment of patients carrying RyR1 mutations.

sentences: A simple Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis We have developed a simple Agrobacterium-mediated method for the stable transformation of the hornwort Anthoceros agrestis, the fifth bryophyte species for which a genetic manipulation technique becomes available. High transformation efficiency was achieved by using thallus tissue grown under low-light conditions. We generated a total of 216 transgenic A. agrestis lines expressing the {beta}-Glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under the control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing, and land plant evolution in general.

sentences: Septin-microtubule association requires a MAP-like motif unique to Sept9 isoform 1 embedded into septin octamers Septins, a family of GTP-binding proteins assembling into higher order structures, interface with the membrane, actin filaments and microtubules, which positions them as important regulators of cytoarchitecture. Septin 9 (Sept9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short MAP-like motif unique to Sept9 isoform 1 (Sept9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring Sept9_i1. Although outside of the MAP-like motif, HNA mutations abrogates this association, identifying a putative regulatory domain. Removal of this domain from Sept9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin associated pathologies.

sentences: Leptospira surface protein LigA plays a multifaceted role in modulating the host innate immune response Leptospira, a zoonotic pathogen, is known to infect various hosts and can establish persistent infection. This remarkable ability of bacteria is attributed to its potential to modulate the host immune response by exploiting its surface proteins. We have identified and characterized the domain of the variable region of Leptospira immunoglobulin-like protein A (LAV) involved in immune modulation. The 11th domain (A11) of the variable region of LigA (LAV) induces a strong TLR4 dependent innate response leading to subsequent induction of humoral and cellular immune responses in mice. A11 is also involved in acquiring complement regulator FH and binds to host protease Plasminogen (PLG), there by mediating functional activity to escape from complement-mediated killing. The deletion of A11 domain significantly impaired TLR4 signaling and subsequent reduction in the innate and adaptive immune response. It also inhibited the binding of FH and PLG thereby mediating killing of bacteria. Our study discovered an unprecedented role of LAV as a nuclease capable of degrading Neutrophil Extracellular Traps (NETs). This nuclease activity was primarily mediated by A11. These results highlighted the moonlighting function of LigA and demonstrated that a single domain of a surface protein is involved in evading a myriad of host innate immune defenses, which might allow the persistence of Leptospira in different hosts for a long term without clearance.

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Baseline psychological traits contribute to Lake Louise Acute Mountain Sickness score at high altitude BackgroundInteroception refers to an individuals ability to sense their internal bodily sensations. Acute mountain sickness (AMS) is a common feature of ascent to high altitude that is only partially explained by measures of peripheral physiology. We hypothesised that interoceptive ability may explain the disconnect between measures of physiology and symptom experience in AMS.

Methods and MaterialTwo groups of 18 participants were recruited to complete a respiratory interoceptive task three times at two-week intervals. The control group remained in Birmingham (140m altitude) for all three tests. The altitude group completed test 1 in Birmingham, test 2 the day after arrival at 2624m, and test 3 at 2728m after an 11-day trek at high altitude (up to 4800m).

ResultsBy measuring changes to metacognitive performance, we showed that acute ascent to altitude neither presented an interoceptive challenge, nor acted as interoceptive training. However, AMS symptom burden throughout the trek was found to relate to sea-level measures of anxiety, agoraphobia, and neuroticism.

ConclusionsThis suggests that the Lake Louise AMS score is not solely a reflection of physiological changes on ascent to high altitude, despite often being used as such by researchers and commercial trekking companies alike.

sentences: Adventive larval parasitoids reconstruct their close association with spotted-wing drosophila in the invaded North American range Two species of larval parasitoids of the globally invasive fruit pest, Drosophila suzukii (Diptera: Drosophilidae), Leptopilina japonica and Ganaspis brasiliensis (both Hymenoptera: Figitidae), were detected in British Columbia, Canada in 2019. Both are presumed to have been unintentionally introduced from Asia, however; the extent of their establishment across different habitats with diverse host plants used by D. suzukii was unclear. In addition, there was no knowledge of the temporal dynamics of parasitism of D. suzukii by these two parasitoids. We repeatedly sampled the fruits of known host plants of D. suzukii over the entire 2020 growing season in British Columbia. We documented the presence of L. japonica and G. brasiliensis and estimated the apparent percentage of D. suzukii parasitized. Across a large region of southwestern British Columbia, both L. japonica and G. brasiliensis were found to be very common across a variety of mostly unmanaged habitats over the entire course of the season (May-October) in the fruits of most host plants known to host D. suzukii larvae. The two parasitoids were responsible for more than 98% of D. suzukii larval parasitism and usually co-existed. Parasitism of D. suzukii was variable among hosts plants and sites (0-66% percent parasitism) and appeared to be time-structured. Our study demonstrates that the close association between the two larval parasitoids and D. suzukii that exists in Asia has evidently been reconstructed in North America, resulting in the highest parasitism levels of D. suzukii yet recorded outside of its area of origin.

sentences: Robust deep learning object recognition models rely on low frequency information in natural images Machine learning models have difficulty generalizing to data outside of the distribution they were trained on. In particular, vision models are usually vulnerable to adversarial attacks or common corruptions, to which the human visual system is robust. Recent studies have found that regularizing machine learning models to favor brain-like representations can improve model robustness, but it is unclear why. We hypothesize that the increased model robustness is partly due to the low spatial frequency preference inherited from the neural representation. We tested this simple hypothesis with several frequency-oriented analyses, including the design and use of hybrid images to probe model frequency sensitivity directly. We also examined many other publicly available robust models that were trained on adversarial images or with data augmentation, and found that all these robust models showed a greater preference to low spatial frequency information. We show that preprocessing by blurring can serve as a defense mechanism against both adversarial attacks and common corruptions, further confirming our hypothesis and demonstrating the utility of low spatial frequency information in robust object recognition.

sentences: Traditional martial arts and shooting training have different effects on auditory fine structure processing ability---Evidence from behavioral tests and fMRI Explore the influence of traditional martial arts and shooting training on the ability of auditory temporal fine structure (TFS) processing. Twenty-five college students participated in the experiment, including 8 traditional martial arts practitioners, 8 high-level shooting athletes, and 9 control groups without any regular exercise habits. The BIC (break in interaural correlation) delay threshold and TFS1 test were used to evaluate the temporary storage capacity and sensitivity of TFS, respectively, and a fMRI test was performed after the test. The results found that the traditional martial arts group had stronger TFS sensitivity, while the shooting group had stronger TFS retention ability, and the performance of the behavioral test of the shooting group is related to the fALFF value of the brain area of interest. Traditional martial arts and shooting training have improved the ability of auditory information processing from different angles, diversified exercise habits will lead to the development of diversity in brain structure and function.

sentences: Pax3 repairs a neural circuit through a program of directed axon outgrowth Repairing damaged or dysfunctional human brain circuits remains an ongoing challenge for biomedical science. While surviving neuronal networks can be reorganised after lesion, for example by neurotrophins, these new connections are disorganised and rarely produce clinical improvement. Here we investigate how to promote axonal growth while retaining correct cellular targeting. We show that, in response to brain-derived neurotrophic factor (BDNF) in target-tissue, potential reinnervating neurons upregulate Pax3. Pax3 in turn increases polysialic acid-neural cell adhesion molecule (PSA-NCAM) on their axon terminals, facilitating their outgrowth and pathfinding, and resulting in correctly-targeted neural circuit repair in the mature nervous system. This is a novel role for Pax3, which we confirmed by showing its expression in afferent neurons is essential for spontaneous and BDNF-induced reinnervation in the developing and mature brains, respectively. Together these results suggest that Pax3 contributes to a repair program, in which axon growth is promoted and direction signaling maintained. These data advance our understanding for accurately rebuilding neural circuits: restricting growth-promotion to potential afferent neurons, as opposed to stimulating the whole circuit, allows axon growth without impairing its guidance.

sentences: Genome assembly and isoform analysis of a highly heterozygous New Zealand fisheries species, the tarakihi (Nemadactylus macropterus) Although being some of the most valuable and heavily exploited wild organisms, few fisheries species have been studied at the whole-genome level. This is especially the case in New Zealand, where genomics resources are urgently needed to assist fisheries management attains its sustainability goals. Here we generated 55 Gb of short Illumina reads (92x coverage) and 73 Gb of long Nanopore reads (122x) to produce the first genome assembly of the marine teleost tarakihi (Nemadactylus macropterus), a highly valuable fisheries species in New Zealand. An additional 300 Mb of Iso-Seq RNA reads were obtained from four tissue types of another specimen to assist in gene annotation. The final genome assembly was 568 Mb long and consisted of 1,214 scaffolds with an N50 of 3.37 Mb. The genome completeness was high, with 97.8% of complete Actinopterygii BUSCOs. Heterozygosity values estimated through k-mer counting (1.00%) and bi-allelic SNPs (0.64%) were high compared to the same values reported for other fishes. Repetitive elements covered 30.45% of the genome and 20,169 protein-coding genes were annotated. Iso-Seq analysis recovered 91,313 unique transcripts (isoforms) from 15,515 genes (mean ratio of 5.89 transcripts per gene), and the most common alternative splicing event was intron retention. This highly contiguous genome assembly along with the isoform-resolved transcriptome will provide a useful resource to assist the study of population genomics, as well as comparative eco-evolutionary studies in other teleost and related organisms.

sentences: Active Anemosensing Hypothesis: How Flying Insects Could Estimate Ambient Wind Direction Through Sensory Integration & Active Movement Estimating the direction of ambient fluid flow is a crucial step during chemical plume tracking for flying and swimming animals. How animals accomplish this remains an open area of investigation. Recent calcium imaging with tethered flying Drosophila has shown that flies encode the angular direction of multiple sensory modalities in their central complex: orientation, apparent wind (or airspeed) direction, and direction of motion. Here we describe a general framework for how these three sensory modalities can be integrated over time to provide a continuous estimate of ambient wind direction. After validating our framework using a flying drone, we use simulations to show that ambient wind direction can be most accurately estimated with trajectories characterized by frequent, large magnitude turns. Furthermore, sensory measurements and estimates of their derivatives must be integrated over a period of time that incorporates at least one of these turns. Finally, we discuss approaches that insects might use to simplify the required computations, and present a list of testable predictions. Together, our results suggest that ambient flow estimation may be an important driver underlying the zigzagging maneuvers characteristic of plume tracking animals trajectories.

sentences: Optimal resistance management for mixtures of high-risk fungicides: robustness to the initial frequency of resistance and pathogen sexual reproduction There is a strong consensus that selection for fungicide resistant pathogen strains can be most effectively limited by using applications of mixtures of fungicides designed to balance disease control against selection. However, how to do this in practice is not entirely characterised. Previous work indicates optimal mixtures of pairs of fungicides which are both at a high risk of resistance can be constructed using pairs of doses which select equally for both single resistant strains in the first year of application. What has not been addressed thus far is the important real-world case in which the initial levels of resistance to each fungicide differ, for example because the chemicals have been available for different lengths of time. We show how recommendations based on equal selection in the first year can be sub-optimal in this case. We introduce a simple alternative approach, based on equalising the frequencies of single resistant strains in the year that achieving acceptable levels of control is predicted to become impossible. We show that this strategy is robust to changes in parameters controlling pathogen epidemiology and fungicide efficacy. We develop our recommendation using a pre-existing, parameterised model of Zymoseptoria tritici (the pathogen causing Septoria leaf blotch on wheat), which exemplifies the range of plant pathogens which predominantly spread clonally, but for which sexual reproduction forms an important component of the life cycle. We show that pathogen sexual reproduction can influence the rate at which fungicide resistance develops, but does not qualitatively affect our optimal resistance management recommendation.

sentences: B1 SINE-binding ZFP266 impedes reprogramming through suppression of chromatin opening mediated by pioneering factors Induced pluripotent stem cell reprogramming is inherently inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 16 novel reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhanced iPSC generation in several iPSC reprogramming settings, emerging as the most robust roadblock. Further analyses revealed that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2 and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with a co-activator domain converted ZFP266 from a reprogramming inhibitor to a potent reprogramming facilitator. This work proposes SINE-KRAB-ZFP interaction to be a critical regulator of chromatin accessibility at enhancers for efficient cellular identity changes and also serves as a resource to further illuminate molecular mechanisms hindering reprogramming.

sentences: TOPLESS promotes plant immunity by repressing auxin signaling and is targeted by the fungal effector Naked1 In plants, the antagonism between growth and defense is hardwired by hormonal signaling. Perception of Pathogen Associated Molecular Patterns (PAMPs) from invading microorganisms inhibits auxin signaling and growth. Conversely, pathogens manipulate auxin signaling to promote disease, but how this hormone inhibits immunity is not fully understood. Ustilago maydis is a maize pathogen that induces auxin signaling in its host. We characterized an U. maydis effector protein, Naked1 (Nkd1), that is translocated into the host nucleus. Through its native EAR motif, Nkd1 binds to the transcriptional co-repressors TOPLESS/TOPLESS related (TPL/TPRs) and prevents the recruitment of transcriptional repressors involved in hormonal signaling, leading to de-repression of auxin and jasmonate signaling. Moderate up-regulation of auxin signaling inhibits an early defense response, PAMP-triggered ROS burst. Thus, our findings establish a clear mechanism for auxin-induced pathogen susceptibility. Manipulation of TPL by Nkd1 is evolutionary well balanced as engineered Nkd1 variants with increased expression or increased EAR-mediated TPL/TPR binding trigger typical salicylic acid mediated defense reactions, leading to pathogen resistance.

sentences: Incomplete bunyavirus particles contribute to within-host spread and between-host transmission Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations that are unable to spread autonomously, are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that incomplete virus particles are capable of co-infecting mosquitoes, resulting in the rescue of infectious virus that is able to disseminate to the mosquito salivary glands. Our findings reveal a significant role of incomplete particles in within-host spread and between-host transmission, reminiscent of the life cycle of multipartite viruses.

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Early exposure to UV radiation causes telomere shortening and poorer condition later in life Determining the contribution of elevated ultraviolet-B radiation (UVBR; 280 - 315 nm) to amphibian population declines is being hindered by a lack of knowledge about how different acute UVBR exposure regimes during early life history stages might affect post-metamorphic stages via long-term carryover effects. We acutely exposed tadpoles of the Australian green tree frog (Litoria caerulea) to a combination of different UVBR irradiances and doses in a multi-factorial experiment, and then reared them to metamorphosis in the absence of UVBR to assess carryover effects in subsequent juvenile frogs. Dose and irradiance of acute UVBR exposure influenced carryover effects into metamorphosis in somewhat opposing manners. Higher doses of UVBR exposure in larvae yielded improved rates of metamorphosis. However, exposure at a high irradiance resulted in frogs metamorphosing smaller in size and in poorer condition than frogs exposed to low and medium irradiance UVBR as larvae. We also demonstrate some of the first empirical evidence of UVBR-induced telomere shortening in vivo, which is one possible mechanism for life-history trade-offs impacting condition post-metamorphosis. These findings contribute to our understanding of how acute UVBR exposure regimes in early life affect later life-history stages, which has implications for how this stressor may shape population dynamics.

SUMMARY STATEMENTWe demonstrate physiological carryover effects in amphibians that link larval UV exposure to detrimental impacts on juvenile frogs, including telomere shortening, which has implications for how UV shapes amphibian populations.

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Multiple bouts of high-intensity interval exercise reverse age-related functional connectivity disruptions without affecting motor learning in older adults Exercise has emerged as an intervention that may mitigate age-related resting state functional connectivity and sensorimotor declines. Here, 42 healthy older adults rested or completed 3 sets of high-intensity interval exercise for a total of 23 minutes, then immediately practiced an implicit motor task with their non-dominant hand across five separate sessions. Participants completed resting stage functional MRI before the first and after the fifth day of practice; they also returned 24-hours and 35-days later to complete short- and long-term retention. Independent component analysis of resting state functional MRI revealed increased connectivity in the frontoparietal, the dorsal attentional, and cerebellar networks and group. seed-based analysis showed strengthened connectivity between the limbic system and right cerebellum, and between the right cerebellum and bilateral middle temporal gyri in the exercise group. There was no motor learning advantage for the exercise group. Our data suggest that exercise paired with an implicit motor learning task in older adults can augment resting state functional connectivity without enhancing behaviour beyond that stimulated by skilled motor practice.

Impact statementFive separate bouts of exercise paired with skilled motor practice strengthens resting state networks in brain regions that are susceptible to declines in older adults without affecting motor acquisition or learning.

sentences: A Genetically Encoded BRET-based SARS-CoV-2 Mpro Protease Activity Sensor The SARS-CoV-2 main protease, Mpro, is critical for its replication and is an appealing target for designing anti-SARS-CoV-2 agents. In this regard, a number of assays have been developed based on its cleavage sequence preferences to monitor its activity. These include the usage of Fluorescence Resonance Energy Transfer (FRET)-based substrates in vitro and a FlipGFP reporter, one which fluoresces after Mpro-mediated cleavage, in live cells. Here, we have engineered a pair of genetically encoded, Bioluminescence Resonance Energy Transfer (BRET)-based sensors for detecting SARS-CoV-2 Mpro proteolytic activity in living host cells as well as in vitro assays. The sensors were generated by sandwiching Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and nanoLuc proteins. Co-expression of the sensor with the Mpro in live cells resulted in its cleavage in a dose- and time-dependent manner while mutation of the critical C145 residue (C145A) in Mpro completely abrogated the sensor cleavage. Importantly, the BRET-based sensors displayed increased sensitivities and specificities as compared to the recently developed FlipGFP-based Mpro sensor. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensor developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

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Signatures of plant defense response specificity mediated by herbivore-associated molecular patterns in legumes I.Chewing herbivores activate plant defense responses through a combination of mechanical wounding and elicitation by herbivore associated molecular patterns (HAMPs). HAMPs are wound response amplifiers; however, specific defense outputs may also exist that strictly require HAMP-mediated defense signaling. To investigate HAMP-mediated signaling and defense responses, we characterized cowpea transcriptome changes following elicitation by inceptin, a peptide HAMP common in Lepidoptera larvae oral secretions. Following inceptin treatment, we observed large-scale reprogramming of the transcriptome consistent with 3 different response categories: 1) amplification of mechanical wound responses, 2) temporal extension through accelerated or prolonged responses, and 3) examples of inceptin-specific elicitation and suppression. At both early and late timepoints, namely 1 and 6 hours, large sets of transcripts specifically accumulated following inceptin elicitation but not wounding alone. Further inceptin-regulated transcripts were classified as reversing changes induced by wounding alone. Within key signaling and defense related gene families, inceptin-elicited responses commonly targeted select subsets of wound-induced transcripts. Transcripts displaying the largest inceptin-elicited fold-changes included terpene synthases (TPS) and peroxidases (POX) that correspond with induced volatile production and increased peroxidase activity in cowpea. Characterization of inceptin-elicited cowpea defenses via heterologous expression in Nicotiana benthamiana demonstrated that specific cowpea TPS and POX were able to confer terpene emission and the reduced growth of beet armyworm (Spodoptera exigua) herbivores, respectively. Collectively, our present findings in cowpea support a model where HAMP-elicitation both amplifies concurrent wound responses and specifically contributes to the activation of selective outputs associated with direct and indirect anti-herbivore defenses.

II. Significance StatementPlants recognize herbivore-associated molecular patterns (HAMPs) to induce defenses, but interactions with the more general wound response are not well-understood. We leveraged a known HAMP-receptor interaction to characterize transcriptomic modulation of the wound response by the peptide HAMP, inceptin. Inceptin not only amplifies wound responses, but can specifically induce or suppress transcripts with demonstrated functions in direct and indirect defense against herbivores. The plant immune system thus recognizes HAMPs to fine-tune wound responses against herbivory.

sentences: Convergent evolution of multiple mutations improves the viral fitness of SARS-CoV-2 variants by balancing positive and negative selection Multiple mutations have been seen to undergo convergent evolution in SARS-CoV-2 variants of concern. One such evolution occurs in Beta, Gamma, and Omicron variants at three amino acid positions K417, E484, and N501 in the receptor binding domain of the spike protein. We examined the physical mechanisms underlying the convergent evolution of three mutations K417T/E484K/N501Y by delineating the individual and collective effects of mutations on binding to angiotensin converting enzyme 2 receptor, immune escape from neutralizing antibodies, protein stability and expression. Our results show that each mutation serves a distinct function that improves virus fitness supporting its positive selection, even though individual mutations have deleterious effects that make them prone to negative selection. Compared to the wild-type, K417T escapes Class 1 antibodies, has increased stability and expression; however, it has decreased receptor binding. E484K escapes Class 2 antibodies; however, it has decreased receptor binding, stability and expression. N501Y increases receptor binding; however, has decreased stability and expression. When these mutations come together, the deleterious effects are mitigated due to the presence of compensatory effects. Triple mutant K417T/E484K/N501Y has increased receptor binding, escapes both Class 1 and Class 2 antibodies, and has similar stability and expression as that of the wild-type. These results show the implications of presence of multiple mutations on virus evolution that enhance viral fitness on different fronts by balancing both positive and negative selection and improves the chances of selection of mutations together.

sentences: The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection Summary ParagraphWe identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone1, a compound in clinical trials for anti-fibrotic and anti-inflammatory applications2, as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry3. We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir4. Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginones translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

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MICOS and F1FO-ATP synthase crosstalk is a fundamental property of mitochondrial cristae Mitochondrial cristae are polymorphic invaginations of the inner membrane that are the fabric of cellular respiration. Both the Mitochondrial Contact Site and Cristae Organization System (MICOS) and the FOF1-ATP synthase are vital for sculpting cristae by opposing membrane bending forces. While MICOS promotes negative curvature at cristae junctions, dimeric FOF1- ATP synthase is crucial for positive curvature at cristae rims. Crosstalk between these two complexes has been observed in bakers yeast, the model organism of the Opisthokonta supergroup. Here, we report that this property is conserved in Trypanosoma brucei, a member of the Discoba supergroup that separated from Opisthokonta [~]2 billion years ago. Specifically, one of the paralogs of the core MICOS subunit Mic10 interacts with dimeric F1FO-ATP synthase, whereas the other core Mic60 subunit has a counteractive effect on F1FO- ATP synthase oligomerization. This is evocative of the nature of MICOS-F1FO-ATP synthase crosstalk in yeast, which is remarkable given the diversification these two complexes have undergone during almost 2 eons of independent evolution. Furthermore, we identified a highly diverged trypanosome homolog of subunit e, which is essential for the stability of F1FO-ATP synthase dimers in yeast. Just like subunit e, it is preferentially associated with dimers, interacts with Mic10 and its silencing results in severe defects to cristae and disintegration of F1FO-ATP synthase dimers. Our findings indicate that crosstalk between MICOS and dimeric F1FO-ATP synthase is a fundamental property impacting cristae shape throughout eukaryotes.

ImportanceMitochondria have undergone profound diversification in separate lineages that have radiated since the last common ancestor of eukaryotes some eons ago. Most eukaryotes are unicellular protists, including etiological agents of infectious diseases like Trypanosoma brucei. Thus, the study of a broad range of protists can reveal fundamental features shared by all eukaryotes and lineage-specific innovations. Here we report that two different protein complexes, MICOS and F1FO-ATP synthase, known to affect mitochondrial architecture, undergo crosstalk in T. brucei, just as in bakers yeast. This is remarkable considering that these complexes have otherwise undergone many changes during their almost two billion years of independent evolution. Thus, this crosstalk is a fundamental property needed to maintain proper mitochondrial structure even if the constituent players considerably diverged.

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Co-clinical FDG-PET Radiomic Signature in Predicting Response to Neoadjuvant Chemotherapy in Triple Negative Breast Cancer PurposeWe sought to exploit the heterogeneity afforded by patient-derived tumor xenografts (PDX) to optimize robust radiomic features associated with response to therapy in the context of a co-clinical trial and implement PDX-optimized image features in the corresponding clinical study to predict and assess response to therapy using machine-learning (ML) algorithms.

MethodsTNBC patients and subtype-matched PDX were recruited into a co-clinical FDG-PET imaging study to predict response to therapy. One hundred thirty-one imaging features were extracted from PDX and human segmented tumors. Robust image features were identified based on reproducibility, cross-correlation, and volume independence. A rank importance of predictors using ReliefF was used to identify predictive radiomic features in the preclinical PDX trial in conjunction with ML algorithms: classification and regression tree (CART), Naive Bayes (NB), and support vector machines (SVM). The top four PDX-optimized image features, defined as radiomic signatures (RadSig), from each task were then used to predict or assess response to therapy. Performance of RadSig in predicting/assessing response was compared to SUVmean, SUVmax, and lean body mass normalized SULpeak measures.

ResultsSixty-four out of 131 preclinical imaging features were identified as robust. NB-RadSig performed highest in predicting and assessing response to therapy in the preclinical PDX trial. In the clinical study, the performance of SVM-RadSig and NB-RadSig to predict and assess response was practically identical and superior to SUVmean, SUVmax, and SULpeak, measures.

ConclusionsWe optimized robust FDG-PET radiomic signatures (RadSig) to predict and assess response to therapy in a context of a co-clinical imaging trial.

DECLARATIONSO_ST_ABSFundingC_ST_ABSThis work was supported by NCI grants U24CA209837, U24CA253531, and U54CA224083; U2CCA233303, and K12CA167540; Siteman Cancer Center (SCC) Support Grant P30CA091842; and Internal funds provided by Mallinckrodt Institute of Radiology.

Conflicts of interest/Competing interests.None.

Availability of data and materialAll the co-clinical data will be available for download through the Washington University School of Medicine Co-Clinical Imaging Research Resource web portal at https://c2ir2.wustl.edu/, co-clinical database (CCDB).

Code availabilityNot applicable.

Authors contributionsConceptualization: SR, FOA, KIS; Methodology: SR, TDW, SL, KIS; Formal analysis and investigation: SR, KIS; Writing - original draft preparation: SR; Writing - review and editing: RLW, FD, KIS; Funding acquisition: RWL, FOA, SL, KIS; Resources: SL; Supervision: FD, KIS. All authors read and approved the final manuscript.

Ethics approvalAll studies were performed with approval from the Washington University Humans subjects research committee and animal studies committee.

Consent to participateInformed consent to participate in the study was obtained from all participants.

Consent for publicationNot applicable.

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AI-guided discovery of the invariant host response to viral pandemics We sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach. Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a  seed gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. Surprisingly, this 166-gene signature was conserved in all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determines severity/fatality. Precise therapeutic goals were formulated and subsequently validated in high-dose SARS-CoV-2-challenged hamsters using neutralizing antibodies that abrogate SARS-CoV-2*ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine tracked with disease severity. Thus, the ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.

One Sentence SummaryThe host immune response in COVID-19.

PANEL: RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSThe SARS-CoV-2 pandemic has inspired many groups to find innovative methodologies that can help us understand the host immune response to the virus; unchecked proportions of such immune response have been implicated in fatality. We searched GEO and ArrayExpress that provided many publicly available gene expression data that objectively measure the host immune response in diverse conditions. However, challenges remain in identifying a set of host response events that are common to every condition. There are no studies that provide a reproducible assessment of prognosticators of disease severity, the host response, and therapeutic goals. Consequently, therapeutic trials for COVID-19 have seen many more  misses than  hits. This work used multiple (> 45,000) gene expression datasets from GEO and ArrayExpress and analyzed them using an unbiased computational approach that relies upon fundamentals of gene expression patterns and mathematical precision when assessing them.

Added value of this studyThis work identifies a signature that is surprisingly conserved in all viral pandemics, including Covid-19, inspiring the nomenclature ViP-signature. A subset of 20-genes classified disease severity in respiratory pandemics. The ViP signatures pinpointed the nature and source of the  cytokine storm mounted by the host. They also helped formulate precise therapeutic goals and rationalized the repurposing of FDA-approved drugs.

Implications of all the available evidenceThe ViP signatures provide a quantitative and qualitative framework for assessing the immune response in viral pandemics when creating pre-clinical models; they serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.

sentences: Burst coding despite unimodal interval distributions The burst coding hypothesis posits that the occurrence of sudden high-frequency patterns of action potentials constitutes a salient syllable of the neural code. Many neurons, however, do not produce clearly demarcated bursts, an observation invoked to rule out the pervasiveness of this coding scheme across brain areas and cell types. Here we ask how identifiable spike-timing patterns have to be to preserve potent transmission of information. Should we expect that neurons avoid ambiguous patterns that are neither clearly bursts nor isolated spikes? We addressed these questions using information theory and computational simulations. By quantifying how information transmission depends on firing statistics, we found that the information transmitted is not strongly influenced by the presence of clearly demarcated modes in the interspike interval distribution, a feature often used to identify the presence of burst coding. Instead, we found that neurons having unimodal interval distributions were still able to ascribe different meanings to bursts and isolated spikes. In this regime, information transmission depends on properties of the synapses as well as the length and relative frequency of bursts. Furthermore, we found that common metrics used to quantify burstiness were also unable to predict the degree with which bursts could be used to carry information. Our results provide guiding principles for the implementation of coding strategies based on spike-timing patterns, and show that even unimodal firing statistics can be consistent with a bivariate neural code.

sentences: Synergistic regulation of bZIP53 and dimerizing partners results in abnormal seed phenotype in Arabidopsis: Use of a designed dominant negative protein A-ZIP53 In Arabidopsis, basic leucine zipper (bZIP) family of transcription factors (TFs) are key proteins to regulate the expression of seed maturation (MAT) genes. bZIPs are functionally redundant and their DNA-binding activity is dependent on dimerization partner. The intervention of loss of function mutation is inadequate to understand and regulate the redundant behavior of TFs and one such example is bZIP53, which is known as a key regulator of seed maturation phenomena. Here, to examine the consequences of hindering the function of bZIP53 and its known and unknown heterodimerizing partners, a transgenic Arabidopsis constitutively expressing a novel dominant negative (DN) protein A-ZIP53 was raised. Transgenic plants demonstrated a delayed growth and retarded seed phenotype. The in vivo inhibition of DNA binding of bZIP53, bZIP10, and bZIP25 to the G-box demonstrated the efficacy of A-ZIP53 protein. In first generation, majority of plants failed to survive beyond four weeks suggesting a pleiotropic nature of bZIP53. Plants expressing A-ZIP53 have small flower, shorter siliques, and small-seeded phenotype. RNA seq analysis of the transgenic lines revealed the reduced expression of target genes of bZIP53 and its heterodimerizing partners. Furthermore, immunoprecipitation followed by mass spectrometry (IP-MS/MS) of transgenic plants helped to identify the additional heterodimerizing partners of the A-ZIP53. The interactions were subsequently confirmed with the transient transfection experiments. Unlike other gene knock out technologies, DN protein can inhibit the function of members of the same group of bZIP TFs.

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Histone demethylase UTX regulates glioblastoma progression through affecting periostin expression The histone H3K27 demethylase UTX participates in regulating multiple cancer types. However, less is known about the UTX function in glioblastoma (GBM). This study aims to define the effect of UTX on GBM. GEPIA2 database analysis showed that UTX expression was significantly increased in GBM and inversely correlated with survival. Knockdown UTX inhibited GBM cell proliferation, migration, and invasion while promoting apoptosis. Moreover, knockdown UTX also hampered tumor growth in the heterotopic xenograft model. RNA-seq combined with qRT-PCR and ChIP-qPCR were used to identify the target genes. The results showed that the UTX-mediated genes were strongly associated with tumor progression and the extracellular environment. Protein-protein interaction analysis suggested that periostin (POSTN) interacted with most of the other UTX-mediated genes. POSTN supplement abolished the effect of UTX knockdown in GBM cells. Furthermore, silencing UTX exhibited similar antitumor effect in patient-derived glioblastoma stem-like cells, while UTX functions were partially restored after exposing POSTN. Our findings may reveal a new insight into the onset of gliomagenesis and progression, providing a promising therapeutic strategy for GBM treatment.

Bullet PointsUTX correlates with survival in glioblastoma. Silencing UTX decreased the levels of H3K27 methylation in the POSTN gene, thereby suppressing POSTN expression. UTX-mediated POSTN expression is crucial for glioblastoma cell growth and tumorigenesis.

O_LIUTX expression is increased in GBM and negatively correlated with survival.
C_LIO_LIUTX knockdown influences proliferation and apoptosis in both GBM cells and GSCs.
C_LIO_LIThe antitumor effect of UTX knockdown is achieved by suppressing POSTN expression.
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sentences: Microbiome signatures in Acropora cervicornis are associated with genotypic resistance to elevated nutrients and heat stress The staghorn coral, Acropora cervicornis, was once abundant in the Caribbean, but now is listed as critically endangered. To recover A. cervicornis populations, restoration efforts have focused on preserving genetic diversity and increasing coral cover. However, identifying stress-resistant corals can help to increase restoration success, by allocating genotypes to reefs where they are more likely to survive. We assessed the performance (growth, survivorship, and photochemical efficiency) and characterized the microbiome (prokaryotes) of six A. cervicornis genotypes that were maintained at control temperatures (~26 {degrees}C) and either ambient nutrients or elevated nutrients (elevated NH4, and elevated NH4 + PO4) for > 2 months. We then compared how these parameters changed when the corals were exposed to heat-stress (3-weeks at ~31.5 {degrees}C). We found that exposure to elevated nutrients reduced A. cervicornis performance under control temperatures and heat stress. However, there was a wide range of variation among genotypes, with three genotypes maintaining relatively higher survivorship and growth rates when exposed to nutrients alone, and nutrients followed by heat stress. Heat stress alone changed the microbial composition among genotypes more than elevated nutrients alone, but heat stress also interacted with nutrient pre-exposure to affect microbial communities. The relative abundance of Midichloriaceae and Spirochaetaceae varied by coral genotype and a high abundance of these bacterial taxa was a positive predictor of coral survivorship rate, suggesting a microbial signature that could aid in identifying resistant A. cervicornis genotypes. Our findings suggest there is significant variation among genotypes in the response of A. cervicornis to elevated nutrients and temperatures. Resistant genotypes may be identifiable via their microbiomes and prioritized for outplanting at sites characterized by high levels of nutrient pollution. Large-scale microbiome screening may help expedite targeted outplanting and could be tested and extended to facilitate the identification of genotypes with other resistance characteristics.

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Distinct Colon Mucosa Microbiomes associated with Tubular Adenomas and Serrated Polyps BackgroundColorectal cancer is the second most deadly and third most common cancer in the world. Its development is heterogenous, with multiple mechanisms of carcinogenesis. Two distinct mechanisms include the adenoma-carcinoma sequence and the serrated pathway. The gut microbiome has been identified as a key player in the adenoma-carcinoma sequence, but its role in serrated carcinogenesis is less clear. In this study, we characterized the gut microbiome of 140 polyp-free and polyp-bearing individuals using colon mucosa and fecal samples to determine if microbiome composition was associated with each of the two key pathways.

ResultsWe discovered significant differences between the microbiomes of colon mucosa and fecal samples, with sample type explaining 14% of the variation observed in the microbiome. Multiple mucosal samples were collected from each individual to investigate whether the gut microbiome differed between polyp and healthy intestinal tissue, but no differences were found. Colon mucosa sampling revealed that the microbiomes of individuals with tubular adenomas and serrated polyps were significantly different from each other and polyp-free individuals, explaining 2-10% of the variance in the microbiome. Further analysis revealed differential abundances of 6 microbes and 1,143 microbial genes across tubular adenoma, serrated polyp, and polyp-free cases.

ConclusionBy directly sampling the colon mucosa and distinguishing between the different developmental pathways of colorectal cancer, this study helps characterize potential mechanistic targets for serrated carcinogenesis. This research also provides insight into multiple microbiome sampling strategies by assessing each methods practicality and effect on microbial community composition.

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Benchmark of data processing methods and machine learning models for gut microbiome-based diagnosis of inflammatory bowel disease BackgroundInflammatory bowel disease (IBD) patients wait months and undergo numerous invasive procedures between the initial appearance of symptoms and receiving a diagnosis. In order to reduce time until diagnosis and improve patient wellbeing, machine learning algorithms capable of diagnosing IBD from the gut microbiomes composition are currently being explored. To date, these models have had limited clinical application due to decreased performance when applied to a new cohort of patient samples. Various methods have been developed to analyze microbiome data which may improve the generalizability of machine learning IBD diagnostic tests. With an abundance of methods, there is a need to benchmark the performance and generalizability of various machine learning pipelines (from data processing to training a machine learning model) for microbiome-based IBD diagnostic tools.

ResultsWe collected fifteen 16S rRNA microbiome datasets (7707 samples) from North America to benchmark combinations of gut microbiome features, data normalization methods, batch effect reduction methods, and machine learning models. Pipeline generalizability to new cohorts of patients was evaluated with four binary classification metrics following leave-one dataset-out cross validation, where all samples from one study were left out of the training set and tested upon. We demonstrate that taxonomic features obtained from QIIME2 lead to better classification of samples from IBD patients than inferred functional features obtained from PICRUSt2. In addition, machine learning models that identify non-linear decision boundaries between labels are more generalizable than those that are linearly constrained. Prior to training a non-linear machine learning model on taxonomic features, it is important to apply a compositional normalization method and remove batch effects with the naive zero-centering method. Lastly, we illustrate the importance of generating a curated training dataset to ensure similar performance across patient demographics.

ConclusionsThese findings will help improve the generalizability of machine learning models as we move towards non-invasive diagnostic and disease management tools for patients with IBD.

sentences: Host-Adapted Strains of Spodoptera frugiperda Hold and Share a Core Microbial Community Across the Western Hemisphere The fall armyworm Spodoptera frugiperda is an important polyphagous agricultural pest in the Western Hemisphere and currently invasive to countries of the Eastern Hemisphere. This species has two host-adapted strains named "rice" and "corn" strains. Our goal was to identify the occurrence of core members in the gut bacterial community of Fall armyworm larvae from distinct geographical distribution and/or host strain. We used next-generation sequencing to identify the microbial communities of S. frugiperda from corn fields in Brazil, Colombia, Mexico, Panama, Paraguay, and Peru, and rice fields from Panama. The larval gut microbiota of S. frugiperda larvae did not differ between the host strains neither was it affected by the geographical distribution of the populations investigated. Our findings provide additional support for Enterococcus and Pseudomonas as core members of the bacterial community associated with the larval gut of S. frugiperda, regardless of the site of collection or strain, suggesting that these bacteria may maintain true symbiotic relationships with the fall armyworm. Further investigations are required for a deeper understanding of the nature of this relationship.

sentences: Relation of inflammatory marker trajectories with frailty and aging in a 20-year longitudinal study Little is known about the development of low-grade inflammation with age and its relationship with the onset of frailty. In this exploratory study, we investigated 18 inflammatory markers measured in blood of 144 individuals aged 65-75 years at study endpoint, collected over 20 years at five-year intervals. IFN{gamma}-induced markers and platelet activation markers changed in synchrony over time. Chronically elevated levels of IL-6-related markers, such as CRP and sIL-6R, were associated with frailty and becoming frail over time, poorer lung function, or less physical strength. Overweight was a possible driver of these associations. More and stronger associations were detected in women, such as between increasing sCD14 levels and frailty, indicating possible monocyte overactivation. Multivariate prediction of frailty showed low accuracy but confirmed the main results. In summary, we documented 20-year temporal changes of inflammatory markers in an aging population, and related these to clinically relevant health outcomes.

sentences: A Mycobacterium tuberculosis effector targets mitochondrion, controls energy metabolism and limits cytochrome c exit. Host metabolism reprogramming is a key feature of Mycobacterium tuberculosis (Mtb) infection that enables the survival of this pathogen within phagocytic cells and modulates the immune response facilitating the spread of the tuberculosis disease. Here, we demonstrate that a previously uncharacterized secreted protein from Mtb, Rv1813c manipulates the host metabolism by targeting mitochondria. When expressed in eukaryotic cells, the protein is delivered to the mitochondrial intermembrane space and enhances host ATP production by boosting the oxidative phosphorylation metabolic pathway. Furthermore, Rv1813c appears to differentially modulate the host cell response to oxidative stress. Expression of Rv1813 in host cells inhibits the release of cytochrome c from mitochondria, an early apoptotic event, in response to short-term oxidative stress. However, Rv1813c expressing cells showed increased sensitivity to prolonged stress. This study reveals a novel class of mitochondria targeting effectors from Mtb and opens new research directions on host metabolic reprogramming and apoptosis control.

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Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution FoF1-ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. Single-molecule Forster resonance energy transfer (smFRET) has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an Anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 complexes uncoupled from the membrane-embedded Fo complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.

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Parameter inference for stochastic biochemical models from perturbation experiments parallelised at the single cell level Understanding and characterising biochemical processes inside single cells requires experimental platforms that allow one to perturb and observe the dynamics of such processes as well as computational methods to build and parameterise models from the collected data. Recent progress with experimental platforms and optogenetics has made it possible to expose each cell in an experiment to an individualised input and automatically record cellular responses over days with fine time resolution. However, methods to infer parameters of stochastic kinetic models from single-cell longitudinal data have generally been developed under the assumption that experimental data is sparse and that responses of cells to at most a few different input perturbations can be observed. Here, we investigate and compare different approaches for calculating parameter likelihoods of single-cell longitudinal data based on approximations of the chemical master equation (CME) with a particular focus on coupling the linear noise approximation (LNA) or moment closure methods to a Kalman filter. We show that, as long as cells are measured sufficiently frequently, coupling the LNA to a Kalman filter allows one to accurately approximate likelihoods and to infer model parameters from data even in cases where the LNA provides poor approximations of the CME. Furthermore, the computational cost of filtering-based iterative likelihood evaluation scales advantageously in the number of measurement times and different input perturbations and is thus ideally suited for data obtained from modern experimental platforms. To demonstrate the practical usefulness of these results, we perform an experiment in which single cells, equipped with an optogenetic gene expression system, are exposed to various different light-input sequences and measured at several hundred time points and use parameter inference based on iterative likelihood evaluation to parameterise a stochastic model of the system.

Author summaryA common result for the modelling of cellular processes is that available data is not sufficiently rich to uniquely determine the biological mechanism or even just to ensure identifiability of parameters of a given model. Perturbing cellular processes with informative input stimuli and measuring dynamical responses may alleviate this problem. With the development of novel experimental platforms, we are now in a position to parallelise such perturbation experiments at the single cell level. This raises a plethora of new questions. Is it more informative to diversify input perturbations but to observe only few cells for each input or should we rather ensure that many cells are observed for only few inputs? How can we calculate likelihoods and infer parameters of stochastic kinetic models from data sets in which each cell receives a different input perturbation? How does the computational efficiency of parameter inference methods scale with the number of inputs and the number of measurement times? Are there approaches that are particularly well-suited for such data sets? In this paper, we investigate these questions using the CcaS/CcaR optogenetic system driving the expression of a fluorescent reporter protein as primary case study.

sentences: Cryo-EM structure of the Rhodobaca bogoriensis RC-LH1-PufX dimeric complex at 2.9 A The reaction centre-light harvesting 1 (RC-LH1) complex is essential for converting light into proton motive force in photosynthetic bacteria. RC-LH1 is a monomer in most purple bacteria, but in Rhodobacter species, it is a dimer. Its assembly depends on an accessory polypeptide (PufX) and, ultimately, on photosynthetic growth. To date, knowledge on the RC-LH1-PufX structure, where the dimer has two incomplete  C-shaped antenna rings surrounding an RC, is mainly limited to the model organism Rhodobacter sphaeroides. Here we present a cryo-electron microscopy structure at 2.9 [A] from Rhodobaca bogorensis strain LBB1. RCs are surrounded by 30 antennas and incorporate protein Y and PufX. RCs are stably connected by PufX, which self-interacts, electrostatically attracts cytochrome c2 (cyt c2) and forms extensive networks with co-factors. This structure underlines coordinated energy transfer in a combinatorial manner, providing a basis to describe bacterial photosynthesis within a dimeric photosynthetic apparatus.

sentences: Sterile Inflammation Alters Neutrophil Kinetics in Mice Neutrophils play a crucial role in establishing inflammation in response to an infection or injury, but their production rates, as well as blood and tissue residence times, remain poorly characterized under these conditions. Herein, using a biomaterial implant model to establish inflammation followed by in vivo tracking of newly formed neutrophils, we determine neutrophil kinetics under inflammatory conditions. To obtain quantifiable information from our experimental observations, we develop an ordinary differential equation-based mathematical model to extract kinetic parameters. Our data show that in the presence of inflammation resulting in emergency granulopoiesis-like conditions, neutrophil maturation time in the bone marrow and half-life in the blood reduces by about 40%, compared to non-inflammatory conditions. Additionally, neutrophil residence time at the inflammatory site increases by two-fold. Together, these data improve our understanding of neutrophil kinetics under inflammatory conditions, which could pave the way for therapies that focus on modulating in vivo neutrophil dynamics.

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PyRodentTracks: flexible computer vision and RFID based system for multiple rodent tracking and behavioral assessment. PyRodentTracks (PRT) is a scalable and customizable computer vision and RFID- based system for multiple rodent tracking and behavior assessment that can be set up within minutes in any user-defined arena at minimal cost. PRT is composed of the online Raspberry Pi-based video and RFID acquisition and the subsequent offline analysis tools. The system is capable of tracking up to 6 mice in experiments ranging from minutes to days. PRT maintained a minimum of 88% detections tracked with an overall accuracy >85% when compared to manual validation of videos containing 1-4 mice in a modified home-cage. As expected, chronic recording in home-cage revealed diurnal activity patterns. Moreover, it was observed that novel non-cagemate mice pairs exhibit more similarity in travel trajectory patterns over a 10-minute period in the openfield than cagemates. Therefore, shared features within travel trajectories between animals may be a measure of sociability that has not been previously reported. Moreover, PRT can interface with open-source packages such as Deeplabcut and Traja for pose estimation and travel trajectory analysis, respectively. In combination with Traja, PRT resolved motor deficits exhibited in stroke animals. Overall, we present an affordable, open-sourced, and customizable/scalable rodent-specific behavior recording and analysis system.

Statement of SignificanceAn affordable, customizable, and easy-to-use open-source rodent tracking system is described. To tackle the increasingly complex questions in neuroscience, researchers need a flexible system to track rodents of different coat colors in various complex experimental paradigms. The majority of current tools, commercial or otherwise, can only be fully automated to track multiple animals of the same type in a single defined environment and are not easily setup within custom arenas or cages. Moreover, many tools are not only expensive but are also difficult to set up and use, often requiring users to have extensive hardware and software knowledge. In contrast, PRT is easy to install and can be adapted to track rodents of any coat color in any user-defined environment with few restrictions. We believe that PRT will be an invaluable tool for researchers that are quantifying behavior in identified animals.

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Genetic epidemiology of blood type, disease and trait variants, and genome-wide genetic diversity in over 11,000 domestic cats In the largest DNA-based study of domestic cat to date, 11,036 individuals (10,419 pedigreed cats from 91 breeds and breed types and 617 non-pedigreed cats) were genotyped via commercial panel testing, elucidating the distribution and frequency of known genetic variants associated with blood type, disease and physical traits across cat breeds. Blood group determining variants, which are relevant clinically and in cat breeding, were genotyped to assess the across breed distribution of blood types A, B and AB. Extensive panel testing identified 13 disease-associated variants in 48 breeds or breed types for which the variant had not previously been observed, strengthening the argument for panel testing across populations. The study also indicates that multiple breed clubs have effectively used DNA testing to reduce disease-associated genetic variants within certain pedigreed cat populations. Appearance-associated genetic variation in all cats is also discussed. Additionally, we combined genotypic data with phenotype information and clinical documentation, actively conducted owner and veterinarian interviews, and recruited cats for clinical examination to investigate the causality of a number of tested variants across different breed backgrounds. Lastly, genome-wide informative SNP heterozygosity levels were calculated to obtain a comparable measure of the genetic diversity in different cat breeds.

This study represents the first comprehensive exploration of informative Mendelian variants in felines by screening over 10,000 domestic cats. The results qualitatively contribute to the understanding of feline variant heritage and genetic diversity and demonstrate the clinical utility and importance of such information in supporting breeding programs and the research community. The work also highlights the crucial commitment of pedigreed cat breeders and registries in supporting the establishment of large genomic databases that when combined with phenotype information can advance scientific understanding and provide insights that can be applied to improve the health and welfare of cats.

sentences: Ribosome-profiling reveals restricted post transcriptional ex-pression of antiviral cytokines and transcription factors during SARS-CoV-2 infection The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions; however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.

sentences: Slow, heterogeneous NFκB single molecule conformational dynamics and implications for function Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-{kappa}B from DNA is achieved through accelerated dissociation by the inhibitor protein I{kappa}B. Using single-molecule FRET, we observe a continuum of conformations of NF-{kappa}B in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-{kappa}B is partially bound, allowing I{kappa}B invasion to facilitate DNA dissociation. I{kappa}B induces a locked conformation where the DNA-binding domains of NF-{kappa}B are too far apart to bind DNA, whereas the loss-of-function I{kappa}B mutant retains the NF-{kappa}B conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.

sentences: 3D-MASNet: 3D Mixed-scale Asymmetric Convolutional Segmentation Network for 6-month-old Infant Brain MR Images Precise segmentation of infant brain MR images into gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) is essential for studying neuroanatomical hallmarks of early brain development. However, for 6-month-old infants, the extremely low-intensity contrast caused by inherent myelination hinders accurate tissue segmentation. Existing convolutional neural networks (CNNs) based segmentation model for this task generally employ single-scale symmetric convolutions, which are inefficient for encoding the isointense tissue boundaries in limited samples of baby brain images. Here, we propose a 3D mixed-scale asymmetric convolutional segmentation network (3D-MASNet) framework for brain MR images of 6-month-old infant. We replaced the traditional convolutional layer of an existing to-be-trained network with a 3D mixed-scale convolution block consisting of asymmetric kernels (MixACB) during the training phase and then equivalently converted it into the original network. Five canonical CNN segmentation models were evaluated using both T1- and T2-weighted images of 23 6-month-old infants from iSeg-2019 datasets, which contained manual labels as ground truth. MixACB significantly enhanced the average accuracy of all five models and obtained the largest improvement in the fully convolutional network model (CC-3D-FCN) and the highest performance in the Dense U-Net model. This approach further obtained Dice coefficient accuracies of 0.931, 0.912, and 0.961 in GM, WM, and CSF, respectively, ranking first among 30 teams on the validation dataset of the iSeg-2019 Grand Challenge. Thus, the proposed 3D-MASNet can improve the accuracy of existing CNNs-based segmentation models as a plug-and-play solution that offers a promising technique for future infant brain MRI studies.

sentences: A MYT1L Syndrome mouse model recapitulates patient phenotypes and reveals altered brain development due to disrupted neuronal maturation Human genetics have defined a new autism-associated syndrome caused by loss-of-function mutations in MYT1L, a transcription factor known for enabling fibroblast-to-neuron conversions. However, how MYT1L mutation causes autism, ADHD, intellectual disability, obesity, and brain anomalies is unknown. Here, we develop a mouse model of this syndrome. Physically, Myt1l haploinsufficiency causes obesity, white-matter thinning, and microcephaly in the mice, mimicking clinical phenotypes. During brain development we discovered disrupted gene expression, mediated in part by loss of Myt1l gene target activation, and identified precocious neuronal differentiation as the mechanism for microcephaly. In contrast, in adults we discovered that mutation results in failure of transcriptional and chromatin maturation, echoed in disruptions in baseline physiological properties of neurons. This results in behavioral anomalies including hyperactivity, muscle weakness and fatigue, and social alterations with more severe phenotypes in males. Overall, our findings provide insight into the mechanistic underpinnings of this disorder and enable future preclinical studies.

sentences: Dynamic influences on static measures of metacognition Humans differ in their capability to judge the accuracy of their own choices via confidence judgments. Signal detection theory has been used to quantify the extent to which confidence tracks accuracy via M-ratio, often referred to as metacognitive efficiency. This measure, however, is static in that it does not consider the dynamics of decision making. This could be problematic because humans may shift their level of response caution to alter the tradeoff between speed and accuracy. Such shifts could induce unaccounted-for sources of variation in the assessment of metacognition. Instead, evidence accumulation frameworks consider decision making, including the computation of confidence, as a dynamic process unfolding over time. We draw on evidence accumulation frameworks to examine the influence of response caution on metacognition. Simulation results demonstrate that response caution has an influence on M-ratio. We then tested and confirmed that this was also the case in human participants who were explicitly instructed to either focus on speed or accuracy. We next demonstrated that this association between M-ratio and response caution was also present in an experiment without any reference towards speed. The latter finding was replicated in an independent dataset. In contrast, when data were analyzed with a novel dynamic measure of metacognition, which we refer to as v-ratio, in all of the three studies there was no effect of speed-accuracy tradeoff. These findings have important implications for research on metacognition, such as its measurement, domain-generality, individual differences, and neural correlates.

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Unravelling processes between phenotypic plasticity and population dynamics in migratory birds O_LIPopulations can rapidly respond to environmental change via adaptive phenotypic plasticity, which can also modify interactions between individuals and their environment, affecting population dynamics. Bird migration is a highly plastic resource-tracking strategy in seasonal environments. However, the link between the population dynamics of migratory birds and migration strategy plasticity is not well understood.
C_LIO_LIThe quality of staging habitats affects individuals migration timing and energy budgets in the course of migration, and can consequently affect individuals breeding and overwintering performance, and impact population dynamics. Given staging habitats being lost in many parts of the world, our goal is to investigate responses of individual migration strategies and population dynamics in the face of loss of staging habitat, and to identify the key processes connecting them.
C_LIO_LIWe started by constructed and analysed a general full-annual-cycle individual-based model with a stylized migratory population to generate hypotheses on how changes in the size of staging habitat might drive changes in individual stopover duration and population dynamics, and to identify the key processes connecting them. Next, through the interrogation of census data, we tested these hypotheses by analysing population trends and stopover duration of migratory waterbirds experiencing loss of staging habitat.
C_LIO_LIWe found empirical support for our modelling-identified hypotheses: the loss of staging habitat generates plasticity in migration strategies, with individuals remaining on the staging habitat for longer to obtain food due to a reduction in per capita food availability. The subsequent increasing population density on the staging habitat has knock on effects on population dynamics in the breeding and overwintering stage.
C_LIO_LIOur results demonstrate how environmental change that impacts one energetically costly life history stage in migratory birds can have population dynamics impacts across the entire annual cycle via phenotypic plasticity.
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Global analysis of multi-mutants to discover stabilizing amino acid substitutions The identification of amino acid substitutions that both enhance the stability and function of a protein is a key challenge in protein engineering. Technological advances have enabled assaying thousands of protein variants in a single high-throughput experiment, and more recent studies use such data in protein engineering. We present a Global Multi-Mutant Analysis (GMMA) that exploits the presence of multiply-substituted variants to identify individual amino acid substitutions that are beneficial for the stability and function across a large library of protein variants. We have applied GMMA to >54,000 variants of green fluorescent protein (GFP), each with known fluorescence output, and each carrying 1-15 amino acid substitutions. The GMMA method achieves a good fit to the data while being analytically transparent. The six top-ranking substitutions are demonstrated to progressively enhance GFP and in general, our analysis recovers nearly all the substitutions previously reported to be beneficial for GFP folding and function, only using a single experiment as input.

Significance StatementProtein engineering is carried out to improve proteins for practical applications by changing one or more amino acid residues in a protein. We present a method termed global multi-mutant analysis (GMMA) that helps solve two problems in protein engineering. First, because many proteins are already highly optimized, it can be difficult to identify individual variants that further improve function. Second, while it is possible to combine variants with small individual effects, such approaches may be hampered by non-additivity. GMMA identifies combinable effects of single substitutions from a large set of variants each carrying multiple substitutions. We demonstrate the approach on a set of 54,000 variants of green fluorescent protein and identify many enhancing single-substitutions from a single experiment.

sentences: Evidence of post-domestication hybridization and adaptive introgression in Western European grapevine varieties Grapevine (Vitis vinifera L.) is one of the most significant crops in the world. Todays richness in grapevine diversity results from a complex domestication history over multiple historical periods. Here, we employed whole genome resequencing to elucidate different aspects of the recent evolutionary history of this crop. Our results support a model in which a central domestication event in grapevine was followed by post-domestication hybridization with local wild genotypes, leading to the presence of an introgression signature in modern wine varieties across Western Europe. The strongest signal was associated with a subset of Iberian grapevine varieties, which show large introgression tracts. We targeted this study group for further analysis, demonstrating how regions under selection in wild populations from the Iberian Peninsula were preferentially passed on to the cultivated varieties by geneflow. Examination of underlying genes suggests that environmental adaptation played a fundamental role in both the evolution of wild genotypes and the outcome of hybridization with cultivated varieties, supporting a case of adaptive introgression in grapevine.

sentences: Piezo1 activation using Yoda1 inhibits macropinocytosis and proliferation of cancer cells Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by macropinosome formation. A certain number of mammalian mechanosensors are sensitive to membrane deformation and tension. However, it remains unclear whether macropinocytosis is regulated by mechanosensors. Focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). Although studies with Piezo1 knockout cells suggest that Piezo1 itself is not physiologically indispensable for macropinocytosis regulation, Yoda1 inhibited ruffle formation depending on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. Moreover, Yoda1 impaired cancer cell proliferation, suggesting the impact of macropinocytosis inhibition. We propose the potential for cancer therapy by macropinocytosis inhibition through the regulation of a mechanosensitive channel activity.

sentences: Tissue-specific and tissue-agnostic effects of genome sequence variation modulating blood pressure Genome-wide association studies (GWAS) have mapped thousands of variants for numerous polygenic traits and diseases. However, with some exceptions, mechanistic understanding of which precise variants affect which genes in which tissues to modulate trait variation is still lacking. To this end, we introduce a novel class of genomic analyses applicable to any complex trait using GWAS together with gene expression and chromatin accessibility data from multiple tissues. Here we identify the transcription factors (TFs) and regulatory variants within active enhancers regulating specific genes in individual tissues to explain trait heritability of blood pressure (BP), a classical polygenic trait. We show that kidney-, adrenal-, heart-, and arterial-specific regulatory variants contribute to 2.5%, 5.3%, 7.7%, and 11.8% of variant heritability, respectively. Collectively, [~]500,000 predicted regulatory variants across these four tissues explain 33.4% of variant heritability. We demonstrate that these variants are enriched in enhancers binding specific TFs in each tissue. Our findings suggest that gene regulatory networks perturbed by common regulatory variants in a tissue relevant to a phenotype are the primary source of interindividual variation of BP. These studies provide an approach to scan each human tissue for its physiological contribution to a trait.

sentences: An evidence-based 3D reconstruction of Asteroxylon mackiei the most complex plant preserved from the Rhynie chert The 407-million-year-old Rhynie chert preserves the earliest terrestrial ecosystem and informs our understanding of early life on land. However, our knowledge of the 3D structure, and development of these plants is still rudimentary. Here we used digital 3D reconstruction techniques to produce the first complete reconstruction of the lycopsid Asteroxylon mackiei, the most complex plant in the Rhynie chert. The reconstruction reveals the organisation of the three distinct axes types - leafy shoot axes, root-bearing axes and rooting axes - in the body plan. Combining this reconstruction with developmental data from fossilised meristems, we demonstrate that the A. mackiei rooting axis - a transitional lycophyte organ between the rootless ancestral state and true roots - developed from root-bearing axes by anisotomous dichotomy. Our discovery demonstrates how this unique organ developed, and highlights the value of evidence-based reconstructions for understanding the development and evolution of the first complex plants on Earth.

sentences: Coordination of -1 Programmed Ribosomal Frameshifting by Transcript and Nascent Chain Features Revealed by Deep Mutational Scanning Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. In the alphavirus structural polyprotein, -1PRF is coordinated by a "slippery" sequence in the transcript, an RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4,530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop disrupt -1PRF, mutagenic effects upstream of the slip-site are far more variable. Molecular dynamics simulations of polyprotein biogenesis suggest many of these mutations alter stimulatory forces on the nascent chain through their effects on translocon-mediated cotranslational folding. Finally, we provide evidence suggesting the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.

sentences: Landscape connectivity alters the evolution of density-dependent dispersal during pushed range expansions AO_SCPLOWBSTRACTC_SCPLOWAs human influence reshapes communities worldwide, many species expand or shift their ranges as a result, with extensive consequences across levels of biological organization. Range expansions can be ranked on a continuum going from pulled dynamics, in which low-density edge populations provide the "fuel" for the advance, to pushed dynamics in which high-density rear populations "push" the expansion forward. While theory suggests that evolution during range expansions could lead pushed expansions to become pulled with time, empirical comparisons of phenotypic divergence in pushed vs. pulled contexts are lacking. In a previous experiment using Trichogramma brassicae wasps as a model, we showed that expansions were more pushed when connectivity was lower. Here we used descendants from these experimental landscapes to look at how the range expansion process and connectivity interact to shape phenotypic evolution. Interestingly, we found no clear and consistent phenotypic shifts, whether along expansion gradients or between reference and low connectivity replicates, when we focused on low-density trait expression. However, we found evidence of changes in density-dependence, in particular regarding dispersal: populations went from positive to negative density-dependent dispersal at the expansion edge, but only when connectivity was high. As positive density-dependent dispersal leads to pushed expansions, our results confirm predictions that evolution during range expansions may lead pushed expansions to become pulled, but add nuance by showing landscape conditions may slow down or cancel this process. This shows we need to jointly consider evolution and landscape context to accurately predict range expansion dynamics and their consequences.

sentences: Transfer learning via multi-scale convolutional neural layers for human-virus protein-protein interaction prediction To predict interactions between human and viral proteins, we combine evolutionary sequence profile features with a Siamese convolutional neural network (CNN) architecture and a multi-layer perceptron (MLP). Our architecture outperforms various feature encodings-based machine learning and state-of-the-art prediction methods. As our main contribution, we introduce two types of transfer learning methods (i.e.,  frozen type and  fine-tuning type) that reliably predict interactions in a target human-virus domain based on training in a source human-virus domain, by retraining CNN layers. Our transfer learning strategies can effectively apply prior knowledge transfer from large source dataset/task to small target dataset/task to improve prediction performance. Finally, we utilize the  frozen type of transfer learning to predict human-SARS-CoV-2 PPIs, indicating that our predictions are topologically and functionally similar to experimentally known interactions. Source code and datasets are available at https://github.com/XiaodiYangCAU/TransPPI/.

sentences: Use of eVLP-based vaccine candidates to broaden immunity against SARS-CoV-2 variants Rapid emergence of SARS-CoV-2 variants is a constant threat and a major hurdle to reach heard immunity. We produced VBI-2905a, an enveloped virus-like particle (eVLP)-based vaccine candidate expressing prefusion spike protein from the Beta variant that contains several escape mutations. VBI-2905a protected hamsters against infection with a Beta variant virus and induced high levels of neutralizing antibodies against Beta RBD. In a heterologous vaccination regimen, a single injection of VBI-2905a in animals previously immunized with VBI-2902, a vaccine candidate expressing S from ancestral SARS-CoV-2, hamsters were equally protected against Beta variant infection. As an alternate strategy to broaden immunity, we produced a trivalent vaccine expressing the prefusion spike protein from SARS-CoV-2 together with unmodifed S from SARS-CoV-1 and MERS-CoV. Relative to immunity induced against the ancestral strain, the trivalent vaccine VBI-2901a induced higher and more consistent antibody binding and neutralizing responses against a panel of variants including Beta, Delta, Kappa, and Lambda, with evidence for broadening of immunity rather than just boosting cross-reactive antibodies.

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Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders.

HighlightsO_LINaMN binds to SARM1 N-terminal allosteric site and inhibits SARM1 NAD+ hydrolase
C_LIO_LINaMN inhibits SARM1 activation by stabilizing its open, inactive structure
C_LIO_LINMN deamidase promotes strong axonal protection by reducing NMN and increasing NaMN
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sentences: Nanometer-Scale Imaging of Compartment-Specific Localization and Dynamics of Voltage-Gated Sodium Channels Membrane excitability and cell-to-cell communication in the brain are tightly regulated by diverse ion channels and receptor proteins localized to distinct membrane compartments. Currently, a major technical barrier in cellular neuroscience is lack of reliable methods to label these membrane proteins and image their sub-cellular localization and dynamics. To overcome this challenge, we devised optical imaging strategies that enable systematic characterization of subcellular composition, relative abundances and trafficking dynamics of membrane proteins at nanometer scales in cultured neurons as well as in the brain. Using these methods, we revealed exquisite developmental regulation of subcellular distributions of voltage-gated sodium channel (VGSC) Nav1.2 and Nav1.6, settling a decade long debate regarding the molecular identity of sodium channels in dendrites. In addition, we discovered a previously uncharacterized trafficking pathway that targets Nav1.2 to unmyelinated fragments in the distal axon. Myelination counteracts this pathway, facilitating the installment of Nav1.6 as the dominant VGSC in the axon. Together, these imaging approaches unveiled compartment-specific trafficking mechanisms underpinning differential membrane distributions of VGSCs and open avenues to decipher how membrane protein localization and dynamics contribute to neural computation in the brain.

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Aspirin Protective Effect Against Cyclophosphamide Hematological Toxicity In Experimental Animals Bone marrow toxicity is the most important factor limiting the use of cytotoxic drugs like alkylating agents in cancer treatment. Recently PG synthase enzyme inhibitors have been reported to potentiate the cytotoxic effects of these agents on cancer cells but little is known if they can affect the toxicity of these agents on bone marrow or other tissues. Cyclophosphamide is one of the most commonly used alkylating agent.

In the present work, the effect of these PG synthase enzyme inhibitors, aspirin on cyclophosphamide myelotoxicity was determined employing the peripheral blood count to reflect bone marrow injury. The effect on body weight changes caused by cyclophosphamide was also determined.

O_LICyclophosphamide in doses of 25, 50 and 75 mg/kg i. v. produced as a dose dependent reduction in total WBC count, granulocyte, non granulocyte, and Hb% which was maximum on second day after injection and still present on 5th day post injection. It also produced a dose dependent reduction in body weight on day 5 after injection.
C_LIO_LIAspirin in doges of 75, 150 and 300 mg/kg i. m. protected against the reduction in WBC counts  measured for 5 days after injection of cyclophosphamide (50 mg/kg). This protection was not dose dependent, though it was more optimum with 300 mg/kg and disappeared largely when a dose of 450 mg/kg was used. Aspirin did not prevent the changes in Hb% but retard the reduction in body weight caused by cyclophosphamide.
C_LIO_LIIt is concluded that aspirin can help to reduce injury and enhance recovery from bone marrow toxicity caused by cytotoxic agents such as the alkylating drugs cyclophosphamide for which no specific antidote is available. Aspirin produces this effect possibly by eliminating the harmful inhibitory effect of excess PGs or leukotrienes, released by bone marrow injury on growth factors of haemopoietic progenitor cells.
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The magnitude of this protection on WBC counts does not seem to differ between either PG synthase enzyme inhibitors or steroids when used alone or in combination although a synergistic effect in protecting erythropoiesis is observed.

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Group size and modularity interact to shape the spread of infection and information through animal societies Social interactions between animals can provide many benefits, including the ability to gain useful environmental information through social learning. However, these social contacts can also facilitate the transmission of infectious diseases through a population. Animals engaging in social interactions must therefore face a trade-off between the potential informational benefits and the risk of acquiring disease. In order to understand how this trade-off can influence animal sociality, it is necessary to quantify the effects of different social structures on individuals likelihood of acquiring information versus infection Theoretical models have suggested that modular social networks, associated with the formation of groups or sub-groups, can slow spread of infection by trapping it within particular groups. However these social structures will not necessarily impact the spread of information in the same way if its transmission is considered as a "complex contagion", e.g. through individuals copying the majority (conformist learning). Here we use simulation models to demonstrate that modular networks can promote the spread of information relative to the spread of infection, but only when the network is fragmented and group sizes are small. We show that the difference in transmission between information and disease is maximised for more well-connected social networks when the likelihood of transmission is intermediate. Our results have important implications for understanding the selective pressures operating on the social structure of animal societies, revealing that highly fragmented networks such as those formed in fission-fusion social groups and multilevel societies can be effective in modulating the infection-information trade-off for individuals within them.

Significance statementRisk of infection is commonly regarded as one of the costs of animal social behaviours, while the potential for acquiring useful information is seen as a benefit. Balancing this risk of infection with the potential to gain useful information is one of the key trade-offs facing animals that engage in social interactions. In order to better understand this trade-off, it is necessary to quantify how different social structures can promote access to useful information while minimising risk of infection. We used simulations of disease and information spread to examine how group sizes and social network fragmentation influences both these transmission processes. Our models find that more subdivided networks slow the spread of disease far more than infection, but only group sizes are small. Our results demonstrate that showing that fragmented social structures can be more effective in balancing the infection-information trade-off for individuals within them.

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Intercellular transport of RNA can limit heritable epigenetic changes RNAs in circulation carry sequence-specific regulatory information between cells in animal, plant, and host-pathogen systems. Double-stranded RNA (dsRNA) delivered into the extracellular space of the nematode C. elegans accumulates within the germline and reaches progeny. Here we provide evidence for spatial, temporal, and substrate specificity in the transport of dsRNA from parental circulation to progeny. Temporary loss of dsRNA transport resulted in the persistent accumulation of mRNA from a germline gene. The expression of this gene varied among siblings and even between gonad arms within one animal. Perturbing RNA regulation of the gene created new epigenetic states that lasted for many generations. Thus, one role for the transport of dsRNA into the germline in every generation is to limit heritable changes in gene expression.

One Sentence SummaryRNA from parental circulation reduces heritable changes in gene expression.

sentences: Hippocampal spatio-temporal cognitive maps adaptively guide reward generalization The brain forms cognitive maps of relational knowledge, an organizing principle thought to underlie our ability to generalize and make inferences. However, how can a relevant map be selected in situations where a stimulus is embedded in multiple relational structures? Here, we find that both spatial and temporal cognitive maps influence generalization in a choice task, where spatial location determines reward magnitude. Mirroring behavior, the hippocampus not only builds a map of spatial relationships but also encodes temporal distances. As the task progresses, participants choices become more influenced by spatial relationships, reflected in a strengthening of the spatial and a weakening of the temporal map. This change is driven by orbitofrontal cortex, which represents the evidence that an observed outcome is generated from the spatial rather than the temporal map and updates hippocampal representations accordingly. Taken together, this demonstrates how hippocampal cognitive maps are used and updated flexibly for inference.

sentences: Colletotrichum higginsianum effectors exhibit cell to cell hypermobility in plant tissues and modulate intercellular connectivity amongst a variety of cellular processes Multicellular organisms exchange information and resources between cells to co-ordinate growth and responses. In plants, plasmodesmata establish cytoplasmic continuity between cells to allow for communication and resource exchange across the cell wall. Some plant pathogens use plasmodesmata as a pathway for both molecular and physical invasion. However, the benefits of molecular invasion (cell-to-cell movement of pathogen effectors) are poorly understood. To begin to investigate this and identify which effectors are cell-to-cell mobile, we performed a live imaging-based screen and identified 15 cell-to-cell mobile effectors of the fungal pathogen Colletotrichum higginsianum. Of these, 6 are "hypermobile", showing cell-to-cell mobility greater than expected for a protein of its size. We further identified 3 effectors that can indirectly modify plasmodesmal aperture. Transcriptional profiling of plants expressing hypermobile effectors implicate them in a variety of processes including senescence, glucosinolate production, cell wall integrity, growth and iron metabolism. However, not all effectors had an independent effect on virulence. This suggests a wide range of benefits to infection gained by the mobility of C. higginsianum effectors that likely interact in a complex way during infection.

sentences: Effects of larval and adult crowding on fitness components in Drosophila populations adapted to larval crowding experienced under different combinations of food amount and egg number Since the realization in the 1970s that simple discrete-time population growth models can show complex unstable dynamics of population size, many explanations were proposed for the evolution of enhanced population stability. The most plausible of these was density-dependent selection, suggested to favour greater stability due to r-K trade-offs. However, the first experiment aimed at testing this prediction revealed that Drosophila melanogaster populations adapted to larval crowding did not evolve greater constancy stability than their ancestral controls. A subsequent study showed that D. ananassae populations adapted to larval crowding had evolved greater constancy and persistence than ancestral controls. These D. ananassae populations had experienced chronic larval crowding in conditions of very low amounts of food, whereas the earlier studied D. melanogaster populations had experienced chronic larval crowding at fairly high food amounts. Further theoretical work also suggested that populations adapting to crowding could evolve greater stability even in the absence of r-K trade-offs. Most recently, studies in our laboratory showed that two sets of crowding adapted D. melanogaster populations, derived from a common ancestral lineage, which differed in the food amounts at which they experienced larval crowding, evolved different patterns of constancy and persistence stability. These two sets of populations also differed in the traits, e.g. larval feeding rate, that evolved as they became more competitive. Here, we examine the response of key fitness components to larval and adult densities in these two sets of populations, to see whether differences in their stability attributes can be explained by variation in how their life-histories respond to crowding at different life stages. Of all traits examined, only pre-adult survivorship responded differently to larval density across the two sets of populations. The populations that adapted to larval crowding at low food amounts showed reduced sensitivity of pre-adult survivorship to larval density, compared to those that adapted to larval crowding at high food amounts. We discuss our results in the context of different ways in which density-dependent selection may facilitate the evolution of greater constancy or persistence, depending on the ecological details of how crowding was experienced.

sentences: Molecular characterization of inversion breakpoints in the Drosophila nasuta species group Chromosomal inversions are fundamental drivers of genome evolution. In the Drosophila genus, inversions have been widely characterized cytologically, and play an important role in local adaptation. Here, we characterize chromosomal inversions in the Drosophila nasuta species group using chromosome-level, reference-quality assemblies of seven species and subspecies in this clade. Reconstruction of ancestral karyotypes allowed us to infer the order in which the 22 identified inversions occurred along the phylogeny. We found a higher rate of inversions on the X chromosome, and heterogeneity in the rate of accumulation across the phylogeny. We molecularly characterize the breakpoints of six autosomal inversions, and found that repeated sequences are associated with inversion breakpoints in four of these inversions, suggesting that ectopic recombination is an important mechanism in generating inversion. Characterization of inversions in this species group provides a foundation for future population genetic and functional studies in this recently diverged species group.

sentences: An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including ribonucleoprotein complexes (RNP). Here we show that orchestration of oskar RNP transport in the Drosophila germline relies on the interplay of two double-stranded RNA binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, enhanced recruitment of Staufen to the RNPs results in dissociation of Egl and reduced minus end-directed transport without overtly affecting overall dynein levels on the cargo. This mechanism allows kinesin-1 to translocate the mRNA to its final destination at the posterior pole of the oocyte. Using in vitro motility assays with purified components, we find that Staufen directly antagonizes Egl-mediated activation of oskar mRNA transport by dynein. We additionally show that Egl associates with staufen mRNA in the nurse cells and mediates enrichment of the mRNA and its protein product in the ooplasm. Our observations identify a feed-forward mechanism, whereby dynein-dependent accumulation of staufen mRNA and protein in the oocyte enables motor switching during oskar mRNA transport by downregulating dynein activity.

sentences: Variation in colour signals among Sarracenia pitcher plants and the potential role of areoles in the attraction of flying Hymenoptera Sarracenia insectivorous plants show a diversity of visual features in their pitchers but their perception by insects and their role in attraction, have received little attention. They also vary in prey composition, with some species trapping more flying Hymenoptera, such as bees. To test the hypothesis of a link between visual signal variability and prey segregation ability, and to identify which signal could attract flying Hymenoptera, we characterised, the colour patterns of 32 pitchers belonging to four taxa, modelled their perception by flying Hymenoptera, and examined the prey they trapped. The pitchers of the four taxa differed in colour patterns, with notably two long-leaved taxa displaying clear areoles, which contrasted strongly in colour and brightness with the vegetative background and with other pitcher areas in the eyes of flying Hymenoptera. These taxa trapped high proportion of flying hymenoptera. This suggests that contrasting areoles may act as a visual lure for flying Hymenoptera, making plants particularly visible to these insects. Prey capture also differed according to pitcher stage, morphology, season and visual characteristics. Further studies on prey visitation are needed to better understand the link between prey capture and attraction feature.

sentences: Transcriptomes across fertilization and seed development in the water lily Nymphaea thermarum (Nymphaeales) reveal dynamic expression of DNA and histone methylation modifiers Studies of gene expression during seed development have been performed for a growing collection of species from a phylogenetically broad sampling of flowering plants (angiosperms). However, attention has mostly been focused on crop species or a small number of  model systems. Information on gene expression during seed development is minimal for those angiosperm lineages whose origins predate the divergence of monocots and eudicots. In order to provide a new perspective on the early evolution of seed development in flowering plants, we sequenced transcriptomes of whole ovules and seeds from three key stages of reproductive development in the waterlily Nymphaea thermarum, an experimentally-tractable member of the Nymphaeales. We first explore general patterns of gene expression, beginning with mature ovules and continuing through fertilization into early- and mid-seed development. We then examine the expression of genes associated with DNA and histone methylation - processes known to be essential for development in distantly-related and structurally-divergent monocots and eudicots. Around 60% of transcripts putatively homologous to DNA and histone methylation modifiers are differentially expressed during seed development in N. thermarum, suggesting that the importance of dynamic epigenetic patterning during seed development dates to the earliest phases of angiosperm evolution. However, genes involved in establishing, maintaining, and removing methylation marks associated with genetic imprinting show a mix of conserved and unique expression patterns between N. thermarum and other flowering plants. Our data suggests that the regulation of imprinting has likely changed throughout angiosperm evolution, and furthermore identifies genes that merit further characterization in any angiosperm system.

sentences: Delayed induction of type I and III interferons and nasal epithelial cell permissiveness to SARS-CoV-2 The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we applied single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrated widespread tropism for nasal epithelial cell types. The host response was dominated by type I and III IFNs and interferon-stimulated gene products. This response was notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response began to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFN{beta} or IFN{lambda}1 induced an efficient antiviral state that potently restricted SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data suggest that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

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Complexity and graded regulation of neuronal cell type-specific alternative splicing revealed by single-cell RNA sequencing The enormous neuronal cellular diversity in the mammalian brain, which is highly prototypical and organized in a hierarchical manner, is dictated by cell type-specific gene regulatory programs at the molecular level. Although prevalent in the brain, contribution of alternative splicing (AS) to the molecular diversity across neuronal cell types is just starting to emerge. Here we systematically investigated AS regulation across over 100 transcriptomically defined neuronal types of the adult mouse cortex using deep single cell RNA-sequencing (scRNA-seq) data. We found distinct splicing programs between glutamatergic and GABAergic neurons and between subclasses within each neuronal class, consisting of overlapping sets of alternative exons showing differential splicing at multiple hierarchical levels. Using an integrative approach, our analysis suggests that RNA-binding proteins (RBPs) Celf1/2, Mbnl2 and Khdrbs3 are preferentially expressed and more active in glutamatergic neurons, while Elavl2 and Qk are preferentially expressed and more active in GABAergic neurons. Importantly, these and additional RBPs also contribute to differential splicing between neuronal subclasses at multiple hierarchical levels, and some RBPs drive splicing dynamics that do not conform to the hierarchical structure defined by the transcriptional profiles. Thus, our results suggest graded regulation of AS across neuronal cell types, which provides a molecular mechanism orthogonal to, rather than downstream of, transcriptional regulation in specifying neuronal identity and function.

SignificanceAlternative splicing (AS) is extensively used in the mammalian brain, but its contribution to the molecular and cellular diversity across neuronal cell types remains poorly understood. Through systematic and integrative analysis of AS regulation across over 100 transcriptomically defined cortical neuronal types, we found neuronal subclass-specific splicing regulatory programs consists of overlapping alternative exons showing differential splicing at multiple hierarchical levels. This graded AS regulation is controlled by unique combinations of RNA-binding proteins (RBPs). Importantly, these RBPs also drive splicing dynamics across neuronal cell types that do not conform to the hierarchical taxonomy established based on transcriptional profiles, suggesting that the graded AS regulation provides a molecular mechanism orthogonal to transcriptional regulation in specifying neuronal identity and function.

sentences: Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19 AUTHOR SUMMARYPhosphatidylserine (PS) receptors are PS binding proteins that mediate uptake of apoptotic bodies. Many enveloped viruses utilize this PS/PS receptor mechanism to adhere to and internalize into the endosomal compartment of cells and this is termed apoptotic mimicry. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. We evaluated if PS receptors serve as cell surface receptors for SARS-CoV-2 and found that the PS receptors, AXL, TIM-1 and TIM-4, facilitated virus infection when low concentrations of the SARS-CoV-2 cognate receptor, ACE2, was present. Consistent with the established mechanism of PS receptor utilization by other viruses, PS liposomes competed with SARS-CoV-2 for binding and entry. We demonstrated that this PS receptor enhances SARS-CoV-2 binding to and infection of an array of human lung cell lines and is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry.

sentences: Gut melange a trois: fluctuating selection modulated by microbiota, host immune system, and antibiotics Iron is critical in host-microbe interactions, and its availability is tightly regulated in the mammalian gut. Antibiotics and inflammation can perturb iron availability in the gut, which could alter host-microbe interactions. Here, we show that an adaptive allele of iscR, a major regulator of iron homeostasis of Escherichia coli, is under fluctuating selection in the mouse gut. In vivo competitions in immune-competent, immune-compromised, and germ-free mice reveal that the selective pressure on an iscR mutant E. coli is modulated by the presence of antibiotics, the microbiota, and the immune system. In vitro assays show that iron availability is an important mediator of the iscR allele fitness benefits or costs. We identify Lipocalin-2, a hosts immune protein that prevents bacterial iron acquisition, as a major host mechanism underlying fluctuating selection of iscR. Our results provide a remarkable example of strong fluctuating selection acting on bacterial iron regulation in the mammalian gut.

sentences: Global, regional, and cryptic population structure in a high gene-flow transatlantic fish 1Lumpfish (Cyclopterus lumpus) is a transatlantic marine fish displaying large population sizes and a high potential for dispersal and gene-flow. These features are expected to result in weak population structure. Here, we investigated population genetic structure of lumpfish throughout its natural distribution in the North Atlantic using two approaches: I) 4393 genome wide SNPs and 95 individuals from 10 locations, and II) 139 discriminatory SNPs and 1,669 individuals from 40 locations. Both approaches identified extensive population genetic structuring with a major split between the East and West Atlantic and a distinct Baltic Sea population, as well as further differentiation of lumpfish from the English Channel, Iceland, and Greenland. The targeted discriminatory loci displayed [~]2-5 times higher divergence than the genome wide approach, revealing further evidence of local population substructures. Lumpfish from Isfjorden in Svalbard were highly distinct but resembled most fish from Greenland. The Kattegat area in the Baltic transition zone, formed a previously undescribed distinct genetic group. Also, further subdivision was detected within North America, Iceland, West Greenland, Barents Sea, and Norway. Although lumpfish have considerable potential for dispersal and gene-flow, the observed high levels of population structuring throughout the Atlantic suggests that this species displays natal homing and potentially local populations with adaptive differences. This fine-scale population structure calls for consideration when defining management units for lumpfish fishing stocks and in decisions related to sourcing and moving lumpfish for farming and cleaner fish use.

sentences: Evaluating an internet-delivered fear conditioning and extinction protocol using response times and affective ratings. Pavlovian fear conditioning is widely used to study mechanisms of fear learning, but high-throughput studies are hampered by the labor-intensive nature of examining participants in the lab. To circumvent this bottle-neck, fear conditioning tasks have been developed for remote delivery. Previous studies have examined remotely delivered fear conditioning protocols using expectancy and affective ratings. Here we replicate and extend these findings using an internet-delivered version of the Screaming Lady paradigm, evaluating the effects on negative affective ratings and response time to an auditory probe during stimulus presentations. In a sample of 80 adults, we observed clear evidence of both fear acquisition and extinction using affective ratings. Response times were faster when probed early, but not later, during presentation of stimuli paired with an aversive scream. The response time findings are at odds with previous lab-based studies showing slower responses to threat-predicting cues. The findings underscore the feasibility of employing remotely delivered fear conditioning paradigms with affective ratings as outcome, and highlight the need for further research examining optimal parameters for concurrent response time measures or alternate modes of non-verbal estimation of conditioned responses in Pavlovian conditioning protocols.

sentences: Long-read sequencing to interrogate strain-level variation among adherent-invasive Escherichia coli isolated from human intestinal tissue Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohns disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohns disease and non-Crohns tissues. We show these strains can colonize the intestine of IBD susceptible mice and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related E. coli and likely other commensal/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.

sentences: Nucleophagy contributes to genome stability though TOP2cc and nucleolar components degradation The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal, healthy cells maintain genome stability avoiding accumulation of nuclear damaged material is less understood. Here we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after Etoposide-induced DNA Double-Stranded Breaks. These nuclear buds and micronuclei co-localize with autophagic proteins BECN1 and LC3 and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and Type II DNA Topoisomerases (TOP2A and TOP2B), or nucleolar protein Fibrillarin, implying they are also targets of nucleophagy. We propose that a basal nucleophagy contributes to genome and nuclear stability and also in response to DNA damage and nucleolar stress.

sentences: A Test of the Pioneer Factor Hypothesis The Pioneer Factor Hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (nonPFs) that activate batteries of silent genes. We tested the PFH by expressing the endodermal PF FoxA1 and nonPF Hnf4a in K562 lymphoblast cells. While co-expression of FoxA1 and Hnf4a activated a burst of endoderm-specific gene expression, we found no evidence for functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and "pioneered" for each other, although FoxA1 required fewer copies of its motif to bind at inaccessible sites. A subset of targets required both TFs, but the mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results we propose an alternative to the PFH where "pioneer activity" depends not on the existence of discrete TF subclasses, but on TF binding affinity and genomic context.

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Comparative genomics of emerging lineages and mobile resistomes of contemporary broiler strains of Salmonella Infantis and E. coli IntroductionCommensal and pathogenic strains of MDR Escherichia coli and non-typhoid strains of Salmonella represent a growing foodborne threat from foods of poultry origin. MDR strains of S. Infantis and E. coli are frequently isolated from broiler chicks and the simultaneous presence of these two enteric bacterial species would potentially allow the exchange of mobile resistance determinants.

ObjectivesIn order to understand possible genomic relations and to obtain a first insight into the potential interplay of resistance genes between enteric bacteria, we compared genomic diversity and mobile resistomes of S. Infantis and E. coli from broiler sources.

ResultsThe cgMLST analysis of 56 S. Infantis and 90 E. coli contemporary strains revealed a high genomic heterogeneity of broiler E. coli. It also allowed the first insight into the genomic diversity of the MDR clone B2 of S. Infantis, which is endemic in Hungary. We also identified new MDR lineages for S. Infantis (ST7081 and ST7082) and for E. coli (ST8702 and ST10088). Comparative analysis of antibiotic resistance genes and plasmid types revealed a relatively narrow interface between the mobile resistomes of E. coli and S. Infantis. The mobile resistance genes tet(A), aadA1 and sul1 were identified at an overall high prevalence in both species. This gene association is characteristic to the plasmid pSI54/04 of the epidemic clone B2 of S. Infantis. Simultaneous presence of these genes and of IncI and IncX plasmids in cohabitant caecal strains of E. coli and S. Infantis suggests an important role of these plasmids in a possible interplay of resistance genes between S. Infantis and E. coli in broilers.

ConclusionsThis is the first comparative genomic analysis of contemporary broiler strains of S. Infantis and E. coli. The diversity of mobile resistomes suggests that commensal E. coli could be potential reservoirs of resistance for S. Infantis, but so far only a few plasmid types and mobile resistance genes could be considered as potentially exchangeable between these two species. Among these, IncI plasmids could make the greatest contribution to the microevolution and genetic interaction between E. coli and S. Infantis.

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PD-1 dependent expansion of Amphregulin+FOXP3+ cells is associated with oral immune dysfunction in HIV patients on therapy Residual systemic inflammation and mucosal immune dysfunction persist in people living with HIV (PLWH) despite treatment with combined anti-retroviral therapy (cART), but the underlying immune mechanisms are poorly understood. Here we report an altered immune landscape involving upregulation of TLR- and inflammasome signaling, localized CD4+ T cell hyperactivation, and counterintuitively, an enrichment of CD4+CD25+FOXP3+ regulatory T cells (Tregs) in the oral mucosa of HIV+ patients on therapy. Using human oral tonsil cultures, we found that HIV infection causes an increase in a unique population of FOXP3+ cells expressing PD-1, IFN-{gamma}, Amphiregulin (AREG), and IL-10. These cells persisted even in the presence of the anti-retroviral drug and underwent further expansion driven by TLR-2 ligands and IL-1{beta}. IL-1{beta} also promoted PD-1 upregulation in AKT1 dependent manner. PD-1 stabilized FOXP3 and AREG expression in these cells through a mechanism requiring the activation of Asparaginyl Endopeptidase (AEP). Importantly, these FOXP3+ cells were incapable of suppressing CD4+ T cells in vitro. Concurrently, HIV+ patients harbored higher levels of PD-1, IFN-{gamma}, Amphiregulin (AREG), and IL-10 expressing FOXP3+ cells, which strongly correlated with CD4+ T cell hyperactivation, suggesting an absence of CD4+ T cell regulation in the oral mucosa. Taken together, this study provides insights into a novel mechanism of FOXP3+ cell dysregulation and reveals a critical link in the positive feedback loop of oral mucosal immune activation events in HIV+ patients on therapy.

One Sentence SummaryHIV-induced immune dysfunction in lymphoid and mucosal tissues

sentences: Feature-reweighted RSA: A method for improving the fit between computational models, brains, and behavior Representational Similarity Analysis (RSA) has emerged as a popular method for relating representational spaces from human brain activity, behavioral data, and computational models. RSA is based on the comparison of representational (dis-)similarity matrices (RDM or RSM), which characterize the pairwise (dis-)similarities of all conditions across all features (e.g. fMRI voxels or units of a model). However, classical RSA treats each feature as equally important. This  equal weights assumption contrasts with the flexibility of multivariate decoding, which reweights individual features for predicting a target variable. As a consequence, classical RSA may lead researchers to underestimate the correspondence between a model and a brain region and, in case of model comparison, may lead them to select an inferior model. The aim of this work is twofold: First, we sought to broadly test feature-reweighted RSA (FR-RSA) applied to computational models and reveal the extent to which reweighting model features improves RSM correspondence and affects model selection. Previous work suggested that reweighting can improve model selection in RSA but it has remained unclear to what extent these results generalize across datasets and data modalities. To draw more general conclusions, we utilized a range of publicly available datasets and three popular deep neural networks (DNNs). Second, we propose voxel-reweighted RSA, a novel use case of FR-RSA that reweights fMRI voxels, mirroring the rationale of multivariate decoding of optimally combining voxel activity patterns. We found that reweighting individual model units markedly improved the fit between model RSMs and target RSMs derived from several fMRI and behavioral datasets and affected model selection, highlighting the importance of considering FR-RSA. For voxel-reweighted RSA, improvements in RSM correspondence were even more pronounced, demonstrating the utility of this novel approach. We additionally show that classical noise ceilings can be exceeded when FR-RSA is applied and propose an updated approach for their computation. Taken together, our results broadly validate the use of FR-RSA for improving the fit between computational models, brain, and behavioral data, possibly allowing us to better adjudicate between competing computational models. Further, our results suggest that FR-RSA applied to brain measurement channels could become an important new method to assess the correspondence between representational spaces.

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Direct observation of the mechanical role of bacterial chaperones in protein folding Protein folding under force is an integral source of generating mechanical energy in various cellular processes, ranging from protein translation to degradation. Although chaperones are well known to interact with proteins under mechanical force, how they respond to force and control cellular energetics remains unknown.

To address this question, we introduce novel real-time magnetic-tweezers technology to mimic physiological force environment on client proteins, keeping the chaperones unperturbed. We studied two structurally distinct client proteins with seven different chaperones, independently and in combination, and proposed novel mechanical activity of chaperones. We found chaperones behave differently, while these client proteins are under force than its previously known functions. For instance, tunnel associated chaperones (DsbA and trigger factor), otherwise working as holdase without force, assist folding under force. This process generates an additional mechanical energy up to [~]147 zJ to facilitate translation or translocation. However, well-known cytoplasmic foldase chaperones (PDI, thioredoxin, or DnaKJE), does not possess the mechanical folding ability under force. Notably, the transferring chaperones (DnaK, DnaJ, SecB), act as unfoldase and slow down folding process, both in the presence and absence of force, to prevent misfolding of the client proteins. This provides an emerging insight of mechanical roles of chaperones: they can generate or consume energy by shifting energy landscape of the client proteins towards folded or unfolded state; suggesting an evolutionary mechanism to minimize the energy consumption in various biological processes.

sentences: Causes and consequences of bacteriophage diversification via genetic exchanges across lifestyles and bacterial taxa Bacteriophages (phages) evolve rapidly by acquiring genes from other phages leading to mosaic genomes. Here, we identify numerous genetic transfers between distantly related phages and aim at understanding their frequency, consequences and the conditions favoring them. Gene flow tends to occur between phages that are enriched for recombinases, transposases and non-homologous end joining, suggesting that both homologous and illegitimate recombination contribute to gene flow. Phage family and host phyla are strong barriers to gene exchange, but phage lifestyle is not. We observe more exchanges between temperate phages even if they tend to have smaller genomes. These acquisitions often include transcription regulators and lysins. Yet, there is also extensive gene flow between temperate and virulent phages, or between the latter. These predominantly involve virulent phages with large genomes previously classed as low gene flux, and lead to the preferential transfer of genes encoding functions involved in cell energetics, nucleotide metabolism, DNA packaging and injection, and virion assembly. Such exchanges may explain the acquisition of genes in virulent phages, which tend to have the largest genomes. We used genetic transfers, which occur upon co-infection of a host, to compare phage host range. We found that virulent phages have broader host ranges and mediate genetic exchanges between narrow host range temperate phages infecting distant bacterial hosts, thus contributing to gene flow between virulent phages, as well as between temperate phages. This gene flow drastically expands the gene repertoires available for phage and bacterial evolution, including the transfer of functional innovations across taxa.

sentences: Tunable human myocardium derived decellularized extracellular matrix for 3D bioprinting and cardiac tissue engineering The generation of 3D tissue constructs with multiple cell types and matching mechanical properties remains a challenge in cardiac tissue engineering. Recently, 3D bioprinting has become a powerful tool to achieve these goals. Decellularized extracellular matrix (dECM) is a common scaffold material due to providing a native biochemical environment. Unfortunately, dECMs low mechanical stability prevents usage for bioprinting applications alone. In this study, we developed bioinks composed of decellularized human heart ECM (dhECM) with either gelatin methacryloyl (GelMA) or GelMA- methacrylated hyaluronic acid (MeHA) hydrogels dual crosslinked with UV light and microbial Transglutaminase (mTGase). We characterized the bioinks mechanical, rheological, swelling, printability and biocompatibility properties. Composite GelMA-MeHA-dhECM (GME) hydrogels demonstrated improved mechanical properties by an order of magnitude, compared to GelMA-dhECM (GE) hydrogels. All hydrogels were extrudable and compatible with human induced pluripotent stem cells derived cardiomyocytes (iCMs) and human cardiac fibroblasts (hCFs). Tissue-like beating of the printed constructs with striated sarcomeric alpha-actinin and Connexin 43 expression was observed. The order of magnitude difference between the elastic modulus of these hydrogel composites offers applications in in vitro modelling of the myocardial infarct boundary. Here, as a proof of concept, we created an infarct boundary region with control over mechanical properties along with cellular and macromolecular content through printing iCMs with GE bioink and hCFs with GME bioink.

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Sex differences in cardio-pulmonary pathology of SARS-CoV2 infected and Trypanosoma cruzi co-infected mice BackgroundCoronavirus disease-2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; CoV2) is a deadly contagious infectious disease. For those who survived COVID-19, post-COVID cardiac damage poses a major threat for the progression of cardiomyopathy and heart failure. Currently, the number of COVID-related cases and deaths are increasing in Latin America, where a major COVID comorbidity is Chagas heart disease (caused by the parasite Trypanosoma cruzi). Here, we investigated the effect of T. cruzi infection on the pathogenesis and severity of CoV2 infection and, conversely, the effect of CoV2 infection on heart pathology during coinfection.

Methodology/findingsWe used transgenic human angiotensin-converting enzyme 2 (huACE2) mice infected with CoV2, T. cruzi, or coinfected with both in this study. Our study shows for the first time that white adipose tissue (WAT) serves as a reservoir for CoV2 and the persistence of CoV2 in WAT alters adipose tissue morphology and adipocyte physiology. Our data demonstrate a correlation between the loss of fat cells and the pulmonary adipogenic signaling (via adiponectin isomers) and pathology in CoV2 infection. The viral load in the lungs is inversely proportional to the viral load in WAT, which differs between male and female mice. Our findings also suggest that adiponectin-PPAR signaling may differently regulate Chagas cardiomyopathy in coinfected males and females.

ConclusionWe conclude that adipogenic signaling may play important roles in cardio-pulmonary pathogenesis during CoV2 infection and T. cruzi coinfection. The levels of adiponectin isomers differ between male and female mice during CoV2 infection and coinfection with T. cruzi, which may differently regulate inflammation, viral load, and pathology in the lungs of both the sexes. Our findings are in line with other clinical observations that reported that males are more susceptible to COVID-19 than females and suffer greater pulmonary damage.

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A platform of patient-derived microtumors identifies individual treatment responses and therapeutic vulnerabilities in ovarian cancer. BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses.

MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format.

ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach.

Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment.

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Red-beet betalain pigment inhibits amyloid-β aggregation and toxicity in amyloid-β expressing Caenorhabditis elegans model BACKGROUNDBetalain pigments are mainly produced by plants in the order Caryophyllales. Recent interest in the biological functions of betalain pigments has increased with antioxidant, anti-inflammatory, and anticancer activities reported.

RESULTSWe investigated the effects of betalain pigments derived from red-beet on amyloid-{beta} (A{beta}) aggregation, a cause of Alzheimers disease. Inhibition of A{beta} aggregation against A{beta}40 and A{beta}42 by betalain pigments in vitro was demonstrated by the Thioflavin T fluorescence assay, circular dichroism spectroscopy analysis and transmission electron microscopic observations. Moreover, we examined the ability of betalain pigments to interfere with A{beta} toxicity by using the transgenic Caenorhabditis elegans strain CL2006, which expresses the human A{beta}42 protein intracellularly within the body wall muscle and responds to A{beta}-toxicity with paralysis. Treatment with 50 M betalain pigments significantly delayed the paralysis of Caenorhabditis elegans.

CONCLUSIONThese results suggest that betalain pigments reduce A{beta}-induced toxicity by inhibiting A{beta} aggregation and may lead to their use as inhibitors of A{beta} aggregation.

sentences: A quantitative synthesis of soil microbial effects on plant species coexistence Soil microorganisms play a major role in shaping plant diversity, not only through their direct effects as pathogens, mutualists, and decomposers, but also by altering interactions between plants. In particular, previous research has shown that the soil community often generates frequency-dependent feedback loops among plants that can either destabilize species interactions, or generate stabilizing niche differences that promote species coexistence. However, recent insights from modern coexistence theory have shown that microbial effects on plant coexistence depend not only on these stabilizing or destabilizing effects, but also on the degree to which they generate competitive fitness differences. While many previous experiments have generated the data necessary for evaluating microbially mediated fitness differences, these effects have rarely been quantified in the literature. Here we present a meta-analysis of data from 50 studies, which we used to quantify the microbially mediated (de)stabilization and fitness differences derived from a classic plant-soil feedback model. Across 518 pairwise comparisons, we found that soil microbes generated both stabilization (or destabilization) and fitness differences, but also that the microbially mediated fitness differences dominated. As a consequence, if plants are otherwise equivalent competitors, the balance of soil microbe-generated (de)stabilization and fitness differences drives species exclusion much more frequently than coexistence or priority effects. Our work shows that microbially mediated fitness differences are an important but overlooked effect of soil microbes on plant coexistence. This finding paves the way for a more complete understanding of the processes that maintain plant biodiversity.

sentences: Causal reasoning over knowledge graphs leveraging drug-perturbed and disease-specific transcriptomic signatures for drug discovery Network-based approaches are becoming increasingly popular for drug discovery as they provide a systems-level overview of the mechanisms underlying disease pathophysiology. They have demonstrated significant early promise over other methods of biological data representation, such as in target discovery, side effect prediction and drug repurposing. In parallel, an explosion of -omics data for the deep characterization of biological systems routinely uncovers molecular signatures of disease for similar applications. Here, we present RPath, a novel algorithm that prioritizes drugs for a given disease by reasoning over causal paths in a knowledge graph (KG), guided by both drug-perturbed as well as disease-specific transcriptomic signatures. First, our approach identifies the causal paths that connect a drug to a particular disease. Next, it reasons over these paths to identify those that correlate with the transcriptional signatures observed in a drug-perturbation experiment, and anti-correlate to signatures observed in the disease of interest. The paths which match this signature profile are then proposed to represent the mechanism of action of the drug. We demonstrate how RPath consistently prioritizes clinically investigated drug-disease pairs on multiple datasets and KGs, achieving better performance over other similar methodologies. Furthermore, we present two applications showing how one can deconvolute the predictions made by RPath as well as predict novel targets. Finally, we have made the source code and data publicly available at https://github.com/enveda/RPath.

sentences: The Development, but not Expression, of Alcohol Front-loading in C57BL/6J Mice Maintained on LabDiet 5001 is Abolished by Maintenance on Teklad 2920x Rodent Diet Excessive alcohol (ethanol) consumption, such as binge-drinking, is extremely commonplace and represents a major health concern. Through modeling excessive drinking in rodents, we are beginning to uncover the neurobiological and neurobehavioral causes and consequences of this pattern of ethanol intake. One important factor for modeling binge drinking in mice is that subjects reliably drink to blood ethanol concentrations (BECs) of 80 mg/dl or higher. Drinking-in-the-dark (DID) is a commonly used mouse model of binge drinking, and we have shown these methods reliably result in robust ethanol front-loading and binge-level BECs in C57BL/6J (B6) mice as well as other ethanol-preferring mouse strains/lines. However, establishing the DID model in a new vivarium space forced us to consider the use of rodent diet formulations we had not previously used. The current set of experiments were designed to investigate the role of two standard rodent diet formulations on binge drinking and the development of ethanol front-loading using DID. We found that BECs in animals maintained on LabDiet 5001 (LD01) were double those found in mice maintained on Teklad 2920x (TL20). Interestingly, this effect was paralleled by differences in the degree of front-loading, such that LD01-fed mice consumed approximately twice as much ethanol in the first 15 minutes of the 2-hour DID sessions compared to TL20-fed mice. Surprisingly however, mice that developed front-loading during maintenance on the LD01 diet continued to display front-loading behavior after being switched to the TL20 diet. These data emphasize the importance of choosing and reporting diet formulations when conducting voluntary drinking studies and support the need for further investigation into the mechanisms behind diet-induced differences in binge drinking, particularly front-loading.

sentences: Evolutionary transcriptomics implicates new genes and pathways in human pregnancy and adverse pregnancy outcomes Evolutionary changes in the anatomy and physiology of the female reproductive system underlie the origins and diversification of pregnancy in Eutherian ("Placental") mammals. This developmental and evolutionary history constrains normal physiological functions and biases the ways in which dysfunction contributes to reproductive trait diseases and adverse pregnancy outcomes. Here, we show that gene expression changes in the human endometrium during pregnancy are associated with the evolution of human-specific traits and pathologies of pregnancy. We found that hundreds of genes gained or lost endometrial expression in the human lineage. Among these are genes that may contribute to human-specific maternal-fetal communication (HTR2B) and maternal-fetal immunotolerance (PDCD1LG2) systems, as well as vascular remodeling and deep placental invasion (CORIN). These data suggest that explicit evolutionary studies of anatomical systems complement traditional methods for characterizing the genetic architecture of disease. We also anticipate our results will advance the emerging synthesis of evolution and medicine ("evolutionary medicine") and be a starting point for more sophisticated studies of the maternal-fetal interface. Furthermore, the gene expression changes we identified may contribute to the development of diagnostics and interventions for adverse pregnancy outcomes.

sentences: Pervasive fold switching in a ubiquitous protein superfamily Hundreds of millions of structured proteins sustain life through chemical interactions and catalytic reactions1. Though dynamic, these proteins are assumed to be built upon fixed scaffolds of secondary structure, -helices and {beta}-sheets. Experimentally determined structures of over >58,000 non-redundant proteins support this assumption, though it has recently been challenged by [~]100 fold-switching proteins2. These "metamorphic3" proteins, though ostensibly rare, raise the question of how many uncharacterized proteins have shapeshifting-rather than fixed-secondary structures. To address this question, we developed a comparative sequence-based approach that predicts fold-switching proteins from differences in secondary structure propensity. We applied this approach to the universally conserved NusG transcription factor family of [~]15,000 proteins, one of which has a 50-residue regulatory subunit experimentally shown to switch between -helical and {beta}-sheet folds4. Our approach predicted that 25% of the sequences in this family undergo similar -helix {rightleftharpoons} {beta}-sheet transitions, a frequency two orders of magnitude larger than previously observed. Our predictions evade state-of-the-art computational methods but were confirmed experimentally by circular dichroism and nuclear magnetic resonance spectroscopy for all 10 assiduously chosen dissimilar variants. These results suggest that fold switching is a pervasive mechanism of transcriptional regulation in all kingdoms of life and imply that numerous uncharacterized proteins may also switch folds.

sentences: Recovery of silver fir (Abies alba Mill.) seedlings from ungulate browsing mirrors soil nitrogen availability Abies alba has a high potential for mitigating climate change in European mountain forests, yet, its natural regeneration is severely limited by ungulate browsing. Here, we simulated browsing in a common garden experiment to study growth and physiological traits, measured from bulk needles, using a randomized block design with two levels of browsing severity and seedlings originating from 19 populations across Switzerland. Genetic factors explained most variation in growth (on average, 51.5%) and physiological traits (10.2%) under control conditions, while heavy browsing considerably reduced the genetic effects on growth (to 30%), but doubled those on physiological traits related to C storage. While browsing reduced seedling height, it also lowered seedlings water use efficiency (decreased{delta} 13C) and N supply by mycorrhizal fungi as indicated by an increase in{delta} 15N. Different populations reacted differently to browsing stress, and for Height, Starch and{delta} 15N, population differences appeared to be the result of natural selection. We found that the fastest growing populations, originating from the warmest regions, decreased their needle starch level the most as a reaction to heavy browsing, suggesting a potential genetic underpinning for a growth-storage trade-off. Further, we found that seedlings originating from mountain populations growing on steep slopes had a significantly lower N discrimination in the common garden than those originating from flat areas, indicating that they have been selected to grow on N poor, potentially drained, soils. This finding was corroborated by the fact that N concentration in adult needles was lower on steep slopes than on flat ground, strongly indicating that steep slopes are the most N poor environments. Seedlings from these poor environments generally had a low growth rate and high storage, thus might be slower to recover from browsing stress than fast growing provenances from the warm environments with developed soils, such as the Swiss plateau.

sentences: Dlc1 controls cardio-vascular development downstream of Vegfa/Kdrl/Nrp1 signaling in the zebrafish embryo. The family of deleted-in-liver-cancer (dlc) genes encodes RhoGTPases and plays pivotal roles in cardiovascular development, but animal models for studying their functions are sparse due to early embryonic lethality. Gain and loss of function of dlc1 and dlc3 severely altered the growth of intersegmental vessels in the trunk of zebrafish embryos. Additionally, overexpression of dlc1 affected the growth of the common cardinal veins, but could rescue the arrest of angiogenesis induced by Vegfr2 inhibition, placing dlc1 downstream of kdrl signaling. Loss of dlc1 negatively affected the lumenization of the first aortic arch arteries and the lateral dorsal aortae. dlc1 mutants displayed a full obstruction in the early outflow tract during cardiac morphogenesis, which models to alterations in DLC1 detected in congenital heart defects in human patients. This study provides a functional in vivo characterization of dlc1 and dlc3 during vertebrate embryogenesis and places dlc1 as a key gene to control vascular development.

sentences: Loss of Elp1 disrupts trigeminal ganglion neurodevelopment in a model of Familial Dysautonomia Familial Dysautonomia (FD) is a sensory and autonomic neuropathy caused by a mutation in Elongator complex protein 1 (ELP1). FD patients have small trigeminal nerves and impaired perception of facial pain and temperature. These signals are relayed by nociceptive neurons in the trigeminal ganglion, a structure comprised of both neural crest- and placode-derived cells. Mice lacking Elp1 in neural crest derivatives ("Elp1 CKO") are born with smaller trigeminal ganglia, suggesting Elp1 is important for trigeminal ganglion development, yet the function of Elp1 in this context is unknown. We demonstrate Elp1 expression in both neural crest- and placode-derived trigeminal neurons, which our data suggest give rise to primarily TrkA- and TrkB/C-expressing neurons, respectively. While Elp1 is not required for initial trigeminal ganglion formation, Elp1 CKO trigeminal neurons exhibit abnormal axon outgrowth and decreased target innervation. Developing nociceptors that express the receptor TrkA are especially vulnerable to Elp1 loss. TrkA expression is decreased in Elp1 CKO trigeminal nerve endings, coinciding with increased cell death. Subsequently, fewer TrkA neurons are present in the Elp1 CKO trigeminal ganglion, indicating Elp1 supports the target innervation and survival of trigeminal nociceptors. These findings explain the loss of facial pain and temperature sensation in FD.

sentences: Aggregation Tool for Genomic Concepts (ATGC): A deep learning framework for sparse genomic measures and its application to tumor mutational burden Deep learning has the ability to extract meaningful features from data given enough training examples. Large scale genomic data are well suited for this class of machine learning algorithms; however, for many of these data the labels are at the level of the sample instead of at the level of the individual genomic measures and features. To leverage the power of deep learning for these types of data we turn to a multiple instance learning framework, and present an easily extensible tool built with TensorFlow and Keras. We show how this tool can be applied to somatic variants (featurizing genomic position, sequence context, and read counts) on a range of artificial tasks (classification, regression, Cox regression). In addition, we confirm the model can achieve high performance on real-world problems, accurately classifying samples according to whether they contain a specific variant (hotspot or tumor suppressor), groups of variants (tumor clonality), or a type of variant (microsatellite instability). Our results suggest this framework will lead to improvements on sample-level tasks that require aggregation of a set of genomic measures.

sentences: Molecular basis for allosteric agonism and G protein subtype selectivity of galanin receptors Peptide hormones and neuropeptides are complex signaling molecules that predominately function through G protein-coupled receptors (GPCRs). Two fundamental questions remained in the field of peptide-GPCR signaling systems are the basis for the diverse binding mode of peptide ligands and the specificity of G protein coupling. Here we report the structures of a neuropeptide, galanin, bound to its receptors, GAL1R and GAL2R, in complex with their primary G protein subtypes Gi and Gq, respectively. The structures reveal a unique binding pose of galanin, which almost  lay flat on the top of the receptor transmembrane domain pocket in an -helical conformation, and acts as an  allosteric-like agonist via a distinct signal transduction cascade. The structures also uncover the important features of intracellular loop 2 (ICL2) that mediate specific interactions with Gq, thus determining the selective coupling of Gq to GAL2R. ICL2 replacement in Gi-coupled GAL1R, OR, 5-HT1AR, and Gs-coupled b2AR and D1R with that of GAL2R promotes Gq coupling of these receptors, highlighting the dominant roles of ICL2 in Gq selectivity. Together our results provide important insights into peptide ligand recognition and allosteric activation of galanin receptors and uncover a general structural element for Gq coupling selectivity.

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Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but inhibited by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.

Significance statementVacuolar electrical excitability and stress-related Ca2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges.

sentences: Glycolic acid protects neurons against ischemia	in vitro and in two animal models of stroke Stroke is the second leading cause of death and disability worldwide. Current treatments, such as pharmacological thrombolysis or mechanical thrombectomy, re-open occluded arteries but do not protect against ischemia-induced damage that has already occurred before reperfusion or ischemia/reperfusion-induced neuronal damage. It has been shown that disrupting the conversion of glyoxal to glycolic acid (GA) results in a decreased tolerance to anhydrobiosis in C. elegans, dauer larva, while GA itself can rescue this phenotype. During the process of desiccation/rehydration, a metabolic stop/start similar to the one observed during ischemia/reperfusion occurs. In this study, we tested the protective effect of GA in different ischemia models, including commonly used stroke models in mice and swine. Our results show that GA, given during reperfusion, strongly protects against ischemic damage and improves the functional outcome. We provide evidence that GA exerts its effect by counteracting the glutamate-dependent increase in intracellular calcium during excitotoxicity. These results suggest that GA treatment has the potential to reduce the mortality and disability caused by stroke in patients.

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Biochemical, structural insights of newly isolated AA16 family of Lytic Polysaccharide Monooxygenase (LPMO) from Aspergillus fumigatus and investigation of its synergistic effect using biomass. The efficient conversion of lignocellulosic biomass into fermentable sugar is a bottleneck for the cheap production of bio-ethanol. The recently identified enzyme Lytic Polysaccharide Monooxygenase (LPMO) family has brought new hope because of its boosting capabilities of cellulose hydrolysis. In this report, we have identified and characterized a new class of auxiliary (AA16) oxidative enzyme LPMO from the genome of a locally isolated thermophilic fungus Aspergillus fumigatus (NITDGPKA3) and evaluated its boosting capacity of biomass hydrolysis. The AfLPMO16 is an intronless gene and encodes the 29kDa protein. While Sequence-wise, it is close to the C1 type of AaAA16 and cellulose-active AA10 family of LPMOs, but the predicted three-dimensional structure shows the resemblance with the AA11 family of LPMO (PDB Id: 4MAH). The gene was expressed under an inducible promoter (AOX1) with C-terminal His tag in the Pichia pastoris. The protein was purified using Ni-NTA affinity chromatography, and we studied the enzyme kinetics with 2,6-dimethoxyphenol. We observed polysaccharides depolymerization activity with Carboxymethyl cellulose (CMC) and Phosphoric acid swollen cellulose (PASC). Moreover, the simultaneous use of cellulase cocktail (commercial) and AfLPMO16 enhances lignocellulosic biomass hydrolysis by 2-fold, which is highest so far reported in the LPMO family.

ImportanceThe auxiliary enzymes, such as LPMOs, have industrial importance. These enzymes are used in cellulolytic enzyme cocktail due to their synergistic effect along with cellulases. In our study, we have biochemically and functionally characterized the new AA16 family of LPMO from Aspergillus fumigatus (NITDGPKA3). The biochemical characterization is the fundamental scientific elucidation of the newly isolated enzyme. The functional characterization, biomass degradation activity of AfLPMO16, and cellulase cocktail (commercial) combination enhancing the activity by 2-fold. This enhancement is the highest reported so far, which gives the enzyme AfLPMO16 enormous potential for industrial use.

sentences: A Potential Novel COVID-19 Vaccine With RBD-HR1/HR2 Hexamer Structure The COVID-19 pandemic and the continued spreading of the SARS-CoV-2 variants have brought a grave public health consequence and severely devastated the global economy with recessions. Vaccination is considered as one of the most promising and efficient methods to end the COVID-19 pandemic and mitigate the disease conditions if infected. Although a few vaccines have been developed with an unprecedented speed, scientists around the world are continuing pursuing the best possible vaccines with innovations. Comparing to the expensive mRNA vaccines and attenuated/inactivated SARS-CoV-2 vaccines, recombinant protein vaccines have certain advantages, including their safety (non-virus components), potential stronger immunogenicity, broader protection, ease of scaling-up production, reduced cost, etc. In this study, we reported a novel COVID-19 vaccine generated with RBD-HR1/HR2 hexamer that was creatively fused with the RBD domain and heptad repeat 1 (HR1) or heptad repeat 2 (HR2) to form a dumbbell-shaped hexamer to target the spike S1 subunit. The novel hexamer COVID-19 vaccine induced high titers of neutralizing antibody in mouse studies (>100,000), and further experiments also showed that the vaccine also induced an alternative antibody to the HR1 region, which probably alleviated the drop of immunogenicity from the frequent mutations of SARS-CoV-2.

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Hexapeptides from mammalian inhibitory hormone hunt activate and inactivate nematode reproduction BackgroundBiopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise.

MethodsReproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling.

ResultsReproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001s bioactivity : MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal.

NOTEPlease see the end of the paper for links to ten files of supplementary information and for an independent peer review.

KEY POINTSO_LISynthetic hexapeptides have upregulated and downregulated nematode reproduction, following similar work in other nematode species and in fish and frogs.
C_LIO_LIPeptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro and influence circulating levels of ovine pituitary hormones in vivo via intracerebroventricular peptide infusion.
C_LIO_LIThe amino acid sequence of the 14mer master peptide arose from a (rat) bioassay- guided fractionation using ovine materials, whose aim was to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination.
C_LIO_LIThe bioactive peptides mimic the non-contiguous six-residue receptor-binding active face of a polypeptide hormone, which in mammals is a derivative of the secretory vesicle prohormone secretogranin II (SgII), taking the form of a novel, structually complex 70mer dubbed  SgII-70.
C_LIO_LIThe ability has been gained to reinforce or block a default reproductive and tissue- building OFF signal, using peptide mimetics.
C_LI

sentences: Maternal separation induces autism spectrum disorder in young rhesus monkeys Autism spectrum disorder (ASD) is a class of severe neurodevelopmental disorders with a high incidence in young children, and its pathogenesis remains elusive. There is no effective treatment, and ASD children usually have a hard time in integrating into society and leading a normal life, which places a heavy burden on the families and society. Studies have shown that in addition to genetic factors, environmental factors are another important risk contributing to the pathogenesis of ASD. Early environmental adversity, which can lead to abnormal brain development and affect cognition and behavior, greatly increases the incidence of a variety of brain developmental diseases including ASD. However, studies on this aspect are inadequate at present, and no clear conclusions can be drawn. We explored whether early adversity could trigger ASD core clinical symptoms in macaques by modeling early adversity through maternal separation. In this study, we conducted a rigorous behavioral analysis of 12 male macaques (1.5-2 years old) that underwent maternal separation and 9 male normal macaques of the same age that had been mother raised, and found that maternal separation could induce a small number of the young individuals to develop three core symptoms of ASD, including social impairment, stereotyped behaviors, and restricted interest simultaneously. According to DSM-V and ASD clinical diagnostic criteria, these individuals should be ASD macaques for having all the three core ASD symptoms at the same time., For the first time, this study reveals that early environmental adversity can lead to ASD pathogenesis in monkeys, and provides a new approach for future ASD studies and modeling ASD monkeys.

sentences: Combinatorial small molecule-mediated activation of CBP/p300 with environmental enrichment in chronic severe experimental spinal cord injury to enable axon regeneration and sprouting for functional recovery The interruption of spinal circuitry following spinal cord injury disrupts neural activity and is followed by a failure to mount an effective regenerative gene expression response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here we will investigate whether the combined epigenetic stimulation of the regenerative gene expression program with neuronal activity-dependent enhancement of neuroplasticity and guidance can overcome the current inability to promote neurological recovery in severe and chronic spinal cord injury. This will be carried out by delivering the small molecule CBP/p300 activator CSP-TTK21 in combination with environmental enrichment. We will deliver the CBP/p300 activator CSP-TTK21 weekly between week twelve and twenty following a severe transection model of spinal cord injury in the mouse. Data analysis will assess modifications in regenerative signalling, in motor and sensory axon sprouting and regeneration, in synaptic plasticity as well as in neurological sensorimotor recovery. This approach will advance our mechanistic understanding of regenerative failure and provide insight of whether combining growth promoting strategies with enhancement of neural activity is an effective therapeutic strategy in chronic spinal cord injury.

sentences: Proteomic phenotyping of stimulated Mueller cells uncovers profound pro-inflammatory signaling and antigen-presenting capacity Muller cells are the main macroglial cells of the retina exerting a wealth of functions to maintain retinal homoeostasis. Upon pathological changes in the retina, they become gliotic with both protective and detrimental consequences. Accumulating data also provide evidence for a pivotal role of Muller cells in the pathogenesis of diabetic retinopathy (DR). While microglial cells, the resident immune cells of the retina are considered as main players in inflammatory processes associated with DR, the implication of activated Muller cells in chronic retinal inflammation remains to be elucidated. In order to assess the signaling capacity of Muller cells and their role in retinal inflammation, we performed in-depth proteomic analysis of Muller cell proteomes and secretomes after stimulation with INF{gamma}, TNF, IL-4, IL-6, IL-10, VEGF, TGF{beta}1, TGF{beta}2 and TGF{beta}3. We used both, primary porcine Muller cells and the human Muller cell line MIO-M1 for our hypothesis generating approach. Our results point towards an intense signaling capacity of Muller cells, which reacted in a highly discriminating manner upon treatment with different cytokines. Stimulation of Muller cells resulted in a primarily pro-inflammatory phenotype with secretion of cytokines and components of the complement system. Furthermore, we observed evidence for mitochondrial dysfunction, implying oxidative stress after treatment with the various cytokines. Finally, both MIO-M1 cells and primary porcine Muller cells showed several characteristics of atypical antigen-presenting cells, as they are capable of inducing MHC class I and MHC class II with co-stimulatory molecules. In line with this, they express proteins associated with formation and maturation of phagosomes. Thus, our findings underline the importance of Muller cell signaling in the inflamed retina, indicating an active role in chronic retinal inflammation underlying the pathogenesis of diabetic retinopathy.

sentences: A brain atlas of the camouflaging dwarf cuttlefish, Sepia bandensis The dwarf cuttlefish, Sepia bandensis, a small cephalopod that exhibits dynamic camouflage, is an emerging model organism in neuroscience. Coleoid cephalopods (cuttlefish, octopus, and squid) evolved large, complex brains capable of learning, problem-solving, and memory. We used high-resolution magnetic resonance imaging (MRI), deep learning, and fluorescent histology to generate a dwarf cuttlefish brain atlas and built an interactive web tool (cuttlebase.org) to host the data. Guided by observations in other cephalopods, we identified 38 brain lobes. The dwarf cuttlefish brain is partially encased in cartilage and includes two large optic lobes (74% the total volume of the brain), chromatophore lobes whose motor neurons directly innervate the skin, and a vertical lobe that has been implicated in learning and memory. Motor neurons emerging from the chromatophore lobe modulate the color, pattern, and texture of the skin to elicit camouflage. This brain atlas provides a valuable tool for exploring the neural basis of cuttlefish behavior.

sentences: Peptidergic signaling controls the dynamics of sickness behavior in Caenorhabditis elegans Pathogenic infection elicits sickness behaviors that promote recovery and survival of the host. For example, following infection with the pathogenic bacterium Pseudomonas aeruginosa PA14, the nematode Caenorhabditis elegans modifies its sensory preferences to avoid the pathogen. Here we identify antagonistic neuromodulatory circuits that shape this sickness behavior. Using an unbiased cell-directed neuropeptide screen, we show that AVK neurons upregulate and release FMRFamide-like FLP-1 neuropeptides during infection to drive pathogen avoidance. Manipulations that increase or decrease AVK signaling accelerate or delay pathogen avoidance, respectively, implicating AVK in the dynamics of sickness behavior. FLP-1 neuropeptides act via the G-protein-coupled receptor DMSR-7 in RIM/RIC neurons to reduce tyraminergic/octopaminergic signaling that opposes pathogen avoidance. RIM/RIC neurons relay parallel signals from neuropeptides and the cytokine TGF-{beta} that represent internal and external regulators of pathogen avoidance. Our results demonstrate that antagonism between neuromodulatory systems results in slow, graded transitions between alternative behavioral states.

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UbE3-APA: A Bioinformatic Strategy to Elucidate Ubiquitin E3 Ligase Activities in Quantitative Proteomics Study MotivationUbiquitination is widely involved in protein homeostasis and cell signaling. Ubiquitin E3 ligases are critical regulators of ubiquitination that recognize and recruit specific ubiquitination targets for the final rate-limiting step of ubiquitin transfer reactions. Understanding the ubiquitin E3 ligase ac-tivities will provide knowledge in the upstream regulator of the ubiquitination pathway and reveal po-tential mechanisms in biological processes and disease progression. Recent advances in mass spec-trometry-based proteomics have enabled deep profiling of ubiquitylome in a quantitative manner. Yet, functional analysis of ubiquitylome dynamics and pathway activity remains challenging.

ResultsHere, we developed a UbE3-APA, a computational algorithm and stand-alone python-based software for Ub E3 ligase Activity Profiling Analysis. Combining an integrated annotation database with statistical analysis, UbE3-APA identifies significantly activated or suppressed E3 ligases based on quantitative ubiquitylome proteomics datasets. Benchmarking the software with published quantitative ubiquitylome analysis confirms the genetic manipulation of SPOP enzyme activity through overexpres-sion and mutation. Application of the algorithm in the re-analysis of a large cohort of ubiquitination proteomics study revealed the activation of PARKIN and the co-activation of other E3 ligases in mito-chondria depolarization-induced mitophagy process. We further demonstrated the application of the algorithm in the DIA-based quantitative ubiquitylome analysis.

AvailabilitySource code and binaries are freely available for download at URL: https://github.com/Chenlab-UMN/Ub-E3-ligase-Activity-Profiling-Analysis, implemented in python and supported on Linux and MS Windows

Contactyuechen@umn.edu

Supplementary informationSupplementary data are available.

sentences: The perception of odor pleasantness is shared across cultures Human sensory experience varies across the globe. Nonetheless, all humans share sensory systems with a common anatomical blueprint. In olfaction, it is unknown to what degree sensory perception, in particular the perception of odor pleasantness, is dictated by universal biological principles versus sculpted by culture. To address this issue, we asked 235 individuals from 9 diverse non-western cultures to rank the hedonic value of monomolecular odorants. We observed substantial global consistency, with molecular identity explaining 41% of the variance in individual pleasantness rankings, while culture explained only 6%. These rankings were predicted by the physicochemical properties of out-of-sample molecules and out-of-sample pleasantness ratings given by a separate group of industrialized western urbanites, indicating human olfactory perception is strongly constrained by universal principles.

sentences: Non-specific adhesive forces between filaments and membraneless organelles Membraneless organelles are liquid-like domains that form inside living cells by phase-separation. While standard physical models of their formation assume their surroundings to be a simple liquid, the cytoplasm is an active viscoelastic environment. To investigate potential coupling of phase separation with the cytoskeleton, we quantify structural correlations of stress granules and microtubules in a human-derived epithelial cell line. We find that microtubule networks are significantly perturbed in the vicinity of stress granules, and that large stress granules conform to the local pore-structure of the microtubule network. When microtubules are depolymerized by nocodazole, tubulin enrichment is localized near the surface of stress granules. We interpret these data using a thermodynamic model of partitioning of particles to the surface and bulk of droplets. This analysis shows that proteins generically have a non-specific affinity for droplet interfaces, which becomes most apparent when they weakly partition to the bulk of droplets and have a large molecular weight. In this framework, our data is consistent with a weak ([lsim] kbT) affinity of tubulin sub-units for stress granule interfaces. As microtubules polymerize their affinity for interfaces increases, providing sufficient adhesion to deform droplets and/or the network. We validate this basic physical phenomena in vitro through the interaction of a simple protein-RNA condensate with tubulin and microtubules.

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Integration of visual motion and pursuit signals in areas V3A and V6+ across cortical depth using 9.4T fMRI Neural mechanisms underlying a stable perception of the world during pursuit eye movements are not fully understood. Both, perceptual stability as well as perception of real (i.e. objective) motion are the product of integration between motion signals on the retina and efference copies of eye movements. Human areas V3A and V6 have previously been shown to have strong objective ( real) motion responses. Here we used high-resolution laminar fMRI at ultra-high magnetic field (9.4T) in human subjects to examine motion integration across cortical depths in these areas. We found an increased preference for objective motion in areas V3A and V6+ i.e. V6 and possibly V6A towards the upper layers. When laminar responses were detrended to remove the upper-layer bias present in all responses, we found a unique, condition-specific laminar profile in V6+, showing reduced mid-layer responses for retinal motion only. The results provide evidence for differential, motion-type dependent laminar processing in area V6+. Mechanistically, the mid-layer dip suggests a special contribution of retinal motion to integration, either in the form of a subtractive (inhibitory) mid-layer input, or in the form of feedback into extragranular or infragranular layers. The results show that differential laminar signals can be measured in high-level motion areas in the human occipitoparietal cortex, opening the prospect of new mechanistic insights using non-invasive brain imaging.

Significance StatementVisual stability and our ability to differentiate between self-induced and real motion are central to our visual sense. Both require the integration of two signals - retinal motion and copies of muscle commands used for eye movements (efference copies). A reasonable assumption is that either the efference copy or the result of integration will be conveyed to high-level visual regions along with visual retinal input, possibly differentially across cortical depth as the input sources differ. Our ultra-high field recordings present the first laminar evidence of differential signal processing of retinal and objective motion signals in area V6+, and present a first window into a mechanistic understanding of visual high-level motion processing.

sentences: Single cell profiling of functionally cured Chronic Hepatitis B patients reveals the emergence of activated innate and an altered adaptive immune response in the intra-hepatic environment Hepatitis B surface antigen (HBsAg) loss or functional cure (FC), is considered the desirable therapeutic outcome for chronic hepatitis B (CHB) patients. However, the immuno-pathological biomarkers and underlying mechanisms remain unclear. Here we present a comprehensive single cell-transcriptomic atlas together with immune-phenotyping of disease-associated cell states (DACS) isolated from intra-hepatic tissue and matched PBMCs of either CHB or FC patients. We find that the intra-hepatic environment displays specific cell identities and molecular signatures that are distinct from PBMCs. FC is associated with emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes (CD4-CTLs), and an activated innate response represented by liver-resident natural killer (LR-NK) cells. Overall, these findings provide novel insights into immuno-pathological cell states associated with FC that could serve as prognostic biomarkers.

sentences: Bmper is required for morphogenesis of the anterior and posterior semicircular canal ducts in the developing zebrafish inner ear BMP signalling is known to have a conserved function in development of the semicircular canal system of the vertebrate inner ear, but its regulation, target genes and effects on cell behaviour during otic morphogenesis are not fully understood. We have characterised the effects of mutations in the zebrafish gene bmper, which codes for a regulator of BMP signalling with both pro- and anti-BMP roles in different developmental contexts. The inner ears of bmper mutant embryos develop with truncations of the anterior and posterior semicircular canal ducts. To image the developing ear in live embryos, we have exploited a new transgenic line, Tg(smad6b:EGFP), which exhibits strong GFP expression in the otic epithelium. Morphometric analysis indicates defects in the bmper mutant ear from early stages of semicircular canal formation, correlating with a specific reduction in BMP signalling activity and specific loss of dlx5a expression in dorsal otic epithelium. Subsequent changes to cell shape occur at the truncation site and the dorsolateral septum. The bmper mutations that we describe are adult viable; truncation of the anterior and posterior semicircular canal ducts persists into adulthood. Our results argue against a major role for Bmper in specification of the pre-placodal region, induction of the otic placode, or development of the neural crest, processes in which Bmper function has previously been implicated. Instead, we conclude that a key requirement for Bmper function in the zebrafish is to promote BMP signalling during patterning and morphogenesis of the semicircular canal system.

sentences: Size and shape regional differentiation during the development of the spine in the nine-banded armadillo (Dasypus novemcinctus) Xenarthrans (armadillos, anteaters, sloths and their extinct relatives) are unique among mammals in displaying a distinctive specialization of the posterior trunk vertebrae - supernumerary vertebral xenarthrous articulations. This study seeks to understand how xenarthry develops through ontogeny and if it may be constrained to appear within pre-existing vertebral regions. Using 3D geometric morphometrics on the neural arches of vertebrae, we explore phenotypic, allometric, and disparity patterns of the different axial morphotypes during ontogeny of nine-banded armadillos. Shape-based regionalisation analyses showed that adult thoracolumbar column is divided into three regions according to the presence or absence of ribs and the presence or absence of xenarthrous articulations. A three-region-division was retrieved in almost all specimens through development, although younger stages (e.g. foetuses, neonates) have more region boundary variability. In size-based regionalisation analyses, thoracolumbar vertebrae are separated into two regions: a pre-diaphragmatic, pre-xenarthrous region, and a post-diaphragmatic xenarthrous region. We show that posterior thoracic vertebrae grow at a slower rate, while anterior thoracics and lumbar grow at a faster rate relatively, with rates decreasing anteroposterioly in the former and increasing anteroposterioly in the latter. We propose that different proportions between vertebrae and vertebral regions might result from differences in growth pattern and timing of ossification.

sentences: CRISPR-VAE: A Method for Explaining CRISPR/Cas12a Predictions, and an Efficiency-aware gRNA Sequence Generator Deep learning has shown great promise in the prediction of the gRNA efficiency, which helps optimize the engineered gRNAs, and thus has greatly improved the usage of CRISPR-Cas systems in genome editing. However, the black box prediction of deep learning methods does not provide adequate explanation to the factors that increase efficiency; rectifying this issue promotes the usage of CRISPR-Cas systems in numerous domains. We put forward a framework for interpreting gRNA efficiency prediction, dubbed CRISPR-VAE, that improves understanding the factors that increase gRNA efficiency, and apply it to CRISPR/Cas12a (formally known as CRISPR/Cpf1). We further lay out a semantic articulation of such factors into position-wise k-mer rules. The paradigm consists of building an efficiency-aware gRNA sequence generator trained on available real data, and using it to generate a large amount of synthetic sequences with favorable traits, upon which the explanation of the gRNA prediction is based. CRISPR-VAE can further be used as a standalone sequence generator, where the user has low-level control ability. The framework can be readily integrated with different CRISPR-Cas tools and datasets, and its efficacy is confirmed. The complete implementation of the methods can be found at github.com/AhmadObeid/CRISPR-VAE.

sentences: Injury-induced Erk1/2 signaling enhances Ca2+ activity and is necessary for regeneration of spinal cord and skeletal muscle The transition of stem cells from quiescence to proliferation enables tissues to self-repair. The signaling mechanisms driving these stem-cell-status decisions are still unclear. Ca2+ and the extracellular signal-regulated kinase (Erk1/2) are two signaling pathways that have the potential to coordinate multiple signals to promote a specific cellular response. They both play important roles during development but their roles during regeneration are not fully deciphered. Here we show in Xenopus laevis larvae that both Ca2+ and Erk1/2 signaling pathways are activated after tail amputation. In response to injury, we find that Erk1/2 signaling is activated in neural and muscle stem cells and is necessary for spinal cord and skeletal muscle regeneration. Finally, we show in vivo that Erk1/2 action is necessary for an injury-induced increase in intracellular store-dependent Ca2+ dynamics in skeletal muscle-associated tissues but that in spinal cord, injury increases Ca2+ influx-dependent Ca2+ activity independent of Erk1/2 signaling. This study suggests that precise temporal and tissue-specific activation of Ca2+ and Erk1/2 pathways is essential for regulating tissue regeneration.

sentences: The anti-sigma factor MucA is required for viability in Pseudomonas aeruginosa During decades-long infections in the cystic fibrosis (CF) airway, Pseudomonas aeruginosa undergoes selection. One bacterial genetic adaptation often observed in CF isolates is mucA mutations. MucA inhibits the sigma factor AlgU. Mutations in mucA lead to AlgU misregulation, resulting in a mucoid phenotype that is associated with poor CF disease outcomes. Due to its ability to be mutated, mucA is assumed to be dispensable for bacterial viability. Here we show that, paradoxically, a portion of mucA is essential in P. aeruginosa. We demonstrate that mucA is no longer required in a strain lacking algU, that mucA alleles encoding for proteins that do not bind to AlgU are insufficient for viability, and that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity. Furthermore, we found that overexpression of algU prevents cell growth in the absence of MucA, and that this phenotype can be rescued by overproduction of RpoD, the housekeeping sigma factor. Together, these results suggest that in the absence of MucA, the inability to regulate AlgU activity results in the loss of bacterial viability. Finally, we speculate that essentiality of anti-sigma factors that regulate envelope function may be a widespread phenomenon in bacteria.

sentences: Entorhinal layer 6b subplate neurons govern spatial learning and memory The mammalian hippocampal formation (HF) plays a key role in several higher brain functions, such as spatial coding, learning and memory. Its simple circuit architecture is often viewed as a "trisynaptic loop", processing input originating from the superficial layers of the entorhinal cortex (EC) in a linear fashion, and sending it back to its deeper layers. Here, we show that contrary to this canonical view, excitatory neurons in the deepest layer of the mouse EC, exhibiting the unique morphology and molecular profile of layer 6b cortical subplate neurons (SPNs), project to all sub-regions comprising the HF, with a preference towards CA3 pyramidal neurons, and receive input from the CA1, thalamus and claustrum. Furthermore, their output is characterized by unique slow-decaying excitatory post-synaptic currents (EPSCs), capable of driving plateau-like potentials in their target cells. Optogenetic inhibition of the EC-6b pathway markedly affects spatial coding in CA1 pyramidal neurons, while cell ablation impairs not only acquisition of new spatial memories, but also degradation of previously acquired ones. Our results provide the first evidence of a functional role for the cortical layer 6b neurons in the adult brain and elucidate a critical novel element determining cortico-hippocampal activity patterns.

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Histone deacetylase inhibition reduces deleterious cytokine release induced by ingenol stimulation IntroductionLatency reversal agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-{kappa}B and, in turn, induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, and rapamycin, an mTOR inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 viral reactivation in vitro. Histone deacetylase inhibitors (HDACi) are known to dampen pro-inflammatory cytokine secretion in the context of other diseases and can synergize with other LRAs to bring dormant proviruses out of latency. In this study we investigated whether a broad panel of epigenetic modifiers, including HDACi, could effectively dampen PKC-induced pro-inflammatory cytokine secretion during latency reversal.

MethodsWe screened an epigenetic modifier library to identify compounds that reduced intracellular IL-6 production induced by the PKC agonist Ingenol-3,20-dibenzoate. We further tested the most promising epigenetic inhibitor class, HDACi, for their ability to reduce a broad panel of pro-inflammatory cytokines and reactivate latent HIV-1 ex vivo.

ResultsWe identified nine epigenetic modulators that reduced PKC-induced intracellular IL-6. In cells from aviremic individuals living with HIV-1, the HDAC1-3 inhibitor, suberohydroxamic acid (SBHA), reduced secretion of pro-inflammatory cytokines TNF-, IL-5, IL-2r, and IL-17 but did not significantly reactivate latent HIV-1 when used in combination with Ingenol-3,20-dibenzoate.

ConclusionThe addition of SBHA to Ingenol-3,20-dibenzoate reduces deleterious cytokine production during latency reversal but does not induce significant viral reactivation in aviremic donor PBMCs. The ability of SBHA to reduce PKC-induced pro-inflammatory cytokines when used in combination with Ingenol-3,20-dibenzoate suggests that SBHA can be used to reduced PKC induced pro-inflammatory cytokines but not to achieve latency reversal in the context of HIV-1.

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Zoom in on Ca2+ pattern and ion flux dynamics to decode spatial and temporal regulation of cotton fiber growth Cotton fibers are single-celled trichomes initiated from ovule epidermis prior to anthesis. Thereafter, the fibers undergo rapid elongation for 20 d before switching to intensive cell wall cellulose synthesis. The final length attained determines fiber yield and quality. As such, cotton fiber represents an excellent single cell model to study regulation of cell growth and differentiation, with significant agronomical implications. One major unresolved question is whether fiber elongation follows a diffusive or a tip growth pattern. We addressed this issue by using cell biology and electrophysiological approaches. Confocal imaging of Ca2+ binding dye, fluo-3 acetoxymethyl (Fluo-3), and in situ microelectrode ion flux measurement revealed that cytosolic Ca2+ was evenly distributed along the elongating fiber cells with Ca2+ and H+ fluxes oscillating from apical to basal regions of the elongating fibers. These findings demonstrate that, contrary to growing pollen tubes or root hairs, cotton fiber growth follows a diffusive, but not the tip growth, pattern. Further analyses showed that the elongating fibers exhibited substantial net H+ efflux, indicating a strong activity of the plasma membrane H+-ATPase required for energy dependent solute uptake. Interestingly, the growing cotton fibers were responding to H2O2 treatment, know to promote fiber elongation, by a massive increase in the net Ca2+ and H+ efflux in both tip and basal zones, while non-growing cells lacked this ability. These observations suggest that desensitization of the cell and a loss of its ability to respond to H2O2 may be causally related to the termination of the cotton fiber elongation.

One sentence summaryConfocal imaging of Ca2+ patterning and in situ microelectrode ion flux measurements demonstrate that, contrary to growing pollen tubes or root hairs, cotton fiber growth follows a diffusive, but not the tip growth, pattern.

sentences: The differentiation of pluripotent stem cells towards transplantable endothelial progenitor cells Endothelial progenitor cells (EPCs) and endothelial cells (ECs) have been applied in the clinic to treat pulmonary arterial hypertension (PAH), a disease characterized by disordered pulmonary vasculature. However, the lack of sufficient transplantable cells before the deterioration of disease condition is a current limitation to apply cell therapy in patients. It is necessary to differentiate pluripotent stem cells (PSCs) into EPCs and identify their characteristics. Comparing previously reported methods of human PSCs-derived ECs, we optimized a highly efficient differentiation protocol to obtain cells that match the phenotype of isolated EPCs from healthy donors. The protocol is compatible with chemically defined medium (CDM), it could produce a large number of clinically applicable cells with low cost. Moreover, we also found PSCs-derived EPCs express CD133, have some characteristics of mesenchymal stem cells and are capable of homing to repair blood vessels in zebrafish xenograft assays. In addition, we further revealed that IPAH PSCs-derived EPCs have higher expression of proliferation-related genes and lower expression of immune-related genes than normal EPCs and PSCs-derived EPCs through microarray analysis. In conclusion, we optimized a highly efficient differentiation protocol to obtain PSCs-derived EPCs with the phenotypic and molecular characteristics of EPCs from healthy donors which distinguished them from EPCs from PAH.

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Alone in a crowd: Effect of a nonfunctional lateral line on expression of the social hormone parathyroid hormone 2. Parathyroid hormone 2 (Pth2) is a vertebrate-specific neuropeptide whose thalamic expression is upregulated by social contact with conspecifics. However, social interactions fail to stimulate pth2 expression in isolated zebrafish whose lateral line hair cells have been chemically ablated. These results suggest that modulation of pth2 by social context is acutely dependent on mechanosensory information from the lateral line. However, it is unclear how a congenital loss of lateral line function influences the ability of zebrafish to interpret their social environment. In this study, we measure pth2 levels in zebrafish mutants lacking hair cell function in either the lateral line only, or in both the inner ear and lateral line. Socially-raised lateral line mutants express lower levels of pth2 relative to wild type siblings, but there is no further reduction when all sensory hair cells are nonfunctional. However, social isolation of hair cell mutants causes a further reduction in pth2 expression, pointing to additional unidentified sensory cues that influence pth2 production. Lastly, we report that social context modulates fluorescent transgenes driven by the pth2 promoter. Altogether, these data suggest that lateral line mutants experience a chronic sense of isolation, even when raised in a social environment.

SUMMARY STATEMENTExpression of the pro-social neuropeptide pth2 is downregulated in larval zebrafish with a congenital loss of lateral line function. Thus, even in social environments, fish with compromised lateral lines may experience chronic loneliness.

sentences: Eusocial insect queens show costs of reproduction and transcriptomic signatures of reduced longevity Eusocial insect queens have been suggested to be counter-examples to the standard evolutionary theory of ageing through lacking costs of reproduction. Using the bumblebee (Bombus terrestris), we tested this hypothesis experimentally against the alternative that costs of reproduction exist in eusocial insect queens, but are latent, resulting in the positive fecundity-longevity relationship typically found in unmanipulated queens. We experimentally increased queens costs of reproduction by removing their eggs, which causes queens to increase their egg-laying rate. Treatment queens had significantly reduced longevity relative to control queens whose egg-laying rate was not increased. In addition, treatment and control queens differed in age-related gene expression based on mRNA-seq in both their overall expression profiles and the expression of ageing-related genes. Remarkably, this occurred principally with respect to relative age, not chronological age. These results provide the first simultaneously phenotypic and transcriptomic experimental evidence of costs of reproduction in queens of eusocial insects and suggest how the genetic pathways underpinning ageing might become remodelled during eusocial evolution.

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Characterization and identification of a novel daidzein reductase involved in (S)-equol biosynthesis in Clostridium C1 (S)-equol is an isoflavone with high estrogen-like activity and no toxic effects in the human body, and is only produced by some gut bacteria in vivo. It plays an important role in maintaining individual health, however, the dearth of resources associated with (S)-equol-producing bacteria has seriously restricted the production and application of (S)-equol. We report here a novel functional gene C1-07020 that was identified from a chick (S)-equol-producing bacterium (Clostridium C1). We found that recombinant protein of C1-07020 possessed similar function to daidzein reductase (DZNR), which can convert daidzein (DZN) into R/S-dihydrodaidzein (R/S-DHD). Interestingly, C1-07020 can reverse convert (R/S)-DHD (DHD oxidases) into DZN even without cofactors or anaerobic conditions. Additionally, high concentrations of (S)-equol can directly promote DHD oxidase but inhibit DZNR activity. Molecular docking and site-directed mutagenesis revealed that the amino acid Arg 75 was the active site of DHD oxidases. Subsequently, an engineered E. coli strain based on C1-07020 was constructed and showed higher yield of (S)-equol than the engineered bacteria from our previous work. Metagenomics analysis and PCR detection surprisingly revealed that C1-07020 and related bacteria may be prevalent in the gut of humans and animals and their (S)-equol production state may cause differed between (S)-equol producer and non-producer. Overall, a novel DZNR from Clostridium C1 was found and identified in this study, and its bidirectional enzyme activities and wide distribution in the gut of humans and animals provide alternative strategies for revealing the individual regulatory mechanisms of (S)-equol-producing bacteria.

Importance(S)-equol is a final product of DZN that metabolized by some enteric bacteria. Although (S)-equol played very important roles in maintaining human health, larger differences in equol production varied between different populations. Here, a novel DZNR gene C1-07020, which related to (S)-equol production, was reported. The bidirectional enzyme functions and wide distribution of C1-07020 in human and animal gut provided additional insights into the metabolic regulation of (S)-equol. Additionally, C1-07020 can be used for improving the production of (S)-equol in vitro.

sentences: Generating accurate 3D gaze vectors using synchronized eye tracking and motion capture Assessing gaze behaviour during real-world tasks is difficult; dynamic bodies moving through dynamic worlds make gaze analysis difficult. Current approaches involve laborious coding of pupil positions. In settings where motion capture and mobile eye tracking are used concurrently in naturalistic tasks, it is critical that data collection be simple, efficient, and systematic. One solution is to combine eye tracking with motion capture to generate 3D gaze vectors. When combined with tracked or known object locations, 3D gaze vector generation can be automated. Here we use combined eye and motion capture and explore how linear regression models generate accurate 3D gaze vectors. We compare spatial accuracy of models derived from four short calibration routines across three pupil data inputs: the efficacy of calibration routines were assessed, a validation task requiring short fixations on taskrelevant locations, and a naturalistic object interaction task to bridge the gap between laboratory and "in the wild" studies. Further, we generated and compared models using spherical and cartesian coordinate systems and monocular (Left or Right) or binocular data. All calibration routines performed similarly, with the best performance (i.e., sub-centimetre errors) coming from the naturalistic task trials when the participant is looking at an object in front of them. We found that spherical coordinate systems generate the most accurate gaze vectors with no differences in accuracy when using monocular or binocular data. Overall, we recommend one-minute calibration routines using binocular pupil data combined with a spherical world coordinate system to produce the highest quality gaze vectors.

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Correlational networking guides the discovery of cryptic proteases for lanthipeptide maturation Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on cryptic enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by cryptic proteases required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and cryptic proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5,209 correlations between precursors and cryptic proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of novel class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of cryptic lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Controllable drug release of pH-sensitive liposomes encapsulating artificial cytosol system The fabrication of cell model containing artificial cytosol is challenging. Herein we constructed an artificial cytosol contained cell model by electroformation method. Agarose was selected as the main component of the artificial cytosol. Sucrose was added into agarose to regulate the sol viscosity and phase transition temperature (Tm). The viscosity of the sol with the mass ratio (agarose-sucrose) 1:9 was closest to the natural cytosolic. DSPC/20 mol% Chol was used to form large unilamellar vesicle (LUV) as cell model compartment. The rhodamine release experiment confirmed that the release of rhodamine from LUVs containing artificial cytosol took more time than that from LUVs containing pure water. The unique release profile makes agarose-sucrose@LUVs suitable as a drug carrier. Doxorubicin (DOX) is loaded in the agarose-sucrose@LUVs, and their half maximum inhibition concentration on HeLa cells is 0.015 mol L-1, which means 31.7 times increase in inhibition efficiency over free DOX.

sentences: Live virus neutralisation of the 501Y.V1 and 501Y.V2 SARS-CoV-2 variants following INO-4800 vaccination of ferrets The ongoing COVID-19 pandemic has resulted in significant global morbidity and mortality on a scale similar to the influenza pandemic of 1918. Over the course of the last few months, a number of SARS-CoV-2 variants have been identified against which vaccine-induced immune responses may be less effective. These "variants-of-concern" have garnered significant attention in the media, with discussion around their impact on the future of the pandemic and the ability of leading COVID-19 vaccines to protect against them effectively. To address concerns about emerging SARS-CoV-2 variants affecting vaccine-induced immunity, we investigated the neutralisation of representative  G614,  501Y.V1 and  501Y.V2 virus isolates using sera from ferrets that had received prime-boost doses of the DNA vaccine, INO-4800. Neutralisation titres against G614 and 501Y.V1 were comparable, but titres against the 501Y.V2 variant were approximately 4-fold lower, similar to results reported with other nucleic acid vaccines and supported by in silico biomolecular modelling. The results confirm that the vaccine-induced neutralising antibodies generated by INO-4800 remain effective against current variants-of-concern, albeit with lower neutralisation titres against 501Y.V2 similar to other leading nucleic acid-based vaccines.

sentences: Single-cell immune profiling with TCR clonotype barcoding identifies biomarker signatures that predict response to immune checkpoint blockade The identification of predictive biomarkers for patient treatment response is urgently needed to increase the probability of success of existing and novel experimental therapies. Single-cell profiling has provided novel biological insights into drug responses in the tumor microenvironment, but its potential for biomarker discovery has not been fully explored for therapeutic purposes. We describe a novel approach to discover predictive response biomarkers from single-cell data from a small patient cohort using the T cell receptor sequence intrinsic to each T cell to match clonotypes between pre- and post-treatment tumor samples. As a result, we have identified a predictive gene expression signature for immune checkpoint blockade and validated its predictive performance using data from three larger clinical studies. Our results demonstrated that applying clonotyping with single-cell genomic profiling is a promising novel approach for biomarker identification that does not require data collected from large patient cohorts. This could increase success rates, reduce clinical trial size, and significantly impact future clinical developments of immunomodulatory therapeutics.

sentences: Transcriptional landscape in BRAF wild type metastatic melanoma Melanoma is a relative rare disease worldwide, nevertheless it has a great relevance in some countries such as in Europe. In order to shed some light upon the transcriptional profile of skin melanoma, we compared the gene expression of 6 independent tumours (all progressed towards metastatic disease and with wild type BRAF) to the expression profile of healthy melanocytes. Paraffin-embedded samples were manually microdissected to obtain enriched samples and then RNA was extracted and analysed through a microarray-based approach. An exhaustive bioinformatics analysis was performed to identify differentially expressed transcripts between the two groups as well as enriched functional terms. Overall, 50 up- and 19 down-regulated transcripts were found to be significantly changed in tumour compared to control tissue. Among the upregulated transcripts, the majority belonged to the immune response group and to proteasome, while most of the downregulated genes were related to cytosolic ribosomes. Interestingly, Gene Set Enrichment Analysis (GSEA), along with the analysis of the expression data retrieved from TCGA/GTEx databases, confirmed the general trend of downregulation affecting cytoribosome proteins. In contrast, transcripts coding for mitori-bosome proteins showed the opposite trend.

sentences: A millennium of increasing ecosystem diversity until the mid-20th century Land-use change is widely regarded as a simplifying and homogenising force in nature. In contrast, analysing global land-use reconstructions from the 10th to 20th centuries, we found progressive increases in the number, evenness, and diversity of ecosystems (including human-modified land-use types) across the globe. Ecosystem diversity increased more rapidly after [~]1700CE, then slowed or partially reversed (depending on the metric) following the mid-20th century acceleration of human impacts. Differentiation also generally increased across space, with homogenization only evident in the presence-absence analysis of ecosystem types at the global scale. Our results suggest that human land-use changes have primarily driven increases in ecosystem diversity over the last millennium.

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tbg - a new file format for genomic data MotivationThe question of determining whether a Single-Nucleotide Polymorphism (SNP) or a variant in general leads to a change in the amino acid sequence of a protein coding gene is often a laborious and time-consuming challenge. Here, we introduce the tbg file format for storing genomic data and tbg-tools, a user-friendly toolbox for the faster analysis of SNPs. The file format stores information for each nucleotide in each gene, allowing to predict which change in the amino acid sequence will be caused by a variant in the nucleotide sequence. Our new tool therefore has the potential to make biological sense of the unprecedented amount of genome-wide genetic variation that researchers currently face.

ResultsThe new tab-separated file for storing the nucleotide data can be easily analyzed and used for a wide variety of biological research. It is also possible to automate some of these analyses using the additional analysis tools from tbg-tools

Availabilitytbg-tools is written in Python and allows the installation from the command line. It can be found on https://github.com/Croxa/tbg-tools.

Contactpschoenn@students.uni-mainz.de

sentences: Passive Exposure Sparsifies Neural Activity in the Primary Visual Cortex A notable feature of neural activity is sparseness - namely, that only a small fraction of neurons in a local circuit have high activity at any moment. Not only is sparse neural activity observed experimentally in most areas of the brain, but sparseness has been proposed as an optimization or design principle for neural circuits. Sparseness can increase the energy efficiency of the neural code as well as allow for beneficial computations to be carried out. But how does the brain achieve sparse-ness? Here, we found that when neurons in the primary visual cortex were passively exposed to a set of images over several days, neural responses became more sparse. Sparsification was driven by a decrease in the response of neurons with low or moderate activity, while highly active neurons retained similar responses. We also observed a net decorrelation of neural activity. These changes sculpt neural activity for greater coding efficiency.

sentences: Chronic release of tailless phage particles from Lactococcus lactis Lactococcus lactis strains residing in the microbial community of a complex dairy starter culture named "Ur" are hosts to prophages belonging to the family Siphoviridae. L. lactis strains (TIFN1 to TIFN7) showed detectable spontaneous phage production and release (109-1010 phage particles/mL) and up to 10-fold increases upon prophage induction, while in both cases we observed no obvious cell lysis, typically described for the lytic life cycle of Siphoviridae phages. Intrigued by this phenomenon, we investigated the host-phage interaction using strain TIFN1 (harboring prophage proPhi1) as a representative. We confirmed that during the massive phage release, all bacterial cells remain viable. Further, by monitoring phage replication in vivo, using a green fluorescence protein reporter combined with flow cytometry, we demonstrated that the majority of the bacterial population (over 80%) is actively producing phage particles when induced with mitomycin C. The released tailless phage particles were found to be engulfed in lipid membranes, as evidenced by electron microscopy and lipid staining combined with chemical lipid analysis. Based on the collective observations, we propose a model of phage-host interaction in L. lactis TIFN1, where the phage particles are engulfed in membranes upon release, thereby leaving the producing host intact. Moreover, we discuss possible mechanisms of chronic, or non-lytic release of LAB Siphoviridae phages and its impact on the bacterial host.

sentences: DNA Supercoiling Drives a Transition between Collective Modes of Gene Synthesis Recent experiments showed that multiple copies of the molecular machine RNA polymerase (RNAP) can efficiently synthesize mRNA collectively in the active state of the promoter. However, environmentally-induced promoter repression results in long-distance antagonistic interactions that drastically reduce the speed of RNAPs and cause a quick arrest of mRNA synthesis. The mechanism underlying this transition between cooperative and antagonistic dynamics remains poorly understood. In this Letter, we introduce a continuum deterministic model for the translocation of RNAPs, where the speed of an RNAP is coupled to the local DNA supercoiling as well as the density of RNAPs on the gene. We assume that torsional stress experienced by individual RNAPs is exacerbated by high RNAP density on the gene and that transcription factors act as physical barriers to the diffusion of DNA supercoils. We show that this minimal model exhibits two transcription modes mediated by the torsional stress: a fluid mode when the promoter is active and a torsionally stressed mode when the promoter is repressed, in quantitative agreement with experimentally observed dynamics of co-transcribing RNAPs. Our work provides an important step towards understanding the collective dynamics of molecular machines involved in gene expression.

sentences: Mechanosensitive Osteogenesis on Native Cellulose Scaffolds for Bone Tissue Engineering. In recent years, plant-derived cellulosic biomaterials have become a popular way to create scaffolds for a variety of tissue engineering applications. Moreover, such scaffolds possess similar physical properties (porosity, stiffness) that resemble bone tissues and have been explored as potential biomaterials for tissue engineering applications. Here, plant-derived cellulose scaffolds were seeded with MC3T3-E1 pre-osteoblast cells. Moreover, to assess the potential of these biomaterials, we also applied cyclic hydrostatic pressure (HP) to the cells and scaffolds over time to mimic a bone-like environment more closely. After one week of proliferation, cell-seeded scaffolds were exposed to HP up to 270 KPa at a frequency of 1Hz, once per day, for up to two weeks. Scaffolds were incubated in osteogenic inducing media (OM) or regular culture media (CM). The effect of cyclic HP combined with OM on cell-seeded scaffolds resulted in an increase of differentiated cells. This corresponded to an upregulation of alkaline phosphatase activity and scaffold mineralization. Importantly, the results reveal that well known mechanosensitive pathways cells which regulate osteogenesis appear to remain functional even on novel plant-derived cellulosic biomaterials.

sentences: Alignment-free Sequence Comparisons via Approximate String Matching Sequence comparison is a fundamental problem in bioinformatics and plays a key role in a wide range of applications. Alignment-free methods provide a computationally efficient alternative to alignment-based methods for large-scale sequence analysis. Several neural network-based methods have recently been developed for this purpose. However, due to the fact that the neural networks employed are not designed specifically for biological sequence analysis, existing methods do not perform well on sequences of varying lengths and are sensitive to the presence of insertions and deletions. In this paper, we describe the development of a new method, referred to as AsMac, that addresses the aforementioned issues. We proposed a novel neural network structure for approximate string matching for the extraction of pertinent information from biological sequences and an efficient gradient computation algorithm for training the constructed neural network. We performed a large-scale benchmark study using real-world data that demonstrated the effectiveness and potential utility of the proposed method.

sentences: Dissecting genomic determinants of positive selection with an evolution-guided regression model In evolutionary genomics, it is fundamentally important to understand how characteristics of genomic sequences, such as gene expression level, determine the rate of adaptive evolution. While numerous statistical methods, such as the McDonald-Kreitman test, are available to examine the association between genomic features and the rate of adaptation, we currently lack a statistical approach to disentangle the independent effect of a genomic feature from the effects of other correlated genomic features. To address this problem, I present a novel statistical model, the MK regression, which augments the McDonald-Kreitman test with a generalized linear model. Analogous to the classical multiple regression model, the MK regression can analyze multiple genomic features simultaneously to infer the independent effect of a genomic feature, holding constant all other genomic features. Using the MK regression, I identify numerous genomic features driving positive selection in chimpanzees. These features include well-known ones, such as local mutation rate, residue exposure level, tissue specificity, and immune genes, as well as new features not previously reported, such as gene expression level and metabolic genes. In particular, I show that highly expressed genes may have a higher adaptation rate than their weakly expressed counterparts, even though a higher expression level may impose stronger negative selection. Also, I show that metabolic genes may have a higher adaptation rate than their non-metabolic counterparts, possibly due to recent changes in diet in primate evolution. Overall, the MK regression is a powerful approach to elucidate the genomic basis of adaptation.

sentences: Proteomics reveals commitment to germination in barley seeds is marked by loss of stress response proteins and mobilisation of nutrient reservoirs Germination is a critical process in the reproduction and propagation of flowering plants, and is also the key stage of industrial grain malting. Germination commences when seeds are steeped in water, followed by degradation of the endosperm cell walls, enzymatic digestion of starch and proteins to provide nutrients for the growing plant, and emergence of the radicle from the seed. Dormancy is a state where seeds fail to germinate upon steeping, but which prevents inappropriate premature germination of the seeds before harvest from the field. This can result in inefficiencies in industrial malting. We used Sequential Window Acquisition of all THeoretical ions Mass Spectrometry (SWATH-MS) proteomics to measure changes in the barley seed proteome throughout germination. We found a large number of proteins involved in desiccation tolerance and germination inhibition rapidly decreased in abundance after imbibition. This was followed by a decrease in proteins involved in lipid, protein and nutrient reservoir storage, consistent with induction and activation of systems for nutrient mobilisation to provide nutrients to the growing embryo. Dormant seeds that failed to germinate showed substantial biochemical activity distinct from that of seeds undergoing germination, with differences in sulfur metabolic enzymes, endogenous alpha-amylase/trypsin inhibitors, and histone proteins. We verified our findings with analysis of germinating barley seeds from two commercial malting facilities, demonstrating that key features of the dynamic proteome of germinating barley seeds were conserved between laboratory and industrial scales. The results provide a more detailed understanding of the changes in the barley proteome during germination and give possible target proteins for testing or to inform selective breeding to enhance germination or control dormancy.

sentences: BDNF signaling in Hebbian and Stentian structural plasticity in the developing visual system During development, patterned neural activity in input neurons innervating their target, instructs topographic map refinement. Axons from adjacent neurons, firing with similar patterns of neural activity, converge onto target neurons and stabilize their synapses with these postsynaptic partners (Hebbian plasticity). On the other hand, non-correlated firing of inputs promotes synaptic weakening and exploratory axonal growth (Stentian plasticity). We used visual stimulation to control the visually-evoked correlation structure of neural activity in ectopic ipsilaterally projecting (ipsi) retinal ganglion cell axons with respect to their neighboring contralateral eye inputs in the optic tectum of albino Xenopus laevis tadpoles. Multiphoton imaging of the ipsi axons in the live tadpole, combined with manipulation of brain-derived neurotrophic factor (BDNF) signaling, revealed that presynaptic p75NTR and TrkB both promoted axonal branch addition during Stentian plasticity, whereas predominantly postsynaptic BDNF signaling mediated activity-dependent Hebbian suppression of axon branch addition. Additionally, we found that BDNF signaling is required for local suppression of branch loss induced by correlated firing.

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The sensitivity of magnetic particle imaging and fluorine-19 magnetic resonance imaging for cell tracking PurposeMagnetic particle imaging (MPI) and fluorine-19 (19F) MRI produce images which allow for quantification of labeled cells. MPI is an emerging instrument for cell tracking, which is expected to have superior sensitivity compared to 19F MRI. Our objective is to assess the cellular sensitivity of MPI and 19F MRI for detection of mesenchymal stem cells (MSC) and breast cancer cells.

MethodsCells were labeled with ferucarbotran or perfluoropolyether, for imaging on a preclinical MPI system or 3 Tesla clinical MRI, respectively. In vivo sensitivity with MPI and 19F MRI was evaluated by imaging MSC that were administered by different routes.

ResultsUsing the same imaging time, as few as 4000 MSC (76 ng iron) and 8000 breast cancer cells (74 ng iron) were reliably detected with MPI, and 256,000 MSC (9.01 x 1016 19F atoms) were detected with 19F MRI, with SNR > 5. In vivo imaging revealed reduced sensitivity compared to ex vivo cell pellets of the same cell number.

ConclusionMPI has the potential to be more sensitive than 19F MRI for cell tracking. We attribute reduced MPI and 19F MRI cell detection in vivo to the effect of cell dispersion among other factors, which are described.

sentences: Annexin A4 senses membrane curvature in a density-dependent manner Annexins (ANXs) are a family of peripheral membrane binding proteins which play a vital role in the maintenance and function of cellular membranes. These proteins are known to be part of the plasma membrane repair machinery where they are known to bind to negatively charged lipids in the cell membrane in a calcium dependent manner. The shape of the plasma membrane is known to be a regulator of protein density and thereby affects several biological functions of the cell such as exo-and endocytosis, cell motility, immune responses and also membrane repair. Membrane deformation and curvature sensing by proteins is a well described phenomenon which can assist in recruitment of specific proteins to certain regions in the cell and facilitate membrane bending. Following ruptures in the plasma membrane, calcium influx assist in the association of ANXs with the membrane around the ruptured area. Due to the expected increase in curvature at the damaged membrane site, it has been suggested that both membrane curvature and Ca2+ participate in the recruitment of ANXs. We have investigated the curvature sensing of ANXA4 in giant unilamellar vesicles (GUVs) by pulling high curvature membrane tethers from the vesicle surface using optical tweezers showing that ANXA4 recruitment increases with higher membrane curvature. We also describe an assay for determining protein density on the plasma membrane by utilizing ANXA4s property as a calcium dependent membrane binding protein. This new assay allows us to investigate the effect of protein density on curvature sensing.

sentences: Improving the Virtual Trichrome Assessment through Bridge Category Models Non-alcoholic steatohepatitis (NASH) is a liver disease characterized by excessive lipid accumulation and disease progression is typically assessed through inspection of a Trichrome stain for Fibrosis staging. As the public health burden of NASH worsens due to evolving lifestyle habits, pathology laboratory resources will become increasingly strained due to rising demand for specialized stains. Virtual staining processes, computational methods which can synthesize the application of chemical staining reagents, can potentially provide resource savings by obviating the need to acquire specialized stains. Virtual staining technologies are assessed by comparing virtual and real tissue stains for their realism and ability to stage. However, these assessment methods are rife with statistical mistreatment of observed phenomena that are difficult to account for. Bridge category ratings represent a phenomenon where a pathologist may assign two adjacent stages simultaneously, which may bias and/or reduce the power of research findings. Such stage assignments were frequently reported in a large-scale assessment of Virtual Trichrome technologies yet were unaccounted for since no statistical adjustment procedures existed. In this work, we provide an updated assessment of Virtual Trichrome technologies using Bridge Category Models, which account for these bridge ratings. We report that two of four pathologists tended to assign lower Fibrosis stages to virtually stained tissue while the other two pathologists assigned similar stages. These research findings differ when bridge ratings are not accounted for. While promising, these results indicate further room for algorithmic finetuning of Virtual Trichrome technologies.

sentences: Large-scale discovery of candidate type VI secretion effectors with antibacterial activity Type VI secretion systems (T6SS) are common bacterial contractile injection systems that inject toxic "effector" proteins into neighboring cells. Effector discovery is generally done manually, and computational approaches used for effector discovery depend on genetic linkage to T6SS genes and/or sequence similarity to known effectors. We bioinformatically investigated T6SS in more than 11,832 genomes of Gram negative bacteria. We found that T6SS encoding bacteria are host-associated and pathogenic, enriched in specific human and plant tissues, while depleted in marine, soil, and engineered environments. Analysis of T6SS cores with C-terminal domains ("evolved" cores) showed "evolved" HCP are rare, overwhelmingly encoded in orphan operons, and are largely restricted to Escherichia. Using the wealth of data generated from our bioinformatic analysis, we developed two algorithms for large-scale discovery of T6SS effector proteins (T6Es). We experimentally validated ten putative antibacterial T6SS effector proteins and one cognate immunity gene from a diverse species. This study provides a systematic genomic perspective of the role of the T6SS in nature, a thorough analysis of T6E evolution and genomic properties, and discovery of a large number of candidate T6Es using new approaches.

sentences: Dopaminergic regulation of vestibulo-cerebellar circuits through unipolar brush cells While multiple monoamines modulate cerebellar output, the mechanistic details of dopaminergic signaling in the cerebellum remain poorly understood. Here we show that Drd1 dopamine receptors are expressed in unipolar brush cells (UBCs) of the cerebellar vermis. Drd1 activation increases UBC firing rate and postsynaptic NMDA receptor-mediated currents. Using anatomical tracing and in situ hybridization, we tested three hypotheses about the source of cerebellar dopamine. We exclude midbrain dopaminergic nuclei and tyrosine hydroxylase-positive Purkinje cells as potential sources, supporting the possibility of dopaminergic co-release from locus coeruleus (LC) axons. Using an optical dopamine sensor GRABDA, electrical stimulation, and optogenetic activation of LC fibers in the acute slice, we find evidence for monoamine release onto Drd1-expressing UBCs. Altogether, we propose that the LC regulates cerebellar cortex activity by co-releasing dopamine onto UBCs to modulate their response to cerebellar inputs. Purkinje neurons directly inhibit these Drd1-positive UBCs, forming a dopamine-sensitive recurrent vestibulo-cerebellar circuit.

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Mechanisms that ensure speed and fidelity in eukaryotic translation termination Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single-molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro-reconstituted yeast translation system. We found that the two eukaryotic release factors bind together to recognize stop codons rapidly and elicit termination via a tightly regulated, multi-step process that resembles tRNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.

One Sentence SummaryDirect visualization of eukaryotic translation reveals the dynamics of termination at stop codons.

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DRDOCK: A Drug Repurposing platform integrating automated docking, simulations and a log-odds-based drug ranking scheme MotivationDrug repurposing, where drugs originally approved to treat a disease are reused to treat other diseases, has received escalating attention especially in pandemic years. Structure-based drug design, integrating small molecular docking, molecular dynamic (MD) simulations and AI, has demonstrated its evidenced importance in streamlining new drug development as well as drug repurposing. To perform a sophisticated and fully automated drug screening using all the FDA drugs, intricate programming, accurate drug ranking methods and friendly user interface are very much needed.

ResultsHere we introduce a new web server, DRDOCK, Drug Repurposing DOcking with Conformation-sampling and pose re-ranKing - refined by MD and statistical models, which integrates small molecular docking and molecular dynamic (MD) simulations for automatic drug screening of 2016 FDA-approved drugs over a user-submitted single-chained target protein. The drugs are ranked by a novel drug-ranking scheme using log-odds (LOD) scores, derived from feature distributions of true binders and decoys. Users can submit a selection of LOD-ranked poses for further MD-based binding affinity evaluation. We demonstrated that our platform can indeed recover one of the substrates for nsp16, a cap ribose 2'-O methyltransferase, and recommends that fluralaner, tegaserod and fenoterol could be repurposed for the COVID19 treatment with the latter two being confirmed in SARS-CoV2 suppression experiments. All the sampled docking poses and trajectories can be 3D-viewed and played via our web interface. This platform shall be easy-to-use for general scientists and medicinal researchers to carry out drug repurposing within a couple of days which should add value to our timely responses to, particularly, emergent disease outbreaks.

Availability and implementationDRDOCK can be freely accessed from https://dyn.life.nthu.edu.tw/drdock/. (Due to the hardware upgrade, the service is NOT available before 7/18, 2021)

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Fitting deterministic population models tostochastic data: from trajectory matching tostate-space models O_LIPopulation and community ecology traditionally has a very strong theoretical foundation with well-known models, such as the logistic and its many variations, and many modification of the classical Lotka-Volterra predator-prey and interspecific competition models. More and more, these classical models are confronted to data via fitting to empirical time-series for purposes of projections or for estimating model parameters of interest. However, the interface between mathematical population or community models and data, provided by a statistical model, is far from trivial.
C_LIO_LIIn order to help empiricists, especially researchers working with experimental laboratory populations in micro- and mesocosms, make informed decisions, we here ask which error structure one should use when fitting classical deterministic ODE models to empirical data, from single species to community dynamics and trophic interactions. We use both realistically simulated data and empirical data from microcosms to answer this question in a Bayesian framework.
C_LIO_LIWe find that many model parameters can be estimated precisely with an appropriate choice of error structure using pure observation error or state-space models, if observation errors are not too high. However, Allee effect models are typically hard to identify and state-space models should be preferred with when model complexity increases.
C_LIO_LIOur work shows that, at least in the context of experimental laboratory populations, deterministic models can be used to describe stochastic population dynamics that include process variability and observation error. Also, many scenarios do not require a complex state-space model formulation and simpler trajectory matching is sufficient for accurate parameter estimates. Finally, we provide a comprehensive tutorial for fitting these models in R.
C_LI

sentences: A novel glucocorticoid and androgen receptor modulator reduces viral entry and innate immune inflammatory responses in the Syrian Hamster model of SARS-CoV-2 Since its initial discovery in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID19, has spread worldwide and despite significant research efforts, treatment options remain limited. Replication of SARS-CoV-2 in lung is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses triggered, in part, by heightened production of interleukin-6 (IL-6) that recruits lymphocytes to the site of infection that amplify tissue injury. Antagonists of the glucocorticoid and androgen receptors have shown promise in experimental models of COVID19 and in clinical studies, because cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2), are transcriptionally regulated by these receptors. We therefore postulated that the glucocorticoid (GR) and androgen receptor (AR) antagonist, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID19. Animals were infected intranasally with 2.5 x 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/ 2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were then examined at days 3, 5 and 7 post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the initiation and progression of SARS-CoV-2 infection. Results of these studies indicated that oral administration of PT150 decreased replication of SARS-CoV-2 in lung, as well as expression of ACE2 and TMPRSS2 protein. Hypercellularity and inflammatory cell infiltration driven by macrophage responses were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular modeling suggested that PT150 binds to the co-activator interface of the ligand binding domain of both AR and GR and thereby acts as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR across multiple species permissive to SARS-CoV-2 infection revealed a high degree of sequence identity maintained across species, including human, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID19 across variants of SARS-CoV-2.

sentences: Host protease activity on bacterial pathogens promotes complement- and antibiotic-directed killing Our understanding of how the host immune system thwarts bacterial evasive mechanisms remains incomplete. Here, we show that host protease neutrophil elastase acts on Acinetobacter baumannii and Pseudomonas aeruginosa to destroy factors that prevent serum-associated, complement-directed killing. The protease activity also enhances bacterial susceptibility to antibiotics in sera. These findings implicate a new paradigm where host protease activity on bacteria acts synergistically with the host complement system and antibiotics to defeat bacterial pathogens.

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Increasing plant group productivity through latent genetic variation for cooperation Historic yield advances in the major crops have to a large part been achieved by selection for improved productivity of groups of plant individuals such as high-density stands. Research suggests that such improved group productivity depends on "cooperative" traits (e.g. erect leaves, short stems) that - while beneficial to the group - decrease individual fitness under competition. This poses a problem for some traditional breeding approaches, especially when selection occurs at the level of individuals, because "selfish" traits will be selected for and reduce yields in high-density monocultures. One approach therefore has been to select individuals based on ideotypes with traits expected to promote group productivity. However, this approach is limited to architectural and physiological traits whose effects on growth and competition are relatively easy to anticipate.

Here, we developed a general and simple method for the discovery of alleles promoting cooperation in plant stands. Our method is based on the game-theoretical premise that alleles increasing cooperation incur a cost to the individual but benefit the monoculture group. Testing the approach using the model plant Arabidopsis thaliana, we found a major effect locus where the rarer allele was associated with increased cooperation and productivity in high-density stands. The allele likely affects a pleiotropic gene, since we find that it is also associated with reduced root competition but higher resistance against disease. Thus, even though cooperation is considered evolutionarily unstable, conflicting selective forces acting on a pleiotropic gene might maintain latent genetic variation for it in nature. Such variation, once identified in a crop, could be rapidly leveraged in modern breeding programs and provide efficient routes to increase yields.

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DeepRank: A deep learning framework for data mining 3D protein-protein interfaces Three-dimensional (3D) structures of protein complexes provide fundamental information to decipher biological processes at the molecular scale. The vast amount of experimentally and computationally resolved protein-protein interfaces (PPIs) offers the possibility of training deep learning models to aid the predictions of their biological relevance.

We present here DeepRank, a general, configurable deep learning framework for data mining PPIs using 3D convolutional neural networks (CNNs). DeepRank maps features of PPIs onto 3D grids and trains a user-specified CNN on these 3D grids. DeepRank allows for efficient training of 3D CNNs with data sets containing millions of PPIs and supports both classification and regression.

We demonstrate the performance of DeepRank on two distinct challenges: The classification of biological versus crystallographic PPIs, and the ranking of docking models. For both problems DeepRank is competitive or outperforms state-of-the-art methods, demonstrating the versatility of the framework for research in structural biology.

sentences: Solute carrier 5A5 regulates systemic glucose homeostasis by mediating glucose absorption in the Drosophila midgut The small intestine is the first organ that is exposed to and absorbs dietary glucose and thus represents the first of a continuum of events that modulates normal systemic glucose homeostasis. A better understanding of the regulation of intestinal glucose transporters is therefore pertinent to our efforts in curbing metabolic disorders. However, so far, the mechanisms known to regulate SGLT1, the primary intestinal glucose transporter, are mainly elucidated from in vitro studies. The Drosophila midgut, functional equivalence of the small intestine, could serve as an efficient in vivo model system for studying intestinal glucose transporter regulation; however, no glucose transporter has yet been identified in the midgut. Here, we report that the Drosophila Solute Carrier 5A5 (dSLC5A5) is homologous to SGLT1 and is highly expressed in the midgut. The knockdown of dSLC5A5 decreases systemic and circulating sugar levels and decreases glucose uptake into the enterocytes. In contrary, the overexpression of dSLC5A5 elevates systemic and circulating sugar levels and promotes glucose uptake into the enterocytes. We show that dSLC5A5 undergoes dynamin-dependent endocytosis in the enterocyte apical membrane, and that dSLC5A5 endocytosis is essential for the glucose uptake capability of dSLC5A5. Moreover, we provide evidence supporting that intracellular lysosomal degradation of endocytosed dSLC5A5 plays a significant role in the maintenance of dSLC5A5 level in the enterocyte apical membrane. We further show that short-term exposure to glucose upregulates SLC5A5 abundance in the enterocyte apical membrane. Finally, we show that the loss or gain of dSLC5A5 ameliorates or exacerbates the high sugar diet (HSD)-mediated glucose metabolic defects. Together, our studies uncovered the first Drosophila glucose transporter in the midgut and reveal new mechanisms that regulate glucose transporters in the enterocyte apical membranes.

sentences: Luminosity thresholds of colored surfaces are determined by their upper-limit luminances empirically internalized in the visual system We typically have a fairly good idea whether a given object is self-luminous or illuminated, but it is not fully understood how we make this judgement. This study aimed to identify determinants of the luminosity threshold, a luminance level at which a surface begins to appear self-luminous. We specifically tested a hypothesis that our visual system knows the maximum luminance level that a surface can reach under the physical constraint that a surface cannot reflect more light than any incident light and applies this prior to determine the luminosity thresholds. Observers were presented with a 2-degree circular test field surrounded by numerous overlapping colored circles and luminosity thresholds were measured as a function of (i) the chromaticity of the test field, (ii) the shape of surrounding color distribution and (iii) the color of the illuminant of the surrounding colors. We found that the luminosity thresholds peaked around the chromaticity of test illuminants and decreased as the purity of the test chromaticity increased. However, the loci of luminosity thresholds across chromaticities were nearly invariant to the shape of the surrounding color distribution and generally resembled the loci drawn from theoretical upper-limit luminances and upper-limit luminance boundaries of real objects. These trends were particularly evident for illuminants on the black-body locus and did not hold well under atypical illuminants such as magenta or green. These results support the idea that our visual system empirically internalizes the gamut of surface colors under natural illuminants and a given object appears self-luminous when its luminance exceeds this internalized upper-limit luminance.

sentences: A neuromechanical model of multiple network rhythmic pattern generators for forward locomotion in C. elegans Multiple mechanisms contribute to the generation, propagation, and coordination of the rhythmic patterns necessary for locomotion in Caenorhabditis elegans. Current experiments have focused on two possibilities: pacemaker neurons and stretch-receptor feedback. Here, we focus on whether it is possible that a chain of multiple network rhythmic pattern generators in the ventral nerve cord also contribute to locomotion. We use a simulation model to search for parameters of the anatomically constrained ventral nerve cord circuit that, when embodied and situated, can drive forward locomotion on agar, in the absence of pacemaker neurons or stretch-receptor feedback. Systematic exploration of the space of possible solutions reveals that there are multiple configurations that result in locomotion that is consistent with certain aspects of the kinematics of worm locomotion on agar. Analysis of the best solutions reveals that gap junctions between different classes of motorneurons in the ventral nerve cord can play key roles in coordinating the multiple rhythmic pattern generators.

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The haplotype-resolved chromosome pairs and transcriptome of a heterozygous diploid African cassava cultivar BackgroundCassava (Manihot esculenta) is an important clonally propagated food crop in tropical and sub-tropical regions worldwide. Genetic gain by molecular breeding is limited because cassava has a highly heterozygous, repetitive and difficult to assemble genome.

FindingsHere we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads. We present two chromosome scale haploid genomes phased with Hi-C technology for the diploid African cassava variety TME204. Genome comparisons revealed extensive chromosome re-arrangements and abundant intra-genomic and inter-genomic divergent sequences despite high gene synteny, with most large structural variations being LTR-retrotransposon related. Allele-specific expression analysis of different tissues based on the haplotype-resolved transcriptome identified both stable and inconsistent alleles with imbalanced expression patterns, while most alleles expressed coordinately. Among tissue-specific differentially expressed transcripts, coordinately and biasedly regulated transcripts were functionally enriched for different biological processes. We use the reference-quality assemblies to build a cassava pan-genome and demonstrate its importance in representing the genetic diversity of cassava for downstream reference-guided omics analysis and breeding.

ConclusionsThe haplotype-resolved genome allows the first systematic view of the heterozygous diploid genome organization in cassava. The completely phased and annotated chromosome pairs will be a valuable resource for cassava breeding and research. Our study may also provide insights into developing cost-effective and efficient strategies for resolving complex genomes with high resolution, accuracy and continuity.

sentences: Ultrastructural features of presynaptic active zones and dense core vesicles of olfactory projection neuron boutons in Drosophila melanogaster In Drosophila melanogaster, olfactory projection neurons (PNs) convey odor information from the peripheral olfactory center to higher brain regions. The anatomical and physiological properties of PNs have been well characterized at the cellular and circuit level. The ultrastructural features of PNs remain unknown however, particularly with respect to presynaptic active zones (PAZs) and dense core vesicles (DCVs). In the current study, membrane-labeled electron microscopy was used to volume-reconstruct 89 PN axonal boutons and identify the internal PAZs and DCVs. Based on ultrastructural parameters, these PN boutons could be classified into three morphological distinct subtypes. Interestingly, the distributions of PAZs and DCVs were distinct within these three subtypes. DCVs were enriched in membrane labeled GH146-positive boutons, suggesting that GH146-positive PNs release both neurotransmitters and neuromodulators. The study identified the detailed distributions of PAZs and DCVs in PN boutons and indicates that neuromodulators mediated by DCVs may play an important role in PNs for olfactory processing.

sentences: Plant-associated Pseudomonas aeruginosa harbor multiple virulence traits essential for mammalian infection Pseudomonas aeruginosa is a leading opportunistic pathogen capable of causing fatal infections in immunocompromised individuals and patients with degenerative lung diseases. Agricultural soil and plants are the vast reservoirs of this dreaded pathogen. However, there have been limited attempts to analyze the pathogenicity of P. aeruginosa strains associated with edible vegetable plants. This study aims to elucidate the virulence attributes of P. aeruginosa strains isolated from the rhizosphere and endophytic niches of cucumber, tomato, eggplant, and chili collected from agricultural fields. Virulence of the agricultural strains was compared to three previously characterized clinical isolates. Our results showed that 50% of the plant-associated strains formed significant levels of biofilm and exhibited swarming motility. Nearly 80% of these strains produced considerable levels of rhamnolipid and exhibited at least one type of lytic activity (hemolysis, proteolysis, and lipolysis). Their virulence was also assessed based on their ability to suppress the growth of plant pathogens (Xanthomonas oryzae, Pythium aphanidermatum, Rhizoctonia solani, and Fusarium oxysporum) and kill a select nematode (Caenorhabditis elegans). The plant-associated strains showed significantly higher virulence against the bacterial phytopathogen whereas the clinical strains had significantly higher antagonism against the fungal pathogens. In C. elegans slow-killing assay, the clinical strains caused 50-100% death while a maximum of 40% mortality was induced by the agricultural strains. This study demonstrates that some of the P. aeruginosa strains associated with edible plants harbor multiple virulence traits. Upon infection of humans or animals, these strains may evolve to be more pathogenic and pose a significant health hazard.

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Evaluation of odorant-binding protein-1 as a molecular marker for identifying biological forms and delimitating sibling species of Anopheles stephensi BackgroundAnopheles stephensi, an invasive malaria vector, has been reported to have three biological forms identifiable based on the number of ridges present on the eggs floats and the dimension of eggs. Recently, these forms have been designated as sibling species based on the fixed differences in the DNA sequence of the first intron of the odorant-binding protein-1 (AsteObp1). In this study, we evaluated the utility of this neutral marker in designating sibling species or identifying biological forms.

MethodsField collected and laboratory-reared An. stephensi were characterized for biological forms based on the number of floats on egg-ridge. DNA sequencing of the partial AsteObp1 gene of An. stephensi individuals were performed by Sangers method, either directly or after cloning with a plasmid vector.

ResultsAsteObp1 intron-1 in Indian An. stephensi populations are highly polymorphic with the presence of more than 12 haplotypes exhibiting nucleotide-as well as length-polymorphism (90-to-121 bp). A majority of the field samples were heterozygous (up to 89% in the field populations). The phasing of haplotypes in heterozygotes through Sangers sequencing was challenging due to indels (1-to-24 bp) at multiple loci. No specific haplotype or monophyletic clade of intron-1 was found associated with a specific biological form. The inbreeding coefficient for this marker was close to zero in field and laboratory populations which refute the existence of sibling species based on the AsteObp1 marker.

ConclusionsAsteObp1 cannot serve as a marker for the identification of biological forms of An. stephensi. The probable existence of sibling species in An. stephensi based on the AsteObp1 intron-1 is refuted.

sentences: Comparing algorithms for assessing upper limb use with inertial measurement units The various existing measures to quantify upper limb use from wrist-worn inertial measurement units (IMU) can be grouped into three categories: (a) Thresholded activity counting, (b) Gross movement score and (c) machine learning. While machine learning algorithms are a promising approach to detect upper limb use, there is currently no knowledge of the information used by these methods, and the data-related factors that influence their performance. A comparison of existing methods was carried out using data from a previous study which was collected from 10 unimpaired and 5 hemiparetic subjects, with annotation to identify periods of functional and non-functional upper limb use. Intra-subject random forest machine learning measures were found to classify upper limb use more accurately than other measures. The random forest measure uses information about the orientation and the amount of movement of the forearm to detect upper limb use. The types of movements and the proportion of functional data included in training/testing set influences the performance of machine learning measures. This study presents the first step towards understanding and optimizing machine learning methods for upper limb use assessment using wearable sensors.

sentences: Discovery of lipotoxicity-sensitive transcription factors in non-alcoholic steatohepatitis Non-alcoholic fatty liver disease is a continuum of disorders among which non-alcoholic steatohepatitis (NASH) is particularly associated with a negative prognosis. Hepatocyte lipotoxicity is one of the main pathogenic factors of liver fibrosis and NASH. However, the molecular mechanisms regulating this process are poorly understood. The main aim of this study was to dissect transcriptional mechanisms regulated by lipotoxicity in hepatocytes. We achieved this aim by combining transcriptomic, proteomic and chromatin accessibility analyses from human liver and mouse hepatocytes. This integrative approach revealed several transcription factor networks deregulated by NASH and lipotoxicity. To validate these predictions, genetic deletion of the transcription factors MAFK and TCF4 was performed, resulting in hepatocytes that were better protected against saturated fatty acid oversupply. MAFK- and TCF4-regulated gene expression profiles suggest a mitigating effect against cell stress, while promoting cell survival and growth. Moreover, in the context of lipotoxicity, some MAFK and TCF4 target genes were to the corresponding differentially regulated transcripts in human liver fibrosis. Collectively, our findings comprehensively profile the transcriptional response to lipotoxicity in hepatocytes, revealing new molecular insights and providing a valuable resource for future endeavours to tackle the molecular mechanisms of NASH.

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SpatialExperiment: infrastructure for spatially resolved transcriptomics data in R using Bioconductor SummarySpatialExperiment is a new data infrastructure for storing and accessing spatially resolved transcriptomics data, implemented within the R/Bioconductor framework, which provides advantages of modularity, interoperability, standardized operations, and comprehensive documentation. Here, we demonstrate the structure and user interface with examples from the 10x Genomics Visium and seqFISH platforms, and provide access to example datasets and visualization tools in the STexampleData, TENxVisiumData, and ggspavis packages.

Availability and ImplementationThe SpatialExperiment, STexampleData, TENxVisiumData, and ggspavis packages are available from Bioconductor. The package versions described in this manuscript are available in Bioconductor version 3.15 onwards.

Contactrisso.davide@gmail.com, shicks19@jhu.edu

Supplementary InformationSupplementary Tables and Figures are available online.

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Lipid Tails Modulate Antimicrobial Peptide Membrane Incorporation and Activity Antimicrobial peptides (AMPs) are cationic, amphipathic peptides that interact directly with lipid bilayers. AMPs generally interact with anionic lipid head groups, but it is less clear how the lipid tail length and saturation modulates interactions with membranes. Here we used native mass spectrometry to measure the stoichiometry of three different AMPs--LL-37, indolicidin, and magainin-2--in lipid nanodiscs. We also measured the activity of these AMPs in large unilamellar vesicle leakage assays. We found that LL-37 formed specific hexamer complexes but with different assembly pathways and affinities that depended on the bilayer thickness. LL-37 was also most active in lipid bilayers containing longer, unsaturated lipids. In contrast, indolicidin incorporated to a higher degree into more fluid lipid bilayers but was more active with thinner, less fluid bilayers. Finally, magainin-2 incorporated to a higher degree into longer, unsaturated bilayers and showed more activity in these same conditions. Together, these data show that higher amounts of peptide incorporation generally led to higher activity and that AMPs tend to incorporate more into longer unsaturated lipid bilayers. However, the activity of AMPs was not always directly related to amount of peptide incorporated.

Graphical Abstract

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CRISPR screening identifies novel PARP inhibitor classification based on distinct base excision repair pathway dependencies DNA repair deficiencies have become an increasingly promising target for novel therapeutics within the realm of clinical oncology. Recently, several inhibitors of Poly(ADP-ribose) Polymerases (PARPs) have received approval for the treatment of cancers primarily with deleterious mutations in the homologous recombination (HR) proteins, BRCA1 and BRCA2. Despite numerous clinical trials which have been completed or are currently ongoing, the mechanism of action by which PARP inhibitors selectively kill tumor cells is poorly understood. While many believe "trapping" of PARP proteins to DNA at sites of damage is the most important determinant driving cytotoxicity by these drugs, clinically effective inhibitors exist with a diverse range of PARP-trapping qualities. These findings suggest that characterization of inhibitors as strong versus weak trappers does not properly capture the intra-class characteristics of these drugs. Here, we use a novel, targeted DNA damage response (DDR) CRISPR/Cas9 screening library to reveal heterogenous genetic dependencies on the base excision repair (BER) pathway for PARP inhibitors, which is not correlated with PARP trapping ability or catalytic inhibition of PARP. These findings demonstrate that inhibition of PARylation and induction of PARP trapping are not the only factors contributing to distinct biological activity for different PARP inhibitors, and they provide insight into the optimal choice of PARP inhibitors for use in the setting of specific DDR defects.

AUTHOR SUMMARYTargeted cancer therapies rely on our general understanding of which genetic mutations are involved in both sensitivity and resistance to such anticancer agents. In this study, we describe the use of functional genetic screening to evaluate the role of various DNA repair proteins in response to inhibitors of PARP, a quintessential example of targeted therapy. While PARP inhibitors are best known for their utility in cancers with homologous recombination defects, we show that some inhibitors within this class may have additional functionality in cancers with deficient base excision repair. These findings highlight not only the importance of PARP inhibitor selection in the appropriate context, but also the mechanistic differences that exist within this class of inhibitors. It is our hope that our findings will inspire future work evaluating the use of specific PARP inhibitor selection in designing clinical trials to further expand the use of PARP inhibitors beyond tumors with homologous recombination deficiencies.

sentences: Lightning Fast and Highly Sensitive Full-Length Single-cell sequencing using FLASH-Seq In the last 10 years, single-cell RNA-sequencing (scRNA-seq) has undergone exponential growth. Emulsion droplets methods1-3, such as those commercialized by 10x Genomics, have allowed researchers to analyze tens of thousands of cells in parallel in a robust and reproducible way. However, in contrast to SMART-based full-length sequencing protocols4,5, these methods interrogate only the outer portion of the transcripts and still lack the required sensitivity for analyzing comprehensively the transcriptome of individual cells. Building upon the existing SMART-seq forerunners protocols4,5, we developed FLASH-Seq (FS), a new scRNA-seq method which displays greater sensitivity while decreasing incubation times and reducing the number of processing steps compared to its predecessors. The entire FS protocol - from lysed cells to pooled cDNA libraries - can be performed in ~4.5 hours, is automation-friendly and can be easily miniaturized to decrease costs.

sentences: Delivering on Biden's 2030 conservation commitment On January 27, 2021, President Biden signed an executive order, Tackling the Climate Crisis at Home and Abroad, committing the United States to various goals within his campaigns major climate policy, the Biden Plan for a Clean Energy Revolution and Environmental Justice. Included in this executive order is a commitment to "conserving at least 30 percent of [the United States] lands and oceans by 2030." This ambitious conservation target signals a promising direction for biodiversity in the United States. However, while the executive order outlines several goals for climate mitigation, the  30x30 target remains vague in its objectives, actions, and implementation strategies for protecting biodiversity. Biodiversity urgently needs effective conservation action, but it remains unclear where and what this 30% target will be applied to. Achieving different climate and biodiversity objectives will require different strategies and, in combination with the associated costs of implementation, will lead to different priority areas for conservation actions. Here, we illustrate what the 30% target could look like across four objectives reflective of the ambitious goals outlined in the executive order. We compile several variations of terrestrial protected area networks guided by these different objectives and examine the trade-offs in costs, ecosystem representation, and climate mitigation potential between each. We find little congruence in priority areas across objectives, emphasizing just how crucial it will be for the Biden administration to develop clear objectives and establish appropriate performance metrics from the outset to maximize both conservation and climate outcomes in support of the 30x30 target. We discuss important considerations that must guide the administrations conservation strategies in order to ensure meaningful conservation outcomes can be achieved over the next decade.

sentences: Cognitive impairment and metabolite profile alterations in the hippocampus and cortex of male and female mice exposed to a fat and sugar-rich diet are normalized by diet reversal Diabetes impacts on brain metabolism, structure and function. Alterations in brain metabolism have been observed in obesity and diabetes models induced by exposure to diets rich in saturated fat and/or sugar, and have been linked to memory impairment. However, it remains to be determined whether brain dysfunction induced by obesogenic diets results from permanent brain alterations. We tested the hypothesis that an obesogenic diet (high-fat and high-sucrose diet; HFHSD) causes reversible changes in hippocampus and cortex metabolism and alterations in behavior. To test this hypothesis, mice were exposed to HFHSD for 24 weeks or for 16 weeks followed by 8 weeks of diet normalization. Development of the metabolic syndrome, changes in behavior, and brain metabolite profiles by 1H magnetic resonance spectroscopy (MRS) were assessed longitudinally. Control mice were fed an ingredient-matched low-fat and low-sugar diet. Mice fed a HFHSD developed obesity, glucose intolerance and insulin resistance, with a more severe phenotype in male than female mice. Relative to controls, both male and female HFHSD-fed mice showed increased anxiety-like behavior, impaired memory in object recognition tasks, but preserved working spatial memory as evaluated by spontaneous alternation in a Y-maze. Alterations in the metabolite profiles were observed both in the hippocampus and cortex, but were more distinct in the former. HFHSD-induced metabolic changes included altered levels of lactate, glutamate, GABA, glutathione, taurine, N-acetylaspartate, creatine and choline. Notably, HFHSD-induced metabolic syndrome, anxiety, memory impairment, and brain metabolic alterations recovered upon diet normalization for 8 weeks. We conclude that cortical and hippocampal derangements induced by long-term HFHSD consumption are reversible rather than being the result of permanent tissue damage.

sentences: Implications Derived from S-Protein Variants of SARS-CoV-2 from Six Continents Spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critical determinants of the infectivity and antigenicity of the virus. Several mutations in the spike protein of SARS-CoV-2 have already been detected, and their effect in immune system evasion and enhanced transmission as a cause of increased morbidity and mortality are being investigated. From pathogenic and epidemiological perspectives, spike proteins are of prime interest to researchers. This study focused on the unique variants of S proteins from six continents Asia, Africa, Europe, Oceania, South America, and North America. In comparison to the other five continents, Africa (29.065%) had the highest percentage of unique S proteins. Notably, only North America had 87% (14046) of the total (16143) specific S proteins available in the NCBI database(across all continents). Based on the amino acid frequency distributions in the S protein variants from all the continents, the phylogenetic relationship implies that unique S proteins from North America were significantly different from those of the other five continents. Overtime, the unique variants originating from North America are most likely to spread to the other geographic locations through international travel or naturally by emerging mutations. Hence it is suggested that restriction of international travel should be considered, and massive vaccination as an utmost measure to combat the spread of COVID-19 pandemic. It is also further suggested that the efficacy of existing vaccines and future vaccine development must be reviewed with careful scrutiny, and if needed, further re-engineered based on requirements dictated by new emerging S protein variants.

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Autonomic arousals contribute to brain fluid pulsations during sleep During sleep, slow waves of neuro-electrical activity engulf the human brain and aid in the consolidation of memories. Recent research suggests that these slow waves may also promote brain health by facilitating the removal of metabolic waste, possibly by orchestrating the pulsatile flow of cerebro-spinal fluid (CSF) through local neural control over vascular tone. To investigate the role of slow waves in the generation of CSF pulsations, we analyzed functional MRI data obtained across the full sleep-wake cycle and during a respiratory task during wakefulness. This revealed a novel generating mechanism that relies on the autonomic regulation of cerebral vascular tone without requiring slow electrocortical activity or even sleep. Therefore, the role of CSF pulsations in brain waste clearance may, in part, depend on proper autoregulatory control of cerebral blood flow.

One-Sentence SummaryAutonomic regulation of cerebral vascular tone mediates CSF flow and may contribute to brain waste clearance.

sentences: Circulating SARS-CoV-2 variants B.1.1.7, 501Y.V2, and P.1 have gained ability to utilize rat and mouse Ace2 and altered in vitro sensitivity to neutralizing antibodies and ACE2-Ig The emergence of SARS-CoV-2 variants poses greater challenges to the control of COVID-19 pandemic. Here, we parallelly investigated three important characteristics of seven SARS-CoV-2 variants, including two mink-associated variants, the B.1.617.1 variant, and the four WHO-designated variants of concerns (B.1.1.7, B.1.351, P.1, and B.1.617.2). We first investigated the ability of these variants to bind and use animal ACE2 orthologs as entry receptor. We found that, in contrast to a prototype variant, the B.1.1.7, B.1.351, and P.1 variants had significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins, suggesting increased susceptibility of these species to these SARS-CoV-2 variants. We then evaluated in vitro neutralization sensitivity of these variants to four monoclonal antibodies in clinical use. We observed that all the variants were partially or completely resistant against at least one of the four tested antibodies, with B.1.351 and P.1 showing significant resistance to three of them. As ACE2-Ig is a broad-spectrum anti-SARS-CoV-2 drug candidate, we then evaluated in vitro neutralization sensitivity of these variants to eight ACE2-Ig constructs previously described in three different studies. All the SARS-CoV-2 variants were efficiently neutralized by these ACE2-Ig constructs. Interestingly, compared to the prototype variant, most tested variants including the variants of concern B.1.1.7, B.1.351, P.1, and B.1.617.2 showed significantly increased (up to [~]15-fold) neutralization sensitivity to ACE2-Ig constructs that are not heavily mutated in the spike-binding interface of the soluble ACE2 domain, suggesting that SARS-CoV-2 evolves toward better utilizing ACE2, and that ACE2-Ig is an attractive drug candidate for coping with SARS-CoV-2 mutations.

sentences: ACTION AFFORDANCE AFFECTS PROXIMAL AND DISTAL GOAL-ORIENTED PLANNING AO_SCPLOWBSTRACTC_SCPLOWSeminal studies on human cognitive behavior have been conducted in controlled laboratory settings, demonstrating that visual attention is mainly goal-directed and allocated based on the action performed. However, it is unclear how far these results generalize to cognition in more naturalistic settings. The present study investigates active inference processes revealed by eye movements during interaction with familiar and novel tools with two levels of realism of the action affordance. We presented participants with 3D tool models that were either familiar or unfamiliar, oriented congruent or incongruent to their handedness, and asked participants to interact with them by lifting or using. Importantly, we used the same experimental design in two setups. In the first experiment, participants interacted with a VR controller in a low realism environment; in the second, they performed the task with an interaction setup that allowed differentiated hand and finger movements in a high realism environment. We investigated the differences in odds of fixations and their eccentricity towards the tool parts before action initiation. The results show that participants fixate more on the tools effector part before action initiation for the use task for unfamiliar tools. Furthermore, with more realistic action affordances, subjects fixate more on the tools handle as a function of the tools orientation, well before the action was executed. Secondly, the spatial viewing bias on the tool reveals early fixations are influenced by the task and the familiarity of the tools. In contrast, later fixations are associated with the manual planning of the interaction. In sum, the findings from the experiments suggest that fixations are made in a task-oriented way to plan the intended action well before action initiation. Further, with more realistic action affordances, fixations are made towards the proximal goal of optimally planning the grasp even though the perceived action on the tools is identical for both experimental setups. Taken together, proximal and distal goal-oriented planning is contextualized to the realism of action/interaction afforded by an environment.

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Extreme hot weather has stronger impacts on avian reproduction in forests than in cities Climate change and urbanization are among the most salient human-induced changes affecting Earths biota. Extreme weather events can have high biological impacts and are becoming more frequent recently. In cities, the urban heat island can amplify the intensity and frequency of hot weather events. However, the joint effects of heat events and urban microclimate on wildlife are unclear, as urban populations may either suffer more from increased heat stress or become adapted to warmer temperatures. Here we test whether the effects of hot weather on reproductive success of great tits (Parus major) are exacerbated or dampened in urban environments compared to forest habitats. By studying two urban and two forest populations over six years, we show that 14-16 days-old nestlings have smaller body mass and tarsus length, and suffer increased mortality when they experience a higher number of hot days during the nestling period. The negative effects of hot weather on body mass and survival are significantly stronger in forests than in urban areas, where these effects are dampened or even reversed. These results suggest that urban birds are less vulnerable to extreme hot weather conditions than their non-urban conspecifics, possibly by adaptively evolving or flexibly adjusting nestling physiology to tolerate heat stress, and/or by adjusting parental behavior in response to heat. This finding highlights that endothermic vertebrates may be able to adapt to heat stress, which may help their populations cope with the joint challenges of climate change and urbanization.

Significance statementExtreme weather events are becoming more frequent due to climate change and can have substantial effects on reproduction and survival of wild animals. Urban heat island can amplify the frequency of extreme hot weather events, making it potentially more harmful for city-dwelling organisms. Alternatively, urban populations living in warmer environments may adapt to better tolerate heat. We investigated these alternatives by comparing nestling development and survival between urban and forest great tit (Parus major) populations. We found that urban populations are less vulnerable to heat: nestling body mass and survival decreased rapidly with the increasing number of hot days in forests, while these effects were dampened in urban broods. Thus, urban populations may become adapted to better tolerate heat events.

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Feedback-related potentials and oscillations during trial and error learning in Parkinson's disease Electrophysiological markers of performance monitoring are thought to reflect functioning of dedicated neural networks and neuromodulatory systems. Whether and how these markers are altered in neurological diseases and whether they can reflect particular cognitive deficits remains to be confirmed. Here we first tested whether the frontal medial feedback-related potential, evoked during a trial and error learning task, is changed in early Parkinsons disease patients compared to control subjects. The potential was not changed in amplitude and discriminated negative and positive feedback as in controls. Feedback-related markers in Parkinsons patients also appeared in time-frequency analyses, unaltered in theta (3-7 Hz) band but reduced in beta (20-30 Hz) oscillations for positive feedback. Beta oscillations power appeared to be dramatically globally reduced during the task. Overall, our results show that Beta oscillation markers of performance monitoring captured by EEG are selectively altered in Parkinsons disease patients, and that they are accompanied by changes in task-related oscillatory dynamics.

Significance StatementFrontal neural activity evoked by outcomes reveal the functioning of neural systems devoted to flexible behaviours. Modulations of such activity in Parkinsons disease (PD) patients could reflect specific alterations of neural systems and cognitive processing. The goal is to evaluate whether such activity can serve as markers of the disease. Here, using EEG and a trial and error learning protocol, we show that mid-frontal midline performance feedback-related potentials for different types of feedback were similar in controls and early diagnosed PD patients. However, task-related oscillations revealed alterations in the beta range accompanied by more global beta activity alteration in PD compared to controls subjects. This study provides data relevant to the search for non-motor biomarkers in early stages of PD.

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Box C/D Small Nucleolar Ribonucleoproteins Regulate Mitochondrial Surveillance and Innate Immunity Monitoring of mitochondrial functions is crucial for organismal survival. This task is performed by mitochondrial surveillance or quality control pathways, which are activated by signals originating from mitochondria and relayed to the nucleus (retrograde response) to start the transcription of protective genes. In Caenorhabditis elegans, several systems exist, including the UPRmt, MAPKmt, and the ESRE pathway. These pathways are highly conserved and their loss results in compromised survival following mitochondrial stress.

In this study, we found a novel interaction between the box C/D snoRNA core proteins (snoRNPs) and mitochondrial surveillance and innate immunity pathways. We showed that C/D snoRNPs are required for the full expressions of UPRmt and ESRE upon stress. Meanwhile, we found that the loss of C/D snoRNPs increased immune responses. Understanding the "molecular switch" mechanisms of interplay between these pathways may be important for understanding of multifactorial processes, including response to infection or aging.

sentences: COT: an efficient Python tool for detecting marker genes among many subtypes We develop an accurate and efficient method to detect marker genes among many subtypes using subtype-enriched expression profiles. We implement a Cosine based One-sample Test (COT) Python software that is easy to use and applicable to multi-omics data. We demonstrate the performance and utility of COT on gene expression and proteomics data acquired from tissue or cell subtypes. Formulated as a one-sample test with Cosine similarity test statistic in scatter space, the detected de novo marker genes will allow biologists to perform a more comprehensive and unbiased molecular characterization, deconvolution and classification of complex tissue or cell subtypes.

sentences: Dynamic Profiling of  Binding and Allosteric Propensities  of the SARS-CoV-2 Spike Protein with Different Classes of Antibodies:  Mutational and Perturbation-Based Scanning Reveal Allosteric Duality of Functionally Adaptable Hotspots Structural and biochemical studies of the SARS-CoV-2 spike complexes with highly potent antibodies have revealed multiple conformation-dependent epitopes and a broad range of recognition modes linked to different neutralization responses In this study, we combined atomistic simulations with mutational and perturbation-based scanning approaches to perform in silico profiling of binding and allosteric propensities of the SARS-CoV-2 spike protein residues in complexes with B38, P2B-2F6, EY6A and S304 antibodies representing three different classes. Conformational dynamics analysis revealed that binding-induced modulation of soft modes can elicit the unique protein response to different classes of antibodies. Mutational scanning heatmaps and sensitivity analysis revealed the binding energy hotspots for different classes of antibodies that are consistent with the experimental deep mutagenesis, showing that differences in the binding affinity caused by global circulating variants in spike positions K417, E484 and N501 are relatively moderate and may not fully account for the observed antibody resistance effects. Through functional dynamics analysis and perturbation-response scanning of the SARS-CoV-2 spike protein residues in the unbound form and antibody-bound forms, we examine how antibody binding can modulate allosteric propensities of spike protein residues and determine allosteric hotspots that control signal transmission and global conformational changes. These results show that residues K417, E484, and N501 targeted by circulating mutations correspond to a group of versatile allosteric centers in which small perturbations can modulate collective motions, alter the global allosteric response and elicit binding resistance. We suggest that SARS-CoV-2 S protein may exploit plasticity of specific allosteric hotspots to generate escape mutants that alter response to antibody binding without compromising activity of the spike protein.

sentences: Microstructural variation in the human striatum using non-negative matrix factorization The striatum is a major subcortical connection hub that has been heavily implicated in a wide array of motor and cognitive functions. Here, we developed a normative multimodal, data-driven microstructural parcellation of the striatum using multiple magnetic resonance imaging-based metrics (mean diffusivity, fractional anisotropy, and the ratio between T1- and T2-weighted structural) from the Human Connectome Project Young Adult dataset (n=329 unrelated participants, age range: 22-35, F/M: 185/144). We further explored the biological and functional relationships of this parcellation by relating our findings to motor and cognitive performance in tasks known to involve the striatum as well as demographics. We identified 5 spatially distinct striatal components, for each hemisphere. We also show the gain in component stability when using multimodal versus unimodal metrics. Our findings suggest distinct microstructural patterns in the human striatum that are largely symmetric and that relate mostly to age and sex. Our work also highlights the putative functional relevance of these striatal components to different designations based on a Neurosynth meta-analysis.

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Babesia divergens egress from host cells is orchestrated by essential and druggable kinases and proteases A unique aspect of apicomplexan biology is the requirement for egress from and invasion into host red blood cells (RBCs). The cellular mechanisms and molecular mediators of RBC egress and invasion remain poorly characterized in Babesia spp., a group of parasites of veterinary importance and emerging cause of zoonotic disease. Through the use of video microscopy, transcriptomics, and chemical genetics we have implicated signaling, proteases and gliding motility in egress and/or invasion by Babesia divergens. We developed CRISPR/Cas9 and two inducible knockdown systems to perform a genetic screen of putative mediators of egress. We found that proteases ASP2 and ASP3 are required for invasion, and the latter is also required for egress. Strikingly, parasites continue to replicate intracellularly in the absence of the protein kinases, PKG or CDPK4, indicating that they are required for exit from the replication cycle and egress. These essential molecules present druggable targets for Babesia spp. All together we have established a molecular framework for the spread of infection through host RBCs, with egress of B. divergens more closely resembling T. gondii than the more evolutionarily related Plasmodium spp.

HighlightsO_LIEgress in Babesia divergens requires host cell lysis and parasite motility
C_LIO_LITranscriptomics can be used to identify egress and invasion proteins
C_LIO_LIKnockdown of the proteases, ASP2 and ASP3, inhibit egress and invasion
C_LIO_LIInhibition of PKG or CDPK4 signaling results in continued intracellular replication
C_LI

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The mechanobiology of craniosynostosis in Crouzon syndrome Children with syndromic forms of craniosynostosis undergo a plethora of surgical interventions to resolve the clinical features caused by the premature fusion of cranial sutures. While surgical correction is reliable, the need for repeated rounds of invasive treatment puts a heavy burden on the child and their family.

This study explores a non-surgical alternative using mechanical loading of the cranial joints to prevent or delay craniofacial phenotypes associated with Crouzon syndrome.

We treated Crouzon syndrome mice before the onset of craniosynostosis by cyclical mechanical loading of cranial joints using a custom designed set-up.

Cranial loading applied to the frontal bone partially restores normal skull morphology, significantly reducing the typical brachycephalic appearance. This is underpinned by the delayed closure of the coronal suture and of the intersphenoidal synchondrosis.

This study provides a novel treatment alternative for syndromic craniosynostosis which has the potential to be an important step towards replacing, reducing or refining the surgical treatment of all craniosynostosis patients.

sentences: Human belief state-based exploration and exploitation in an information-selective symmetric reversal bandit task Humans often face sequential decision-making problems, in which information about the environmental reward structure is detached from rewards for a subset of actions. In the current exploratory study, we introduce an information-selective symmetric reversal bandit task to model such situations and obtained choice data on this task from 24 participants. To arbitrate between different decision-making strategies that participants may use on this task, we developed a set of probabilistic agent-based behavioral models, including exploitative and explorative Bayesian agents, as well as heuristic control agents. Upon validating the model and parameter recovery properties of our model set and summarizing the participants choice data in a descriptive way, we used a maximum likelihood approach to evaluate the participants choice data from the perspective of our model set. In brief, we provide quantitative evidence that participants employ a belief state-based hybrid explorative-exploitative strategy on the information-selective symmetric reversal bandit task, lending further support to the finding that humans are guided by their subjective uncertainty when solving exploration-exploitation dilemmas.

sentences: WATLAS: high resolution and real-time tracking of many small birds in the Dutch Wadden Sea Movement is a fundamental aspect of life and tracking wild animals under natural conditions has become central to animal behaviour, ecology, and conservation science. Data from tracked animals have provided novel scientific insights on extreme migratory journeys, mechanisms of navigation, space use, and early warning signals of environmental change. Studying movement is therefore important, particularly in systems that may be vulnerable to anthropogenic effects. Technological advancements, and chiefly the development of GPS tags, have enabled animal tracking at high spatiotemporal resolution, yet trade-offs between cost, sampling frequency, tag weight and data retrieval limit the use of GPS tags to relatively few individuals and large species. A new  reverse-GPS wildlife tracking system, ATLAS, employs an array of receiver stations that detect and localise small ([~]0.6 g without battery), low-cost ([~]25 euro) tags by calculating differences in the arrival time of tag signal at minimally three stations. In this study, we introduce the Wadden Sea ATLAS system (WATLAS), implemented in the Dutch Wadden Sea, the Netherlands only natural UNESCO World Heritage Site, yet affected by a suite of anthropogenic activities, such as commercial fishing, mining, shipping, as well as sea level rise. From July 2017 to July 2021, we tracked 821 red knots, 182 sanderlings, 33 bar-tailed godwits, and 6 common terns. With four examples, we illustrate how WATLAS opens-up possibilities for studying space-use, among-individual variation in movement, and intra-specific interactions, and inter-specific (community) space use in the wild. We additionally argue that WATLAS could provide a tool for impact assessment, and thus aid nature conservation and management of the globally important Wadden Sea ecosystem.

sentences: A role for heritable transcriptomic variation in maize adaptation to temperate environments Transcription bridges genetic information and phenotypes. Here, we evaluated how changes in transcriptional regulation enable maize (Zea mays), a crop originally domesticated in the tropics, to adapt to temperate environments. We generated 572 unique RNA-seq datasets from the roots of 340 maize genotypes. Genes involved in core processes such as cell division, chromosome organization and cytoskeleton organization showed lower heritability of gene expression. While genes involved in anti-oxidation activity exhibited higher expression heritability. An expression genome-wide association study (eGWAS) identified 19,602 expression quantitative trait loci (eQTLs) associated with the expression of 11,444 genes. A GWAS for alternative splicing identified 49,897 splicing QTLs (sQTLs) for 7,614 genes. Rare allele burden within genomic intervals with trans-eQTLs correlated with extremes of expression in target genes as previously reported for cis-eQTLs. Genes harboring both cis-eQTLs and cis-sQTLs in linkage disequilibrium were disproportionately likely to encode transcription factors or were annotated as responding to one or more stresses. Independent component analysis of gene expression data identified loci regulating co-expression modules involved in phytohormone pathways, cell wall biosynthesis, lipid metabolism and stress response. Several genes involved in cell proliferation, flower development, DNA replication and gene silencing showed lower gene expression variation explained by genetic factors between temperate and tropical maize lines. A GWAS of 27 previously published phenotypes identified several candidate genes overlapping with genomic intervals showing signatures of selection during adaptation to temperate environments. Our results illustrate how maize transcriptional regulatory networks enable changes in transcriptional regulation to adapt to temperate regions.

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Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans Microbial pathogens grow in a wide range of different morphologies that provide distinct advantages for virulence. In the fungal pathogen Candida albicans, adenylyl cyclase (Cyr1) is thought to be a master regulator of the switch to invasive hyphal morphogenesis and biofilm formation. However, faster growing cyr1{Delta}/{Delta} pseudorevertant (PR) mutants were identified that form hyphae in the absence of cAMP. Isolation of additional PR mutants revealed that their improved growth was due to loss of one copy of BCY1, the negative regulatory subunit of protein kinase (A) from the left arm of chromosome 2. Furthermore, hyphal morphogenesis was improved in some of PR mutants by multigenic haploinsufficiency resulting from loss of large regions of the left arm of chromosome 2, including global transcriptional regulators. Interestingly, hyphal-associated genes were also induced in a manner that was independent of cAMP. This indicates that basal protein kinase A activity is an important prerequisite to induce hyphae, but activation of the cAMP pathway is not needed. Instead, phosphoproteomic analysis indicated that the Cdc28 cyclin-dependent kinase and the casein kinase 1 family member Yck2 play key roles in promoting polarized growth. In addition, integrating transcriptomic and proteomic data reveals hyphal stimuli induce increased production of key transcription factors that contribute to polarized morphogenesis.

AUTHOR SUMMARYThe human fungal pathogen Candida albicans switches between budding and filamentous hyphal morphologies to gain advantages for virulence and survival in the host. Although adenylyl cyclase has been thought to be a master regulator that controls this switch, we identified C. albicans pseudorevertant mutants that grow better and form hyphae in the absence of adenylyl cyclase and cAMP. The mutant cells were also able to induce hyphal-associated genes in the absence of cAMP that are needed for virulence. Integrating information from different omics approaches identified cAMP-independent mechanisms that promote hyphal growth. This includes phosphoproteomic studies that revealed key roles for the Cdc28 cyclin-dependent kinase and casein kinase 1 in promoting hyphal growth. In addition, integrating transcriptomic and proteomic data revealed that post-transcriptional mechanisms regulate the levels of a set of key transcription factors that are important for hyphal induction, suggesting a novel form of translational regulation. These studies better define the pathways that stimulate C. albicans to switch from budding to hyphal growth, which is important for invasion into tissues, escape from the immune system, and biofilm formation.

sentences: Hybridization underlies localized trait evolution in cavefish Compared to selection on new mutations and standing genetic variation, the role of gene flow in generating adaptive genetic variation has been subject to much debate. Theory predicts that gene flow constrains adaptive evolution via natural selection by homogenizing allele frequencies among populations and introducing migrant alleles that may be locally maladaptive1. However, recent work has revealed that populations can diverge even when high levels of gene flow are present2-4 and that gene flow may play an underappreciated role in facilitating local adaptation by increasing the amount of genetic variation present for selection to act upon5-8. Here, we investigate how genetic variation introduced by gene flow contributes to adaptive evolution of complex traits using an emerging eco-evolutionary model system, the Mexican tetra (Astyanax mexicanus). The ancestral surface form of the Mexican tetra has repeatedly invaded and adapted to cave environments. The Chica cave is unique in that it contains several pool microenvironments inhabited by putative hybrids between surface and cave populations9, providing an opportunity to investigate the dynamics of complex trait evolution and gene flow on a local scale. Here we conduct high-resolution genomic mapping and analysis of eye morphology and pigmentation in fish from multiple pools within Chica cave. We demonstrate that hybridization between cave and surface populations contributes to highly localized variation in behavioral and morphological traits. Analysis of sleep and locomotor behaviors between individual pools within this cave revealed reduced sleep associated with an increase in ancestry derived from cave populations, suggesting pool-specific ecological differences may drive the highly-localized evolution of sleep and locomotor behaviors. Lastly, our analyses uncovered a compelling example of convergent evolution in a core circadian clock gene in multiple independent cavefish lineages and burrowing mammals, indicating a shared genetic mechanism underlying circadian disruption in subterranean vertebrates. Together, our results provide insight into the evolutionary mechanisms that promote adaptive genetic variation and the genetic basis of complex behavioral phenotypes involved in local adaptation.

sentences: Identification of enhancer regulatory elements that direct epicardial gene expression during zebrafish heart regeneration The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in the case of zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with 5 of 6 elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in hearts of larval animals. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration.

sentences: Calling the Amino Acid Sequence of a Protein/Peptide from the Nanospectrum Produced by a Sub-nanometer Diameter Pore The blockade current that develops when a protein translocates across a thin membrane through a sub-nanometer diameter pore (i.e., a nanospectrum) informs with extreme sensitivity on the sequence of amino acids that constitute the protein. Whereas mass spectrometry (MS) is still the dominant technology for protein identification, it suffers limitations. In proteome-wide studies, MS fails to sequence proteins de novo, but merely classifies a protein and it is not very sensitive requiring about a femtomole to do that. Compared with MS, a sub-nanometer diameter pore (i.e. a sub-nanopore) directly reads the amino acids constituting a single protein molecule, but efficient computational tools are still required for processing and interpreting the blockade current. Here, we delineate computational methods for processing sub-nanopore nanospectra and predicting electrical blockade currents from protein sequences, which are essential for protein identification.

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Dopamine release in nucleus accumbens is under tonic inhibition by adenosine A1 receptors regulated by astrocytic ENT1 and dysregulated by ethanol Striatal adenosine A1 receptor (A1R) activation can inhibit dopamine release. A1Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol-sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A1Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A1R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A1R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor NBTI reduced dopamine release and promoted A1R-mediated inhibition, and conversely, virally-mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A1R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor GRAB-Ado, we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mM) promoted A1R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function.

SIGNIFICANCE STATEMENTDopamine axons in the mammalian striatum are emerging as strategic sites where neuromodulators can powerfully influence dopamine output in health and disease. We found that ambient levels of the neuromodulator adenosine tonically inhibit dopamine release in nucleus accumbens core via adenosine A1 receptors (A1Rs), to a variable level that promotes the contrast in dopamine signals released by different frequencies of activity. We reveal that the equilibrative nucleoside transporter 1 (ENT1) on astrocytes limits this tonic inhibition, and that ethanol promotes it by diminishing adenosine uptake via ENT1. These findings support the hypotheses that A1Rs on dopamine axons inhibit DA release and, furthermore, that astrocytes perform important roles in setting the level of striatal dopamine output, in health and disease.

sentences: Cortical Magnification in Human Visual Cortex Parallels Task Performance around the Visual Field Human vision has striking radial asymmetries, with performance on many tasks varying sharply with stimulus polar angle. Performance is generally better on the horizontal than vertical meridian, and on the lower than upper vertical meridian, and these asymmetries decrease gradually with deviation from the vertical meridian. Here we report cortical magnification at a fine angular resolution around the visual field. This precision enables comparisons between cortical magnification and behavior, between cortical magnification and retinal cell densities, and between cortical magnification in twin pairs. We show that cortical magnification in human primary visual cortex, measured in 163 subjects, varies substantially around the visual field, with a pattern similar to behavior. These radial asymmetries in cortex are larger than those found in the retina, and they are correlated between monozygotic twin pairs. These findings indicate a tight link between cortical topography and behavior, and suggest that visual field asymmetries are partly heritable.

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Nematode histone H2A variant evolution reveals diverse histories of retention and loss, and evidence for conserved core-like variants Histone variants replace canonical histones in nucleosomes, sometimes changing nucleosome function. Histone variant evolution is poorly characterized, and, as we show here, reconstruction of histone protein evolution can be challenging given large differences in rates across gene lineages and across sites. The positions of introns that interrupt genes can provide complementary phylogenetic information. We combined sequence and intron data to reconstruct the evolution of three histone H2A variants in Caenorhabditis elegans to reveal disparate histories. For the variant HIS-35 (which differs from H2A by only a single glycine-to-alanine C-terminal change), we find no evidence for the hypothesis of distinct protein function: the HIS-35 alanine is ancestral and common across canonical Caenorhabditis H2A sequences, with one species encoding identical HIS-35 and canonical H2A proteins. We propose instead that HIS-35 allows for H2A expression outside of the S-phase. Genes encoding such "backup" functions could be functionally important yet readily replaceable; consistent with this notion, both HTAS-1 and HIS-35 exhibit phylogenetic patterns that combine long-term evolutionary persistence and recurrent loss. Finally, the H2A.Z homolog, HTZ-1, exhibits recurrent intron loss and gain, suggesting that it is intron presence, rather than a specific intron sequence or position, that may be important in histone variant expression.

SIGNIFICANCEHistone variants are proteins that replace the core canonical histones in the nucleosomes and often confer specific structural and functional features. Histone variants represent a unique opportunity to study the functional significance of gene duplicates. The origin of new variants by core histone gene duplication is generally a highly asymmetrical affair marked by variant-specific changes in gene processing signals and accelerated evolution. The functions and evolutionary significance of the diverse variants found in available eukaryotic genomes remain almost entirely obscure, with only a few characterized case studies. By using a novel method that leverages intron position conservation, we reconstructed the evolutionary history of H2A variants within C. elegans and relatives. Our findings provide several insights that run counter to current paradigms of histone variant function and evolution.

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Mapping the allosteric effects that define functional activity of SARS-CoV-2 specific antibodies Previous studies on the structural relationship between human antibodies and SARS-CoV-2 have focused on generating static snapshots of antibody complexes with the Spike trimer. However, antibody-antigen interactions are dynamic, with significant binding-induced allosteric effects on conformations of antibody and its target antigen. In this study, we employ hydrogen-deuterium exchange mass spectrometry, in vitro assays, and molecular dynamics simulations to investigate the allosteric perturbations linked to binding events between a group of human antibodies with differential functional activities, and the Spike trimer from SARS-CoV-2. Our investigations have revealed key dynamic features that define weakly or moderately neutralizing antibodies versus those with strong neutralizing activity. These results provide mechanistic insights into the functional modes of human antibodies against COVID-19, and provide a rationale for effective antiviral strategies.

TeaserDifferent neutralizing antibodies induce site-specific allosteric effects across SARS-CoV-2 Spike protein

sentences: Exploratory analysis of multiple traits co-adaptations in the population history During the history of range expansion, the populations encounter with variety of environments. They respond to the local environments by modifying the mutually interacting traits. Therefore, to understand the whole life history of the populations, it is ideal to capture the history of their range expansion with reference to the series of surrounding environments and to infer the coadaptation of the multiple traits. Toward this end, we provide an exploratory analysis based on the features of populations: site frequency spectra of populations, population-specific FST, association between genes and environments, positive selections on traits mapped on the admixture graph, and GWAS results. Correspondence analysis of genes, environments, and traits provides a birds-eye view of the history of population differentiation and range expansion and various types of environmental selections at the times. Principal component analysis of the estimated trait-specific polygenic adaptations mapped on the admixture graph enables to understand the coadaptation of multiple traits. The potential usefulness was confirmed by analyzing a public dataset of wild poplar in northwestern America. In response to the northern cold temperature and longer daylength, the populations increased the photosynthetic activity and nutrient use efficiency at the expense of the risk of pathogen invasion, and in response to warm temperature, they increased the growth. At higher altitude, they shifted the maximum activity to earlier period in spring to reduce the activity in dry summer. The R codes for our representation method and simulations of population colonization used in this study are available as supplementary script.

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Global and regional ecological boundaries drive abrupt changes in avian frugivory interactions Species interactions can propagate disturbances across space, though ecological and biogeographic boundaries may limit this spread. We tested whether large-scale ecological boundaries (ecoregions and biomes) and human disturbance gradients increase dissimilarity among ecological networks, while accounting for background spatial and elevational effects and differences in network sampling. We assessed network dissimilarity patterns over a broad spatial scale, using 196 quantitative avian frugivory networks (encompassing 1,496 plant and 1,003 bird species) distributed across 67 ecoregions and 11 biomes. Dissimilarity in species and interactions, but not in network structure, increased significantly across ecoregion and biome boundaries and along human disturbance gradients. Our findings suggest that ecological boundaries contribute to maintaining the worlds biodiversity of interactions and mitigating the propagation of disturbances at large spatial scales.

One-Sentence SummaryEcoregions and biomes delineate the large-scale distribution of plant-frugivore interactions.

sentences: Circulating miRNA diversity, origin and response to changing metabolic and reproductive states, new insights from the rainbow trout Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well established biomarkers of many human pathologies. The aim of the present study was to investigate the potential of c-miRNAs as biomarkers of reproductive and metabolic states in fish, a question that has received little attention. Plasma was collected throughout the reproductive cycle from rainbow trout females subjected to two different feeding levels to trigger contrasting metabolic states; ovarian fluid was sample at ovulation. Fluid samples were subjected to small RNA-seq analysis followed by quantitative PCR validation for a subset of promising c-miRNA biomarkers. A comprehensive miRNA repertoire, which was lacking in trout, was first established to allow subsequent analysis. We first showed that biological fluids miRNAomes are complex and encompass a high proportion of the overall species miRNAome. While sharing a high proportion of common miRNAs, plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further showed that the plasma miRNAome exhibited major significant changes depending on metabolic and reproductive state. We subsequently identified three (miR-1-1/2-3p, miR-133-a-1/2-3p and miR-206-3p) evolutionarily conserved muscle-specific miRNA that accumulate in the plasma in response to high feeding rates, making these myomiRs strong candidate biomarkers of active myogenesis. We also identified miR-202-5p as a candidate biomarker for reproductive success that could be used to predict ovulation and/or egg quality. These highly promising results reveal the high potential of c-miRNAs as physiologically relevant biomarkers and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species.

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pop-1/TCF, ref-2/ZIC and T-box factors regulate the development of anterior cells in the C. elegans embryo Patterning of the anterior-posterior axis is fundamental to animal development. The Wnt pathway plays a major role in this process by activating the expression of posterior genes in animals from worms to humans. This observation raises the question of whether the Wnt pathway or other regulators control the expression of the many anterior-expressed genes. We found that the expression of five anterior-specific genes in Caenorhabditis elegans embryos depends on the Wnt pathway effectors pop-1/TCF and sys-1/{beta}-catenin. We focused further on one of these anterior genes, ref-2/ZIC, a conserved transcription factor expressed in multiple anterior lineages. Live imaging of ref-2 mutant embryos identified defects in cell division timing and position in anterior lineages. Cis-regulatory dissection identified three ref-2 transcriptional enhancers, one of which is necessary and sufficient for anterior-specific expression. This enhancer is activated by the T-box transcription factors TBX-37 and TBX-38, and surprisingly, concatemerized TBX-37/38 binding sites are sufficient to drive anterior-biased expression alone, despite the broad expression of TBX-37 and TBX-38. Taken together, our results highlight the diverse mechanisms used to regulate anterior expression patterns in the embryo.

Graphical Abstract

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sentences: Movement is governed by rotational population dynamics in spinal motor networks Although the generation of movements is a fundamental function of the nervous system, the underlying neural principles remain unclear. Since flexor- and extensor-muscles alternate during rhythmic movements like walking, it is often assumed that the responsible neural circuitry is similarly displaying alternating activity. Here, we present ensemble-recordings of neurons in the lumbar spinal cord that indicate that, rather than alternating, the population is performing a low-dimensional "rotation" in neural space, in which the neural activity is cycling through all phases continuously during the rhythmic behavior. The radius of rotation correlates with the intended muscle force and a perturbation of the low-dimensional trajectory can modify the motor behavior. Since existing models of spinal motor control offer an inadequate explanation of rotation, we propose a new theory of neural generation of movements from which this and other unresolved issues, such as speed regulation, force control, and multi-functionalism, are readily explained.

sentences: HIV-1 drug resistance profiling using amino acid sequence space cartography Human immunodeficiency virus (HIV) drug resistance is a global healthcare issue. The emergence of drug resistance demands treatment adaptation. Computational methods predicting the drug resistance profile from genomic data of HIV isolates are advantageous for monitoring drug resistance in patients. Yet, the currently existing computational methods for drug resistance prediction are either not suitable for complex mutational patterns in emerging HIV strains or lack interpretability of prediction results which is of paramount importance in clinical practice. Hence, to overcome these limitations, new approaches for the HIV drug resistance prediction combining high accuracy and interpretability are required. In this work, a new methodology for the analysis of protein sequence data based on the application of generative topographic mapping was developed and applied for HIV drug resistance profiling. It allowed achieving high accuracy of resistance predictions and intuitive interpretation of prediction results. The developed approach was successfully applied for the prediction of HIV resistance towards protease, reverse-transcriptase and integrase inhibitors and in-depth analysis of HIV resistance-inducing mutation patterns. Hence, it can serve as an efficient and interpretable tool to suggest optimal treatment regimens.

sentences: Characterization of the DNA binding domain of StbA, a key protein of a new type of DNA segregation system Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 [A] resolution. It folds into an HTH DNA-binding motif, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.

sentences: Deficiency of p38&α-MAPK in myeloid cells ameliorates symptoms and pathology of APP-transgenic AD mice Microglial activation is a hall marker of Alzheimers disease (AD); its pathogenic role and regulating mechanisms are unclear. p38-MAPK, a stress-responding kinase, is activated in AD brain in early disease stages. In APP-transgenic mice, we deleted p38-MAPK in whole myeloid cells from birth or specifically in microglia from 9 months, and analysed AD pathology at the age of 4, 9 and 12 months. In both experimental settings, p38-MAPK deficiency decreased cerebral A{beta} and improved cognitive function of AD mice; however, p38-MAPK-deficient myeloid cells were more effective than p38-MAPK-deficient microglia in preventing AD pathogenesis. Deficiency of p38-MAPK in myeloid cells inhibited the inflammatory activation of individual microglia by 4 months, but enhanced it by 9 months. Inflammatory activation was essential for p38-MAPK deficiency to promote microglial internalization of A{beta}. Interestingly, p38-MAPK deficiency in peripheral myeloid cells reduced il-17a transcription in CD4-positive spleen cells. By cross-breeding APP-transgenic mice and IL-17a knockout mice, we further observed that IL-17a deficiency activated microglia and decreased A{beta} deposits in AD mouse brain. Thus, p38-MAPK deficiency in myeloid cells prevents AD pathogenesis, perhaps through reducing IL-17a-expressing T lymphocytes, and promoting A{beta} clearance in the brain. Our study supports p38-MAPK as a novel target for AD therapy.

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Measuring nonapoptotic caspase activity with a transgenic reporter in mice The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that nonapoptotic caspase activity influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathological models.

Significance StatementCaspase-3 is an enzyme that can cause cell death when highly active but can also perform important cellular functions, such as maturation and structural changes, when only weakly or transiently active. Despite the importance of this nonlethal type of caspase activity, there is no straightforward method to measure it in live rodents. We therefore developed mice that have a fluorescent reporter that is sensitive enough to detect nonlethal caspase activity. Surprisingly, we found that weak caspase activity influences the synchrony of neuronal firing across different brain regions. We also observed increased caspase activity in female mice after severe stress. This simple, live-animal caspase activity reporter can subserve multiple applications in behavior and pathology research.

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Natural loss of function of ephrin-B3 shapes spinal flight circuitry in birds Flight in birds evolved through patterning of the wings from forelimbs and transition from alternating gait to synchronous flapping. In mammals, the spinal midline guidance molecule ephrin-B3 instructs the wiring that enables limb alternation, and its deletion leads to synchronous hopping gait. Here we show that the ephrin-B3 protein in birds lacks several motifs present in other vertebrates, diminishing its affinity for the EphA4 receptor. The avian ephrin-B3 gene lacks an enhancer that drives midline expression, and is missing in Galliformes. The morphology and wiring at brachial levels of the chick spinal cord resemble those of ephrin-B3 null mice. Importantly, dorsal midline decussation, evident in the mutant mouse, is apparent at the chick brachial level, and is prevented by expression of exogenous ephrin-B3 at the roof plate. Our findings support a role for loss of ephrin-B3 function in shaping the avian brachial spinal cord circuitry and facilitating synchronous wing flapping.

TeaserWalking vs flying: Deciphering the organization and evolution of the neuronal network that controls wing flapping in birds.

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Adaptive coding of pain prediction error in the anterior insula BackgroundTo reduce the computational demands of the task of determining values, the brain is thought to engage in  adaptive coding, where the sensitivity of some neurons to value is modulated by contextual information. There is good behavioural evidence that pain is coded adaptively, but controversy regarding the underlying neural mechanism. Additionally, there is evidence that reward prediction errors are coded adaptively, but no parallel evidence regarding pain prediction errors.

MethodsWe tested the hypothesis that pain prediction errors are coded adaptively by scanning 19 healthy adults with fMRI while they performed a cued pain task. Our analysis followed an axiomatic approach.

ResultsWe found that the left anterior insula was the only region which was sensitive both to predicted pain magnitudes and the unexpectedness of pain delivery, but not to the magnitude of delivered pain.

ConclusionsThis pattern suggests that the left anterior insula is part of a neural mechanism that serves the adaptive prediction error of pain.

Significance statementThere is behavioural evidence that pain is coded adaptively but controversy as to the neural mechanism that serves this process, and limited understanding of how adaptive coding of pain relates to adaptively-coded pain prediction error. We provide evidence that the left anterior insula complies with axioms that describe adaptive coding of pain prediction error, adding to the understanding of the role it plays in subjective pain representation.

sentences: Evolutionary Inference Predicts Novel ACE2 Protein Interactions Relevant to COVID-19 Pathologies Angiotensin-converting enzyme 2 (ACE2) is the cell receptor that the coronavirus SARS-CoV-2 binds to and uses to enter and infect human cells. COVID-19, the pandemic disease caused by the coronavirus, involves diverse pathologies beyond those of a respiratory disease, including micro-thrombosis (micro-clotting), cytokine storms, and inflammatory responses affecting many organ systems. Longer-term chronic illness can persist for many months, often well after the pathogen is no longer detected. A better understanding of the proteins that ACE2 interacts with can reveal information relevant to these disease manifestations and possible avenues for treatment. We have undertaken an approach to predict candidate ACE2 interacting proteins which uses evolutionary inference to identify a set of mammalian proteins that "coevolve" with ACE2. The approach, called evolutionary rate correlation (ERC), detects proteins that show highly correlated evolutionary rates during mammalian evolution. Such proteins are candidates for biological interactions with the ACE2 receptor. The approach has uncovered a number of key ACE2 protein interactions of potential relevance to COVID-19 pathologies. Some proteins have previously been reported to be associated with severe COVID-19, but are not currently known to interact directly with ACE2, while additional predicted novel ACE2 interactors are of potential relevance to the disease. Using reciprocal rankings of protein ERCs, we have identified strongly interconnected ACE2 associated protein networks relevant to COVID-19 pathologies. ACE2 has clear connections to coagulation pathway proteins, such as Coagulation Factor V and fibrinogen components FGG, FGB, and FGA, the latter possibly mediated through ACE2 connections to Clusterin (which clears misfolded extracellular proteins) and GPR141 (whose functions are relatively unknown). ACE2 also connects to proteins involved in cytokine signaling and immune response (e.g. IFNAR2, XCR1, and TLR8), and to Androgen Receptor (AR). The ERC prescreening approach has also elucidated possible functions for previously uncharacterized proteins and possible new functions for well-characterized ones. Suggestions are made for the validation of ERC predicted ACE2 protein interactions. We propose that ACE2 has novel protein interactions that are disrupted during SARS-CoV-2 infection, contributing to the spectrum of COVID-19 pathologies.

sentences: The population genetics of adaptation through copy-number variation in a fungal plant pathogen Microbial pathogens can rapidly adapt to changing environments such as the application of pesticides or host resistance. Copy number variations (CNV) are a major source of adaptive genetic variation for recent adaptation. Here, we analyze how a major fungal pathogen of barley, Rhynchosporium commune, has adapted to host environment, fungicide and temperature challenges. We screen the genomes of 126 isolates sampled across a worldwide set of populations and identify a total of 7879 gene duplications and 116 gene deletions. Most gene duplications result from segmental chromosomal duplications. We find that genes showing recent gains or losses are enriched in functions related to host exploitation (i.e. effectors and cell wall degrading enzymes). We perform a phylogeny-informed genome-wide association study (GWAS) and identify 191 copy-number variants associated with different pathogenesis and temperature related traits, including a large segmental duplication of CYP51A that has contributed to the emergence of azole resistance. Additionally, we use a genome-wide SNP dataset to replicate the GWAS and contrast it with the CNV-focused analysis. We find that frequencies of adaptive CNV alleles show high variation among populations for traits under strong selection such as fungicide resistance. In contrast, adaptive CNV alleles underpinning temperature adaptation tend to be near fixation. Finally, we show that transposable elements are important drivers of recent gene copy-number variation. Loci showing signatures of recent positive selection are enriched in miniature inverted repeat transposons. Our findings show how extensive segmental duplications create the raw material for recent adaptation in global populations of a fungal pathogen.

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Symmetry processing in the macaque visual cortex Symmetry is a highly salient feature of the natural world that is perceived by many species of the animal kingdom and that impacts a large array of behaviours such as partner selection or food choice. In humans, the cerebral areas processing symmetry are now well identified from neuroimaging measurements. However, we currently lack an animal model to explore the underlying neural mechanisms. Macaque is a potentially good candidate, but a previous comparative study (1) found that functional magnetic resonance imaging (fMRI) responses to mirror symmetry in this species were substantially weaker than those observed in humans under similar experimental conditions. Here, we re-examined symmetry processing in macaques from a broader perspective, using both rotation (experiment 1) and reflection (experiment 2) symmetry. Our experimental design was directly derived from that of a previous human fMRI study (2), in order to facilitate the comparison between the two primate species. Highly consistent responses to symmetry were found in a large network of areas (notably V3, V3A, V4, V4A and PITd), in line with what has been observed in humans. Within this network, response properties in areas V3 and V4 (notably their dependency on the rotation symmetry order) were strikingly similar to those observed in their human counterparts. Our results suggest that the cortical networks that process symmetry in humans and macaques are much more similar than previously reported and point toward macaque as a relevant model for understanding symmetry processing.

Significance statementSymmetry processing is an important aspect of human visual perception. We currently lack an animal model for characterizing the neural mechanisms that underlie it at the microscopic scale. Here, we use fMRI measurements in macaques to demonstrate that the cortical responses to symmetry in this species are comparable to those observed in humans under similar experimental conditions to a much higher extent than previously documented. Our results call for a re-examination of the relevance of the macaque model for symmetry processing in humans and open the door to an exploration of the underlying neural mechanisms at the single-cell level, notably in V3, an area often neglected in most current models of visual processing.

sentences: SPARC Data Structure: Rationale and Design of a FAIR Standard for Biomedical Research Data The NIH Common Funds Stimulating Peripheral Activity to Relieve Conditions (SPARC) initiative is a large-scale program that seeks to accelerate the development of therapeutic devices that modulate electrical activity in nerves to improve organ function. Integral to the SPARC program are the rich anatomical and functional datasets produced by investigators across the SPARC consortium that provide key details about organ-specific circuitry, including structural and functional connectivity, mapping of cell types and molecular profiling. These datasets are provided to the research community through an open data platform, the SPARC Portal. To ensure SPARC datasets are Findable, Accessible, Interoperable and Reusable (FAIR), they are all submitted to the SPARC portal following a standard scheme established by the SPARC Curation Team, called the SPARC Data Structure (SDS). Inspired by the Brain Imaging Data Structure (BIDS), the SDS has been designed to capture the large variety of data generated by SPARC investigators who are coming from all fields of biomedical research. Here we present the rationale and design of the SDS, including a description of the SPARC curation process and the automated tools for complying with the SDS, including the SDS validator and Software to Organize Data Automatically (SODA) for SPARC. The objective is to provide detailed guidelines for anyone desiring to comply with the SDS. Since the SDS are suitable for any type of biomedical research data, it can be adopted by any group desiring to follow the FAIR data principles for managing their data, even outside of the SPARC consortium. Finally, this manuscript provides a foundational framework that can be used by any organization desiring to either adapt the SDS to suit the specific needs of their data or simply desiring to design their own FAIR data sharing scheme from scratch.

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Gut transcriptome reveals differential gene expression and enriched pathways linked to immune activation in response to weaning in pigs. Weaning represents one of the most critical periods in pig production associated with increase in disease risk, reduction in performance and economic loss. Physiological changes faced by piglets during the weaning period have been well characterised, however little is currently known about the underlying molecular pathways involved in these processes. As pig meat remains one of the most consumed sources of protein worldwide, understanding how these changes are mediated is critical to improve pig production and consequently sustainable food production globally. In this study, we evaluated the effect of weaning on transcriptomic changes in the colon of healthy piglets over time using an RNA-sequencing approach.

The findings revealed a complex and coordinated response to weaning with the majority of genes found to be rapidly differentially expressed within one day post weaning. Multiple genes and pathways affected by weaning in the colon were associated with immune regulation, cell signalling and bacterial defence. NOD-like receptors, Toll-like receptor and JAK-STAT signalling pathways were amongst the pathways significantly enriched. Immune activation was evidenced by the enrichment of pathways involved in interferon response, cytokines interactions, oxidoreductase activities and response to microbial invasion. Biosynthesis of amino acids, in particular arginine, was also amongst the most enriched KEGG pathways in weaned pigs, reinforcing the critical role of arginine in gut homeostasis under stress conditions.

Overall, transcriptomic and physiological results suggest that pigs going through the weaning transition undergo a transient period of inflammatory state with a temporary breakdown of barrier functions in the gut. These findings could provide valuable tools to monitor host response post weaning, and may be of particular relevance for the investigation and development of intervention strategies aimed to reduce antibiotic use and improve pig health and performance.

sentences: G9a/GLP methyltransferases inhibit autophagy by methylation-mediated ATG12 protein degradation Previous studies have shown that G9a, a lysine methyltransferase, inhibits autophagy by repressing the transcription of autophagy genes. Here, we demonstrate a novel mechanism whereby G9a/GLP inhibit autophagy through post-translational modification of ATG12, a protein critical for the initiation of autophagosome formation. Under non-stress conditions, G9a/GLP directly methylate ATG12. The methylated ATG12 undergoes ubiquitin-mediated protein degradation, thereby inhibiting autophagy induction. By contrast, under stress conditions that elevate intracellular Ca2+ levels, the activated calpain system cleaves the G9a/GLP proteins, leading to G9a/GLP protein degradation. The reduced G9a/GLP levels allow ATG12 to accumulate and form the ATG12-ATG5 conjugate, thus expediting autophagy initiation. Collectively, our findings reveal a distinct signaling pathway that links cellular stress responses involving Ca2+/calpain to G9a/GLP-mediated autophagy regulation. Moreover, our model proposes that the methylation status of ATG12 is a molecular rheostat that controls autophagy induction.

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The interaction between a parasite and sub-optimal temperatures contributes to honey bee decline Global insect decline and, in particular, honey bee colony losses are related to multiple stress factors, including landscape deterioration, pollution, parasites and climate change. However, the implications of the interaction among different stress factors for insect health are still poorly understood; in particular, little is known on how challenging environmental conditions can influence the impact of parasites. Here we exploited the honey bee as a model system to approach this problem and carried out extensive lab and field work aiming at assessing how suboptimal temperatures and parasitic challenges can alter the homeostatic balance of individual bees and the whole colony, leading to individual death and colony collapse.

We found that mite infestation further than increasing the mortality of bees, induces an anorexia that in turn reduces the capacity of bees to thermoregulate, thus exposing them to the detrimental effect of lower temperatures. This, in turn, has important implications for the colony as a whole. The results highlight the role that abiotic factors can have in shaping the effect of parasitic challenges on honey bees. Furthermore, the multilevel and holistic approach adopted here can represent a useful template for similar studies on other insect species, which are particularly urgent in view of climate change and the continuous pressure of natural and exotic parasites on insect populations.

sentences: Genome editing in the unicellular holozoan Capsaspora owczarzaki suggests a premetazoan role for the Hippo pathway in multicellular morphogenesis Animal development is mediated by a surprisingly small set of canonical signaling pathways such as Wnt, Hedgehog, TGF-beta, Notch and Hippo pathways. Although once thought to be present only in animals, recent genome sequencing has revealed components of these pathways in the closest unicellular relatives of animals. These findings raise questions about the ancestral functions of these developmental pathways and their potential role in the emergence of animal multicellularity. Here, we provide the first functional characterization of any of these developmental pathways in unicellular organisms by developing techniques for genetic manipulation in Capsaspora owczarzaki, a close unicellular relative of animals that displays aggregative multicellularity. We then use these tools to characterize the Capsaspora ortholog of the Hippo signaling nuclear effector YAP/TAZ/Yorkie (coYki), a key regulator of tissue size in animals. In contrast to what might be expected based on studies in animals, we show that coYki is dispensable for cell proliferation but regulates cytoskeletal dynamics and the three-dimensional (3D) shape of multicellular structures. We further demonstrate that the cytoskeletal abnormalities of individual coYki mutant cells underlie the abnormal 3D shape of coYki mutant aggregates. Taken together, these findings implicate an ancestral role for the Hippo pathway in cytoskeletal dynamics and multicellular morphogenesis predating the origin of animal multicellularity, which was co-opted during evolution to regulate cell proliferation.

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Performance of a phylogenetic independent contrast method and an improved pairwise comparison under different scenarios of trait evolution after speciation and duplication O_LIDespite the importance of gene function to evolutionary biology, the applicability of comparative methods to gene function is poorly known. A specific case which has crystalized methodological questions is the "ortholog conjecture", the hypothesis that function evolves faster after duplication (i.e., in paralogs), and conversely is conserved between orthologs. Since the mode of functional evolution after duplication is not well known, we investigate under which reasonable evolutionary scenarios phylogenetic independent contrasts or pairwise comparisons can recover a putative signal of different functional evolution between orthologs and paralogs.
C_LIO_LIWe investigate three different simulation models, which represent reasonable but simplified hypotheses about the evolution of a gene function trait. These are time dependent trait acceleration, correlated changes in rates of both sequence and trait evolution, and asymmetric trait jump. For each model we tested phylogenetic independent contrasts and an improved pairwise comparison method which accounts for interactions between events and node age.
C_LIO_LIBoth approaches lose power to detect the trend of functional evolution when the functional trait accelerates for a long time following duplication for trees with many duplications, with better power of phylogenetic contrasts under intermediate scenarios. Concomitant increase in evolutionary rates of sequence and of trait after duplication can lead to both an incorrect rejection of the null under null simulations of trait evolution, and a false rejection of the ortholog conjecture under ortholog conjecture simulations, by phylogenetic independent contrasts. Improved pairwise comparisons are robust to this bias. Both approaches perform equally well under rapid shifts in traits.
C_LIO_LIConsidering our ignorance of gene function evolution, and the potential for bias under simple models, we recommend methodological pluralism in studying gene family evolution. Functional phylogenomics is complex and results supported by only one method should be treated with caution.
C_LI

sentences: Nutrigonometry I: using right-angle triangles to quantify nutritional trade-offs in multidimensional performance landscapes Animals regulate their diet in order to maximise the expression of fitness traits that often have different nutritional needs. These nutritional trade-offs have been experimentally uncovered using the Geometric framework for nutrition (GF). However, current analytical methods to measure such responses rely on either visual inspection or complex models applied to multidimensional performance landscapes, making these approaches subjective, or conceptually difficult, computationally expensive, and in some cases inaccurate. This limits our ability to understand how animal nutrition evolved to support life-histories within and between species. Here, we introduce a simple trigonometric model to measure nutritional trade-offs in multidimensional landscapes ( Nutrigonometry). Nutrigonometry is both conceptually and computationally easier than current approaches, as it harnesses the trigonometric relationships of right-angle triangles instead of vector calculations. Using landmark GF datasets, we first show how polynomial (Bayesian) regressions can be used for precise and accurate predictions of peaks and valleys in performance landscapes, irrespective of the underlying structure of the data (i.e., individual food intakes vs fixed diet ratios). Using trigonometric relationships, we then identified the known nutritional trade-off between lifespan and reproductive rate both in terms of nutrient balance and concentration. Nutrigonometry enables a fast, reliable and reproducible quantification of nutritional trade-offs in multidimensional performance landscapes, thereby broadening the potential for future developments in comparative research on the evolution of animal nutrition.

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High-dimension to high-dimension screening for detecting genome-wide epigenetic regulators of gene expression MotivationThe advancement of high-throughput technology characterizes a wide range of epigenetic modifications across the genome involved in disease pathogenesis via regulating gene expression. The high-dimensionality of both epigenetic and gene expression data make it challenging to identify the important epigenetic regulators of genes. Conducting univariate test for each epigenetic-gene pair is subject to serious multiple comparison burden, and direct application of regularization methods to select epigenetic-gene pairs is computationally infeasible. Applying fast screening to reduce dimension first before regularization is more efficient and stable than applying regularization methods alone.

ResultsWe propose a novel screening method based on robust partial correlation to detect epigenetic regulators of gene expression over the whole genome, a problem that includes both high-dimensional predictors and high-dimensional responses. Compared to existing screening methods, our method is conceptually innovative that it reduces the dimension of both predictor and response, and screens at both node (epigenetic features or genes) and edge (epigenetic-gene pairs) levels. We develop data-driven procedures to determine the conditional sets and the optimal screening threshold, and implement a fast iterative algorithm. Simulations and two applications to long non-coding RNA and DNA methylation regulation in Kidney cancer and Glioblastoma Multiforme illustrate the validity and advantage of our method.

AvailabilityThe R package, related source codes and real data sets used in this paper are provided at https://github.com/kehongjie/rPCor.

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MyD88-dependent TLR signaling oppositely regulates hematopoietic progenitor and stem cell formation in the embryo Hemogenic endothelial (HE) cells in the dorsal aorta undergo an endothelial to hematopoietic transition (EHT) to form lympho-myeloid biased progenitors (LMPs), pre-hematopoietic stem cells (pre-HSCs) and adult-repopulating HSCs. These briefly accumulate in intra-arterial hematopoietic clusters (IAHCs) before being released into the circulation. It is generally assumed that the number of IAHC cells correlates with the number of HSCs. Here we show that changes in the number of IAHC cells, LMPs, and HSCs can be uncoupled. Mutations impairing MyD88-dependent toll-like receptor (TLR) signaling decreased the number of IAHC cells and LMPs but increased the number of HSCs in the aorta-gonad-mesonephros region of mouse embryos. TLR4 -deficient embryos generated normal numbers of HE cells but the proliferation of IAHC cells was decreased. Loss of MyD88-dependent TLR signaling in innate immune myeloid cells had no effect on IAHC cell numbers. Instead, TLR4 deletion in endothelial cells recapitulated the phenotype observed with germline deletion, demonstrating that MyD88-dependent TLR signaling in endothelial cells and/or in IAHCs regulates the balance between generating LMPs and HSCs.

Summary StatementToll-like receptor signaling in endothelial cells restricts the number of hematopoietic stem cells but increases the number of committed progenitors and intra-arterial hematopoietic cluster cells by promoting their proliferation.

sentences: A chemical-genetic map of the pathways controlling drug potency in Mycobacterium tuberculosis Mycobacterium tuberculosis (Mtb) infection is notoriously difficult to treat. Treatment efficacy is limited by Mtbs intrinsic drug resistance, as well as its ability to evolve acquired resistance to all antituberculars in clinical use. A deeper understanding of the bacterial pathways that govern drug efficacy could facilitate the development of more effective therapies to overcome resistance, identify new mechanisms of acquired resistance, and reveal overlooked therapeutic opportunities. To define these pathways, we developed a CRISPR interference chemical-genetics platform to titrate the expression of Mtb genes and quantify bacterial fitness in the presence of different drugs. Mining this dataset, we discovered diverse and novel mechanisms of intrinsic drug resistance, unveiling hundreds of potential targets for synergistic drug combinations. Combining chemical-genetics with comparative genomics of Mtb clinical isolates, we further identified numerous new potential mechanisms of acquired drug resistance, one of which is associated with the emergence of a multidrug-resistant tuberculosis (TB) outbreak in South America. Lastly, we make the unexpected discovery of an "acquired drug sensitivity." We found that the intrinsic resistance factor whiB7 was inactivated in an entire Mtb sublineage endemic to Southeast Asia, presenting an opportunity to potentially repurpose the macrolide antibiotic clarithromycin to treat TB. This chemical-genetic map provides a rich resource to understand drug efficacy in Mtb and guide future TB drug development and treatment.

sentences: Structural dynamics of DNA strand break sensing by PARP-1 at a single-molecule level Single stranded breaks (SSBs) are the most frequent DNA lesions threatening genomic integrity. A highly kinked DNA structure in complex with human PARP-1 led to the proposal that SSB sensing in Eukaryotes relies on dynamics of both the broken DNA double helix and PARP-1s multi-domain organization. Here, we directly probe this fundamental yet poorly understood process at the single-molecule level. Quantitative SM-FRET and structural ensemble calculations reveal how PARP-1 binding converts DNA SSBs from a largely unperturbed conformation into the highly kinked state. Binding of the second N-terminal zinc finger yields an intermediate DNA conformation that can be recognized by the first zinc finger. Thus, the data is inconsistent with a conformational selection model, instead an induced fit mechanism via a multi-domain assembly cascade drives SSBs sensing. Interestingly, a clinically used PARP-1 inhibitor niraparib shifts the equilibrium towards the unkinked state, whereas the inhibitor EB47 stabilizes the kinked state.

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Holobiont Evolution: Population Theory for the Hologenome This article develops mathematical theory for the population dynamics of microbiomes with their hosts and theory for microbiome evolution caused by holobiont selection.

A holobiont is the union of a host with its microbiome. Holobiont selection is natural selection on the combined host and microbiome as a unit. The hologenome is the union of genes in the microbiome with genes in the host nucleus. The deep integration within many holobionts of microbiomes with their hosts extends to development, metabolism, physiology and behavior. The kind of eco-evolutionary processes producing this integration is unclear.

For a microbe to join a hosts microbiome, its population-dynamic parameters must sufficiently align with the hosts population-dynamic parameters. Accordingly, a microbe might be excluded from the microbiome, might enter the microbiome in a way leading to its subsequent gradual shedding from the holobiont, might join the microbiome and coexist with the host, or might, for a deleterious microbe, enter the microbiome culminating in the extinction of both it and the host.

A horizontally transmitted microbiome constitutes a genetic system that features "collective inheritance" capable of underwriting Darwinian descent with modification. The microbial source pool in the environment is the counterpart for the microbiome of the gamete pool for the nuclear genome. Poisson sampling of the microbial source pool is the counterpart of binomial sampling of the gamete pool. However, holobiont selection on the microbiome does not lead to a counterpart of the Hardy-Weinberg Law, a concept of selective neutrality, or directional selection that produces fixation of the microbial gene with the highest fitness. The effectiveness of holobiont selection on microbial genes is limited compared to the effectiveness of natural selection on nuclear genes. Nonetheless, holobiont selection can affect the composition of the microbiome by permitting the coexistence of a competitively inferior microbial strain with a superior strain in a colonization-extinction equilibrium and by permitting a polymorphism between altruistic and selfish microbial strains.

An altruistic microbe might strike an optimal balance between lowering within-host fitness to increase holobiont fitness. However, altruistic microbes are replaced by otherwise identical microbes that contribute nothing to holobiont fitness. This replacement can be reversed by hosts that discriminate against colonization by selfish microbes--likely brought about by hosts making antibodies against selfish microbes. This discrimination leads to a process of microbial species sorting orchestrated by the host immune system. Holobiont assembly with host-orchestrated species sorting (HOSS) followed by microbial competition, rather than coevolution or multi-level selection, is the likely cause of host-microbiome integration.

sentences: Widespread false gene gains caused by duplication errors in genome assemblies False duplications in genome assemblies lead to false biological conclusions. We quantified false duplications in previous genome assemblies and their new counterparts of the same species (platypus, zebra finch, Annas hummingbird) generated by the Vertebrate Genomes Project (VGP). Whole genome alignments revealed that 4 to 16% of the sequences were falsely duplicated in the previous assemblies, impacting hundreds to thousands of genes. These led to overestimated gene family expansions. The main source of the false duplications was heterotype duplications, where the haplotype sequences were more divergent than other parts of the genome leading the assembly algorithms to classify them as separate genes or genomic regions. A minor source was sequencing errors. Although present in a smaller proportion, we observed false duplications remaining in the VGP assemblies that can be identified and purged. This study highlights the need for more advanced assembly methods that better separates haplotypes and sequence errors, and the need for cautious analyses on gene gains.

sentences: On the efficacy of restoration in stream networks: comments, critiques, and prospective recommendations Swan and Brown (2017) recently addressed the effects of restoration on stream communities under the meta-community framework. Using a combination of headwater and mainstem streams, Swan and Brown (2017) evaluated how position within a stream network affected the outcome of restoration on invertebrate communities. Ostensibly, their hypotheses were partially supported as restoration had stronger effects in headwater streams: invertebrate taxonomic richness was increased and temporal variability decreased in restored reaches; however, these results were not consistent upon closer scrutiny for both the original paper (Swan and Brown 2017) and the later erratum (Swan and Brown 2018). Here, I provide a secondary analysis of the data, with hypotheses and interpretations in the context of stream, meta-community, and restoration ecology. Swan and Brown (2017, 2018) evaluated the effect of restoration on sites receiving various combinations of in-channel manipulation and riparian reforestation treatments. Given the difference in the relative importance of environmental filtering and dispersal between headwaters and mainstems and the structure of river networks, I contend that different restoration treatments have differential effects between headwaters and mainstems. I hypothesized in-channel manipulations would have more consistent effects between headwaters and mainstems compared to riparian reforestation, and I used this hypothesis to guide site selection in the re-analysis. I then compared results from the re-analysis to those presented by Swan and Brown (2017, 2018). I did not find any effects of restoration on local diversity, spatial dissimilarity, or temporal variability, let alone differential effects of restoration between headwaters and mainstems; these results are contrary Swan and Brown (2017, 2018), who reported that restoration increased taxonomic richness, increased spatial dissimilarity, and decreased temporal variability in restored headwater streams. I demonstrate further that the statistical tests conducted by Swan and Brown (2017, 2018) were invalid and, therefore, recommend the use of the results presented here. More broadly, I suggest, in agreement with Swan and Brown (2017, 2018) and a growing body of research, that river and stream restoration will likely have greater success if a regional approach is taken to designing and implementing restoration projects.

sentences: PanGraph: scalable bacterial pan-genome graph construction The genomic diversity of microbes is commonly parameterized as population genetic polymorphisms relative to a reference genome of a well-characterized, but arbitrary, isolate. Reference genomes contain a fraction of the microbial pangenome, the set of genes observed within all isolates of a given species, and are thus blind to both the dynamics of the accessory genome, as well as variation within gene order and copy number. With the wide-spread usage of long-read sequencing, the number of high-quality, complete genome assemblies has increased dramatically. Traditional computational approaches towards whole-genome analysis either scale poorly, or treat genomes as dissociated bags of genes, and thus are not suited for this new era. Here, we present PanGraph, a Julia based library and command line interface for aligning whole genomes into a graph, wherein each genome is represented as an undirected path along vertices, which in turn, encapsulate homologous multiple sequence alignments. The resultant data structure succinctly summarizes population-level nucleotide and structural polymorphisms and can be exported into a several common formats for either downstream analysis or immediate visualization.

sentences: Lack of Cdk5 activity is involved on Dopamine Transporter expression and function: Evidences from an animal model of Attention-Deficit Hyperactivity Disorder Attention deficit/Hyperactivity disorder (ADHD) is one of the most diagnosed psychiatric disorders nowadays. The core symptoms of the condition include hyperactivity, impulsiveness and inattention. The main pharmacological treatment consists of psychostimulant drugs affecting Dopamine Transporter (DAT) function. We have previously shown that genetically modified mice lacking p35 protein (p35KO), which have reduced Cdk5 activity, present key hallmarks resembling those described in animal models useful for studying ADHD. The p35KO mouse displays spontaneous hyperactivity and shows a calming effect of methylphenidate or amphetamine treatment. Interestingly, dopaminergic neurotransmission is altered in these mice as they have an increased Dopamine (DA) content together with a low DA turnover. This led us to hypothesize that the lack of Cdk5 activity affects DAT expression and/or function in this animal model. In this study, we performed biochemical assays, cell-based approaches, quantitative fluorescence analysis and functional studies that allowed us to demonstrate that p35KO mice exhibit decreased DA uptake and reduced cell surface DAT expression levels in the striatum (STR). These findings are supported by in vitro observations in which the inhibition of Cdk5 activity in N2a cells induced a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization to recycling endosomes. Taken together, these data provide evidences regarding the role of Cdk5/p35 in DAT expression and function, thus contributing to the knowledge of DA neurotransmission physiology and also providing therapeutic options for the treatment of DA pathologies such as ADHD.

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Systematic identification of a CDYL1-dependent decrease in lysine crotonylation at DNA double-strand break sites functionally uncouples transcriptional silencing and repair Previously, we showed that CDYL1 is recruited to DNA double-strand breaks (DSBs) to promote homology-directed repair (HDR) and foster transcriptional silencing. Yet, how CDYL1 elicits DSB-induced silencing is not fully understood. Here, we systematically identify a CDYL1-dependent local decrease in the transcriptionally active marks lysine crotonylation (PanKcr) and crotonylated histone residue H3K9cr at AsiSI-induced DSBs, which correlates with transcriptional silencing. Mechanistically, we reveal that CDYL1 crotonyl-CoA hydratase activity counteracts PanKcr and H3K9cr at AsiSI sites, which triggers the eviction of the transcriptional elongation factor ENL and foster transcriptional silencing. Furthermore, genetic inhibition of CDYL1 hydratase activity blocks the reduction in H3K9cr and alleviates DSB-induced silencing, while HDR efficiency unexpectedly remains intact. Therefore, our results functionally uncouple the repair and silencing activity of CDYL1 at DSBs. In a broader context, we address a long-standing question concerning the functional relationship between HDR and DSB-induced transcriptional silencing, suggesting that they may occur independently.

HighlightsO_LISystematic identification of a local decrease in lysine crotonylation PanKcr and H3K9cr at AsiSI-induced DSBs that correlates with transcriptional silencing.
C_LIO_LICDYL1 crotonyl-CoA hydratase activity downregulates Kcr at DSBs.
C_LIO_LIKcr reduction at DSBs promotes ENL eviction and fosters transcriptional silencing.
C_LIO_LICDYL1 roles in DSB-induced transcriptional silencing and HDR are functionally uncoupled.
C_LI

sentences: The population frequency of human mitochondrial DNA variants is highly dependent upon mutational bias Next-generation sequencing can quickly reveal genetic variation potentially linked to heritable disease. As databases encompassing human variation continue to expand, rare variants have been of high interest, since the frequency of a variant is expected to be low if the genetic change leads to a loss of fitness or fecundity. However, the use of variant frequency when seeking genomic changes linked to disease remains very challenging. Here, we explore the role of selection in controlling human variant frequency using the HelixMT database, which encompasses hundreds of thousands of mitochondrial DNA (mtDNA) samples. We find that a substantial number of synonymous substitutions, which have no effect on protein sequence, were never encountered in this large study, while many other synonymous changes are found at very low frequencies. Further analyses of human and mammalian mtDNA datasets indicate that the population frequency of synonymous variants is predominantly determined by mutational biases rather than by strong selection acting upon nucleotide choice. Our work has important implications that extend to the interpretation of variant frequency for non-synonymous substitutions.

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The cardiac STAT3 intercalated disc specific expression in tail suspension rat BackgroundThe cardiovascular system is significantly agitated by loss of gravity. In microgravity, the body fluids shift toward the thoracic cavity, induced the heart becomes more spherical. This further increased the cardiac preload with an increasing of transmural central venous pressure, affects the right heart ventricles to tolerating the enhanced preload on the right ventricular wall.

MethodIn this study we investigated the rat right ventricle remodeling in simulating persistent microgravity by using tail-suspension model, examined the remolding of the heart and the specific STAT3 expression in right heart myocardium.

ResultThe results indicated that microgravity induced heart remodeling included a significant increasing of the ventricular weight in the left. However, the right ventricle was not increased significantly in the microgravity simulation rats. The histological study demonstrated that the outstanding development on right ventricular wall which included the gap junction remodeling and STAT3 signaling protein specific accumulation in the right ventricles.

ConclusionThe results existed that the right cardiac ventricle has a distinctive remodeling process during microgravity simulation which was not the muscular hypertrophy and relative weight increasing, but manifested the STAT3 accumulation and the electrical gap junction remodeling. The effect of microgravity induced right ventricle remodeling and the STAT3 specific accumulation can be used for multi-purpose research.

sentences: drda: An R package for dose-response data analysis Analysis of dose-response data is an important step in many scientific disciplines, including but not limited to pharmacology, toxicology, and epidemiology. The R package drda is designed to facilitate the analysis of dose-response data by implementing efficient and accurate functions with a familiar interface. With drda, it is possible to fit models by the method of least squares, perform goodness of fit tests, and conduct model selection. Compared to other similar packages, drda provides, in general, more accurate estimates in the least-squares sense. This result is achieved by a smart choice of the starting point in the optimization algorithm and by implementing the Newton method with a trust region with analytical gradients and Hessian matrices. In this article, drda is presented through the description of its methodological components and examples of its user-friendly functions. Performance is finally evaluated using a real, large-scale drug sensitivity screening dataset.

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Functional substitutability of native herbivores by livestock for long-term soil carbon sequestration depends on microbial decomposers. The influence of native mammalian herbivores on ecosystems is declining as livestock have displaced them to become the dominant grazers across the world. Alongside rewilding, restoration, and conservation, it is now important to address whether livestock can assume the functional roles of displaced native herbivores in grasslands, savannas, and steppes. If yes, this has implications for the future of biodiversity, ecosystem functions and services, food-security, and other social and environmental dimensions. We test competing alternative hypotheses with a replicated, long-term, landscape-level experiment to ask whether livestock in the Trans-Himalayan ecosystem can match decadal-scale (2006-17) soil-C sequestration under native herbivores. Despite comparable settings, livestock were imperfect substitutes and attained three-fourths of soil-C stocks under native herbivores. We evaluate multiple lines of evidence surrounding this difference with a grazing-exclusion experiment (nutrient-cycling, microbial metabolism, and e-DNA), and asses their inter-relationships. Livestock and native herbivores differed in their effects on several soil microbial variables. Structural equation models showed that livestock alter soil microbial communities towards lower fungal abundance, which is detrimental for microbial carbon use efficiency (CUE), and ultimately also for soil-C. Herbivore-identity influenced ecosystem functions even when total amount of herbivory was comparable. Lags in soil-C storage can be addressed through microbial restoration and rewilding of soil. Therefore, the idea of functional substitutability must be leveraged alongside microbial rewilding to restore and conserve ecosystem functions. Together they can reconcile conflicting demands from food-security and ecosystem services in ways that have eluded conventional approaches to become an integral part of nature-based climate solutions.

Significance StatementGrazing by large mammalian herbivores affords an important natural climate solution via soil-C storage. But native grazers are progressively being replaced by domestic livestock to accommodate changing human diets. Even though livestock are now the dominant grazers around the world, it remains unclear whether they can be functional substitutes for native herbivores in sequestering soil-C. And if so, then to what extent? We find that livestock could only attain three-fourths of soil-C stocks under native herbivores, because they shift the microbial decomposer community in soil towards lower fungal abundance. This reduces microbial carbon use efficiency, and ultimately is detrimental for soil-C storage. So, microbial rewilding may help livestock production systems to emulate the ecosystem services provided by native herbivores.

sentences: Immune activation state modulates retrieval of infant engrams Infantile amnesia is possibly the most ubiquitous form of memory loss in mammals. Despite its widespread relevance, little is known about the biological conditions for infantile amnesia to occur and its effect on the engram cells that encode a memory. We investigated how memories are stored in the brain throughout development by integrating engram labeling technology with mouse models of infantile amnesia. We discovered a sex-specific phenomenon in which male offspring in maternal immune activation models of autism spectrum disorder do not experience infantile amnesia. We rescued the same apparently forgotten infantile memories in mice by optogenetically reactivating dentate gyrus engram cells that were labeled during complex experiences in infant development. Further, we were able to permanently reinstate lost infantile memories by artificially updating the memory engram, demonstrating that infantile amnesia is a reversible process. Our findings suggest that immune activation during development modulates innate, and reversible, forgetting switches that determine whether infantile amnesia will occur.

sentences: Essential functions of MLL1 and MLL2 in retinal development and cone cell maintenance MLL1 (KMT2A) and MLL2 (KMT2B) are homologous members of the mixed-lineage leukemia (MLL) family of histone methyltransferases involved in epigenomic transcriptional regulation. Their sequence variants have been associated with neurological and psychological disorders, but little is known about their roles and mechanism of action in CNS development. Using mouse retina as a model, we previously reported MLL1s role in retinal neurogenesis and horizontal cell maintenance. Here we determine roles of MLL2 and MLL1/MLL2 together in retinal development using conditional knockout (CKO) mice. Deleting Mll2 from Chx10+ retinal progenitors resulted in a similar phenotype as Mll1 CKO, but removal of both alleles produced much more severe deficits than each single CKO: 1-month double CKO mutants displayed null light responses in electroretinogram; thin retinal layers, including shorter photoreceptor outer segments with impaired phototransduction gene expression; and reduced numbers of M-cones, horizontal and amacrine neurons, followed by fast retinal degeneration. Despite moderately reduced progenitor cell proliferation at P0, the neurogenic capacity was largely maintained in double CKO mutants. However, upregulated apoptosis and reactive gliosis were detected during postnatal retinal development. Finally, the removal of both MLLs in fated rods produced a normal phenotype, but the CKO in M-cones impaired M-cone function and survival, indicating both cell non-autonomous and autonomous mechanisms. Altogether, our results suggest that MLL1/MLL2 play redundant roles in maintaining specific retinal neurons after cell fate specification and are essential for establishing functional neural networks.

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COVID-19 lung disease shares driver AT2 cytopathic features with Idiopathic pulmonary fibrosis BackgroundIn the aftermath of Covid-19, some patients develop a fibrotic lung disease, i.e., post-COVID-19 lung disease (PCLD), for which we currently lack insights into pathogenesis, disease models, or treatment options.

MethodUsing an AI-guided approach, we analyzed > 1000 human lung transcriptomic datasets associated with various lung conditions using two viral pandemic signatures (ViP and sViP) and one covid lung-derived signature. Upon identifying similarities between COVID-19 and idiopathic pulmonary fibrosis (IPF), we subsequently dissected the basis for such similarity from molecular, cytopathic, and immunologic perspectives using a panel of IPF-specific gene signatures, alongside signatures of alveolar type II (AT2) cytopathies and of prognostic monocyte-driven processes that are known drivers of IPF. Transcriptome-derived findings were used to construct protein-protein interaction (PPI) network to identify the major triggers of AT2 dysfunction. Key findings were validated in hamster and human adult lung organoid (ALO) pre-clinical models of COVID-19 using immunohistochemistry and qPCR.

FindingsCOVID-19 resembles IPF at a fundamental level; it recapitulates the gene expression patterns (ViP and IPF signatures), cytokine storm (IL15-centric) and the AT2 cytopathic changes, e.g., injury, DNA damage, arrest in a transient, damage-induced progenitor state, and senescence-associated secretory phenotype (SASP). These immunocytopathic features were induced in pre-clinical COVID models (ALO and hamster) and reversed with effective anti-CoV-2 therapeutics in hamsters. PPI-network analyses pinpointed ER stress as one of the shared early triggers of both diseases, and IHC studies validated the same in the lungs of deceased subjects with COVID-19 and SARS-CoV-2-challenged hamster lungs. Lungs from tg-mice, in which ER stress is induced specifically in the AT2 cells, faithfully recapitulate the host immune response and alveolar cytopathic changes that are induced by SARS-CoV-2.

InterpretationLike IPF, COVID-19 may be driven by injury-induced ER stress that culminates into progenitor state arrest and SASP in AT2 cells. The ViP signatures in monocytes may be key determinants of prognosis. The insights, signatures, disease models identified here are likely to spur the development of therapies for patients with IPF and other fibrotic interstitial lung disease.

FundingThis work was supported by the National Institutes for Health grants R01-GM138385 and AI155696 and funding from the Tobacco-Related disease Research Program (R01RG3780).

One Sentence SummarySevere COVID-19 triggers cellular processes seen in fibrosing Interstitial Lung Disease

sentences: Single cell transcriptome mapping identifies a local innate B cell population driving local antibody production and chronic rejection after lung transplantation Bronchiolitis obliterans syndrome (BOS) due to chronic rejection is the main reason for poor outcomes after lung transplantation (LTx). We and others have recently identified B cells as major contributors to BOS after LTx. The extent of B cell heterogeneity, however, as well as those B cell populations that determine chronic rejection of the lung, remain entirely unclear. Here, we provide a map of cell population heterogeneity and their gene expression patterns during chronic rejection after orthotopic LTx in mice. Out of a total of 14 major comprehensive cell clusters, corresponding to 11 known major cell subpopulations, Mzb1-expressing plasma cells (PCs) were the most prominently increased cell population in lungs with BOS. Scgb1a1-expressing bronchial epithelial cells were depleted, while Cd14-expressing monocytes and Pdgfra-expressing fibroblasts were enriched in BOS grafts. These findings were validated in two different cohorts of human BOS after LTx. MZB1-IgG-double positive PCs infiltrated the airways of patients with BOS, while they were absent from healthy lungs. IgG, but not IgA, IgD, IgE or IgM, were significantly increased in bronchoalveolar lavage from BOS patients, compared with LTx patients who did not develop BOS. Pseudotime and trajectory analysis revealed that a Bhlhe41, Cxcr3, Itgb1-triple positive-B cell subset, also expressing classical markers of the innate-like B-1 B cell population, served as the progenitor pool for Mzb1+ PCs. This progenitor B cell subset accounted for the increase in IgG2c expression and production in BOS lung grafts. Importantly, Aicda-/- mice, which lack all isotypes of Ig (except IgM) were protected from the onset of BOS after LTx. In summary, we provide a detailed atlas of cell population changes of chronic rejection after LTx. We have identified IgG-positive PCs and their progenitors - an innate B cell subpopulation characterized by a specific subset of markers - as the major source of local antibody production and a major contributor to both mouse and human BOS after LTx.

sentences: Trends in Pacific Canadian groundfish stock status We assembled estimated biomass (B) time series from stock assessments for 24 Pacific Canadian groundfish stocks and modelled average and stock status through 2020 based on biomass relative to each stocks (1) Limit Reference Point (B/LRP), (2) Upper Stock Reference (B/USR), and (3) biomass at maximum sustainable yield (B/BMSY). The overall mean B/LRP in 2020 was 3.2 (95% credible interval [CI]: 2.6-3.9). The overall mean B/USR and B/BMSY in 2020 was 1.5 (95% CI: 1.3-1.9) and 1.4 (95% CI: 1.1-1.7), respectively. Average stock status declined from 1950 to around 2000 and has remained relatively stable since then. The change around 2000 followed the implementation of ITQs (individual transferable quotas) for the trawl fleet and the commencement of the synoptic trawl surveys. As of their last assessment, four stocks (Strait of Georgia Lingcod [Area 4B], coastwide Bocaccio, and inside and outside Quillback Rockfish) had a greater than 5% probability of being below their LRP (i.e., in the " critical zone"); Pacific Cod in Area 3CD had a 4.6% probability. Roughly one-third of stocks had a greater than 1 in 4 chance of being below their USR (i.e., in the " cautious zone"). Conversely, two-thirds of assessed groundfish stocks had a high (>75%) probability of being above the USR (i.e., in the " healthy zone").

sentences: Placenta DNA methylation at ZNF300 is associated with fetal sex and placental morphology Fetal sex-specific differences in placental morphology and physiology have been associated with sexually dimorphic health outcomes. However, the molecular mechanisms underlying these sex differences are not well understood. We performed whole genome bisulfite sequencing in 133 placenta samples and discovered a significant difference in DNA methylation (DNAm) at the ZNF300 gene locus between male and female offspring and replicated this result in 6 independent datasets. Additionally, the sex-specific pattern appears to be placenta-specific, is robust to a wide range of gestational ages and adverse health outcomes and is present in sorted placenta villous cytotrophoblast cells. Integration of DNAm, genetic, and placental morphology data from the same individuals revealed ZNF300 methylation is also associated with placenta area, perimeter, and max diameter, genetic variants on chromosomes 5 and X, and may mediate the effects of genetic variation on placental area.

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BioProfiling.jl: Profiling biological perturbations with high-content imaging in single cells and heterogeneous populations MotivationHigh-content imaging screens provide a cost-effective and scalable way to assess cell states across diverse experimental conditions. The analysis of the acquired microscopy images involves assembling and curating morphological measurements of individual cells into morphological profiles suitable for testing biological hypotheses. Despite being a critical step, there is currently no standard approach to morphological profiling and no solution is available for the high-performance Julia programming language.

ResultsHere, we introduce BioProfiling.jl, an efficient end-to-end solution for compiling and filtering informative morphological profiles in Julia. The package contains all the necessary data structures to curate morphological measurements and helper functions to transform, normalize and visualize profiles. Robust statistical distances and permutation tests enable quantification of the significance of the observed changes despite the high fraction of outliers inherent to high-content screens. This package also simplifies visual artifact diagnostics, thus streamlining a bottleneck of morphological analyses. We showcase the features of the package by analyzing a chemical imaging screen, in which the morphological profiles prove to be informative about the compounds mechanisms of action and can be conveniently integrated with the network localization of molecular targets.

AvailabilityThe Julia package is available on GitHub: https://github.com/menchelab/BioProfiling.jl We also provide Jupyter notebooks reproducing our analyses: https://github.com/menchelab/BioProfilingNotebooks

Contactjoerg.menche@univie.ac.at

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Helminth infection is associated with dampened cytokine responses to viral and bacterial stimulations in Tsimane hunter-horticulturalists. BackgroundSoil-transmitted helminth (STH) infections can catalyze immunological changes that affect the response to subsequent infections, particularly those that elicit strong inflammatory responses. As globalization heightens the risk that remote communities with high STH prevalence will encounter novel pathogens, understanding how STHs shape immune responses to these downstream infections becomes increasingly crucial.

MethodologyWe worked with Tsimane forager-horticulturalists in the Bolivian Amazon, where STHs are prevalent. We tested whether STHs and eosinophil levels--most likely indicative of infection in this population--are associated with dampened immune responses to in vitro stimulation with H1N1 and lipopolysaccharide (LPS) antigens. Whole blood samples (n = 179) were treated with H1N1 vaccine and LPS and assayed for 13 cytokines (interferon gamma [INF-{gamma}], interleukin [IL]-1{beta}, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, Granulocyte-macrophage colony-stimulating factor [GM-CSF], and Tumor necrosis factor-alpha [TNF-]). We evaluated how STHs and eosinophil levels affected cytokine responses and T helper (Th) 1 and Th2-cytokine suite responses to stimulation.

ResultsInfection with Ascaris lumbricoides was significantly (p [≤] 0.05) associated with lower response of some cytokines to H1N1 and LPS in women. Eosinophils were significantly negatively associated with some cytokine responses to H1N1 and LPS, with the strongest effects in women, and associated with a reduced Th1- and Th2-cytokine response to H1N1 and LPS in women and men.

Conclusions and implicationsWe find that STHs were associated with dampened cytokine responses to certain viral and bacterial antigens, and suggest that this mitigation of host-induced damage may reduce the incidence of cytokine storms in populations with high STH prevalence.

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Phylogeny, evolution, and classification of the ant genus Lasius, the tribe Lasiini, and the subfamily Formicinae (Hymenoptera: Formicidae) Within the Formicidae, the higher classification of nearly all subfamilies has been recently revised due to the findings of molecular phylogenetics. Here, we integrate morphology and molecular data to holistically address the evolution and classification of the ant genus Lasius, its tribe Lasiini, and their subfamily Formicinae. We accomplish this through a critical re-examination of morphology of extant and fossil taxa, molecular phylogenetic analyses, total-evidence dating under fossilized birth-death process, phylogeography, and ancestral state estimation. We use these results to provide revised taxonomic definitions for the Lasiini and select genera, and we provide a key to the genera of the Lasiini with emphasis on the Lasius genus group. We find that the crown Lasiini originated around the end of the Cretaceous on the Eurasian continent and is divisible into four morphologically distinct clades: Cladomyrma, the Lasius genus group, the Prenolepis genus group, and a previously undetected lineage we name XXX gen. n. The crown of the Lasius genus group is considerably younger than that of the Prenolepis genus group, indicating that extinction has played a major role in the evolution of the former clade. Lasius itself is divided into two well-supported monophyletic groups which are approximately equally speciose. We present evidence that temporary social parasitism and fungiculture arose in Lasius two times independently. Additionally, we recover the paraphyly of three Lasius subgenera and propose replacing all subgenera with an informal species group classification: Lasius = Acanthomyops syn. rev., = Austrolasius syn. n., = Cautolasius syn. n., = Chthonolasius syn. n., = Dendrolasius syn. n. Total-evidence analysis reveals that the Baltic-region amber fossil species {dagger}Lasius pumilus and {dagger}Pseudolasius boreus are misplaced to genus; we therefore designate {dagger}XXX gen. n. for the former and {dagger}XXX gen. n. for the latter. Further, we transfer {dagger}XXX and {dagger}Glaphyromyrmex out of the tribe, considering the former to be incertae sedis in the subfamily, and the latter a member of the Formicini (tribal transfer). Two final taxonomic actions are deemed necessary: synonymy of Lasius escamole Reza, 1925 with Liometopum apiculatum Mayr, 1870 syn. n. (subfamilial transfer), and transfer of Paratrechina kohli to Anoplolepis (tribal transfer, forming A. kohli (Forel, 1916) n. comb.).

Summary of taxonomic actionsO_LISubgenera of Lasius synonymized: Lasius = Acanthomyops syn. rev. = Austrolasius syn. n. = Cautolasius syn. n. = Chthonolasius syn. n. = Dendrolasius syn. n.
C_LIO_LILasius myrmidon transferred to XXX gen. n. (Lasiini, XXX genus group).
C_LIO_LI{dagger}Lasius pumilus transferred to {dagger}XXX gen. n. (Lasiini, XXX genus group).
C_LIO_LI{dagger}Pseudolasius boreus transferred to {dagger}XXX gen. n. (incertae sedis in Formicinae) (tribal transfer).
C_LIO_LI{dagger}Glaphyromyrmex transferred to the Formicini from the Lasiini (tribal transfer).
C_LIO_LILasius escamole Reza, 1925 synonymized with Liometopum apiculatum Mayr, 1870, syn. n. (subfamilial transfer).
C_LIO_LIParatrechina kohli (Forel, 1916) transferred to Anoplolepis (Plagiolepidini) (genus and tribal transfer).
C_LI

sentences: A hardware/software system for electrophysiology "supersessions" in marmosets We introduce a straightforward, robust method for recording and analyzing spiking activity over timeframes longer than a single session, with primary application to the marmoset (Callithrix jacchus). Although in theory the marmosets smooth brain allows for broad deployment of powerful tools in primate cortex, in practice marmosets do not typically engage in long experimental sessions akin to rhesus monkeys. This potentially limits their value for detailed, quantitative neurophysiological study. Here we describe chronically-implanted arrays with a 3D arrangement of electrodes yielding stable single and multi-unit responses, and an analytic method for creating "supersessions" combining that array data across multiple experiments. We could match units across different recording sessions over several weeks, demonstrating the feasibility of pooling data over sessions. This could be a key tool for extending the viability of marmosets for dissecting neural computations in primate cortex.

sentences: Motor Protein MYO1C is Critical for Photoreceptor Opsin Trafficking and Visual Function Unconventional myosins linked to deafness are also proposed to play a role in retinal cell physiology. However, their direct role in photoreceptor function remains unclear. We demonstrate that systemic loss of the unconventional myosin MYO1C in mice specifically affected opsin trafficking, leading to loss of visual function. Electroretinogram analysis of Myo1c knockout (Myo1c-KO) mice showed a progressive loss of photoreceptor function. Immunohistochemistry and binding assays demonstrated MYO1C localization to photoreceptor inner and outer segments (OS) and identified a direct interaction of rhodopsin with the MYO1C cargo domain. In Myo1c-KO retinas, rhodopsin mislocalized to rod inner segments (IS) and cell bodies, while cone opsins in OS showed punctate staining. In aged mice, the histological and ultrastructural examination of the phenotype of Myo1c-KO retinas showed progressively shorter photoreceptor OS. These results demonstrate that MYO1C is critical for opsin trafficking to the photoreceptor OS and for normal visual function.

sentences:
Landscapes of transposable elements in Apis species by meta-analysis Transposable elements (TEs) are grouped into several classes with diverse sequences. Owing to their diversity, studies involving the detection, classification, and annotation of TEs are difficult tasks. Moreover, simple comparisons of TEs among different species with different methods can lead to misinterpretations. The genome data of several honey bee (Apis) species are available in public databases. Therefore, we conducted a meta-analysis of TEs, using 10 sets of genome data for Apis species, in order to establish the basal TE data (termed here as the  landscape of TEs). Consensus TE sequences were constructed and their distributions in the Apis genomes were determined. Our results showed that TEs from several limited families mainly consisted of Apis TEs and that more DNA/TcMar-Mariner consensus sequences and copies were present in all Apis genomes tested. In addition, more consensus sequences and copy numbers of DNA/TcMar-Mariner were detected in Apis mellifera than in other Apis species. These results suggest that TcMar-Mariner might exert A. mellifera-specific effects on the host A. mellifera species. In conclusion, our unified approach enabled comparison of Apis genome sequences to determine the TE landscape, which provide novel evolutionary insights into Apis species.

Simple SummaryStudies on the detection of transposable elements and their annotations have posed several challenges. For example, simple comparisons of transposable elements in different species using different methods can lead to misinterpretations. Thus, assembling data for transposable elements analyzed by unified methods is important for comparison purposes. Therefore, we performed a meta-analysis of transposable elements identified using genome datasets from 4 Apis species (10 sets of genome data) and specific software to detect the transposable elements, which revealed the landscapes of transposable elements. We examined the types and locations of transposable elements in the Apis genomes. The landscapes of transposable elements showed that several limited transposable element families consisted mainly of Apis-associated transposable elements. These limited families include DNA/TcMar-Mariner and DNA/CMC-EnSpm. In addition, more DNA/TcMar-Mariner consensus sequences and copies were detected in Apis mellifera than in other Apis species. These data suggest that TcMar-Mariner might exert A. mellifera-specific effects in the host A. mellifera species. Our landscape data provide new insights into Apis transposable elements; furthermore, detailed analyses of our data could pave the way for new biological insights in this field.

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Tissue specific dual RNA-seq defines host-parasite interplay in murine visceral leishmaniasis caused by Leishmania donovani and Leishmania infantum Visceral leishmaniasis is associated with hepato-splenomegaly and altered immune and haematological parameters in both pre-clinical animal models and humans. We studied mouse experimental visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani in BALB/c mice using dual RNA-seq to investigate the transcriptional response of host and parasite in liver and spleen. We identified only 4 species-specific parasite expressed genes (SSPEGs; log2FC >1, FDR <0.05) in the infected spleen, and none in the infected liver. For the host transcriptome, we found 789 differentially expressed genes (DEGs; log2FC >1, FDR <0.05) in the spleen that were common to both infections, with IFN{gamma} signaling and complement and coagulation cascade pathways highly enriched, and an additional 286 and 186 DEGs that were selective to L. donovani and L. infantum infection respectively. Among those, there were network interactions between genes of amino acid metabolism and PPAR signaling in L. donovani infection and increased IL1{beta} and positive regulation of fatty acid transport in L. infantum infection, although no pathway enrichment was observed. In the liver, there were 1939 DEGs in mice infected with either L. infantum or L. donovani in comparison to uninfected mice, and the most enriched pathways were IFN{gamma} signaling, neutrophil mediated immunity, complement and coagulation, cytokine-chemokine responses and hemostasis. Additionally, 221 DEGs were selective in L. donovani and 429 DEGs in L. infantum infections. These data show that the host response for these two visceral leishmaniasis infection models is broadly similar, and [~]10% of host DEGs vary in infections with either parasite species.

ImportanceVisceral leishmaniasis (VL) is caused by two species of Leishmania parasites, L. donovani in the Old World and L. infantum in the New World and countries bordering the Mediterranean. Although cardinal features such as hepato-splenomegaly and alterations in blood and immune function are evident, clinical presentation may vary by geography, with for example severe bleeding often associated with VL in Brazil. Although animal models of both L. donovani and L. infantum have been widely used to study disease pathogenesis, a direct side-by-side comparison of how these parasites species impact the infected host and/or how they might respond to the stresses of mammalian infection has not been previously reported. Identifying common and distinct pathways to pathogenesis will be important to ensure that new therapeutic or prophylactic approaches will be applicable across all forms of VL.

sentences: Transcription initiation mapping in 31 bovine tissues reveals complex promoter activity, pervasive transcription, and tissue-specific promoter usage Characterizing transcription start sites is essential for understanding the regulatory mechanisms that control gene expression. Recently, a new bovine genome assembly (ARS-UCD1.2) with high continuity, accuracy, and completeness was released; however, the functional annotation of the bovine genome lacks precise transcription start sites and contains a low number of transcripts in comparison to human and mouse. Using the RAMPAGE approach, this study identified transcription start sites at high resolution in a large collection of bovine tissues. We found several known and novel transcription start sites attributed to promoters of protein coding and lncRNA genes that were validated through experimental and in silico evidence. With these findings, the annotation of transcription start sites in cattle reached a level comparable to the mouse and human genome annotations. In addition, we identified and characterized transcription start sites for antisense transcripts derived from bidirectional promoters, potential lncRNAs, mRNAs, and pre-miRNAs. We also analyzed the quantitative aspects of RAMPAGE to produce a promoter activity atlas, reaching highly reproducible results comparable to traditional RNA-seq. Co-expression networks revealed considerable use of tissue specific promoters, especially between brain and testicle, which expressed several genes in common from alternate loci. Furthermore, regions surrounding co-expressed modules were enriched in binding factor motifs representative of each tissue. The comprehensive annotation of promoters in such a large collection of tissues will substantially contribute to our understanding of gene expression in cattle and other mammalian species, shortening the gap between genotypes and phenotypes.

sentences: Deriving accurate molecular indicators of protein synthesis through Raman-based sparse classification Raman spectroscopy has the ability to retrieve molecular information from live biological samples non-invasively through optical means. Coupled with machine learning, it is possible to use the large amount of information contained in a Raman spectrum to create models that can predict the state of new samples based on statistical analysis from previous measurements. Furthermore, in case of linear models, the separation coefficients can be used to interpret which bands are contributing to the discrimination between experimental conditions, which correspond here to single-cell measurements of macrophages under in vitro immune stimulation. We here evaluate a typical linear method using discriminant analysis and PCA, and compare it to regularized logistic regression (Lasso). We find that the use of PCA is not beneficial to the classification performance. Furthermore, the Lasso approach yields sparse separation vectors, since it suppresses spectral coefficients which do not improve classification, making interpretation easier. To further evaluate the approach, we apply the Lasso technique to a well-defined case where protein synthesis is inhibited, and show that the separating features are consistent with RNA accumulation and protein levels depletion. Surprisingly, when Raman features are selected purely in terms of their classification power (Lasso), the selected coefficients are contained in side bands, while typical strong Raman peaks are not present in the discrimination vector. We propose that this occurs because large Raman bands are representative of a wide variety of cellular molecules and are therefore less suited for accurate classification.

sentences: Placental uptake and metabolism as determinants of pregnancy vitamin D status Pregnancy 25-hydroxyvitamin D (25(OH)D) concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy.

sentences: Type I photosynthetic reaction center in an anoxygenic phototrophic member of the Chloroflexota Phototrophic members of the Chloroflexota phylum are enigmas in the evolution of phototrophy. Although all previously characterized phototrophic Chloroflexota members use a Type II reaction center (RCII) to perform light energy conversion1,2, many members host chlorosomes, a light-harvesting apparatus only otherwise reported among Type I reaction center (RCI)-containing phototrophs3. Here we report the discovery and cultivation of "Candidatus Chlorohelix allophototropha", the first known member of the Chloroflexota to use RCI, rather than RCII, for phototrophy. Cultivated from an iron-rich Boreal Shield lake, "Ca. Chx. allophototropha" cells contain chlorosomes and encode a novel fifth clade of RCI protein. Phylogenomic data demonstrate that "Ca. Chx. allophototropha" shares closest ancestry to RCII-utilizing relatives for multiple photosynthesis accessory genes, including chlorosomes. Thus, we propose that the enigmatic distribution of chlorosomes in the Chloroflexota phylum resulted from genetic interaction of RCI- and RCII-utilizing Chloroflexota members. Although undetected previously, metatranscriptome data demonstrate that RCI-utilizing Chloroflexota members can be among the most active populations in illuminated anoxic waters of Boreal Shield lakes, which number in the millions globally4. Our results establish the Chloroflexota as the only bacterial phylum containing RCI- and RCII-utilizing anoxygenic phototrophs, providing new context for understanding the origins of phototrophic life on Earth.

sentences: Distinct Roles of Dopamine D1 and D2 Receptor-expressing Neurons in the Nucleus Accumbens for a Strategy Dependent Decision Making To optimize decision making, animals need to execute not only a strategy to choose a good option but sometimes also one to avoid a bad option. A psychological study indicates that positive and negative information is processed in a different manner in the brain. The nucleus accumbens (NAc) contains two different types of neurons, dopamine D1 and D2 receptor-expressing neurons which are implicated in reward-based decision making and aversive learning. However, little is known about the neural mechanisms by which D1 or D2 receptor-expressing neurons in the NAc contribute to the execution of the strategy to choose a good option or one to avoid a bad option under decision making. Here, we have developed two novel visual discrimination tasks for mice to assess the strategy to choose a good option and one to avoid a bad option. By chemogenetically suppressing the subpopulation of the NAc neurons, we have shown that dopamine D2 receptor-expressing neurons in the NAc selectively contribute to the strategy to avoid a bad option under reward-based decision making. Furthermore, our optogenetic and calcium imaging experiments indicate that dopamine D2 receptor-expressing neurons are activated by error choices and the activation following an error plays an important role in optimizing the strategy in the next trial. Our findings suggest that the activation of D2 receptor-expressing neurons by error choices through learning enables animals to execute the appropriate strategy.

sentences: Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells Are Controlled by pH Balance Many biological processes are controlled by cell death and proliferation. The present study found that intracellular pH positively correlated with proliferation but negatively with apoptosis. Acid treatments lowered intracellular pH and preferentially depleted highly proliferating lymphocytes, whereas alkaline treatments increased intracellular pH and enhanced lymphocyte proliferation. Likewise, acid treatments induced apoptosis and alkaline treatments maintained proliferation of tumor cells. These findings support a unified theory of regulation of cell death and proliferation where an intracellular pH balance controls both events, and the mitochondria act as the pH-stats of the cell. Accordingly, the Warburg effect is explained as necessary for proliferating cells to have a high intracellular pH environment to accelerate proliferation and avoid low pH-induced death.

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Functional Characterization of an Electromagnetic Perceptive Protein Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and in-vivo. Here, we report for the first time the cellular location and orientation of the EPG protein. We utilized a differential labelling technique to determine that the EPG protein is a membrane anchored protein with an N-terminal extracellular domain. The kinetics and diffusion dynamics of the EPG protein in response to magnetic stimulation was also elucidated using single particle imaging and tracking. Pulse chase labelling and Total Internal Reflection Fluorescence (TIRF) imaging revealed an increase in EPG kinetics post magnetic activation at a single particle level. Trajectory analysis show notably different EPG protein kinetics before and after magnetic stimulation in both 2 (free vs bound particle) and 3 state (free vs intermediate vs bound particle) tracking models. This data provides additional information to support and understand the underlying biophysical mechanisms behind EPG activation by magnetic fields and provides evidence for the basis of magnetoreception in the EPG protein that will aid in future studies that seek to further understand this novel mechanism. This study is important for understanding magnetoreception as well as developing new technologies for magnetogenetics - the utilization of electromagnetic fields to remotely control cellular function.

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sentences: CGRP Administration into the Cerebellum Evokes Migraine-like Behaviors Predominately in Female Mice The neuropeptide calcitonin gene-related peptide (CGRP) is a major player in migraine pathophysiology. Previous preclinical studies demonstrated that intracerebroventricular administration of CGRP caused migraine-like behaviors in mice, but the sites of action in the brain remain unidentified. The cerebellum has the most CGRP binding sites in the central nervous system and is increasingly recognized as both a sensory and motor integration center. The objective of this study was to test whether the cerebellum, particularly the medial cerebellar nuclei (MN), might be a site of CGRP action. In this study, CGRP was directly injected into the right MN of C57BL/6J mice via a cannula. A battery of behavioral tests was done to assess migraine-like behaviors. CGRP caused light aversion measured as decreased time in the light zone even with dim light. The mice also spent more time resting in the dark zone, but not the light, along with decreased rearing and transitions between zones. These behaviors were similar for both sexes. In contrast, significant responses to CGRP were seen only with female mice in the open field assay, von Frey test, and automated squint assay, indicating anxiety, tactile hypersensitivity, and spontaneous pain, respectively. In male mice, the responses had the same trend as females but did not reach statistical significance. No detectable effect of CGRP on gait was observed in either sex. These results suggest that CGRP in the MN causes light aversion in males, while in females, light aversion is accompanied by increased anxiety, tactile hypersensitivity, and spontaneous pain. A caveat is that we cannot exclude contributions from other cerebellar regions in addition to the MN due to diffusion of the injected peptide. These results reveal the cerebellum as a new site of CGRP actions that may contribute to migraine pathophysiology and possibly its prevalence in females.

sentences: The minimum land area requiring conservation attention to safeguard biodiversity More ambitious conservation efforts are needed to stop the global biodiversity crisis. Here, we estimate the minimum land area to secure important sites for terrestrial fauna, ecologically intact areas, and the optimal locations for representation of species ranges and ecoregions. We discover that at least 64 million km2 (44% of terrestrial area) requires conservation attention. Over 1.8 billion people live on these lands so responses that promote agency, self-determination, equity, and sustainable management for safeguarding biodiversity are essential. Spatially explicit land-use scenarios suggest that 1.3 million km2 of land requiring conservation could be lost to intensive human land-uses by 2030, which requires immediate attention. However, there is a seven-fold difference between the amount of habitat converted under optimistic and pessimistic scenarios, highlighting an opportunity to avert this crisis. Appropriate targets in the post-2020 Global Biodiversity Framework to ensure conservation of the identified land would contribute substantially to safeguarding biodiversity.

sentences: The highly conserved stem-loop II motif is important for the lifecycle of astroviruses but dispensable for SARS-CoV-2 The stem-loop II motif (s2m) is an RNA element present in viruses from divergent viral families, including astroviruses and coronaviruses, but its functional significance is unknown. We created deletions or substitutions of the s2m in astrovirus VA1 (VA1), classic human astrovirus 1 (HAstV1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For VA1, recombinant virus could not be rescued upon partial deletion of the s2m or substitutions of G-C base pairs. Compensatory substitutions that restored the G-C base-pair enabled recovery of VA1. For HAstV1, a partial deletion of the s2m resulted in decreased viral titers compared to wild-type virus, and reduced activity in a replicon system. In contrast, deletion or mutation of the SARS-CoV-2 s2m had no effect on the ability to rescue the virus, growth in vitro, or growth in Syrian hamsters. Our study demonstrates the importance of the s2m is virus-dependent.

sentences: Polyphyly of the Niphargus stygius species group (Crustacea, Amphipoda, Niphargidae) in the Southern Limestone Alps AO_SCPLOWBSTRACTC_SCPLOWThe Niphargus stygius species complex is a groundwater group of large-sized, sexually dimorphic species inhabiting mainly caves and, less frequently, wells and springs. According to the taxonomists of the last century, this species complex was supposed to be present in the whole Southern Limestone Alps of Italy as well as in peninsular Italy and Slovenia. Considering the large presumed distribution area, we tested the contrasting hypotheses of monophyly versus paraphyly of this subterranean species complex, taking in account the presence of putative cryptic species. For this reason, we sampled the type localities of all the described species in the complex present in the Italian Southern Limestone Alps and neighbouring areas, and used nuclear (28S, ITS region) and mtDNA (COI) sequences to assess their phylogenetic relationships and species richness. Phylogenetic analysis confirmed that the Niphargus stygius complex in the Southern Limestone Alps is not monophyletic but comprises an eastern clade (the N. kenki clade, present in NW Italy, northern Slovenia, and southern Austria) and two western clades (the N. brixianus and N. montellianus clades), _These two clades are not closely related to the eastern one but rather form a monophyletic group together with a widely distributed Apennine clade (N. speziae clade). None of these clades is closely related to typical N. stygius. Three different molecular species delimitation methods applied to COI and ITS recognized different number of putative species, richer for the COI marker, suggesting that each clade is a species complex in itself. Bayesian time-calibrated phylogeny revealed that most clades began to split up during Miocene and Pliocene, ruling out the effect of Pleistocene glaciations in explaining their evolutionary history and justifying the presence of several putative cryptic species.

sentences: Declining population sizes and loss of genetic diversity in commercial fishes: a simple method for a first diagnostic Exploited fish species may have or are experiencing declines in population sizes coupled with a decrease in genetic diversity. This can lead to the loss of adaptive potential to face current and future environmental changes. However, little is known about this subject while research on it is urgently needed. Thus, this study aims to answer a simple, even naive question, given the complexity of the subject: Could we use a simple method to obtain information on the loss of genetic diversity in exploited fish species? We investigated the use of the levels of genetic diversity in the widely used genetic marker Cytochrome C Oxidase subunit I (COI) mitochondrial gene. Estimates of genetic diversity in COI were obtained for populations of seven fish species with different commercial importance from the East China Sea. These estimates were contrasted against a large dataset of fish species distributed worldwide (N=1426), a dataset of East-Asian fish species (N=118), two farmed species with expected low genetic diversity, and four long-term managed species from the Mediterranean Sea. We found that estimates of genetic diversity in COI match the expectations from theoretical predictions, known population declines, and fishing pressures. Thus, the answer to our question is affirmative and we conclude that estimates of genetic diversity in COI provide an effective first diagnostic of the conservation status of exploited fish species. This simple and cost-effective tool can help prioritize research, management, and conservation on species with suspected loss of genetic diversity potentially eroding their adaptive potential to global change.

sentences: Novel Object Detection and Multiplexed Motion Representation in Retinal Bipolar Cells Antagonistic interactions between the center and surround receptive field (RF) components lie at the heart of the computations performed in the visual system. Center-surround RFs are thought to enhance responses to spatial contrasts (i.e., edges), but how they contribute to motion processing is unknown. Here, we addressed this question in retinal bipolar cells, the first visual neuron with classic center-surround interactions. We found that bipolar glutamate release emphasizes objects that emerge in the RF; their responses to continuous motion are smaller, slower, and cannot be predicted by signals elicited by stationary stimuli. The alteration in signal dynamics induced by novel objects dwarfs the enhancement of spatial edges and can be explained by priming of RF surround during continuous motion. These findings echo the salience of human visual perception and demonstrate an unappreciated capacity of the center-surround architecture to facilitate novel object detection and multiplexed encoding of distinct sensory modalities.

sentences: Circulatory systems and mortality rates Aging is a complex process involving multiple factors and subcellular processes, ultimately leading to the death of an organism. The microscopic processes that cause aging are relatively well understood and effective macroscopic theories help explain the universality of aging in complex systems. However, these theories fail to explain the diversity of aging observed for various lifeforms. As such, more complete "mesoscopic" theories of aging are needed, combining the biophysical details of microscopic failure and the macroscopic structure of complex systems. Here we explore two models: (1) a network theoretic model, and (2) a convection diffusion model emphasizing the biophysical details of communicated signals. The first model allows us to explore the effects of connectivity structures on aging. In our second model, cells interact through cooperative and antagonistic factors. We find by varying the ratio at which these factors affect cell death, as well as the reaction kinetics, diffusive and flow parameters, we obtain a wide diversity of mortality curves. As such, the connectivity structures as well as the biophysical details of how various factors are transported in an organism may explain the diversity of aging observed across different lifeforms.

sentences: Differentiable molecular simulation can learn all the parameters in a coarse-grained force field for proteins Finding optimal parameters for force fields used in molecular simulation is a challenging and time-consuming task, partly due to the difficulty of tuning multiple parameters at once. Automatic differentiation presents a general solution: run a simulation, obtain gradients of a loss function with respect to all the parameters, and use these to improve the force field. This approach takes advantage of the deep learning revolution whilst retaining the interpretability and efficiency of existing force fields. We demonstrate that this is possible by parameterising a simple coarse-grained force field for proteins, based on training simulations of up to 2,000 steps learning to keep the native structure stable. The learned potential matches chemical knowledge and PDB data, can fold and reproduce the dynamics of small proteins, and shows ability in protein design and model scoring applications. Problems in applying differentiable molecular simulation to all-atom models of proteins are discussed along with possible solutions. The learned potential, simulation scripts and training code are made available at https://github.com/psipred/cgdms.

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Ozone nanobubble treatments improve survivability of Nile tilapia (Oreochromis niloticus) challenged with a pathogenic multidrug-resistant Aeromonas hydrophila Multidrug-resistant (MDR) bacteria has rapidly increased in aquaculture, which highlights the risk of production losses due to diseases and potential public health concerns. Previously, we reported that ozone nanobubbles (NB-O3) were effective at reducing concentrations of pathogenic bacteria in water and modulating fish immunity against pathogens; however, multiple treatments with direct NB-O3 exposures caused alterations to the gills of exposed-fish. Here, we set up a modified recirculation system (MRS) assembled with an NB-O3 device (MRS-NB-O3) to investigate whether MRS-NB-O3 were 1) safe for tilapia (Oreochromis niloticus), 2) effective at reducing bacterial load in rearing water, and 3) improved survivability of Nile tilapia following an immersion challenge with a lethal dose of MDR Aeromonas hydrophila. The results indicated no behavioral abnormalities or mortality of Nile tilapia during the 14 day study using the MRS-NB-O3 system. In the immersion challenge, although high bacterial concentration (~2 x 107 CFU/mL) was used, multiple NB-O3 treatments in the first two days reduced the bacteria between 15.9% to 35.6% of bacterial load in water while bacterial concentration increased 13.1% to 27.9% in the untreated control. There was slight up-regulation of non-specific immune-related genes in the gills of the fish receiving NB-O3 treatments. Most importantly, this treatment significantly improved survivability of Nile tilapia with relative percent survival (RPS) of 64.7 - 66.7% in treated fish and surviving fish developed specific antibody against MDR A. hydrophila. In summary, the result suggests that NB-O3 is a promising alternative to antibiotics to control bacterial diseases, including MDR bacteria, and has high potential for application in recirculation aquaculture system (RAS).

HighlightsO_LIMultiple treatments of NB-O3 in a modified recirculation system (MRS) were relatively safe for juvenile Nile tilapia
C_LIO_LINB-O3 treatments in MRS significantly improved survivability of Nile tilapia challenged with multidrug-resistant (MDR) A. hydrophila with RPS of 64.7 - 66.7%
C_LIO_LIConcentration of MDR A. hydrophila in MRS was reduced by 15.9 to 35.6% following each NB-O3 treatment, and increased by 13.1 to 27.9 % in untreated control
C_LIO_LISurviving fish developed specific antibody IgM against MDR A. hydrophila
C_LIO_LINB-O3 is a promising non-antibiotic approach to control diseases caused by MDR A. hydrophila
C_LI

sentences: Sensory and choice responses in MT distinct from motion encoding Macaque area MT is well known for its visual motion selectivity and relevance to motion perception, but the possibility of it also reflecting non-sensory functions has largely been ignored. Manipulating subjects temporal evidence weighting revealed multiple components of MT responses that were, surprisingly, not interpretable as behaviorally-relevant modulations of motion encoding, nor as consequences of readout of motion direction. MTs time-varying motion-driven responses were starkly changed by our strategic manipulation, but with timecourses opposite the subjects temporal weighting strategies. Furthermore, large choice-correlated signals were represented in population activity distinctly from motion responses (even after the stimulus) with multiple phases that both lagged psychophysical readout and preceded motor responses. These results reveal multiple cognitive contributions to MT responses that are task-related but not functionally relevant to encoding or decoding of motion for psychophysical direction discrimination, calling into question its nature as a simple sensory area.

sentences: Astrocyte plasticity ensures continued endfoot coverage of cerebral blood vessels and integrity of the blood brain barrier, with plasticity declining with normal aging. Astrocytes extend endfeet that enwrap the vasculature. Disruptions to this association in disease coincide with breaches in blood-brain barrier (BBB) integrity, so we asked if the focal ablation of an astrocyte is sufficient to disrupt the BBB. 2Phatal ablation of astrocytes induced a plasticity response whereby surrounding astrocytes extended processes to cover vascular vacancies. This occurred prior to endfoot retraction in young mice yet occurred with significant delay in aged animals. Laser-stimulating replacement astrocytes showed them to induce constrictions in pre-capillary arterioles indicating that replacement astrocytes are functional. Inhibition of EGFR and pSTAT3 significantly reduced astrocyte replacement post-ablation yet without perturbations to BBB integrity. Identical endfoot replacement following astrocyte cell death due to reperfusion post-stroke supports the conclusion that astrocyte plasticity ensures continual vascular coverage so as to retain the BBB. Together, these studies uncover the ability of astrocytes to maintain cerebrovascular coverage via substitution from nearby cells and may represent a novel therapeutic target for vessel recovery post-stroke.

sentences: Efficient Generative Modelling of Protein Structure Fragments using a Deep Markov Model Fragment libraries are often used in protein structure prediction, simulation and design as a means to significantly reduce the vast conformational search space. Current state-of-the-art methods for fragment library generation do not properly account for aleatory and epistemic uncertainty, respectively due to the dynamic nature of proteins and experimental errors in protein structures. Additionally, they typically rely on information that is not generally or readily available, such as homologous sequences, related protein structures and other complementary information. To address these issues, we developed BIFROST, a novel take on the fragment library problem based on a Deep Markov Model architecture combined with directional statistics for angular degrees of freedom, implemented in the deep probabilistic programming language Pyro. BIFROST is a probabilistic, generative model of the protein backbone dihedral angles conditioned solely on the amino acid sequence. BIFROST generates fragment libraries with a quality on par with current state-of-the-art methods at a fraction of the run-time, while requiring considerably less information and allowing efficient evaluation of probabilities.

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Microbiome structure of a wild Drosophila community along tropical elevational gradients and comparison to laboratory lines While the biogeography of free-living microbial communities is well-studied, community turnover along environmental gradients in host-associated communities is not well understood. In particular, patterns of host-microbiome diversity along elevational gradients remain largely uncharacterized. Because elevational gradients may serve as natural proxies for climate change, understanding these temperature-influenced patterns can inform our understanding of the threats facing hosts and their microbes in a warming world. In this study, we analysed microbiomes from pupae & adults of four Drosophila species native to Australian tropical rainforests. We sampled wild individuals at high and low elevation along two mountain gradients, to determine natural diversity patterns, and sampled laboratory-reared individuals from isofemale lines established from the same localities, to see if any natural patterns would be retained in the lab. In both environments, we controlled for diet to help elucidate other deterministic patterns of microbiome composition. Microbiome community composition differed radically between laboratory-reared and field-caught flies but did not significantly differ across elevation. We found some notable taxonomic differences in Drosophila microbiomes between different species and elevations. We also found similar microbiome composition from both types of provided food, and we therefore suggest the significant differences in richness are the products of environments with different bacterial species pools. We conclude that elevational differences in temperature are not a major factor in determining Drosophila microbiome composition and we caution against determining microbiome composition from lab-only specimens, particularly long-term cultures.

ImportanceBacteria form microbiome communities inside many different hosts. These communities vary widely in diversity, and form differently, depending on a wide variety of factors. Diet is often an important factor for microbiome composition. We controlled diet in our study by providing the same food sources. We looked at how host-microbiome communities differed in four Drosophila species over two mountain gradients in tropical Australia, to see if temperature-driven differences in elevation affected host-microbiomes. We also compared these results to individuals kept in the laboratory to understand how different settings changed microbiome communities. We found that field samples had substantially greater microbiome diversity than those from the lab and found minimal differences in microbiome communities over elevation. Our study shows that environmental sources of bacteria matter for Drosophila microbiome composition, and caution should be exercised when interpreting microbiome results from lab-only studies.

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Decreased in vitro dihydroartemisinin sensitivity in malaria parasites infecting sickle cell disease patients BackgroundPartial ACTs treatment failure in Plasmodium falciparum malaria has been previously reported in sickle cell patients. The main purpose of this study was to investigate the in vitro susceptibility of clinical isolates to DHA to find out hypothesis backing up the reason of this poor therapeutic response.

ResultsA total of 134 clinical isolates from patients attending health centers in Abidjan with uncomplicated Plasmodium falciparum malaria were selected. Hemoglobin HbAS, HbSS, HbAC, HbSC and HbAA were identified. Parasitemia and hemoglobin level at inclusion were lower in sickle cell patients with major forms than in patients with normal phenotype. A significant number of parasites with survival rates ranging from 14.68 to 33.75% were observed in clinical isolates from the SS phenotype. At inclusion, these resistant clinical isolates showed lower parasite densities, and patients had lower red blood cell count and hematocrit levels compared to those with susceptible clinical isolates. A low rate of parasitic growth has more often occurred with AS sickle cell phenotype. However, the decrease in in vitro sensitivity to DHA was not associated with Kelch 13-Propeller gene polymorphism.

ConclusionThis study highlights an in vitro decreased sensitivity to DHA, for clinical isolates collected from sickle cell SS patients living in Abidjan (Cote dIvoire), which is not related to the Pfkelch13 gene mutations. These clinical isolates may represent a health threat for sickle cell disease patients especially during crisis. Moreover, these results could suggest additional mechanisms of artemisinin resistance that need to be explored.

sentences: Investigation of extramammary sources of Group B Streptococcus reveals its unusual ecology and epidemiology in camels Camels are vital to food production in the drylands of the Horn of Africa, with milk as their main contribution to food security. A major constraint to camel milk production is mastitis, inflammation of the mammary gland. The condition negatively impacts milk yield and quality as well as household income. The leading cause of mastitis in dairy camels is Streptococcus agalactiae, group B Streptococcus (GBS), which is also a commensal and pathogen of humans. It has been suggested that extramammary reservoirs for this pathogen may contribute to the occurrence of mastitis in camels. We explored the molecular epidemiology of GBS in camels using a cross-sectional study design for sample collection and phenotypic, genomic and phylogenetic analysis of isolates. Among 88 adult camels and 93 calves from six herds in Laikipia County, Kenya, GBS was detected in 20% of 50 milk samples, 25% of 152 nasal swabs, 8% of 90 oral swabs and 3% of 90 rectal swabs, but not in vaginal swabs. Per camel herd, two to four sequence types (ST) were present. More than half of the isolates belonged to ST617 or its single-locus variant, ST1652, with these STs found across all sample types. Serotype VI was detected in 30 of 58 isolates. In three herds, identical STs were detected in milk and swab samples, suggesting that extramammary sources of GBS may contribute to the maintenance and spread of GBS within camel herds. This needs to be considered when developing prevention and control strategies. In addition, the high nasal carriage rate, low recto-vaginal carriage rate, and high prevalence of serotype VI for GBS in camels are in stark contrast to the distribution of GBS in humans and reveal hitherto unknown ecological and molecular features of this bacterial species.

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Environmental stress determines the colonization and impact of an endophytic fungus on invasive knotweed O_LIThere is increasing evidence that microbes play a key role in some plant invasions. A diverse and widespread but little understood group of plant-associated microbes are the fungal root endophytes of the order Sebacinales. They are associated with exotic populations of invasive knotweed (Reynoutria ssp.) in Europe, but their effects on the invaders are unknown.
C_LIO_LIWe used the recently isolated Sebacinales root endophyte Serendipita herbamans to experimentally inoculate invasive knotweed and study root colonisation and effects on knotweed growth under different environmental conditions. We verified the inoculation success and fungal colonisation through immunofluorescence microscopy and qPCR.
C_LIO_LIWe found that S. herbamans strongly colonized invasive knotweed in low-nutrient and shade environments, but much less under drought or benign conditions. At low nutrients, the endophyte had a positive effect on plant growth, whereas the opposite was true under shaded conditions.
C_LIO_LISynthesis. Our study demonstrates that the root endophyte S. herbamans has the potential to colonize invasive knotweed fine roots and impact its growth, and it could thus also play a role in natural populations. Our results also show that effects of fungal endophytes on plants can be strongly environment-dependent, and may only be visible under stressful environmental conditions.
C_LI

sentences: Evidence that Nek1 does not phosphorylate Rad54-S572 during recovery from IR A main focus of the work in our lab is on the activity of Tousled Like Kinase 1 (TLK1) in the area of DNA Damage and Repair. As one of its key interactor, TLK1 phosphorylates NIMA related kinase 1 (Nek1), and Nek1 was reported by Spies et al. to phosphorylate and regulate the activity of the key HRR protein Rad541, which suggested an intriguing signal transduction pathway: TLK1>Nek1>Rad54. In an effort to confirm such relations, we now report that we have not been able to reproduce key findings from that study. Specifically, we found that Nek1 does not phosphorylate RAD54-S572 as was reported. We generated Nek1-KO mouse NT1 cells2 and Nek1-Knock-down in Hek293 (same cells as in Spies et al.), and the pRAD54-S572 signal does not change with our custom Ab, with or w/o IR. When we used an Ab from Lobrichs lab, it detected an immunoreactive band of wrong size for RAD54, which also did not change after IR even in synchronized G2 cells, contrary to their report. We also note that their P-assignment was based on guessing a weak consensus Nek1 sequence, and that site-directed mutagenesis of RAD54-S572 failed to yield biological effects in their in in vitro studies1. To conclusively establish that S572 is not a site of phosphorylation of Nek1, we carried out a IVK with purified Nek1 and RAD54 followed by MS analysis of the phosphatides, which revealed several but not S572. We also could not reproduce their copurification of Nek1-RAD54 by coIP, calling into question this interaction.

sentences: MHC class IIa haplotypes derived by high throughput SNP screening in an isolated sheep population Investigating the current evolutionary processes acting on a highly polymorphic gene region, such as the major histocompatibility complex (MHC), requires extensive population data for both genotypes and phenotypes. The MHC consists of several tightly linked loci with both allelic and gene content variation, making it challenging to genotype. Eight class IIa haplotypes have previously been identified in the Soay sheep (Ovis aries) of St. Kilda using Sanger sequencing and cloning, but no single locus is representative of all haplotypes. Here, we exploit the closed nature of the island population of Soay sheep and its limited haplotypic variation to identify a panel of SNPs that enable imputation of MHC haplotypes. We compared MHC class IIa haplotypes determined by Sanger sequence-based genotyping of 135 individuals to their SNP profiles generated using the Ovine Infinium HD BeadChip. A panel of 11 SNPs could reliably determine MHC diplotypes, and two additional SNPs within the DQA1 gene enabled detection of a recombinant haplotype affecting only the SNPs downstream of the expressed genes. The panel of 13 SNPs was genotyped in 5951 Soay sheep, of which 5349 passed quality control. Using the Soay sheep pedigree, we were able to trace the origin and inheritance of the recombinant SNP haplotype. This SNP-based method has enabled the rapid generation of locus-specific MHC genotypes for large numbers of Soay sheep. This volume of high-quality genotypes in a well-characterized population of free-living sheep will be valuable for investigating the mechanisms maintaining diversity at the MHC.

sentences: Cells with Stochastically Increased Methyltransferase to Restriction Endonuclease Ratio Provide an Entry for Bacteriophage into Protected Cell Population The action of type II restriction-modification (RM) systems depends on restriction endonuclease (REase), which cleaves foreign DNA at specific sites, and methyltransferase (MTase), which protects host genome from restriction by methylating the same sites. We show that protection from phage infection increases as the copy number of plasmids carrying the Esp1396l RM system is increased. However, since increased plasmid copy number leads to both increased absolute intracellular REase and MTase levels and decreased MTase to REase ratio, it is impossible to determine which factor determines resistance/susceptibility to infection. By controlled expression of Esp1396I MTase or REase genes in cells carrying the Esp1396I system, we show that a shift in the MTase to REase ratio caused by overproduction of MTase or REase leads, respectively, to decreased or increased protection from infection. Consistently, due to stochastic variation of MTase and REase amount in individual cells, bacterial cells that are productively infected by bacteriophage have significantly higher MTase to REase ratios than cells that ward off the infection. Our results suggest that cells with transiently increased MTase to REase ratio at the time of infection serve as entry points for unmodified phage DNA into protected bacterial populations.

sentences: In-phase and in-antiphase connectivity in EEG Zero-lag synchrony is generally discarded from functional connectivity studies to eliminate the confounding effect of volume conduction. Demonstrating genuine and significant unlagged synchronization between distant brain regions would indicate that most electroencephalography (EEG) connectivity studies neglect an important mechanism for neuronal communication. We previously demonstrated that local field potentials recorded intracranially tend to synchronize with no lag between homotopic brain regions. This synchrony occurs most frequently in antiphase, potentially supporting corpus callosal inhibition and interhemispheric rivalry. We are now extending our investigation to EEG. By comparing the coherency in a recorded and a surrogate dataset, we confirm the presence of a significant proportion of genuine zero-lag synchrony unlikely to be due to volume conduction or to recording reference artifacts. These results stress the necessity for integrating zero-lag synchrony in our understanding of neural communication and for disentangling volume conduction and zero-lag synchrony when estimating EEG sources and their functional connectivity.

sentences: Unique features of transcription termination and initiation at closely spaced tandem human genes The synthesis of RNA Polymerase II (Pol2) products, which include messenger RNAs or long noncoding RNAs, culminates in transcription termination. How the transcriptional termination of a gene impacts the activity of promoters found immediately downstream of it, and which can be subject to potential transcriptional interference, remains largely unknown. We examined in an unbiased manner features of the intergenic region of pairs of tandem and closely spaced (<2kb) genes found on the same strand. Intergenic regions separating tandem genes are enriched with Guanines and are characterized by binding of several proteins, including AGO1 and AGO2 of the RNA interference pathway. Additionally, we found that Pol2 with a specific modification pattern is particularly enriched in this region, and it is lost upon perturbations affecting splicing or transcriptional elongation. Perturbations of genes involved in Pol2 pausing and R loop biology preferentially affect expression of downstream genes in tandem gene pairs. Overall, we find that features associated with potential Pol2 recycling rather than those associated with avoidance of transcriptional interference are the predominant driving force shaping these regions.

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An integrative transcriptional logic model of hepatic insulin resistance Abnormalities of lipid/lipoprotein and glucose metabolism are hallmarks of hepatic insulin resistance in type 2 diabetes. The former antedate the latter, but the latter become progressively refractory to treatment and contribute to therapeutic failures. Its unclear whether the two processes share a common pathogenesis and what underlies their progressive nature. In this study, we investigated the hypothesis that genes in the lipid/lipoprotein pathway and those in the glucose metabolic pathway are governed by different transcriptional logics that affect their response to physiologic (fasting/refeeding) as well as pathophysiologic cues (insulin resistance and hyperglycemia). To this end, we obtained genomic and transcriptomic maps of the key insulin-regulated transcription factor, FoxO1, and integrated them with those of CREB, PPAR, and glucocorticoid receptor. We found an enrichment of glucose metabolic genes among those regulated by intergenic and promoter enhancers in a fasting-dependent manner, while lipid genes were enriched among fasting-dependent intron enhancers and fasting-independent enhancer-less introns. Glucose genes also showed a remarkable transcriptional resiliency, i.e., an enrichment of active marks at shared PPAR/FoxO1 regulatory elements when FoxO1 was inactivated. Surprisingly, the main features associated with insulin resistance and hyperglycemia were a "spreading" of FoxO1 binding to enhancers, and the emergence of target sites unique to this condition. We surmise that this unusual pattern correlates with the progressively intractable nature of hepatic insulin resistance. This transcriptional logic provides an integrated model to interpret the combined lipid and glucose abnormalities of type 2 diabetes.

Significance StatementThe liver is a source of excess lipid, atherogenic lipoproteins, and glucose in patients with type 2 diabetes. These factors predispose to micro- and macrovascular complications. The underlying pathophysiology is not well understood, and mechanistic insight into it may provide better tools to prevent, treat, and reverse the disease. Here we propose an alternative explanation for this pathophysiologic conundrum by illustrating a transcriptional "logic" underlying the regulation of different classes of genes. These findings can be interpreted to provide an integrated stepwise model for the coexistence of lipid and glucose abnormalities in hepatic insulin resistance.

HighlightsO_LIFoxo1 regulates liver metabolism through active enhancers, and hepatocyte maintenance through core promoters
C_LIO_LIFoxo1 regulates glucose genes through fasting-dependent intergenic enhancers
C_LIO_LIBipartite intron regulation of lipid genes is partly fasting-independent
C_LIO_LIPpar contributes to the transcriptional resiliency of Foxo1 metabolic targets
C_LIO_LIInsulin resistance causes de novo recruitment of Foxo1 to active enhancers
C_LIO_LIA stepwise model of insulin resistance
C_LI

sentences: Assumptions about frequency-dependent architectures of complex traits bias measures of functional enrichment Linkage-Disequilibrium Score Regression (LDSC) is a popular framework for analyzing GWAS summary statistics that allows for estimating SNP heritability, confounding, and functional enrichment of genetic variants with different annotations. Recent work has highlighted the influence of implicit and explicit assumptions of the model on the biological interpretation of the results. In this work, we explored a formulation of LDSC that replaces the r2 measure of LD with a recently-proposed unbiased estimator of the D2 statistic. In addition to modest statistical difference across estimators, this derivation highlighted implicit and unrealistic assumptions about the relationship between allele frequency, effect size, and annotation status. We carry out a systematic comparison of alternative LDSC formulations by applying them to summary statistics from 47 GWAS traits. Our results show that commonly used models likely underestimate functional enrichment. These results highlight the importance of calibrating the LDSC model to achieve a more robust understanding of polygenic traits.

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HTSplotter: an end-to-end data processing, analysis and visualisation tool for chemical and genetic in vitro perturbation screening Biomedical researchers are moving towards high-throughput screening, as this allows for automatization, better reproducibility and more and faster results. High-throughput screening experiments encompass drug, drug combination, genetic perturbagen or a combination of genetic and chemical perturbagen screens. These experiments are conducted in real-time assays over time or in an endpoint assay. The data analysis consists of data cleaning and structuring, as well as further data processing and visualisation, which, due to the amount of data, can easily become laborious, time consuming, and error-prone. Therefore, several tools have been developed to aid researchers in this data analysis, but they focus on specific experimental set-ups and are unable to process data of several time points and genetic-chemical perturbagen screens together. To meet these needs, we developed HTSplotter, available as web tool and Python module, that performs automatic data analysis and visualisation of either endpoint or real-time assays from different high-throughput screening experiments: drug, drug combination, genetic perturbagen and genetic-chemical perturbagen screens. HTSplotter implements an algorithm based on conditional statements in order to identify experiment type and controls. After appropriate data normalization, HTSplotter executes downstream analyses such as dose-response relationship and drug synergism by the Bliss independence method. All results are exported as a text file and plots are saved in a PDF file. The main advantage of HTSplotter over other available tools is the automatic analysis of genetic-chemical perturbagen screens and real-time assays where results are plotted over time. In conclusion, HTSplotter allows for the automatic end-to-end data processing, analysis and visualisation of various high-throughput in vitro cell culture screens, offering major improvements in terms of versatility, convenience and time over existing tools.

Author summaryAcademic researchers are moving towards high-throughput screenings, where the experiments execution follows an automatic approach, such as robotic seeding, liquid dispensing and/or automatics readouts. This grants more flexible and reproducible experimental set ups. The type of high-throughput experiment can vary from drug, drug combination, genetic perturbagen to genetic-chemical perturbagen screens. These can be assessed through endpoint assays, measuring the effect at one time point, or real-time assays, assessing the phenotypic effect over time. High-throughput screening results in large amounts of data, requiring laborious, time consuming and error-prone data handling and analysis. These challenges hamper the biological interpretation of data and the fast progress in drug development programs. Hence, we developed HTSplotter, a web tool and Python module, to allow researchers to conduct a fast and flexible end-to-end data processing, analysis and visualisation of different high-throughput experiments, assessed either by endpoint or real-time assays, relieving the high-throughput analysis bottleneck. In this way, HTSplotter, directly contributes to a faster identification of drugs and drug combinations, with potential practical applications, or novel targets for therapies.

sentences: Cinnamon bark extract suppresses metastatic dissemination of cancer cells through inhibition of glycolytic metabolism Metastasis, a leading contributor to the morbidity of cancer patients, occurs through multiple steps. As each of these steps is promoted by different molecular mechanisms, blocking metastasis needs to target each of these steps. Here we report that cinnamon bark extract (CBE) has a suppressor effect on metastatic dissemination of cancer cells. Though a zebrafish embryo screen which utilizes conserved mechanisms between metastasis and zebrafish gastrulation for identifying anti-metastasis drugs, CBE was identified to interfere with gastrulation progression of zebrafish. A zebrafish xenotransplantation model of metastasis validated that CBE suppressed metastatic dissemination of human cancer cells (MDA-MB-231). Interestingly, quantitative metabolome analyses revealed that CBE-treated MDA-MB-231 cells disrupted the production of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P), which are intermediate metabolites of glycolytic metabolism. CBE decreased the expression of hexokinase 2 (HK2), which catalyzes G6P production, and pharmacological inhibition of HK2 suppressed cell invasion and migration of MDA-MB-231 cells. Taken together, CBE suppressed metastatic dissemination of human cancer cells by inhibiting glycolytic metabolism.

sentences: Is there magnocellular facilitation of early neural processes underlying visual word recognition? Evidence from masked repetition priming with ERPs An influential theory in the field of visual object recognition proposes that fast magnocellular (M) information facilitates neural processing of spatially more fine-grained but slower parvocellular (P) information. While written words can be considered as a special type of visual objects, it is unknown whether magnocellular facilitation also plays a role in reading. We used a masked priming paradigm that has been shown to result in neural facilitation in visual word processing and tested whether these facilitating effects are mediated by the magnocellular system. In two experiments, we manipulated the influence of magnocellular and parvocellular systems on visual processing of a contextually predictable target character by contrasting high versus low spatial frequency and luminance versus color contrast, respectively. In addition, unchanged (normal) primes were included in both experiments as a manipulation check. As expected, unchanged primes elicited typical repetition effects in the N1, N250 and P3 components of the ERP in both experiments. In the experiment manipulating spatial contrast, we obtained repetition effects only for the N1 component for both M- and P-biased primes. In the luminance versus color contrast experiment, repetition effects were found in N1 and N250 for both M- and P- biased primes. Furthermore, no interactions were found between M- vs. P-biased prime types and repetition. Together these results indicate that M- and P- information contributes jointly to early neural processes underlying visual word recognition.

sentences: Flow cytometry-based determination of ploidy from dried leaf specimens in genomically complex collections of the tropical forage grass Urochloa s. l. We aimed to develop an optimized approach to determine ploidy for dried leaf material in a germplasm collection of a tropical forage grass group, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. The methods enable robust identification of ploidy levels (coefficient of variation, CV, typically <5%). Ploidy of some 353 forage grass accessions (ploidy range from 2 to 9), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes used in the current breeding programs at CIAT and EMBRAPA: the  brizantha and  humidicola agamic complexes are variable, with multiple ploidy levels and DNA content. U. brizantha has odd level of ploidy (x=5), and the relative differences in nuclear DNA content between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed some aneuploidy.

sentences: Visual category representations in the infant brain Visual categorization is a human core cognitive capacity1,2 that depends on the development of visual category representations in the infant brain3-7. However, the exact nature of infant visual category representations and their relationship to the corresponding adult form remains unknown8. Our results clarify the nature of visual category representations from electroencephalography (EEG) data in 6- to 8-month-old infants and their developmental trajectory towards adult maturity in the key characteristics of temporal dynamics2,9, representational format10-12, and spectral properties13,14. Temporal dynamics change from slowly emerging, developing representations in infants to quickly emerging, complex representations in adults. Despite those differences, infants and adults already partly share visual category representations. The format of infants representations is visual features of low to intermediate complexity, whereas adults representations also encode high complexity features. Theta band neural oscillations form the basis of visual category representations in infants, and these representations are shifted to the alpha/beta band in adults. Together, we reveal the developmental neural basis of visual categorization in humans, show how information transmission channels change in development, and demonstrate the power of advanced multivariate analysis techniques in infant EEG research for theory building in developmental cognitive science.

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

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spiralize: an R Package for Visualizing Data on Spirals SummarySpiral layout has two major advantages for data visualization. First, it is able to visualize data with long axes, which greatly improves the resolution of visualization. Second, it is efficient for time series data to reveal periodic patterns. Here we present the R package spiralize that provides a general solution for visualizing data on spirals. spiralize implements numerous graphics functions so that self-defined high-level graphics can be easily implemented by users. The flexibility and power of spiralize are demonstrated by five examples from real-world datasets.

Availability and implementationThe spiralize package and documentations are freely available at the Comprehensive R Archive Network (CRAN) https://CRAN.R-project.org/package=spiralize.

Contactz.gu@dkfz.de or d.huebschmann@dkfz.de

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Parallel pathways for rapid odor processing in lateral entorhinal cortex: Rate and temporal coding by layer 2 subcircuits Olfactory information is encoded in lateral entorhinal cortex (LEC) by two classes of layer 2 (L2) principal neurons: fan and pyramidal cells. However, the functional properties of L2 neurons are unclear. Here, we show in awake mice that L2 cells respond rapidly to odors during single sniffs and that LEC is essential for discrimination of odor identity and intensity. Population analyses of L2 ensembles reveals that while rate coding distinguishes odor identity, firing rates are weakly concentration-dependent and changes in spike timing represent odor intensity. L2 principal cells differ in afferent olfactory input and connectivity with local inhibitory circuits and the relative timing of pyramidal and fan cell spikes underlies odor intensity coding. Downstream, intensity is encoded purely by spike timing in hippocampal CA1. Together, these results reveal the unique processing of odor information by parallel LEC subcircuits and highlight the importance of temporal coding in higher olfactory areas.

sentences: Time cell encoding in deep reinforcement learning agents depends on mnemonic demands The representation of "what happened when" is central to encoding episodic and working memories. Recently discovered hippocampal time cells are theorized to provide the neural substrate for such representations by forming distinct sequences that both encode time elapsed and sensory content. However, little work has directly addressed to what extent cognitive demands and temporal structure of experimental tasks affect the emergence and informativeness of these temporal representations. Here, we trained deep reinforcement learning (DRL) agents on a simulated trial-unique nonmatch-to-location (TUNL) task, and analyzed the activities of artificial recurrent units using neuroscience-based methods. We show that, after training, representations resembling both time cells and ramping cells (whose activity increases or decreases monotonically over time) simultaneously emerged in the same population of recurrent units. Furthermore, with simulated variations of the TUNL task that controlled for (1) memory demands during the delay period and (2) the temporal structure of the episodes, we show that memory demands are necessary for the time cells to encode information about the sensory stimuli, while the temporal structure of the task only affected the encoding of "what" and "when" by time cells minimally. Our findings help to reconcile current discrepancies regarding the involvement of time cells in memory-encoding by providing a normative framework. Our modelling results also provide concrete experimental predictions for future studies.

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Fresh extension of Vibrio cholerae competence type IV pili predisposes them for motor-independent retraction Bacteria utilize dynamic appendages called type IV pili (T4P) to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can either occur with the aid of an ATPase motor, or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remains poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken, but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization.

SIGNIFICANCEExtracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions, however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the external pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.

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Collective polymerase dynamics emerge from DNA Supercoiling Mediated Transcription All biological processes ultimately come from physical interactions. The mechanical properties of DNA play a critical role in transcription. RNA polymerase can over or under twist DNA (referred to as DNA supercoiling) when it moves along a gene resulting in mechanical stresses in DNA that impact its own motion and that of other polymerases. For example, when enough supercoiling accumulates, an isolated polymerase halts and transcription stops. DNA supercoiling can also mediate non-local interactions between polymerases that shape gene expression fluctuations. Here, we construct a comprehensive model of transcription that captures how RNA polymerase motion changes the degree of DNA supercoiling which in turn feeds back into the rate at which polymerases are recruited and move along the DNA. Surprisingly, our model predicts that a group of three or more polymerases move together at a constant velocity and sustain their motion (forming what we call a polymeton) whereas one or two polymerases would have halted. We further show that accounting for the impact of DNA supercoiling on both RNA polymerase recruitment and velocity recapitulates empirical observations of gene expression fluctuations. Finally, we propose a mechanical toggle switch whereby interactions between genes are mediated by DNA twisting as opposed to proteins. Understanding the mechanical regulation of gene expression provides new insights into how endogenous genes can interact and informs the design of new forms of engineered interactions.

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sentences: Long-term Transverse Imaging of the Hippocampus with Glass Microperiscopes The hippocampus consists of a stereotyped neuronal circuit repeated along the septal-temporal axis. This transverse circuit contains distinct subfields with stereotyped connectivity that support crucial cognitive processes, including episodic and spatial memory. However, comprehensive measurements across the transverse hippocampal circuit in vivo are intractable with existing techniques. Here, we developed an approach for two-photon imaging of the transverse hippocampal plane in awake mice via implanted glass microperiscopes, allowing optical access to the major hippocampal subfields and to the dendritic arbor of pyramidal neurons. Using this approach, we tracked dendritic morphological dynamics on CA1 apical dendrites and characterized spine turnover. We then used calcium imaging to quantify the prevalence of place and speed cells across subfields. Finally, we measured the anatomical distribution of spatial information, finding a non-uniform distribution of spatial selectivity along the DG-to-CA1 axis. This approach extends the existing toolbox for structural and functional measurements of hippocampal circuitry.

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Sexual dimorphism in epilepsy and comorbidities in Dravet syndrome mice carrying a targeted deletion of exon 1 of the Scn1a gene ObjectiveDravet Syndrome (DS) is a catastrophic form of paediatric epilepsy associated with multiple comorbidities mainly caused by mutations in the SCN1A gene. DS progresses in three different phases termed febrile, worsening and stabilization stage. Mice that are haploinsufficient for Scn1a faithfully model each stage of DS, although various aspects have not been fully described, including the temporal appearance and sex differences of the epilepsy and comorbidities. The aim of the present study was to investigate the epilepsy landscape according to the progression of DS and the long-term co-morbidities in the Scn1a(+/-)tm1Kea DS mouse line that are not fully understood yet.

MethodsMale and female F1.Scn1a(+/+) and F1.Scn1a(+/-)tm1Kea mice were assessed in the hyperthermia model or monitored by video electroencephalogram (vEEG) and wireless video-EEG according to the respective stage of DS. Long-term comorbidities were investigated through a battery of behaviour assessments in [~]6 month-old mice.

ResultsAt P18, F1.Scn1a(+/-)tm1Kea mice showed the expected sensitivity to hyperthermia-induced seizures. Between P21 and P28, EEG recordings in F1.Scn1a(+/-)tm1Kea mice combined with video monitoring revealed a high frequency of SRS and SUDEP. Power spectral analyses of background EEG activity also revealed that low EEG power in multiple frequency bands was associated with SUDEP risk in F1.Scn1a(+/-)tm1Kea mice during the worsening stage of DS. Later, SRS and SUDEP rates stabilized and then declined in F1.Scn1a(+/-)tm1kea mice. SRS and SUDEP in F1.Scn1a(+/-)tm1kea mice displayed variations with the time of day and sex, with female mice displaying higher numbers of seizures and greater SUDEP risk. F1.Scn1a(+/-)tm1kea mice [~]6 month- old displayed fewer behavioural impairments than expected including hyperactivity, impaired exploratory behaviour and poor nest building performance.

SignificanceThese results reveal new features of this model that will optimize use and selection of phenotype assays for future studies on the mechanisms, diagnosis, and treatment of DS.

Key point boxO_LIScn1a(+/-)tm1kea DS mouse model faithfully reproduces the three stages of DS
C_LIO_LISex of F1.Scn1a(+/-)tm1kea mice influences the epilepsy phenotype
C_LIO_LIF1.Scn1a(+/-)tm1kea develop some of the long-term comorbidities of DS
C_LI

sentences: Perceived and mentally rotated contents are differentially represented in cortical depth of V1 Primary visual cortex (V1) in humans is known to represent both veridically perceived external input and internally-generated contents underlying imagery and mental rotation. However, it is unknown how the brain keeps these contents separate thus avoiding a mixture of the perceived and the imagined which could lead to potentially detrimental consequences. Inspired by neuroanatomical studies showing that feedforward and feedback connections in V1 terminate in different cortical layers, we hypothesized that this anatomical compartmentalization underlies functional segregation of external and internally-generated visual contents, respectively. We used high-resolution layer-specific fMRI to test this hypothesis in a mental rotation task. We found that rotated contents were predominant at outer cortical depth bins (i.e. superficial and deep). At the same time perceived contents were represented stronger at the middle cortical bin. These results identify how through cortical depth compartmentalization V1 functionally segregates rather than confuses external from internally-generated visual contents. These results indicate that feedforward and feedback manifest in distinct subdivisions of the early visual cortex, thereby reflecting a general strategy for implementing multiple cognitive functions within a single brain region.

sentences: Mapping the vocal circuitry of Alston's singing mouse with pseudorabies virus Vocalizations, like many social displays, are often elaborate, rhythmically structured behaviors that are modulated by a complex combination of cues. Vocal motor patterns require close coordination of neural circuits governing the muscles of the larynx, jaw, and respiratory system. In the elaborate vocalization of Alstons singing mouse (Scotinomys teguina), for example, each note of its rapid, frequency-modulated trill is accompanied by equally rapid modulation of breath and gape. To elucidate the neural circuitry underlying this behavior, we introduced the polysynaptic retrograde neuronal tracer pseudorabies virus (PRV) into the cricothyroid and digastricus muscles, which control frequency modulation and jaw opening respectively. Each virus singly labels ipsilateral motoneurons (nucleus ambiguous for cricothyroid, and motor trigeminal nucleus for digastricus). We find that the two isogenic viruses heavily and bilaterally co-label neurons in the gigantocellular reticular formation, a putative central pattern generator. The viruses also show strong co-labeling in compartments of the midbrain including the ventrolateral periaqueductal grey and the parabrachial nucleus, two structures strongly implicated in vocalizations. In the forebrain, regions important to social cognition and energy balance both exhibit extensive co-labeling. This includes the paraventricular and arcuate nuclei of the hypothalamus, the lateral hypothalamus, preoptic area, extended amygdala, central amygdala, and the bed nucleus of the stria terminalis. Finally, we find doubly labeled neurons in M1 motor cortex previously described as laryngeal, as well as in the prelimbic cortex, which indicate these cortical regions play a role in vocal production. Although we observe some novel patterns of double-labelling, the progress of both viruses is broadly consistent with vertebrate-general patterns of vocal circuitry, as well as with circuit models derived from primate literature.

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Toxicity Profile of Parenteral Artesunate following Subacute Treatment in Rats and Dogs AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSOBJECTIVESC_ST_ABSThe objectives of these studies were to investigate the toxicity, safety and toxicokinetics of single and multiple doses of artesunate for injection in rats and dogs.

METHODSSprague-Dawley rats and Beagle dogs were treated intravenously or intramuscularly for 28 consecutive days with doses of up to 30 mg/kg artesunate, evaluating toxicity, kinetics, genotoxicity, and cardiovascular and central nervous safety parameters after single and 4-week repeated administrations. Furthermore, respiratory parameters were evaluated after a single intravenous administration in rats.

RESULTSArtesunate was well tolerated with no mortality and only minor effects on clinical pathology parameters. Following repeated intramuscular administration, local reactions at the injection site became evident.

CONCLUSIONSThe results obtained in these studies support the safe use of artesunate in humans.

Subchronic data for repeated intravenous and intramuscular artesunate in animals are available in the literature for a maximum duration of 14-days. Published results of genotoxicity studies in vitro in bacteria or cells of animal or human origin and in vivo in animals are equivocal, with a couple of negative results following oral administration and positive results in human cells.

With the studies performed in rats and dogs for a treatment period of 28-days including measures of safety pharmacology, toxicokinetic evaluation and in vivo genotoxicity data, a comparison of toxicity and exposure of intravenous versus intramuscular artesunate for a prolonged treatment period is possible, delivering important information, when a decision between both routes has to be made for use in patients. Results of the peripheral blood micronucleus assay in rats were positive, suggesting genotoxic potential for the intravenous route of administration of artesunate, what is, beside patients, likewise relevant for production and health care personnel.

sentences: Sequencing SARS-CoV-2 in a malaria research laboratory in Mali, West Africa: the road to sequencing the first SARS-CoV-2 genome in Mali Next generation sequencing (NGS) has become a necessary tool for genomic epidemiology. Even though the utility of genomics in human health has been proved, the genomic surveillance has never been so important until the COVID 19 pandemic. This has been evidenced with the detection of new variants of SARS-CoV-2 in the United Kingdom, South Africa and Brazil recently using genomic surveillance. Until recently, Malian scientists did not have access to any local NGS platform and samples had to be shipped abroad for sequencing. Here, we report on how we adapted a laboratory setup for Plasmodium research to generate the first complete SARS-CoV-2 genome locally. Total RNA underwent a library preparation using an Illumina TruSeq stranded RNA kit. A metagenomics sequencing was performed on an Illumina MiSeq platform following by bioinformatic analyses on a local server in Mali. We recovered a full genome of SARS-CoV-2 of 29 kb with an average depth coverage of 200x. We have demonstrated our capability of generating a high quality genome with limited resources and highlight the need to develop genomics capacity locally to solve health problems. We discuss challenges related to access to reagents during a pandemic period and propose some home-made solutions.

sentences: A mathematical framework for dissecting normative foundations of objective-driven conservation decisions Perspectives in conservation are based on a variety of value systems. Such differences in how people value nature and its components lead to different evaluations of the morality of conservation goals and approaches, and often underlie disagreements in the formulation and implementation of environmental management policies. Specifically, whether a conservation action (e.g. killing feral cats to reduce predation on bird species threatened with extinction) is viewed as appropriate or not can vary among people with different value systems. Here, we present a conceptual, mathematical framework intended as a tool to systematically explore and clarify core value statements in conservation approaches. Its purpose is to highlight how fundamental differences between these value systems can lead to different prioritizations of available management options and offer a common ground for discourse. The proposed equations decompose the question underlying many controversies around management decisions in conservation: what or who is valued, how, and to what extent? We compare how management decisions would likely be viewed under three different idealised value systems: ecocentric conservation, which aims to preserve biodiversity; new conservation, which considers that biodiversity can only be preserved if it benefits humans; and sentientist conservation, which aims at minimising suffering for sentient beings. We illustrate the utility of the framework by applying it to case studies involving invasive alien species, rewilding, and trophy hunting. By making value systems and their consequences in practice explicit, the framework facilitates debates on contested conservation issues, and complements philosophical discursive approaches about moral reasoning. We believe dissecting the core value statements on which conservation decisions are based will provide an additional tool to understand and address conservation conflicts.

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Structural basis of transcription antitermination by Q{lambda}: NusA induces refolding of Q{lambda} to form nozzle for RNA polymerase exit channel Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element to yield a Q-loading complex, and translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein from bacteriophage 21 (Q21) functions by forming a "nozzle" that narrows and extends the RNAP RNA-exit channel, preventing formation of pause and termination RNA hairpins. Here, we report atomic structures of four states on the pathway of antitermination by the Q protein from bacteriophage {lambda} (Q{lambda}), a Q protein that shows no sequence similarity to Q21 and that, unlike Q21, requires the transcription elongation factor NusA for efficient antipausing and antitermination. We report structures of Q{lambda}, the Q{lambda}-QBE complex, the NusA-free "pre-engaged" Q{lambda}-loading complex, and the NusA-containing "engaged" Q{lambda}-loading complex. The results show that Q{lambda}, like Q21, forms a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of RNA hairpins. However, the results show that Q{lambda} has no three-dimensional structural similarity to Q21, employs a different mechanism of QBE recognition than Q21, and employs a more complex process for loading onto RNAP than Q21, involving recruitment of Q{lambda} to form a "pre-engaged" loading complex, followed by NusA-facilitated refolding of Q{lambda} to form an "engaged" loading complex. The results establish Q{lambda} and Q21 are not structural homologs and are solely functional analogs.

SIGNIFICANCE STATEMENTBacteriophage Q proteins are textbook examples of regulators of gene expression that function at the level of transcription antitermination. Here, we report structures defining the mechanism of antitermination by the Q protein of bacteriophage {lambda} (Q{lambda}). The results show Q{lambda} forms a "nozzle" that narrows and extends the RNA polymerase RNA-exit channel, precluding the formation of terminator RNA hairpins. The results show Q{lambda} exhibits no structural similarity to the Q protein of bacteriophage 21 (Q21), employs a different mechanism for DNA binding than Q21, and employs a more complex process of loading onto RNA polymerase than Q21. We conclude Q{lambda} and Q21 are not structural homologs and are solely functional analogs, akin to a bird wing and a bat wing.

sentences: The coupling between healthspan and lifespan in Caenorhabditis depends on complex interactions between compound intervention and genetic background Aging is characterized by declining health that results in decreased neuromuscular function and cellular resilience. The relationship between lifespan and health, and the influence of genetic background on that relationship, has important implications in the development of anti-aging interventions. Here we combined survival under thermal and oxidative stress with swimming performance, to evaluate health effects across a nematode genetic diversity panel for three compounds previously studied in the Caenorhabditis Intervention Testing Program - NP1, propyl gallate, and resveratrol. We show that oxidative stress resistance and thermotolerance vary with compound intervention, genetic background, and age. The effects of tested compounds on swimming locomotion, in contrast, are largely species-specific. Additionally, thermotolerance, but not oxidative stress or swimming ability, correlates with lifespan. Our results demonstrate the importance of assessing health and lifespan across genetic backgrounds in the effort to identify reproducible aging interventions.

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Population-level inference for home-range areas O_LIHome-range estimates are a common product of animal tracking data, as each range informs on the area needed by a given individual. Population-level inference on home-range areas--where multiple individual home-ranges are considered to be sampled from a population--is also important to evaluate changes over time, space, or covariates, such as habitat quality or fragmentation, and for comparative analyses of species averages. Population-level home-range parameters have traditionally been estimated by first assuming that the input tracking data were sampled independently when calculating home ranges via conventional kernel density estimation (KDE) or minimal convex polygon (MCP) methods, and then assuming that those individual home ranges were measured exactly when calculating the population-level estimates. This conventional approach does not account for the temporal autocorrelation that is inherent in modern tracking data, nor for the uncertainties of each individual home-range estimate, which are often large and heterogeneous.
C_LIO_LIHere, we introduce a statistically and computationally efficient framework for the population-level analysis of home-range areas, based on autocorrelated kernel density estimation (AKDE), that can account for variable temporal autocorrelation and estimation uncertainty.
C_LIO_LIWe apply our method to empirical examples on lowland tapir (Tapirus terrestris), kinkajou (Potos flavus), white-nosed coati (Nasua narica), white-faced capuchin monkey (Cebus capucinus), and spider monkey (Ateles geoffroyi), and quantify differences between species, environments, and sexes.
C_LIO_LIOur approach allows researchers to more accurately compare different populations with different movement behaviors or sampling schedules, while retaining statistical precision and power when individual home-range uncertainties vary. Finally, we emphasize the estimation of effect sizes when comparing populations, rather than mere significance tests.
C_LI

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Protein Vaccine Induces a Durable, More Broadly Neutralizing Antibody Response in Macaques than Natural Infection with SARS-CoV-2 P.1 FDA-approved and Emergency Use Authorized (EUA) vaccines using new mRNA and viral-vector technology are highly effective in preventing moderate to severe disease, however, information on their long-term efficacy and protective breadth against SARS-CoV-2 Variants of Concern (VOCs) is currently scarce. Here we describe the durability and broad-spectrum VOC immunity of a prefusion-stabilized spike (S) protein adjuvanted with liquid or lyophilized CoVaccine HT in cynomolgus macaques. This recombinant subunit vaccine is highly immunogenic and induces robust spike-specific and broadly neutralizing antibody responses effective against circulating VOCs (B.1.351 [Beta], P.1 [Gamma], B.1.617 [Delta]) for at least 3 months after the final boost. Protective efficacy and post-exposure immunity were evaluated using a heterologous P.1 challenge nearly 3 months after the last immunization. Our results indicate that while immunization with both high and low S doses shorten and reduce viral loads in the upper and lower respiratory tract, a higher antigen dose is required to provide durable protection against disease as vaccine immunity wanes. Histologically, P.1 infection causes similar COVID-19-like lung pathology as seen with early pandemic isolates. Post-challenge IgG concentrations were restored to peak immunity levels and vaccine-matched and cross-variant neutralizing antibodies were significantly elevated in immunized macaques indicating an efficient anamnestic response. Only low levels of P.1-specific neutralizing antibodies with limited breadth were observed in control (non-vaccinated but challenged) macaques suggesting that natural infection may not prevent reinfection by other VOCs. Overall, these results demonstrate that a properly dosed and adjuvanted recombinant subunit vaccine can provide long-lasting and protective immunity against circulating VOCs.

One Sentence SummaryA recombinant subunit protein formulated with CoVaccine HT adjuvant induces superior immunity than natural infection and reduces viral load while protecting cynomolgus macaques from COVID-19-like disease caused by late SARS-CoV-2 P.1 (Gamma) challenge.

sentences: A neural network model of hippocampal contributions to category learning In addition to its critical role in encoding individual episodes, the hippocampus is capable of extracting regularities across experiences. This ability is central to category learning, and a growing literature indicates that the hippocampus indeed makes important contributions to this kind of learning. Using a neural network model that mirrors the anatomy of the hippocampus, we investigated the mechanisms by which the hippocampus may support novel category learning. We simulated three category learning paradigms and evaluated the networks ability to categorize and to recognize specific exemplars in each. We found that the trisynaptic pathway within the hippocampus--connecting entorhinal cortex to dentate gyrus, CA3, and CA1--was critical for remembering individual exemplars, reflecting the rapid binding and pattern separation functions of this circuit. The monosynaptic pathway from entorhinal cortex to CA1, in contrast, was responsible for detecting the regularities that define category structure, made possible by the use of distributed representations and a slower learning rate. Together, the simulations provide an account of how the hippocampus and its constituent pathways support novel category learning.

sentences: Spatial organization of chromosomes leads to heterogeneous chromatin motion and drives the liquid- or gel-like behavior of chromatin Chromosome organization and dynamics are involved in regulating many fundamental processes such as gene transcription and DNA repair. Experiments unveiled that chromatin motion is highly heterogeneous inside cell nuclei, ranging from a liquid-like, mobile state to a gel-like, rigid regime. Using polymer modeling, we investigate how these different physical states and dynamical heterogeneities may emerge from the same structural mechanisms. We found that the formation of topologically-associating domains (TADs) is a key driver of chromatin motion heterogeneity. In particular, we demonstrated that the local degree of compaction of the TAD regulates the transition from a weakly compact, fluid state of chromatin to a more compact, gel state exhibiting anomalous diffusion and coherent motion. Our work provides a comprehensive study of chromosome dynamics and a unified view of chromatin motion enabling to interpret the wide variety of dynamical behaviors observed experimentally across different biological conditions, suggesting that the  liquid or  solid behaviour of chromatin are in fact two sides of the same coin.

sentences: Energy content of anchovy and sardine using surrogate calorimetry methods European anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) are crucial species for the marine ecosystem of the Northwestern Mediterranean Sea. They account for a high percentage of fish landings and they represent an important economic income. Concerns over their stock status are rising in recent years as biomass, growth, reproductive capacity and body condition of both species are declining. Therefore, there is an urgent need for a continuous and fast body condition monitoring scheme. Energy storage variability has important implications for both fish recruitment and population structure. Direct condition indices, such as bomb calorimetry, are highly reliable for measuring the energy content, but extremely time-consuming. Alternatively, fatmeter analysis and relative condition index (Kn) have been proposed as effective indirect methods. The aim of this study is to test the application of fatmeter as a surrogate of bomb calorimetry to infer the energy content of both small pelagic fishes. For the validation, fatmeter values were compared with both energy density (ED; via bomb calorimetry) and Kn values. Individuals of both species were sampled monthly in Barcelona harbor for a year in order to assess seasonal variations in energy content. Our results highlight that fatmeter measurements are strongly correlated with calorimetry for sardine, while a weaker but significant correlation was found for anchovy. The observed differences between the two species are related to their breeding strategies. Based on this study, fatmeter analysis appears to be a faster and suitable method to evaluate the energy content of both species routinely., In addition, we provide a linear model to infer ED from fatmeter values of both small pelagic fish. Eventually, these findings could allow for the avoidance of bomb calorimetry and could be used to implement body condition monitoring protocols, and to boost continuous large-scale monitoring.

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The C-type Lectin Receptor Dectin-2 is a receptor for  Aspergillus fumigatus  galactomannan Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly amongst immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient sera and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the innate immune system. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of purified Aspergillus galactomannan to identify a C-type lectin host receptor responsible for binding this fungal carbohydrate. We identified a novel interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2 dependent signaling including activation of spleen tyrosine kinase and potent TNFa production. Deficiency of Dectin-2 increased neutrophil recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for Aspergillus galactomannan which leads to a pro-inflammatory response.

SUMMARYThe Aspergillus carbohydrate galactomannan is the primary clinical biomarker for diagnosis of invasive aspergillosis. In this manuscript, we identify a novel interaction between galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan induces Dectin-2 dependent proinflammatory signaling in macrophages.

sentences: A mitochondrial long-chain fatty acid oxidation defect leads to uncharged tRNA accumulation and activation of the integrated stress response in the mouse heart The heart relies mainly on mitochondrial fatty acid {beta}-oxidation (FAO) for its high energy requirements. Cardiomyopathy and arrhythmias can be severe complications in patients with inherited defects in mitochondrial long-chain FAO, reinforcing the importance of FAO for cardiac health. However, the pathophysiological mechanisms that underlie the cardiac abnormalities in long-chain FAO disorders remain largely unknown. Here, we investigated the cardiac transcriptional adaptations to the FAO defect in the long-chain acyl-CoA dehydrogenase (LCAD) knockout (KO) mouse. We found a prominent activation of the integrated stress response (ISR) mediated by the eIF2/ATF4 axis in both fed and fasted states, accompanied by a reduction in cardiac protein synthesis during a short period of food withdrawal. Notably, we found an accumulation of uncharged tRNAs in LCAD KO hearts, consistent with a reduced availability of cardiac amino acids, in particular, glutamine. We replicated the activation of the cardiac ISR in hearts of mice with a muscle-specific deletion of carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). Our results show that perturbations in amino acid metabolism caused by long-chain FAO deficiency impact cardiac metabolic signaling, in particular the ISR, and may play a role in the associated cardiac pathology.

sentences: Beyond category-supervision: instance-level contrastive learning models predict human visual system responses to objects Anterior regions of the ventral visual stream have substantial information about object categories, prompting theories that category-level forces are critical for shaping visual representation. The strong correspondence between category-supervised deep neural networks and ventral stream representation supports this view, but does not provide a viable learning model, as these deepnets rely upon millions of labeled examples. Here we present a fully self-supervised model which instead learns to represent individual images, where views of the same image are embedded nearby in a low-dimensional feature space, distinctly from other recently encountered views. We find category information implicitly emerges in the feature space, and critically that these models achieve parity with category-supervised models in predicting the hierarchical structure of brain responses across the human ventral visual stream. These results provide computational support for learning instance-level representation as a viable goal of the ventral stream, offering an alternative to the category-based framework that has been dominant in visual cognitive neuroscience.

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Photoaffinity capture compounds to profile the Magic Spot Nucleotide interactomes Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival when confronted with adverse external challenges. In times of antibiotic crisis, a detailed understanding of the stringent response is of critical importance, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium cell lysates applying a family of trifunctional photoaffinity capture compounds. We introduce different MSN probes covering diverse phosphorylation patterns, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of diverse putative MSN receptors both from cytosolic and membrane fractions that, upon validation, unveil new MSN targets. We find, for example, that the dinucleoside polyphosphate hydrolase activity of the non-Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The photoaffinity capture compounds described herein will be useful to identify MSN interactomes under varying conditions and across bacterial species.

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org.highwire.dtl.DTLVardef@15cfc02org.highwire.dtl.DTLVardef@1a4ec1borg.highwire.dtl.DTLVardef@88fd27org.highwire.dtl.DTLVardef@1a42248_HPS_FORMAT_FIGEXP  M_FIG Molecular fishing: a family of trifunctional photoaffinity capture compounds enables the identification of Magic Spot Nucleotide receptors by a chemoproteomics approach.

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Sustained upregulation of widespread hippocampal-neocortical coupling following memory encoding Systems consolidation of new experiences into lasting episodic memories involves interactions between hippocampus and the neocortex. Evidence of this process is seen already during early awake post-encoding rest periods. Functional MRI (fMRI) studies have demonstrated increased hippocampal coupling with task-relevant perceptual regions and reactivation of stimulus-specific encoding patterns following intensive encoding tasks. Here we investigate the spatial and temporal characteristics of these hippocampally anchored post-encoding neocortical modulations. Eighty-nine adults participated in an experiment consisting of interleaved memory task- and resting-state periods. As expected, we observed increased post-encoding functional connectivity between hippocampus and individually localized neocortical regions responsive to stimulus categories encountered during memory encoding. Post-encoding modulations were however not restricted to stimulus-selective cortex, but manifested as a nearly system-wide upregulation in hippocampal coupling with all major functional networks. The spatial configuration of these extensive modulations resembled hippocampal-neocortical interaction patterns estimated from active encoding operations, suggesting hippocampal post-encoding involvement by far exceeds reactivation of perceptual aspects. This reinstatement of encoding patterns during immediate post-encoding rest was not observed in resting-state scans collected 12 hours later, nor in control analyses estimating post-encoding neocortical modulations in functional connectivity using other candidate seed regions. The broad similarity in hippocampal functional coupling between online memory encoding and offline post-encoding rest suggests reactivation in humans may involve a spectrum of cognitive processes engaged during experience of an event.

Significance statementStabilization of newly acquired information into lasting memories occurs through systems consolidation - a process which gradually spreads the locus of memory traces from hippocampus to more distributed neocortical representations. One of the earliest signs of consolidation is the upregulation of hippocampal-neocortical interactions during periods of awake rest following an active encoding task. We here show that these modulations involve much larger parts of the brain than previously reported in humans. Comparing changes in hippocampal coupling during post-encoding rest with those observed under active encoding, we find evidence for encoding-like hippocampal reinstatement throughout cortex during task-free periods. This suggests early systems consolidation of an experience involves reactivating not only core sensory details but multiple additional aspects of the encoding event.

sentences: Automatically tracking feeding behavior in populations of foraging worms C. elegans feeds on bacteria and other small microorganisms which it ingests using its pharynx, a neuromuscular pump. Currently, measuring feeding behavior requires tracking a single animal, indirectly estimating food intake from population-level metrics, or using restrained animals. Therefore, to enable large throughput feeding measurements of unrestrained, crawling worms on agarose plates, we developed an imaging protocol and a complementary image analysis tool called PharaGlow. We image up to 50 freely moving worms simultaneously and extract locomotion and feeding behaviors. Our tool reliably detects pharyngeal pumping in adult worms with a maximum deviation of 5% in the number of pumps compared to an expert annotator. We demonstrate the tools robustness and high-throughput capabilities by measuring feeding in different use-case scenarios. This includes tracing pharyngeal dynamics during development, revealing their highly conserved nature throughout all life cycle stages. We also observed pumping after food deprivation, corroborating previous studies in which starvation time strongly influences pumping. Finally, we further validated our behavioral tracker by exploring two previously characterized pumping defective mutants: unc-31 and eat-18. Remarkably, our analysis of eat-18 mutants identified unreported defects in pumping and overall locomotion regulation, highlighting the potential of this toolkit. Pharaglow therefore enables the observation and analysis of the temporal dynamics of food intake with high-throughput and precision in a user-friendly system.

sentences: Interrogation of the Dynamic Properties of Higher-Order Heterochromatin Using CRISPR/dCas9 Eukaryotic chromosomes feature large regions of compact, repressed heterochromatin hallmarked by Heterochromatin Protein 1 (HP1). HP1 proteins play multi-faceted roles in shaping heterochromatin, and in cells, HP1 tethering to individual gene promoters leads to epigenetic modifications and silencing. However, emergent properties of HP1 at supranucleosomal scales remain difficult to study in cells due to lack of appropriate tools. Here, we develop CRISPR-Engineered Chromatin Organization (EChO), combining live cell CRISPR imaging with inducible large-scale recruitment of chromatin proteins to native genomic targets. We demonstrate that human HP1 tiling across kilobase-scale genomic DNA forms novel contacts with natural heterochromatin, integrates two distantly targeted regions, and reversibly changes chromatin from a diffuse to compact state. The compact state exhibits delayed disassembly kinetics and represses transcription across over 600 kilobases. These findings support a polymer model of HP1-mediated chromatin regulation and highlight the utility of CRISPR-EChO in studying supranucleosomal chromatin organization in living cells.

sentences: Data-driven extraction of human kinase-substrate relationships from omics datasets Phosphorylation forms an important part of the signalling system that cells use for decision making and regulation of processes such as celll division and differentiation. To date, a large portion of identified phosphosites are not known to be targeted by any kinase. At the same time around 30% of kinases have no known target. This knowledge gap stresses the need to make large scale, data-driven computational predictions. In this paper, we have created a machine learning-based model to derive a probabilistic kinase-substrate network from omics datasets. We show that our methodology displays improved performance compared to other state of the art kinase-substrate predictions, and provides predictions for more kinases than most of them. Importantly, it better captures new experimentally-identified kinase-substrate relationships. It can therefore allow the improved prioritisation of kinase-substrate pairs for illuminating the dark human cell signalling space.

sentences: How Hippocampus Solves Inverse Problems: A Circuit Hypothesis This paper proposes an explicit computational process which reproduces what are considered to be two essential experimentally observed hippocampus capabilities. These are path planning and determining the chain of ordered relationships which bind two concepts. These are both inverse problems. The paper presents algorithmic simulations of the process and a neuronal circuit (and variants) with the same dataflow, both consistent with the known neuroanatomy of the hippocampus. The process described here is purpose-built for hippocampus

sentences: MethodsJ2: A Software Tool to Improve Microscopy Methods Reporting Proper reporting of metadata is essential to reproduce microscopy experiments, interpret results and share images. Experimental scientists can report details about sample preparation and imaging conditions while imaging scientists have the expertise required to collect and report the image acquisition, hardware and software metadata information. MethodsJ2 is an ImageJ/Fiji based software tool that gathers metadata and automatically generates text for the methods section of publications.

sentences: Nuclear HMGB1 protects from non-alcoholic fatty liver diseases through negative regulation of liver X receptor Dysregulations of lipid metabolism in the liver may trigger steatosis progression leading to potentially severe clinical consequences such as non-alcoholic fatty liver diseases (NAFLD). Molecular mechanisms underlying liver lipogenesis are very complex and fine-tuned by chromatin dynamics and the activity of multiple key transcription factors. Here, we demonstrate that the nuclear factor HMGB1 acts as a strong repressor of liver lipogenesis during metabolic stress in NAFLD. Mice with liver-specific Hmgb1-deficiency display exacerbated liver steatosis and hepatic insulin resistance when subjected to a high-fat diet or after fasting/refeeding. Global transcriptome and functional analysis revealed that the deletion of Hmgb1 gene enhances LXR activity resulting in increased lipogenesis. HMGB1 repression is not mediated through nucleosome landscape re-organization but rather via a preferential DNA occupation in region carrying genes regulated by LXR. Together these findings suggest that hepatocellular HMGB1 protects from liver steatosis development. HMGB1 may constitute a new attractive option to therapeutically target LXR axis during NAFLD.

sentences: An atlas of gene expression variation across the Caenorhabditis elegans species Phenotypic variation in diverse organism-level traits have been studied in Caenorhabditis elegans wild strains, but differences in gene expression and the underlying variation in regulatory mechanisms are largely unknown. Here, we use natural variation in gene expression to connect genetic variants to differences in organismal- level traits, including drug and toxicant responses. We performed transcriptomic analysis on 207 genetically distinct C. elegans wild strains to study natural regulatory variation of gene expression. Using this massive dataset, we performed genome-wide association mappings to investigate the genetic basis underlying gene expression variation and revealed complex genetic architectures. We found a large collection of hotspots enriched for expression quantitative trait loci across the genome. We further used mediation analysis to understand how gene expression variation could underlie organism-level phenotypic variation for a variety of complex traits. These results reveal the natural diversity in gene expression and possible regulatory mechanisms in this keystone model organism, highlighting the promise of gene expression variation in shaping phenotypic diversity.

sentences: PlantNexus: A Gene Co-expression Network Database and Visualization Tool for Barley and Sorghum Global gene co-expression networks (GCNs) are powerful tools for functional genomics whereby putative functions and regulatory mechanisms can be inferred by gene co-expression. With the recent accumulation of RNA-seq data sets, the construction of RNA-seq-based GCNs has now become possible. Cereal crops, such as Hordeum vulgare (barley) and Sorghum bicolor (sorghum), are among the most important plants to humanity and contribute significantly to our food supply. However, co-expression network tools for these plants are outdated or lacking. In this study, we constructed global GCNs for barley and sorghum using 500 and 774 RNA-seq data sets, respectively. In addition, we curated the meta-information of these RNA-seq data sets and categorized them into four main tissue types, leaf, root, shoot, and flower/seed, and built tissue-specific GCNs. To enable GCN searching and visualization, we implemented a website and database named PlantNexus, offering an immersive environment for the exploration and visualization of gene expressions and co-expressions of barley and sorghum at the global and tissue-specific levels. PlantNexus is freely available at https://plantnexus.ohio.edu/.

sentences: Regenerative and non-regenerative transcriptional states of the human epicardium: from foetus to adult and backagain Epicardial activation appears to be required for cardiac regeneration. Although reverting quiescent adult epicardium to an active neonatal or foetal state will likely represent a key therapeutic approach for human cardiac regeneration, the exact molecular differences between human adult and foetal epicardium are not understood. We used single-cell RNA sequencing to compare epicardial cells from both foetal and adult hearts. We found two foetal epicardial cell types, mesothelial and fibroblast-like, with only the mesothelial population present in adults. We also identified foetal-specific epicardial genes associated with regeneration and angiogenesis, and found that adult epicardium may be primed for immune and inflammatory responses. We predict that restoring the foetal epicardial state in human hearts would increase adult angiogenic potential. Finally, we demonstrated that human embryonic stem-cell derived epicardium is a valid model for the foetal epicardium and for investigating epicardial-mediated cardiac regeneration in humans. Our study defines regenerative programs in human foetal epicardium that are absent in the adult, brings human context to animal studies, and provides a roadmap for directing the epicardium in human heart regeneration.

sentences: Dysgu: efficient structural variant calling using short or long reads Structural variation (SV) plays a fundamental role in genome evolution and can underlie inherited or acquired diseases such as cancer. Long-read sequencing technologies have led to improvements in the characterization of structural variants (SVs), although paired-end sequencing offers better scalability. Here, we present dysgu, which calls SVs or indels using paired-end or long reads. Dysgu detects signals from alignment gaps, discordant and supplementary mappings, and generates consensus contigs, before classifying events using machine learning. Additional SVs are identified by remapping of anomalous sequences. Dysgu outperforms existing state-of-the-art tools using paired-end or long-reads, offering high sensitivity and precision whilst being among the fastest tools to run. We find that combining low coverage paired-end and long-reads is competitive in terms of performance with long-reads at higher coverage values.

sentences: Sequential increase of PHGDH expression with Alzheimer's pathology and symptoms We report consistent increases in phosphoglycerate dehydrogenase (PHGDH) expression in mouse models of Alzheimers disease (AD) (3xTg-AD) and related tauopathy (PS19), particularly in hippocampal astrocytes. Human single-cell RNA-sequencing data reveal a sequential increase of PHGDH expression in people with no, early, and late AD pathology, which is corroborated by protein mass spectrometry and immunohistochemical analyses of three independent cohorts. A sequential increase of PHGDH expression also correlates with increasing clinical AD symptoms and worsening cognitive decline in patients. The consistent increase of PHGDH expression in six AD cohorts (Mayo, ROSMAP, Mount Sinai, Baltimore, Amsterdam, and UCSD/UCI) corroborates with the recent report of PHGDH extracellular RNA in blood plasma as a candidate biomarker for early diagnosis of AD and offers a caution to the suggested use of L-serine as a potential therapy of AD.

sentences: Present and Future Ecological Niche Modeling of Rift Valley fever in East Africa in Response to Climate Change Rift Valley fever (RVF) has been linked with recurrent outbreaks among humans and livestock in several parts of the globe. Predicting RVFs habitat suitability under different climate scenarios offers vital information for developing informed management schemes. The present study evaluated the probable impacts of climate change on the distribution of RVF disease in East Africa (E. A.), using the maximum entropy (MaxEnt) model and the disease outbreak cases. Considering the potential of the spread of the disease in the East Africa region, we utilized two representative concentration pathways (RCP 4.5 and RCP 8.5) climate scenarios in the 2050s and 2070s (average for 2041-2060, and 2061-2080), respectively. All models had satisfactory AUC values of more than 0.809, which are considered excellent. Jackknife tests revealed that Bio4 (temperature seasonality), land use, and population density were the main factors influencing RVF distribution in the region. From the risk maps generated, we infer that, without regulations, this disease might establish itself across more extensive areas in the region, including most of Rwanda and Burundi. The ongoing trade between East African countries and changing climates could intensify RVF spread into new geographic extents with suitable habitats for the important zoonosis. The predicted suitable areas for RVF in eastern Kenya, southern Tanzania, and Somalia overlaps to a large extent where cattle keeping and pastoralism are highly practiced, thereby signifying the urgency to manage and control the disease. This work validates RVF outbreak cases effectiveness to map the diseases distribution, thus contributing to enhanced ecological modeling and improved disease tracking and control efforts in East Africa.

sentences: YAPP-CD: Yet another protein-peptide complex database Protein-peptide interactions are of great interest to the research community not only because they serve as mediators in many protein-protein interactions but also because of the increasing demand for peptide-based pharmaceutical products. Protein-peptide docking is a major tool for studying protein-peptide interactions, and several docking methods are currently available. Among various protein-peptide docking algorithms, template-based approaches, which utilize known protein-peptide complexes or templates to predict a new one, have been shown to yield more reliable results than template-free methods in recent comparative research. To obtain reliable results with a template-based docking method, the template database must be comprehensive enough; that is, there must be similar templates of protein-peptide complexes to the protein and peptide being investigated. Thus, the template database must be updated to leverage recent advances in structural biology. However, the template database distributed with GalaxyPepDock, one of the most widely used peptide docking programs, is outdated, limiting the prediction quality of the method. Here, we present an up-to-date protein-peptide complex database called YAPP-CD, which can be directly plugged into the GalaxyPepDock binary package to improve GalaxyPepDocks prediction quality by drawing on recent discoveries in structural biology. Experimental results show that YAPP-CD significantly improves GalaxyPepDocks prediction quality, e.g., the average Ligand/Interface RMSD of a benchmark set is reduced from 7.60 [A]/3.62 [A] to 3.47 [A]/1.71 [A].

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A unified mechanism to support innate and learned use of visual landmark guidance in insects Insects can navigate efficiently in both novel and familiar environments, and this requires flexiblity in how they are guided by sensory cues. A prominent landmark, for example, can ellicit strong innate behaviours (attraction or menotaxis) but can also be used, after learning, as a specific directional cue as part of a navigation memory. However, the mechanisms that allow both pathways to co-exist, interact or override each other are largely unknown. Here we propose a model for the behavioural integration of innate and learned guidance based on the neuroanatomy of the central complex (CX), adapted to control landmark guided behaviours. We consider a reward signal provided either by an innate attraction to landmarks or a long-term visual memory in the mushroom bodies (MB) that modulates the formation of a local vector memory in the CX. Using an operant strategy for a simulated agent exploring a simple world containing a single visual cue, we show how the generated short-term memory can support both innate and learned steering behaviour. In addition, we show how this architecture is consistent with the observed effects of unilateral MB lesions in ants that cause a reversion to innate behaviour. We suggest the formation of a directional memory in the CX can be interpreted as transforming rewarding (positive or negative) sensory signals into a mapping of the environment that describes the geometrical attractiveness (or repulsion). We discuss how this scheme might represent an ideal way to combine multisensory information gathered during the exploration of an environment and support optimized cue integration.

1 Author summaryIn this paper, we modeled the neural pathway allowing insects to perform landmark guided behaviours using their internal compass. First, we observed the intrinsic property of the connectome, extracted from drosophila online database, between the internal compass neurons and the steering neurons to support an oriented behaviour towards a single landmark. Then, we proposed and evaluated an adaptation of the bees path integration neural circuit, to sustain flexible landmark guidance behaviours such as attraction or menotaxis. We showed the model ability to form a memory during the exploration of the local environment to support both innate or learned navigation behaviour using a single landmark in the environment. In addtion, we demonstrated the transformation of a simple goodness/badness signal, from innate or long-term memory pathways, into an oriented steering signal that could be applied to other sensory pathways. Furthermore, by reproducing lesion experiments in the mushroom bodies of wood ants we highlight the consistency of the model with biological observation. We then discuss the different emergent properties and the potential outcome that this local, and operant, memory supports.

sentences: MuSiC2: cell type deconvolution for multi-condition bulk RNA-seq data Cell type composition of intact bulk tissues can vary across samples. Deciphering cell type composition and its changes during disease progression is an important step towards understanding disease pathogenesis. To infer cell type composition, existing cell type deconvolution methods for bulk RNA-seq data often require matched single-cell RNA-seq (scRNA-seq) data, generated from samples with similar clinical conditions, as reference. However, due to the difficulty of obtaining scRNA-seq data in diseased samples, only limited scRNA-seq data in matched disease conditions are available. Using scRNA-seq reference to deconvolve bulk RNA-seq data from samples with different disease conditions may lead to biased estimation of cell type proportions. To overcome this limitation, we propose an iterative estimation procedure, MuSiC2, which is an extension of MuSiC [1], to perform deconvolution analysis of bulk RNA-seq data generated from samples with multiple clinical conditions where at least one condition is different from that of the scRNA-seq reference. Extensive benchmark evaluations indicated that MuSiC2 improved the accuracy of cell type proportion estimates of bulk RNA-seq samples under different conditions as compared to the traditional MuSiC [1] deconvolution. MuSiC2 was applied to two bulk RNA-seq datasets for deconvolution analysis, including one from human pancreatic islets and the other from human retina. We show that MuSiC2 improves current deconvolution methods and provides more accurate cell type proportion estimates when the bulk and single-cell reference differ in clinical conditions. We believe the condition-specific cell type composition estimates from MuSiC2 will facilitate downstream analysis and help identify cellular targets of human diseases.

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Polyunsaturated fatty acid structure determines the strength of potentiation of Acid-sensing ion channels Acid-sensing ion channels (ASICs) are sensitized to activation by inflammatory mediators like the polyunsaturated fatty acid (PUFA) arachidonic acid (AA). Previous work has shown that AA can potentiate ASIC currents at subsaturating proton concentrations, but the structural mechanisms of this change in gating are not understood. Here we show that PUFAs cause multiple gating changes in ASIC3 including shifting the pH dependence of activation, slowing the rate of desensitization, and increasing the current even at a saturating pH. The impact on gating depends on the nature of both the head and tail of the lipid with the head group structure primarily determining the magnitude of the effect on the channel. An N-acyl amino acid (NAAA), arachidonoyl glycine (AG), is such a strong regulator that it can act as a ligand at neutral pH. Mutation of an arginine in the outer segment of TM1 (R64) eliminated the effect of docosahexaenoic acid (DHA) even at high concentrations suggesting a potential interaction site for the lipid on the channel. Our results suggest a model where PUFAs bind to ASICs both via their tail group as well as an electrostatic interaction between the negatively charged PUFA head group and the positively charged arginine side chain. These data provide the first look at the structural features of lipids that are important for modulating ASICs and suggest a potential binding site for PUFAs on the channel.

Short SummaryArachidonic acid (AA) is a known modulator of ASICs. Bankston and Klipp examine the structural requirements for ASIC modulation by a number of lipids related to AA. Negatively charged head groups are stronger potentiators and may interact with an arginine in TM1 near the outer leaflet of the plasma membrane.

sentences: High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin. The unprecedented number of high-resolution structures of a single myosin finally enabled us to assemble a nearly complete structural model of the myosin-V motor cycle and describe the molecular principles of force production.

sentences: White matter connections of high-level visual areas predict cytoarchitecture better than category-selectivity Ventral temporal cortex (VTC) consists of high-level visual regions that are arranged in consistent anatomical locations across individuals. This consistency has led to several hypotheses about the factors that constrain the functional organization of VTC. A prevailing theory is that white matter connections influence the organization of VTC, however, the nature of this constraint is unclear. Here, we test two hypotheses: (1) white matter tracts are specific for each category or (2) white matter tracts are specific to cytoarchitectonic areas of VTC. To test these hypotheses, we used diffusion magnetic resonance imaging (dMRI) to identify white matter tracts and functional magnetic resonance imaging (fMRI) to identify category-selective regions in VTC in children and adults. We find that in childhood, white matter connections are linked to cytoarchitecture rather than category-selectivity. In adulthood, however, white matter connections are linked to both cytoarchitecture and category-selectivity. These results suggest a rethinking of the view that category-selective regions in VTC have category-specific white matter connections early in development. Instead, these findings suggest that the neural hardware underlying the processing of categorical stimuli may be more domain-general than previously thought, particularly in childhood.

sentences: Extracellular vesicles mediate the intercellular exchange of nanoparticles In order to exert their therapeutic effects, nanoparticles (NPs) often need to travel into the tissues composed of multilayered cells. Accumulative evidence has revealed the crucial role of transcellular transport route (entry into one cell, exocytosis, and re-entry into another) in this process. While NP endocytosis and subcellular transport have been intensively characterized, the exocytosis and re-entry steps are poorly understood, which becomes a barrier to improve NP delivery into complex tissues. Here, we termed the exocytosis and re-entry steps together as intercellular exchange. We developed a collagen-based 3D assay to specifically monitor and quantify the intercellular exchange events of NPs, and distinguish the contributions of several potential mechanisms. Our results showed that NPs can be exocytosed freely or enclosed inside extracellular vesicles (EVs) for re-entry, while direct cell-cell contact is hardly involved. EVs account for a significant fraction of NP intercellular exchange, and its importance in NP transport was demonstrated in vitro and in vivo. Intriguingly, while freely released NPs engage with the same cellular receptors for re-entry, EV-enclosed ones bypass this dependence. These studies provide an easy and precise system to investigate the intercellular exchange stage of NP delivery, and shed the first light in the importance of EVs in NP transport between cells and across complex tissues.

sentences: Microbiota responses to different prebiotics are conserved within individuals and associated with habitual fiber intake Background: Short-chain fatty acids (SCFAs) derived from gut bacteria are associated with protective roles in diseases ranging from obesity to colorectal cancers. Intake of microbially accessible dietary fibers (prebiotics) lead to varying effects on SCFA production in human studies, and gut microbial responses to nutritional interventions vary by individual. It is therefore possible that prebiotic therapies will require customizing to individuals. Results: Here, we explored prebiotic personalization by conducting a three-way crossover study of three prebiotic treatments in healthy adults. We found that within individuals, metabolic responses were correlated across the three prebiotics. Individual identity, rather than prebiotic choice, was also the major determinant of SCFA response. Across individuals, prebiotic response was inversely related to basal fecal SCFA concentration, which, in turn, was associated with habitual fiber intake. Experimental measures of gut microbial SCFA production for each participant also negatively correlated with fiber consumption, supporting a model in which individuals' gut microbiota are limited in their overall capacity to produce fecal SCFAs from fiber. Conclusions: Our findings support developing personalized prebiotic regimens that focus on selecting individuals who stand to benefit, and that such individuals are likely to be deficient in fiber intake.

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On the Gestural Origin of Language Lateralisation: Manual Communication reflects Broca's Asymmetry in Monkeys Manual gestures and speech recruit a common neural network, involving Brocas area in the left hemisphere. Such speech-gesture integration gave rise to theories on the critical role of manual gesturing in the origin of language.

Within this evolutionary framework, research on gestural communication in our closer primate relatives has received renewed attention for investigating its potential language-like features.

Here, using in-vivo anatomical MRI in 50 baboons, we found that communicative gesturing is related to Broca homologues marker in monkeys, namely the ventral portion of the Inferior Arcuate sulcus (IA sulcus). In fact, both direction and degree of gestural communications handedness - but not handedness for object manipulation - are associated and correlated with contralateral depth asymmetry at this exact IA sulcus portion. In other words, baboons that prefer to communicate with their right hand have a deeper left-than-right IA sulcus, than those preferring to communicate with their left hand and vice versa. Interestingly, in contrast to handedness for object manipulation, gestural communications lateralisation is not associated to the Central sulcus depth asymmetry, suggesting a double dissociation of handedness types between manipulative action and gestural communication.

It is thus not excluded that this specific gestural lateralisation signature within the baboons frontal cortex might reflect a phylogenetical continuity with language-related Broca lateralisation in humans.

sentences: Effects of forced cohesin eviction and retention on X-inactivation and autosomes Depletion of architectural factors globally alters chromatin structure, but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Interestingly, cohesin loss disrupts Xi superstructure, unveiling superloops between escapee genes, with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure and compromises spreading of Xist RNA-Polycomb complexes, attenuating Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss created an "Xi superloop" and cohesin retention created "Xi megadomains" on the active X. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X-inactivation and inter-chromosomal interactions across the genome.

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Dynamic Allostery Highlights the Evolutionary Differences between the CoV-1 and CoV-2 Main Proteases The SARS-CoV-2 coronavirus has become one of the most immediate and widely-studied systems since its identification and subsequent global outbreak from 2019-2020. In an effort to understand the biophysical changes as a result of mutations, the mechanics of multiple different proteins within the SARS-CoV-2 virus have been studied and compared with SARS-CoV-1. Focusing on the main protease (mPro), we first explored the long range dynamic-relationship, particularly in cross-chain dynamics, using the Dynamic Coupling Index (DCI) to investigate the dynamic coupling between the catalytic site residues and the rest of the protein, both inter and intra chain for the CoV-1 and CoV-2 mPro. We found that there is significant cross-chain coupling between these active sites and distal residues in the CoV-2 mPro but it was missing in CoV-1. The enhanced long distance interactions, particularly between the two chains, suggest subsequently enhanced cooperativity for CoV-2. A further comparative analysis of the dynamic flexibility using the Dynamic Flexibility Index (DFI) between the CoV-1 and CoV-2 mPros shows that the inhibitor binding near active sites induces change in flexibility to a distal region of the protein, opposite in behavior between the two systems; this region becomes more flexible upon inhibitor binding in CoV-1 while it becomes less flexible in the CoV-2 mPro. Upon inspection, we show that, on average, the dynamic flexibility of the sites substituted from CoV-1 to CoV-2 changes significantly less than the average calculated across all residues within the structure, indicating that the differences in behaviors between the two systems is likely the result of allosteric influence, where the new substitutions in COV-2 induce flexibility and dynamical changes elsewhere in the structure.

SIGNIFICANCEHere we have conducted a comparative analysis between the SARS-CoV-1 and SARS-CoV-2 mPro systems to shed mechanistic insight on the biophysical changes associated with the mutations between these two enzymes. Our work shows that the CoV-2 mPro system exhibits enhanced cross-chain communication between catalytic site residues and the rest of the structure. Further, both dynamic coupling and dynamic flexibility analyses indicates that, largely, the dynamic changes as evaluated by DCI and DFI occur at sites other than the mutation sites themselves, indicating that the functional differences between these two proteins are a result of dynamic allostery

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Antimicrobial resistance and virulence characteristics of Klebsiella pneumoniae isolates in Kenya Klebsiella pneumoniae is a globally significant opportunistic pathogen causing healthcare-associated and community-acquired infections. This study examined the epidemiology and the distribution of resistance and virulence genes in clinical K. pneumoniae strains in Kenya. Eighty-nine K. pneumoniae isolates were collected over six years from five counties in Kenya and were analyzed using whole genome sequencing and bioinformatics. These isolates were obtained from community-acquired (62/89) and healthcare-associated infections (21/89), and the hospital environment (6/89). Genetic analysis revealed the presence of blaNDM-1 and blaOXA-181 carbapenemase genes and the armA and rmtF genes known to confer pan-aminoglycoside resistance. The most abundant extended-spectrum beta-lactamase genes identified were blaCTX-M-15 (36/89), blaTEM (35/89), and blaOXA (18/89). In addition, one isolate had a mobile colistin resistance gene (mcr-8). Fluoroquinolone resistance-conferring mutations in gyrA and parC genes were also observed. The most notable virulence factors were those associated with hyper-virulence (rmpA/A2 and magA), yersiniabactin (ybt), salmochelin (iro), and aerobactin (iuc and iutA). Thirty-eight distinct sequence types were identified, including known global lineages ST14, ST15, ST147, and ST307, and a regional clone ST17 implicated in regional outbreaks. In addition, this study genetically characterized two potential hypervirulent isolates and two community-acquired ST147 high-risk clones that contained carbapenemase genes, yersiniabactin, and other multidrug resistance genes. These results demonstrate that the resistome and virulome of Kenyan clinical and hospital environmental K. pneumoniae isolates are diverse. The reservoir of high risk-clones capable of spreading resistance and virulence factors have the potential to cause unmanageable infection outbreaks with high morbidity and mortality.

AUTHOR SUMMARYKlebsiella pneumoniae is one of the human-disease-causing bacteria that easily acquires and spreads antibiotic resistance genes and is thus a serious threat to human health. We studied both the antibiotic resistance genes and the genes it uses to cause disease (virulence). Forty-two percent of our isolates were multidrug resistant (MDR). They carried several resistance and virulence genes bound in mobile circular DNA molecules called plasmids which easily migrate and spread the genes between bacteria. We identified 38 distinct K. pneumoniae strains (STs) distributed within the study sites. Fifteen isolates were classified under the groups of K. pneumoniae strains known to cause global infection outbreaks such as ST14, ST15 and ST147 collected from Nairobi and Kisumu, hotspot areas for spread of resistance. In particular, two ST147 isolates were resistant to carbapenems and one isolate to colistin, which are last line antibiotics. We also identified two isolates with the potential to cause high levels of disease. We concluded that the presence of highly resistant and virulent strains in the hospital and community demonstrates a need for the continuous monitoring and management of MDR K. pneumoniae infections to prevent disease outbreaks that are difficult to control and that lead to high death rate.

sentences: Relatedness modulates reproductive competition among queens in ant societies with multiple queens Reproductive sharing in animal groups with multiple breeders, insects and vertebrates alike, contains elements of both conflict and cooperation, and depends on both relatedness between co-breeders, as well as their internal and external conditions. We studied how queens of the ant Formica fusca adjust their reproductive efforts in response to experimental manipulations of the kin competition regime in their nest, as well as their own reproductive status. Queens respond to the presence of competitors by increasing their egg laying efforts, but only if the competitors are highly fecund and distantly related. Furthermore, queens only engage in cannibalism of eggs when the risk of erroneously destroying own offspring is absent. We demonstrate that queens of Formica fusca fine-tune their behaviours in response to kinship and fecundity of others as well as their own physiological state in an unusually precise manner.

sentences: Characterization of the different behaviours exhibited by juvenile grey mullet (Mugil cephalus) under rearing conditions This research described the common behaviour of grey mullet (Mugil cephalus) under rearing conditions. The different behaviours exhibited by mullets were videorecorded with submersible cameras installed inside of three tanks. A total of 690 minutes per day (07:30 - 18:30 hours) were recorded per tank during a week. Afterwards, an ethogram was elaborated to organize the different behaviours exhibited by juvenile M. cephalus, with two general categories: a) locomotion, including three different observed behaviours (resting, swimming and fast swimming) b) feeding, including three behaviours (surface feeding, bottom feeding and rubbing). The video recordings showed that M. cephalus is a species with a constant locomotion associated to feeding, since they showed constantly movement during most of day light period the opposite to dark periods. Mullets were observed to be a non-aggressive fish species, due to the absence of dominance and aggression towards conspecifics, resulting in a high predisposition for adaptation to captivity. Finally, behavioural frequencies of grey mullets juveniles were not significantly different among the three tanks for most of the behavioural variables analysed (p>0.05) except for the variable bottom feeding (p=0.02). Results from this study could be of interest to the aquaculture industry to implement protocols and to optimize rearing techniques for the production of grey mullet.

sentences: Response to nitrogen and salinity in Rhizophora mangle propagules varies by maternal family and population of origin Many coastal foundation plant species thrive across a range of environmental conditions, often displaying dramatic phenotypic variation in response to environmental variation. We characterized the response of propagules from six populations of the foundation species Rhizophora mangle L. to full factorial combinations of two levels of salt (15 ppt and 45 ppt) reflecting the range of salinity measured in the field populations, and two levels of nitrogen (N; no addition and amended at approximately 3 mg N per pot each week) equivalent to comparing ambient N to a rate of addition of 75 kg per hectare per year. The response to increasing salt included significant plasticity in succulence. Propagules also showed plasticity in maximum photosynthetic rate in response to N amendment, but the responses depended on the level of salt and varied by population of origin. Generally, survival was lower in high salt and high N, but the impact varied among populations. Overall, this study revealed significant phenotypic plasticity in response to salt and N level. Propagules from different populations differed in all traits measured. Variation in phenotypic plasticity and propagule survival in R. mangle may contribute to adaptation to a complex mosaic of environmental conditions and response to climate change.

sentences: Experimental Confirmation of Multiple Co-Existent DNA Secondary Structures using Low-Yield Bisulfite Sequencing The prediction of DNA secondary structures from DNA sequences using thermodynamic models is imperfect for many biological sequences, both due to insufficient experimental data for training and to the kinetics of folding that lead to metastable structures. Here, we developed low-yield bisulfite sequencing (LYB-seq) to query the secondary structure states of cytosine (C) nucleotides in thousands of different DNA oligonucleotides on a single-molecule level. We observed that the reaction kinetics between bisulfite and C nucleotides is highly dependent on the secondary structure state of the C nucleotides, with the most accessible C nucleotides (those in small hairpin loops) reacting 70-fold faster than those in stable duplexes. Next, we developed a statistical model to evaluate the likelihood of an NGS read being consistent with a particular proposed secondary structure. By analyzing thousands of NGS reads for each DNA species, we can infer the distribution of secondary structures adopted by each species in solution. We find that 84% of 1,057 human genome subsequences studied here adopt 2 or more stable secondary structures in solution.

sentences: Actin-related protein 5 functions as a novel modulator of MyoD and MyoG in skeletal muscle and in rhabdomyosarcoma Myogenic regulatory factors (MRFs) are pivotal transcription factors in myogenic differentiation. MyoD commits cells to the skeletal muscle lineage by inducing myogenic genes through recruitment of chromatin remodelers to its target loci. This study showed that Actin-related protein 5 (Arp5) acts as an inhibitory regulator of MyoD and MyoG by binding to their cysteine-rich (CR) region, which overlaps with the region essential for their epigenetic functions. Arp5 expression was faint in skeletal muscle tissues. Excessive Arp5 in mouse hind limbs caused skeletal muscle fiber atrophy. Further, Arp5 overexpression in myoblasts inhibited myotube formation by diminishing myogenic gene expression, whereas Arp5 depletion augmented myogenic gene expression. Arp5 disturbed MyoD-mediated chromatin remodeling through competition with the three-amino-acid-loop-extension-class homeodomain transcription factors the Pbx1-Meis1 heterodimer for binding to the CR region. This antimyogenic function was independent of the INO80 chromatin remodeling complex, although Arp5 is an important component of that. In rhabdomyosarcoma (RMS) cells, Arp5 expression was significantly higher than in normal myoblasts and skeletal muscle tissue, probably contributing to MyoD and MyoG activity dysregulation. Arp5 depletion in RMS partially restored myogenic properties while inhibiting tumorigenic properties. Thus, Arp5 is a novel modulator of MRFs in skeletal muscle differentiation.

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Functional Analysis of the Expanded Phosphodiesterase Gene Family in Toxoplasma gondii Tachyzoites Toxoplasma motility is both activated and suppressed by 3-5 cyclic nucleotide signaling. Cyclic GMP (cGMP) signaling through TgPKG activates motility, whereas cyclic AMP (cAMP) signaling through TgPKAc1 inhibits motility. Despite their importance, it remains unclear how cGMP and cAMP levels are maintained in Toxoplasma. Phosphodiesterases (PDEs) are known to inactivate cyclic nucleotides and are highly expanded in the Toxoplasma genome. Here we analyzed the expression and function of the 18-member TgPDE family in tachyzoites, the virulent life-stage of Toxoplasma. We detected the expression of 11 of 18 TgPDEs, confirming prior expression studies. A knockdown screen of the TgPDE family revealed four TgPDEs that contribute to lytic Toxoplasma growth (TgPDE1, TgPDE2, TgPDE5, and TgPDE9). Depletion of TgPDE1 or TgPDE2 caused severe growth defects, prompting further investigation. While TgPDE1 was important for extracellular motility, TgPDE2 was important for host cell invasion, parasite replication, host cell egress, and extracellular motility. TgPDE1 displayed a plasma membrane/cytomembranous distribution, whereas TgPDE2 displayed an endoplasmic reticulum/cytomembranous distribution. Biochemical analysis of TgPDE1 and TgPDE2 purified from Toxoplasma lysates revealed that TgPDE1 hydrolyzes both cGMP and cAMP, whereas TgPDE2 was cAMP-specific. Interactome studies of TgPDE1 and TgPDE2 indicated that they do not physically interact with each other or other TgPDEs but may be regulated by kinases and proteases. Our studies have identified TgPDE1 and TgPDE2 as central regulators of tachyzoite cyclic nucleotide levels and enable future studies aimed at determining how these enzymes are regulated and cooperate to control Toxoplasma motility and growth.

ImportanceApicomplexan parasites require motility to actively infect host cells and cause disease. Cyclic nucleotide signaling governs apicomplexan motility, but it is unclear how cyclic nucleotide levels are maintained in these parasites. In search of novel regulators of cyclic nucleotides in the model apicomplexan Toxoplasma, we identified and characterized two catalytically active phosphodiesterases, TgPDE1 and TgPDE2, that are important for Toxoplasmas virulent tachyzoite lifecycle. Enzymes that generate, sense, or degrade cyclic nucleotides make attractive targets for therapies aimed at paralyzing and killing apicomplexan parasites.

sentences: Memory destabilization during reconsolidation - A consequence of homeostatic plasticity? Remembering is not a static process: when retrieved, a memory can be destabilized and become prone to modifications. This phenomenon has been demonstrated in a number of brain regions, but the neuronal mechanisms that rule memory destabilization and its boundary conditions remain elusive. Using two distinct computational models that combine Hebbian plasticity and synaptic downscaling, we show that homeostatic plasticity can function as a destabilization mechanism, accounting for behavioral results of protein synthesis inhibition upon reactivation with different reexposure times. Furthermore, by performing systematic reviews, we identify a series of overlapping molecular mechanisms between memory destabilization and synaptic downscaling, although direct experimental links between both phenomena remain scarce. In light of these results, we propose a theoretical framework where memory destabilization can emerge as an epiphenomenon of homeostatic adaptations prompted by memory retrieval.

sentences: Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions. Characterization of the size and material properties of particles in liquid suspensions is in very high demand, e.g., for the analysis of colloidal samples or of bodily fluids such as urine or blood plasma. However, the existing methods are limited in deciphering the constituents of realistic samples. Here, we introduce iNTA as a new method, which combines interferometric detection of scattering with nanoparticle tracking analysis, to reach an unprecedented sensitivity and precision in determining the size and refractive index distributions of nanoparticles in suspensions. After benchmarking iNTA with samples of colloidal gold, we present its remarkable ability to resolve the constituents of various multi-component and polydisperse samples of known origin. Furthermore, we showcase the method by elucidating the refractive index and size distributions of extracellular vesicles from Leishmania parasites and nanoparticles in human urine. The current performance of iNTA already enables advances in several important applications, but we also discuss possible improvements.

sentences: USPNet: unbiased organism-agnostic signal peptidepredictor with deep protein language model Signal peptide is a short peptide located in the N-terminus of proteins. It plays an important role in targeting and transferring transmembrane proteins and secreted proteins to correct positions. Compared with traditional experimental methods to identify and discover signal peptides, the computational methods are faster and more efficient, which are more practical for the analysis of thousands or even millions of protein sequences in reality, especially for the metagenomic data. Therefore, computational tools are recently proposed to classify signal peptides and predict cleavage site positions, but most of them disregard the extreme data imbalance problem in these tasks. In addition, almost all these methods rely on additional group information of proteins to boost their performances, which, however, may not always be available. To deal with these issues, in this paper, we present Unbiased Organism-agnostic Signal Peptide Network (USPNet), a signal peptide prediction and cleavage site prediction model based on deep protein language model. We propose to use label distribution-aware margin (LDAM) loss and evolutionary scale modeling (ESM) embedding to handle data imbalance and object-dependence problems. Extensive experimental results demonstrate that the proposed method significantly outperforms all the previous methods on the classification performance. Additional study on the simulated metagenomic data further indicates that our model is a more universal and robust tool without dependency on additional group information of proteins, with the Matthews correlation coefficient improved by up to 17.5%. The proposed method will be potentially useful to discover new signal peptides from the abundant metagenomic data.

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Columnar localization and laminar origin of cortical surface electrical potentials Electrocorticography (ECoG) methodologically bridges basic neuroscience and understanding of human brains in health and disease. However, the localization of ECoG signals across the surface of the brain and the spatial distribution of their generating neuronal sources are poorly understood. To address this gap, we recorded from rat auditory cortex using customized ECoG, and simulated cortical surface electrical potentials with a full-scale, biophysically detailed cortical column model. Experimentally, ECoG-derived auditory representations were tonotopically organized and signals were anisotropically localized to [≤]{+/-}200 m, i.e., a single cortical column. Biophysical simulations reproduce experimental findings, and indicate that neurons in cortical layers V and VI contribute [~]85% of evoked high-gamma signal recorded at the surface. Cell number and synchronicity were the primary biophysical properties determining laminar contributions to evoked ECoG signals, while distance was only a minimal factor. Thus, evoked ECoG signals primarily originate from neurons in the infragranular layers of a single cortical column.

In BriefBaratham et al., investigated the localization and origins of sensory evoked ECoG responses. They experimentally found that ECoG responses were anisotropically localized [≤]{+/-}200 m, i.e., a single cortical column. Biophysically detailed simulations revealed that neurons in layers V &VI were the primary sources of evoked ECoG responses, in contrast to common thinking.

HighlightsEvoked ECoG signals are localized on the surface to a cortical column.

Neurons in cortical layers V and VI constitute the vast majority of the signal recorded at the surface.

Different laminar contributions to ECoG signal are driven by cell density and synchronicity.

sentences: Preliminary report of preclinical efficacy and safety analysis of gamma-irradiated inactivated SARS-CoV-2 vaccine candidates, SK-01 version 1 and OZG-3861 version 1 COVID-19 outbreak caused by SARS-CoV-2 created an unprecedented health crisis since there is no vaccine for this novel virus. Therefore, SARS-CoV-2 vaccines have become crucial for reducing morbidity and mortality. In this study, in vitro and in vivo safety and efficacy analyzes of lyophilized vaccine candidates inactivated by gamma-irradiation were performed. The candidate vaccines in this study were OZG-3861 version 1 (V1), an inactivated SARS-CoV-2 virus vaccine, and SK-01 version 1 (V1), a GM-CSF adjuvant added vaccine. The candidate vaccines were applied intradermally to BALB/c mice to assess toxicity and immunogenicity. Preliminary results in vaccinated mice are reported in this study. Especially, the vaccine models containing GM-CSF caused significant antibody production with neutralization capacity in absence of the antibody-dependent enhancement feature, when considered in terms of T and B cell responses. Another important finding was that the presence of adjuvant was more important in T cell in comparison with B cell response. Vaccinated mice showed T cell response upon restimulation with whole inactivated SARS-CoV-2 or peptide pool. This study shows that the vaccines are effective and leads us to start the challenge test to investigate the gamma-irradiated inactivated vaccine candidates for infective SARS-CoV-2 virus in humanized ACE2+ mice.

sentences: The role of the angular gyrus in semantic cognition - A synthesis of five functional neuroimaging studies Semantic knowledge is central to human cognition. The angular gyrus (AG) is widely considered a key brain region for semantic cognition. However, the role of the AG in semantic processing is controversial. Key controversies concern response polarity (activation vs. deactivation) and its relation to task difficulty, lateralization (left vs. right AG), and functional-anatomical subdivision (PGa vs. PGp subregions). Here, we combined the fMRI data of five studies on semantic processing (n = 172) and analyzed the response profiles from the same anatomical regions-of-interest for left and right PGa and PGp. We found that the AG was consistently deactivated during non-semantic conditions, whereas response polarity during semantic conditions was inconsistent. However, the AG consistently showed relative response differences between semantic and non-semantic conditions, and between different semantic conditions. A combined analysis across all studies revealed that AG responses could be best explained by separable effects of task difficulty and semantic processing demand. Task difficulty effects were stronger in PGa than PGp, regardless of hemisphere. Semantic effects were stronger in left than right AG, regardless of subregion. These results suggest that the AG is engaged in both domain-general task-difficulty-related processes and domain-specific semantic processes. In semantic processing, we propose that left AG acts as a "multimodal convergence zone" that binds different semantic features associated with the same concept, enabling efficient access to task-relevant features.

sentences: RNA-binding protein Mub1 and the nuclear RNA exosome act to fine-tune environmental stress response The nuclear RNA exosome plays a key role in mediating degradation and processing of multiple cellular RNAs. Recognition of the specific RNA substrates by the exosome is mediated by the RNA-binding co-factors. Transient binding of co-factors either to the exosome or the substrate RNAs as well as their rapid decay make identification of the co-factors challenging. Here, we employ a comparative poly(A)+ RNA interactome capture approach in the fission yeast exosome mutants to identify proteins that interact with poly(A)+ RNA in an exosome-dependent manner. Our analyses identified multiple proteins whose occupancy on RNA is altered in the exosome mutants including zinc-finger protein Mub1. Mub1 is required to maintain the levels of a subset of the exosome RNA substrates including mRNAs encoding for stress-responsive proteins. Removal of the zinc finger domain leads to loss of RNA suppression under non-stressed conditions, altered expression of heat shock genes in response to stress, and reduced growth at elevated temperature. These findings highlight importance of exosome-dependent mRNA degradation in buffering gene expression networks to mediate cellular adaptation to stress.

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Brainprints: identifying individuals from magnetoencephalograms Magnetoencephalography (MEG) is used to study a wide variety of cognitive processes. Increasingly, researchers are adopting principles of open science and releasing their MEG data. While essential for reproducibility, sharing MEG data has unforeseen privacy risks. Individual differences may make a participant identifiable from their anonymized recordings. However, our ability to identify individuals based on these individual differences has not yet been assessed. Here, we propose interpretable MEG features to characterize individual difference. We term these features brainprints (brain fingerprints). We show through several datasets that brainprints accurately identify individuals across days, tasks, and even between MEG and Electroencephalography (EEG). Furthermore, we identify consistent brainprint components that are important for identification. We study the dependence of identifiability on the amount of data available. We also relate identifiability to the level of preprocessing, the experimental task. Our findings reveal specific aspects of individual variability in MEG. They also raise concerns about unregulated sharing of brain data, even if anonymized.

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Identifying which subject a segment of MEG data belongs to is strikingly easy when other data from the same session is available for every subject. We propose three types of interpretable features that can also be used to identify individuals across sessions with high accuracy. Identifiability of individuals is influenced by factors such as resting state vs. task state, components of each feature, the sample size and the level of preprocessing. Our results reveal aspects of individual variability in MEG signals and highlight privacy risks associated with MEG data sharing.

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sentences: The skeletal muscle circadian clock regulates sarcomere length homeostasis through titin splicing Circadian rhythms are maintained by a cell autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression through both RNAseq and LC-MS. This alteration in titins spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. Finally, we identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.

sentences: Does pollen limitation limit plant ranges? Evidence and implications Range limits often involve declines in sexual reproduction, reducing fitness, dispersal, and adaptive potential at range edges. For plants, sexual reproduction is frequently limited by inadequate pollination. While case studies show that pollen limitation can limit plant distributions, the extent to which pollination commonly declines toward plant range edges is unknown. Here, we leverage global databases of pollen-supplementation experiments and plant occurrence data to test whether pollen limitation increases toward plant range edges, using a phylogenetically controlled meta-analysis. While there was significant pollen limitation across studies, we found little evidence that pollen limitation increases toward plant range edges. Pollen limitation was not stronger toward the tropics, nor at species equatorward vs poleward range limits. Meta-analysis results are consistent with results from targeted experiments, in which pollen limitation increased significantly toward only 14% of 14 plant range edges, suggesting that pollination contributes to range limits less often than do other interactions. Together, these results suggest pollination is one of the rich variety of potential ecological factors that can contribute to range limits, rather than a generally important constraint on plant distributions.

sentences: Whole-genome resequencing reveals the population structure, genomic diversity, and demographic history of American chestnut (Castanea dentata) American chestnut (Castanea dentata) was once the most economically and ecologically important hardwood species in the United States. In the first half of the 20th century, an exotic fungal pathogen - Cryphonectria parasitica - decimated the species, killing approximately four billion trees. Two approaches to developing blight resistant American chestnut populations show promise, but both will require introduction of adaptive genomic diversity from wild germplasm to produce diverse, locally adapted reforestation populations. Here we characterize population structure, demographic history, and genomic diversity in a range-wide sample of 384 wild American chestnuts to inform conservation and breeding with blight resistant varieties. Population structure analyses with DAPC and ADMIXTURE suggest that the chestnut range can be roughly divided into northeast, central, and southwest populations. Within-population genomic diversity estimates revealed a clinal pattern with the highest diversity in the southwest, which likely reflects bottleneck events associated with Quaternary glaciation. Finally, we identified genomic regions under positive selection within each population, which suggests that defense against fungal pathogens is a common target of selection across all populations. Taken together, these results show that American chestnut underwent a postglacial expansion from the southern portion of its range leading to three extant populations. These populations will serve as management units for breeding adaptive genetic variation into the blight-resistant tree populations for targeted reintroduction efforts.

sentences: A COVID Moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.

sentences: Restricting a single amino acid cross-protects D. melanogaster from nicotine poisoning through mTORC1 and GCN2 signalling. Dietary interventions that restrict protein intake have repeatedly been shown to offer beneficial health outcomes to the consumer. Benefits such as increased stress tolerance can be observed in response to restricting individual amino acids, thus mimicking dietary protein restriction. Here, we sought to further understand the relationship between dietary amino acids and stress tolerance using Drosophila melanogaster. Utilising a chemically defined medium for Drosophila, we found that transiently restricting adult flies of a single essential amino acid generally protects against a lethal dose of the naturally occurring insecticide, nicotine. This protection was conferred during the pre-treatment window, was specific for individual amino acids and depended on the identity of the focal amino acid, as well as the duration and intensity of its restriction. For instance, complete isoleucine deprivation for 7 days maximised its protective effect - increasing survival during nicotine exposure by 100%. However, a dose of 25% threonine was required to maximise its protective effect (53% enhanced survival). To understand the molecular basis of these effects, we modified the signalling of two cellular sensors of amino acids, GCN2 (General control non-derepressible) and mTORC1 (mechanistic Target of Rapamycin Complex 1) in combination with amino acid restriction. We found that GCN2 was necessary for diets to protect against nicotine, whereas suppression of mTORC1 was sufficient to induce nicotine resistance. This finding implies that amino acid restriction acts via amino acid signalling to cross-protect against seemingly unrelated stressors. Altogether, our study offers new insights into the physiological responses to restriction of individual amino acids that confer stress tolerance. This has broad potential for application in animal and human health.

sentences: ATP6AP2-to-MMP14, a key pathway for osteoblast to osteocyte transition Osteocytes, derived from osteoblast (OB)-lineage, occupy lacunae within bone matrix and exhibit unique morphology with dendrite-like projections to form an inter-connected network. Such a network is essential for osteocytes to monitor and orchestrate bone homeostasis. Thus, it is of considerable interest to investigate how osteocytes are formed and how they built the network. Here we provide evidence for ATP6AP2, an accessory subunit of V-ATPase, in OB-lineage cells to be critical for OB-to-osteocyte transition, and osteocyte distribution, maturation, and morphogenesis. Mice (ATP6AP2Ocn-Cre) that selectively deplete ATP6AP2 in OB-lineage cells results in altered osteocyte distribution and morphology, impaired osteocyte maturation and dendrite-like processes, increased osteocyte cell death and cortical woven bone formation. Further mechanistic studies identify MMP14 (matrix metalloproteinase-14) as a critical downstream of ATP6AP2 for osteocyte differentiation. ATP6AP2 interacts with MMP14 and promotes MMP14 surface distribution largely in immature- or osteoid-osteocytes, where this pathway regulates bone matrix remodeling and osteocyte differentiation. Expression of MMP14 into ATP6AP2 knock out OB-lineage cells in the mouse cortical bone could diminish the deficits in osteocyte maturation, dendrite-like process formation, and survival. These results thus demonstrate an un-recognized function of ATP6AP2 in promoting OB-to-osteocyte transition and uncover a pathway from ATP6AP2-to-MMP14 in osteocyte differentiation.

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Smart sealants for prevention and monitoring of gastrointestinal anastomotic leaks using portable smartphone-controlled ultrasound transducers Millions of patients every year undergo gastrointestinal surgery. While often lifesaving, sutured and stapled reconnections leak in around 10% of the cases. Penetration of digestive fluids into the peritoneal cavity may lead to dreadful complications, including sepsis and premature death. Modern suture supports and tissue adhesives only insufficiently address the issue. Due to the scarcity of alternatives, surgeons rely on monitoring surrogate markers and clinical symptoms, which oftentimes lack sensitivity and specificity, hence only offering late-stage detection of already fully developed leaks.

Here, a first-of-its-kind, modular, intelligent suture support patch capable of sealing and monitoring leaks under harsh gastrointestinal conditions is presented. The smart adhesive layered hydrogel patch provides, in addition to unprecedented tissue sealing under most demanding conditions, unique leak-detection capabilities based on pH and/or enzyme-responsive sensing elements, which can be read out by non-invasive point-of-need ultrasound imaging. Reliable detection of the breaching of sutures in as little as 3 hours in intestinal leak and 15 minutes in gastric leak conditions, and before an actual leak develops, is demonstrated. This technology paves the way for next-generation suture support materials that offer disambiguation in cases of anastomotic leaks based on point-of-need monitoring, without reliance on complex electronics or bulky (bio)electronic implantables.

SummaryElectronic-free smart surgical hydrogel sealants leveraging tissue-penetrating polymer networks and trigger-responsive echogenic entities to enable point-of-need monitoring and early anastomotic leak detection using a hand-held ultrasound transducer and a smartphone.

sentences: iGEM comes of age: trends in its research output. The international Genetically Engineered Machine (iGEM) is an educational benchmark in synthetic biology. Eighteen years after its inception, it has also catalysed the infusion of synthetic biology with interdisciplinary fundamental and translational research as well as with inspired young scientists. Here, we communicate a quantitative analysis of compiled published work associated to iGEM projects, highlighting trends in their dissemination and versatility. As iGEM comes of age, we anticipate it will continue to revolutionise, alongside SynBio, several disciplines of science and industries through the development of synthetic biological systems towards a sustainable future.

sentences: A biophotoelectrochemical approach to unravelling the role of cyanobacterial cell structures in exoelectrogenesis Photosynthetic microorganisms can export electrons outside their cells, a phenomenon called exoelectrogenesis, which can be harnessed for solar energy conversion. However, the route electrons take from thylakoid membranes to the cell exterior is not understood. Electrochemistry is a powerful analytical technique for studying electron transfer pathways. Here, we show how photoelectrochemistry can be used to compare electron flux from cyanobacterial cells of different growth stages, species and with the outer layers systematically removed. We show that the periplasmic space contributes significantly to the photocurrent profile complexity of whole cells, indicating that it gates electron transfer in exoelectrogenesis. We found that although components of the type IV pili machinery do not have a role in exoelectrogenesis, they contribute significantly to cell-electrode adherence. This study establishes that analytical photoelectrochemistry and molecular microbiology provide a powerful combination to study exoelectrogenesis, enabling future studies to answer biological questions and advance solar energy conversion applications.

sentences: Ras protein abundance correlates with Ras isoform mutation patterns in cancer. Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS>NRAS>>HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. However, they challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage.

sentences: Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

sentences: Evolutionary implications of recombination differences across diverging populations of Anopheles Recombination is one of the main evolutionary mechanisms responsible for changing the genomic architecture of populations; and in essence, it is the main mechanism by which novel combinations of alleles, haplotypes, are formed. A clear picture that has emerged across study systems is that recombination is highly variable, even among closely related species. However, it is only until very recently that we have started to understand how recombination variation between populations of the same species impact genetic diversity and divergence. Here, we used whole-genome sequence data to build fine-scale recombination maps for nine populations within two species of Anopheles, Anopheles gambiae and Anopheles coluzzii. The genome-wide recombination averages were on the same order of magnitude for all populations except one. Yet, we identified significant differences in fine-scale recombination rates among all population comparisons. We report that effective population sizes, and presence of a chromosomal inversion has major contribution to recombination rate variation along the genome and across populations. We identified over 400 highly variable recombination hotspots across all populations, where only 9.6% are shared between two or more populations. Additionally, our results are consistent with recombination hotspots contributing to both genetic diversity and absolute divergence (dxy) between populations and species of Anopheles. However, we also show that recombination has a small impact on population genetic differentiation as estimated with FST. The minimal impact that recombination has on genetic differentiation across populations represents the first empirical evidence against recent theoretical work suggesting that variation in recombination along the genome can mask or impair our ability to detect signatures of selection. Our findings add new understanding to how recombination rates vary within species, and how this major evolutionary mechanism can maintain and contribute to genetic variation and divergence within a prominent malaria vector.

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AGO1 regulates major satellite transcripts and H3K9me3 distribution at pericentromeric regions in mESCs Argonaute proteins (AGOs), that play an essential role in cytosolic post-transcriptional gene silencing, have been also reported to function in nuclear processes like transcriptional activation or repression, alternative splicing and, chromatin organization. As most of these studies have been conducted in human cancer cell lines, the relevance of AGOs nuclear functions in the context of mouse early embryonic development remains uninvestigated. Here, we examined a possible role of the AGO1 protein on the distribution of constitutive heterochromatin in mouse Embryonic Stem Cells (mESCs). We observed a specific redistribution of the repressive histone mark H3K9me3 and the heterochromatin protein HP1, away from pericentromeric regions upon Ago1 depletion. Furthermore, we demonstrated that major satellite transcripts are strongly upregulated in Ago1_KO mESCs and that their levels are partially restored upon AGO1 rescue. We also observed a similar redistribution of H3K9me3 and HP1 in Drosha_KO mESCs, suggesting a role for microRNAs (miRNAs) in the regulation of heterochromatin distribution in mESCs. Finally, we showed that specific miRNAs with complementarity to major satellites can partially regulate the expression of these transcripts.

Summary blurbDepletion of AGO1 in mESCs leads to a redistribution of H3K9me3 and HP1 away from pericentromeric regions and is accompanied by an upregulation of major satellites transcripts.

sentences: Nr2f1-dependent shaping of mitochondrial architecture in newborn mouse hippocampal neurons unravels novel insights into the neurodevelopmental disorder BBSOAS The nuclear receptor Nr2f1 plays a key role during cortical development by acting as a strong transcriptional regulator in embryonic and postnatal neural cells. In humans, its mutations cause the Bosch-Boonstra-Schaaf optic atrophy-intellectual syndrome (BBSOAS), a rare neurodevelopmental disorder characterized by multiple clinical features including optic nerve atrophy, intellectual disability, and autistic traits. In this study by genome-wide and in silico analyses we found that key factors for mitochondrial function and dynamics represent potential genomic targets under direct Nr2f1 transcriptional control in neurons. Although mitochondrial dysfunction is increasingly implicated in neurodevelopmental disorders, whether and how Nr2f1 can regulate mitochondria function in neural cells is still completely unknown. To address these questions, we have combined mouse genetics, neuroanatomical and imaging approaches. To genetically manipulate Nr2f1 function in newborn neurons we focused on the adult mouse hippocampal dentate gyrus, a site of persistent neurogenesis where Nr2f1 is highly expressed. We showed that conditional Nr2f1 loss-of-function within the hippocampal neurogenic niche lead to a reduced mitochondrial mass associated to mitochondrial fragmentation in newborn neurons, likely reflecting alteration in mitochondrial dynamics. Accordingly, we showed that the fusion factor Mfn2, which is as a putative Nr2f1 target, is downregulated following both Nr2f1 adult depletion and embryonic haploinsufficiency in mice. Overall, our study provides the first evidence of a crucial role of Nr2f1 in shaping mitochondria in neurons and opens a promising avenue for the identification of new mechanisms implicated in BBSOAS pathogenesis involving mitochondrial dysfunction.

sentences: Detection of functional activity in brain white matter using fiber architecture informed synchrony mapping A general linear model is widely used for analyzing fMRI data, in which the blood oxygenation-level dependent (BOLD) signals in gray matter (GM) evoked in response to neural stimulation are modeled by convolving the time course of the expected neural activity with a canonical hemodynamic response function (HRF) obtained a priori. The maps of brain activity produced reflect the magnitude of local BOLD responses. However, detecting BOLD signals in white matter (WM) is more challenging as the BOLD signals are weaker and the HRF is different, and may vary more across the brain. Here we propose a model-free approach to detect changes in BOLD signals in WM by measuring task-evoked increases of BOLD signal synchrony in WM fibers. The proposed approach relies on a simple assumption that, in response to a functional task, BOLD signals in relevant fibers are modulated by stimulus-evoked neural activity and thereby show greater synchrony than when measured in a resting state, even if their magnitudes do not change significantly. This approach is implemented in two technical stages. First, for each voxel a fiber-architecture-informed spatial window is created with orientation distribution functions constructed from diffusion imaging data. This provides the basis for defining neighborhoods in WM that share similar local fiber architectures. Second, a modified principal component analysis (PCA) is used to estimate the synchrony of BOLD signals in each spatial window. The proposed approach is validated using a 3T fMRI dataset from the Human Connectome Project (HCP) at a group level. The results demonstrate that neural activity can be reliably detected as increases in fMRI signal synchrony within WM fibers that are engaged in a task with high sensitivities and reproducibility.

sentences: Implications of the evolution of agriculture and resource foraging for the maintenance of species diversity and community structure Agriculture is found in numerous taxa such as humans, ants, beetles, fishes and even bacteria. This type of niche construction has evolved independently from hunting, though many species remain primarily predators. When a consumer has a positive effect on its resource, we can expect an allocative cost of agriculture, as the agricultural care diverts time and energy from other activities. Defending the resource against predators may divert time from its consumption (exploitation cost). The cost may also occur on the foraging of alternative resources, for instance if the consumer spends more time nearby the farmed resource and underexploiting resources elsewhere (opportunity cost). We here investigate transitions from predation to agriculture in a simple three-species model of a farmer that consumes two resources and has a positive effect on one. We study the conditions for the (co)evolution of the investment into agriculture and specialization on the two resources, and its consequences on the ecological dynamics of the community. Eco-evolutionary dynamics generate a feedback between the evolution of agriculture and specialization on the helped resource, that can lead to varying selected intensity of agriculture, from generalist strategies with no agriculture, to specialist farmers, with possible coexistence between these two extreme strategies.

sentences: Role of Ori in Thermococcus barophilus The mechanisms underpinning replication of genomic DNA in Archaea have recently been challenged. Species belonging to two different taxonomic orders grow well in the absence of an origin of replication, challenging the role of the replication origin in these organisms. Here, we pursue the investigation of the particular way some archaea manage their DNA replication with Thermococcus barophilus and the role of Ori in this Archaea. Surprisingly we discovered that T. barophilus uses its Ori all along the growth curve with marked increase at the end of exponential phase. Through gene deletion, we show that Ori utilization requires Cdc6, and that origin deletion results in increased time in lag phase and a moderate decrease of growth rate in mutants. The number of chromosomes are quite similar between both strains during exponential and early stationary phases but differs after 24h of growth where {Delta}TbOriC has only 6 chromosomes/cell compared to 10 for the reference strain (WT). Following 1hr of growth in fresh media, {Delta}TbOriC strains contains 3 chromosome copies/cell, whereas the WT contains only 1. We hypothesize that the T. barophilus might degrade DNA to obtain energy to start replication and cell division, whereas the {Delta}TbOriC must maintain more chromosomal copies in order to initiate DNA replication in the absence of an origin or replication. Finally, we analyzed the role of Ori at temperatures above or below the optimal temperature, revealing that Ori is important to start growth at those temperatures, suggesting that replication origins may be involved in stress response.

sentences: Copy number variation of the putative speciation genes in the European house mouse hybrid zone AO_SCPLOWBSTRACTC_SCPLOWHybrid zones have long been described as "windows on the evolutionary process", and studying them has become even more important since the advance in the genome analysis tools. The hybrid zone between two subspecies of the house mouse (Mus musculus musculus and Mus m. domesticus) is a unique model speciation system to study fine scale interactions of recently diverged genomes. Here, we explore the role of gene Copy Number Variation in shaping the barrier to introgression in the hybrid zone within a previously established transect in Central Europe. The CNV of seven pre-selected candidate genes was determined via droplet-digital PCR and analyzed in the context of ~500k SNPs, with the ancestral population (i.e. musculus or domesticus) of every SNP allele previously inferred in the admixed individuals (Baird et al., in prep.). The copy numbers of five genes were clearly associated with the prevalence of either musculus or domesticus genomes across the hybrid zone. In three cases, the highest and/or outlying levels of association were observed at or very close to the annotated positions of the respective gene amplicons, demonstrating the power of our approach in confirming the reference locations of copy number variants. Notably, several other reference locations were recognized as positive outliers in the association with particular CNV genes, possibly representing the extra gene copies and/or their epistatic interaction sites.

sentences: Directed information sharing in the brain distinguishes perception from its consequences during perceptual bistability In the search for the neural basis of conscious experience, perception and its cognitive consequences are typically confounded as neural activity is recorded while participants explicitly report what they experience. Here we present a novel way to disentangle perception from report using eye-movement analysis techniques based on convolutional neural networks and neurodynamical analyses based on information theory. We use a visual bistable stimulus that instantiates two well-known properties of conscious perception: integration and differentiation. At any given moment, observers either experience the stimulus as one integrated unitary percept or as two differentiated objects that are clearly distinct from each other. Using electroencephalography, we show that measures of neural information dynamics of directed information integration and differentiation closely follow participants experience when perceptual transitions were reported. We observed increased directed information from anterior to posterior electrodes (front to back) leading up to the moment the stimulus was reported to switch to the integrated percept and higher information differentiation of anterior signals leading up to reporting the differentiated percept. Crucially, neural integration dynamics were closely linked to perception and even observed in a no-report condition when perceptual transitions were inferred from eye-movements. In contrast, the link between perceptual transitions and neural differentiation was observed solely in the active report condition. Our results therefore suggest a differential role of anterior-posterior network communication vs anterior information differentiation during perception and reporting: while front to back directed information is associated with changes in the content of perception, frontal information differentiation reflects cognitive processes that are consequential of perceptual transitions, not perception per se.

sentences: Semi-field evaluations of three botanically derived repellents against the blacklegged tick, Ixodes scapularis (Acari: Ixodidae) Three compounds derived from botanicals sources, ethyl perillyl carbonate, geranyl isovalerate, and citronellyl cyclobutane carboxylate, were tested for repellent activity against Ixodes scapularis Say in a semi-field trial. Tick drags were treated with the compounds or with N, N-diethyl-m-toluamide (DEET) at high (0.25mg/cm2) or low (0.15mg/cm2) concentrations. Negative controls included untreated drags and drags treated with acetone, the carrier for all repellents. Freshly treated drags (within 20 minutes) were used to collect I. scapularis ticks at a county park in Wisconsin. To assess effectiveness, we measured tick encounter rates, detachment rate, and time to detachment. None of the repellent treatments resulted in significantly fewer encounters compared to both control treatments. However, the percentage of ticks that detached within 3 min was significantly higher on drags treated with repellents compared to controls. DEET was the most effective, repelling 69.7 - 87% of ticks by 3 min, but the effectiveness of the three test compounds was still high, ranging from 42% to 87% of ticks detaching by 3 min. For time to detachment, there were no significant differences between DEET and the three test compounds. We conclude that these botanically-derived repellents were effective against I. scapularis in a semi-field trial and could be viable alternatives to DEET.

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ACTIN Anchors the Highly Oligomeric DRP1 at Mitochondria-Sarcoplasmic Reticulum Contact Sites in Adult Murine Heart: Its Functional Implication RationaleMitochondrial fission and fusion are relatively infrequent in adult cardiomyocytes compared to another cell types1-3. This is surprising considering that proteins involved in mitochondrial dynamics are highly expressed in the heart. It has been previously reported that dynamin-related protein 1 (DRP1) has a critical role in mitochondrial fitness and cardiac protection1, 4. Cardiac DRP1 ablation in the adult heart evokes a progressive dilated cardiac myopathy and lethal heart failure1. Nevertheless, the conditional cardiac-specific DRP1 knock-out animals present a significantly longer survival rate compared with global DRP1 KO models1, 4, 5. We have described before the great importance for cardiac physiology of the strategic positioning of mitochondrial proteins in the cardiac tissue6, 7. Therefore, we hypothesize that DRP1 plays a regulatory role in cardiac physiology and mitochondrial fitness by preferentially accumulating at mitochondria and junctional sarcoplasmic reticulum (jSR) contact sites, where the high Ca2+ microdomain is formed during excitation-contraction (EC) coupling.

ObjectiveThis study aims to determine whether mitochondria-associated DRP1 is preferentially accumulated in the mitochondria and jSR contact sites, the mechanism responsible for such a biased distribution, and its functional implication.

Methods and ResultsUsing high-resolution imaging approaches, we found that mitochondria-associated DRP1 in cardiomyocytes was localized in the discrete regions where T-tubule, jSR, and mitochondria are adjacent to each other. Western blot results showed that mitochondria-bound DRP1 was restricted to the mitochondria-associated membranes (MAM), with undetectable levels in purified mitochondria. Furthermore, in comparison to the cytosolic DRP1, the membrane-bound DRP1 in SR and MAM fractions formed high molecular weight oligomers demosntratd by 2D blue native technique. In both electrically paced adult cardiomyocytes and Langendorff-perfused beating hearts, the oscillatory Ca2+ pulses preserved MAM-associated DRP1 accumulation. Interestingly, similar to DRP1, all mitochondria-bound {beta}-ACTIN only exists in MAM and not in the purified mitochondria.

Additionally, co-immunoprecipitation pulls down both DRP1 and {beta}-ACTIN together. Inhibition of {beta}-ACTIN polymerization with Cytochalasin D disrupts the tight association between DRP1 and {beta}-ACTIN. In cardiac-specific DRP1 knock-out mouse after 6 weeks of tamoxifen induction (DRP1icKo), the cardiomyocytes show disarray of sarcomere, a decrease of cardiac contraction, loss of mitochondrial membrane potential, significantly decreased spare respiratory capacity, and frequent occurrence of early after contraction (EAC), suggesting the heart is susceptible to arrhythmias and heart failure. Despite of this phenotype, DRP1icKo animals have longer life span than other DRP1 KO models. Strikingly, DRP1 levels are only modestly decreased in the MAM when compared with the rest of the cellular fractions. These preserved levels were accompanied by the preservation of the mitochondrial pool in the MAM fraction obtained from the DRP1icKO hearts.

ConclusionsThe results show that in adult cardiomyocytes, mitochondria bound DRP1 clusters in high molecular weight protein complexes at MAM. This clustering is fortified by EC coupling mediated Ca2+ transients and requires its interaction with {beta}-ACTIN. Together with the better preserved DRP1 levels in the DRP1icKO model in the MAM, we conclude that DRP1 is anchored at the mitochondria-SR interface through {beta}-ACTIN and positions itself to play a fundamental role in regulating mitochondrial quality control in the working heart.

sentences: Analysis of B-cell receptor repertoires in COVID-19 patients using deep embedded representations of protein sequences Analyzing B cell receptor (BCR) repertoires is immensely useful in evaluating ones immunological status. Conventionally, repertoire analysis methods have focused on comprehensive assessments of clonal compositions, including V(D)J segment usage, nucleotide insertions/deletions, and amino acid distributions. Here, we introduce a novel computational approach that applies deep-learning-based protein embedding techniques to analyze BCR repertoires. By selecting the most frequently occurring BCR sequences in a given repertoire and computing the sum of the vector representations of these sequences, we represent an entire repertoire as a 100-dimensional vector and eventually as a single data point in vector space. We demonstrate that this new approach enables us to not only accurately cluster BCR repertoires of coronavirus disease 2019 (COVID-19) patients and healthy subjects but also efficiently track minute changes in immune status over time as patients undergo treatment. Furthermore, using the distributed representations, we successfully trained an XGBoost classification model that achieved a mean accuracy rate of over 87% given a repertoire of CDR3 sequences.

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Vectorial Channeling as a Mechanism for Translational Control by Functional Prions and Condensates Translation of messenger RNA is regulated through a diverse set of RNA-binding proteins. A significant fraction of RNA-binding proteins contain prion-like domains which form functional prions. This raises the question of how prions can play a role in translational control. Local control of translation in dendritic spines by prions has been invoked in the mechanism of synaptic plasticity and memory. We show how channeling through diffusion and processive translation cooperate in highly ordered mRNA/prion aggregates as well as in less ordered mRNA/protein condensates depending on their sub-structure. We show the direction of translational control, whether it is repressive or activating, depends on the polarity of the mRNA distribution in mRNA/prion assemblies which determines whether vectorial channeling can enhance recycling of ribosomes. Our model also addresses the effect of changes of substrate concentration in assemblies that have been suggested previously to explain translation control by assemblies through the introduction of a potential of mean force biasing diffusion of ribosomes inside the assemblies. The results from the model are compared with the experimental data on translational control by two functional RNA-binding prions, CPEB involved in memory and Rim4 involved in gametogenesis.

Significance StatementmRNA/protein assemblies such as functional prions and condensates are involved in locally regulating translation in eukaryotic cells. The mode of regulation depends on the structure of these assemblies. We show that the vectorial processive nature of translation can couple to transport via diffusion so as to repress or activate translation depending on the structure of the RNA protein assembly. We find that multiple factors including diffusivity changes and free energy biases in the assemblies can regulate the translation rate of mRNA by changing the balance between substrate recycling and competition between mRNAs. We mainly focus on the example of CPEB, a functional prion that has been implicated in the mechanism of synaptic plasticity of neurons and in memory.

sentences: Ultrashort treatment with telacebec alone and with companion drugs in immunocompetent and immunosuppressed mouse footpad models of Buruli ulcer The antimicrobial treatment of Mycobacterium ulcerans infection, or Buruli ulcer (BU), has a long duration and is therefore burdensome and linked to indirect costs for affected patients. The new antimycobacterial drug telacebec (Q203) has previously shown promising treatment-shortening potential in mouse models of BU. In the present study, we investigated the potential of Q203 to reduce the treatment duration further. The first experiment investigated the possibility of cure by one, three or five doses of Q203 (2 mg/kg) with or without a companion drug (bedaquiline, BDQ, clofazimine, CFZ, or clarithromycin, CLR) in immunocompetent BALB/c mice. The second experiment assessed the effect of five doses of Q203 with or without BDQ or CFZ on Mycobacterium ulcerans infection of immunocompromised SCID-beige mice with the aim to evaluate the contribution of host immunity to treatment efficacy. In BALB/c mice, a treatment duration as short as 3 days was sufficient to prevent relapse in nearly all footpads and a single dose of Q203 with or without BDQ or CFZ prevented relapse in approximately 50% of footpads. Unlike in BALB/c mice, a small percentage of SCID-beige mouse footpads were culture-positive after a treatment duration of five days, highlighting an important role of host immunity for M. ulcerans clearance. Our results confirm the marked potency and prolonged bactericidal and sterilizing effects of Q203, even in immunocompromised SCID-beige mice.

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Size-dependent protein segregation creates a spatial switch for Notch signaling and function Aberrant cleavage of Notch by {gamma}-secretase is implicated in numerous diseases, but how cleavage is regulated in space and time is unclear. Here, we report that cadherin-based adherens junctions (cadAJs) are sites of high cell-surface {gamma}-secretase activity, as well as sites of constrained physical space that excludes {gamma}-secretase substrates having large extracellular domains (ECDs) like Notch. ECD shedding initiates drastic spatial relocalization of Notch to cadAJs, allowing enzyme-substrate interactions and downstream signaling. Spatial mutations by adjusting the ECD size or the physical constraint alter signaling. Dysregulation of this spatial switch promotes precocious differentiation of ventricular zone neural progenitor cells in vivo. We show the generality of this spatial switch for amyloid precursor protein proteolysis. Thus, cadAJs create spatially distinct biochemical compartments regulating cleavage events involving {gamma}-secretase and preventing aberrant activation of receptors.

One Sentence SummaryNotch cleavage by {gamma}-secretase is regulated through dynamic spatial control of receptors, adhesion molecules, and activating proteases

sentences: Contrasting impacts of environmental variability on the breeding biology of two sympatric small procellariiform seabirds in south-eastern Australia Seabirds play a vital role in marine ecosystems and are determinant sentinels of the productivity of their environments. The long-term study of their breeding biology and their responses to environmental variations can be used to monitor the effects of climate change on marine fauna. However, the ecological and physiological differences among seabirds induce a large range of responses complicating our understanding of the effects of environmental changes on marine ecosystems. The present study investigated the impact of environmental variability on breeding biology in two sympatric small Procellariiform species, the fairy prion (Pachyptila turtur) and the common diving petrel (Pelecanoides urinatrix), over four reproductive seasons (2017-2020) in Bass Strait, south-eastern Australia. Marine heatwaves had a negative effect on chick growth, breeding success, and induced a delay in laying dates in both species. While fairy prions maintained a relatively high breeding success and broadly constant breeding phenology, common diving petrels delayed the start of the breeding season by up to 50 days and experienced dramatic collapses in breeding success in years of high marine heat wave occurrence. The high wing loading and absence of stomach oils in the common diving petrel are likely to have limited the capacity of this species to increase foraging effort in years of low food availability.

sentences: Positive Feedback Regulation of fzd7 Expression Robustly Shapes Wnt Signaling Range in Early Heart Development Secreted molecules called morphogens govern tissue patterning in a concentration-dependent manner. However, it is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when patterning differentiation of a thin region. Wnt is a morphogen that organizes cardiac development; especially Wnt6 patterns the cardiogenic mesoderm to induce differentiation of a thin pericardium in Xenopus. It is, however, unclear how Wnt6 can pattern such a thin tissue. In this study, we reveal that a Wnt receptor, frizzled7, is expressed in a Wnt-dependent manner in the prospective heart region, and that this receptor-feedback is essential for shaping a steep gradient of Wnt. In addition, the feedback imparts robustness against fluctuations of Wnt ligand production and allows the system to reach a steady state quickly. We also found a Wnt antagonist sFRP1, which is expressed at the opposite side of Wnt source, accumulates on a novel type of heparan sulfate (HS), N-acetyl-rich HS, which is highly presented in the outer of cardiogenic mesoderm, achieving local inhibition of Wnt signaling by restricting sFRP1 spreading. These two intricate regulatory systems restrict Wnt signaling and ensure reproducible patterning of a thin pericardium tissue.

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Positive Selection and Heat-Response Transcriptomes Reveal Adaptive Features of the Arabidopsis Desert Relative, Anastatica hierochuntica O_LIPlant adaptation to a desert environment and its endemic heat stress is poorly understood at the molecular level. The naturally heat-tolerant Brassicaceae species Anastatica hierochuntica is an ideal extremophyte model to identify genetic adaptations that have evolved to allow plants to tolerate heat stress and thrive in deserts.
C_LIO_LIWe generated an A. hierochuntica reference transcriptome and pinpointed extremophyte adaptations by comparing Arabidopsis thaliana and A. hierochuntica transcriptome responses to heat and identifying positively selected genes in A. hierochuntica.
C_LIO_LIThe two species exhibit similar transcriptome adjustment in response to heat and the A. hierochuntica transcriptome does not exist in a constitutive heat "stress-ready" state. Furthermore, the A. hierochuntica global transcriptome as well as heat-responsive orthologs, display a lower basal and higher heat-induced expression than in A. thaliana. Genes positively selected in multiple extremophytes are associated with stomatal opening, nutrient acquisition, and UV-B induced DNA repair while those unique to A. hierochuntica are consistent with its photoperiod-insensitive, early-flowering phenotype.
C_LIO_LIWe suggest that evolution of a flexible transcriptome confers the ability to quickly react to extreme diurnal temperature fluctuations characteristic of a desert environment while positive selection of genes involved in stress tolerance and early flowering could facilitate an opportunistic desert lifestyle.
C_LI

sentences: Mechanical morphotype switching as an adaptive response in mycobacteria Bacterial stress tolerance during infection is commonly thought to be encoded by discrete biochemical stress-specific mechanisms 1. However, increasing evidence suggests that fluctuating conditions of stress drive biomechanical changes to bacterial components on varying cellular scales 2-5. Here, we use atomic-force microscopy (AFM) 6 to demonstrate that during macrophage infection, intracellular mycobacteria diverge into two mechanically distinct subpopulations exhibiting either a two-fold increase or decrease in mean cell surface stiffness. We describe this process as "mechanical morphotype switching", which we define as a phenotypic differentiation into two distinct, semi-stable mechanical cell states. Mycobacterial mechanical morphotype states are enriched by gene mutation and cytokine modulation of host macrophage responses, suggesting a molecular basis for switching and a role for host immunity in driving selection of discrete mechanical morphotypes. Crucially, mycobacterial mechanical morphotype switching enhances survival in macrophages of both non-pathogenic Mycobacterium smegmatis and pathogenic, M. abscessus. These results reveal that mycobacteria mechanically adapt to enhance intracellular viability. Our study highlights the importance of bacterial mechanical properties for optimised survival in host intracellular environments during pathogenesis.

sentences: Polymorphism of Genetic Ambigrams Double synonyms in the genetic code can be used as a tool to test competing hypotheses regarding ambigrammatic narnavirus genomes. Applying the analysis to recent observations of Culex narnavirus 1 and Zhejiang mosquito virus 3 ambigrammatic viruses indicates that the open reading frame on the complementary strand of the segment coding for RNA-dependent RNA polymerase does not code for a functional protein. Culex narnavirus 1 has been shown to possess a second segment, also ambigrammatic, termed  Robin. We find a comparable segment for Zhejiang mosquito virus 3, a moderately diverged relative of Culex narnavirus 1. Our analysis of Robin polymorphisms suggests that its reverse open reading frame also does not code for a functional protein. We make a hypothesis about its role.

sentences: Epigenetic clocks reveal a rejuvenation event during embryogenesis followed by aging The notion that germline cells do not age goes back to the 19th century ideas of August Weismann. However, being in a metabolically active state, they accumulate damage and other age-related changes over time, i.e., they age. For new life to begin in the same young state, they must be rejuvenated in the offspring. Here, we developed a new multi-tissue epigenetic clock and applied it, together with other aging clocks, to track changes in biological age during mouse and human prenatal development. This analysis revealed a significant decrease in biological age, i.e. rejuvenation, during early stages of embryogenesis, followed by an increase in later stages. We further found that pluripotent stem cells do not age even after extensive passaging and that the examined epigenetic age dynamics is conserved across species. Overall, this study uncovers a natural rejuvenation event during embryogenesis and suggests that the minimal biological age (the ground zero) marks the beginning of organismal aging.

sentences: The Mycobacterium tuberculosis protein O-phosphorylation landscape Bacterial phosphosignaling has long been synonymous with the histidine kinases of the two component systems, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignaling enzymes in eukaryotes, but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria remains unclear. Here, we explored the global signaling capacity of the STPKs in Mtb. We generated STPK loss-and gain-of-function strains and measured the resulting O-phosphorylation and transcriptional changes. This deep phosphoproteome shows that O-phosphorylation in Mtb is an underexplored protein modification that affects >70% of the proteome. The substrate-STPK interactions show an extensive interface with the transcriptional machinery, resulting in regulation of gene expression of [~]30% of Mtb genes. Mtb O-phosphorylation gives rise to an expansive, distributed, and cooperative network of a complexity that has previously only been associated with eukaryotic phosphosignaling networks.

sentences: Unbiased screen of human transcriptome reveals an unexpected role of 3'UTRs in translation initiation Although most eukaryotic mRNAs require a 5'-cap for translation initiation, some can also be translated through a poorly studied cap-independent pathway. Here we developed a circRNA-based system and unbiasedly identified more than 10,000 sequences in the human transcriptome that contain Cap-independent Translation Initiators (CiTIs). Surprisingly, most of the identified CiTIs are located in 3'UTRs, which mainly promote translation initiation in mRNAs bearing highly structured 5'UTR. Mechanistically, CiTI recruits several translation initiation factors including eIF3 and DHX29, which in turn unwind 5'UTR structures and facilitate ribosome scanning. Functionally, we showed that the translation of HIF1A mRNA, an endogenous DHX29 target, is antagonistically regulated by its 5'UTR structure and a new 3'-CiTI in response to hypoxia. Therefore, deletion of 3'-CiTI suppresses cell growth in hypoxia and tumor progression in vivo. Collectively, our study uncovers a new regulatory mode for translation where the 3'UTR actively participate in the translation initiation.

sentences: Cell Types of Origin in the Cell Free Transcriptome in Human Health and Disease Cell-free RNA (cfRNA) can be used to noninvasively measure dynamic and longitudinal physiological changes throughout the body. While there is considerable effort in the liquid biopsy field to determine disease tissue-of-origin, pathophysiology occurs at the cellular level. Here, we describe two approaches to identify cell type contributions to cfRNA. First we used Tabula Sapiens, a transcriptomic cell atlas of the human body to computationally deconvolve the cell-free transcriptome into a sum of cell type specific transcriptomes, thus revealing the spectrum of cell types readily detectable in the blood. Second, we used individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset to create cell type signature scores which can be used to infer the implicated cell types from cfRNA for a variety of diseases. Taken together, these results demonstrate that cfRNA reflects cellular contributions in health and disease from diverse cell types, potentially enabling determination of pathophysiological changes of many cell types from a single blood test.

sentences: Bacterial metatranscriptomes in wastewater can differentiate virally infected human populations Monitoring wastewater samples at building-level resolution screens large populations for SARS-CoV-2, prioritizing testing and isolation efforts. Here we perform untargeted metatranscriptomics on virally-enriched wastewater samples from 10 locations on the UC San Diego campus, demonstrating that resulting bacterial taxonomic and functional profiles discriminate SARS-CoV-2 status even without direct detection of viral transcripts. Our proof-of-principle reveals emergent threats through changes in the human microbiome, suggesting new approaches for untargeted wastewater-based epidemiology.

sentences: Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host-pathogen interactions. We chose to study further Yor1, a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1{Delta} strains revealed defects in non-lytic exocytosis and capsule size, when compared to wild-type strains. We detected no difference in growth rates, cell body size and vesicle secretion. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal-macrophage interaction.

sentences: The impact of SNP density on quantitative genetic analyses of body size traits in a wild population of Soay sheep Understanding the genetic architecture underpinning quantitative traits in wild populations is pivotal to understanding the processes behind trait evolution. The  animal model is a popular method for estimating quantitative genetic parameters such as heritability and genetic correlation and involves fitting an estimate of relatedness between individuals in the study population. Genotypes at genome-wide markers can be used to estimate relatedness; however, relatedness estimates vary with marker density, potentially affecting results. Increasing density of markers is also expected to increase the power to detect quantitative trait loci (QTL). We estimated heritability and performed genome-wide association studies (GWAS) on five body size traits in an unmanaged population of Soay sheep using two different SNP densities: a dataset of 37,037 genotyped SNPs, and an imputed dataset of 417,373 SNPs. Heritability estimates did not differ between the two SNP densities, but the high-density imputed SNP dataset revealed five new SNP-trait associations that were not found with the lower density dataset. Conditional GWAS analyses after fitting the most significant SNPs revealed two more novel SNP-trait associations.

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

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Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations ObjectiveTo determine if mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis.

DesignThe ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis.

SettingThe vivarium of a research laboratory

SubjectsBalb/c mice

Interventions: Mice were challenged with an LD80 dose of either lipopolysaccharide (LPS / endotoxin), TNF, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis.

Measurement and Main ResultsEach group of mice was checked daily for survivors, and Kaplan-Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs (1) were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time and dose dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animal if administered early and at a high dose.

ConclusionsOur conclusions are MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAbs efficacy is both time and dose dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens-of-thousands of lives annually and could result in improved antibiotic stewardship.

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Nonsynonymous mutations in fepR are associated with Listeria adaptation to low concentrations of benzalkonium chloride, but not increased survival in use level concentrations of benzalkonium chloride. Presence of and selection for Listeria spp. and Listeria monocytogenes that are less effectively inactivated by quaternary ammonium compounds (such as benzalkonium chloride [BC]) is a food safety concern, including in fresh produce environments. An initial MIC assay on 67 produce-associated Listeria strains showed that strains carrying BC resistance genes bcrABC (n=10) and qacH (n=1) showed higher MIC (4-6 mg/L BC) compared to strains lacking these genes (MIC of 1-2 mg/L BC). Serial passaging experiments that exposed the 67 strains to increasing BC concentrations revealed that 62/67 strains showed growth in BC concentrations above their parent MIC (range of 4-20 mg/L). Two serially passaged isolates were obtained for each parent strain and substreaked onto BHI agar in the absence of BC for seven rounds; 105/134 substreaked isolates showed higher substreaked MIC (range of 4 - 6 mg/L) compared to parent MIC. These results suggested isolates acquired genetic adaptations that confer BC resistance. Substreaked isolates were characterized by a combination of whole genome sequencing and Sanger sequencing of fepR, a local repressor of the MATE family efflux pump FepA. These data identified nonsynonymous fepR mutations in 48/67 isolates including 24 missense, 16 nonsense, and 8 frameshift mutations. The mean inactivation of substreaked isolates after exposure to use level concentrations of BC (300 mg/L) was 4.48 log, which was not significantly different from inactivation observed in parent strains. Serial passage experiments performed on cocultures of Listeria strains containing bcrABC or qacH did not yield growth at higher BC concentrations than monoculture experiments.

IMPORTANCEListeria resistance to quaternary ammonium compounds has been raised as a concern with regards to Listeria persistence in food environments, which can increase risk of product contamination, food recalls and foodborne illness outbreaks. Findings from our study show that individual Listeria strains can acquire genetic adaptations that confer resistance to low concentrations of benzalkonium chloride, but these genetic adaptations dont increase Listeria survival when exposed to concentrations of benzalkonium chloride used for food contact surface sanitation (300 mg/L). Our study also suggests that there is limited risk of benzalkonium chloride resistance genes bcrABC and qacH spreading through horizontal gene transfer and conferring enhanced resistance of L. monocytogenes to benzalkonium chloride. Overall, our study suggests that emergence of benzalkonium chloride resistant Listeria strains in food processing environments is of limited concern, as even strains adapted to gain higher MIC in vitro maintain full sensitivity to use level concentrations of benzalkonium chloride.

sentences: Plant immune system activation is necessary for efficient interaction with auxin secreting beneficial bacteria Plants continuously monitor the presence of microorganisms through their immune system to establish an adaptive response. Unlike immune recognition of pathogenic bacteria, how beneficial bacteria interact with the plant immune system is not well understood. Analysis of colonization of Arabidopsis thaliana by auxin producing beneficial bacteria revealed that activation of the plant immune system is necessary for efficient bacterial colonization and auxin secretion. A feedback loop is established in which bacterial colonization triggers an immune reaction and production of reactive oxygen species, which, in turn, stimulate auxin production by the bacteria. Auxin promotes bacterial survival and efficient root colonization, allowing the bacteria to inhibit fungal infection and promote plant health.

sentences: Generation of bright autobioluminescent bacteria by chromosomal integration of the improved lux operon ilux2 The bacterial bioluminescence system enables light production in living cells without an external luciferin. Due to its relatively low levels of light emission, many applications of bioluminescence imaging would benefit from an increase in brightness of this system. In this report a new approach of mutagenesis and screening of the involved proteins is described that is based on the identification of mutants with improved properties under rate-limiting reaction conditions. Multiple rounds of screening in Escherichia coli resulted in the operon ilux2 that contains 26 new mutations in the fatty acid reductase complex which provides the aldehyde substrate for the bioluminescence reaction. Chromosomal integration of ilux2 yielded an autonomously bioluminescent E. coli strain with 7-fold increased brightness compared to the previously described ilux operon. The ilux2 strain produces sufficient signal for the robust detection of individual cells and enables highly sensitive long-term imaging of bacterial propagation without a selection marker.

sentences: Characterization of peptide-protein relationships in protein ambiguity groups via bipartite graphs In bottom-up proteomics, proteins are enzymatically digested into peptides before measurement with mass spectrometry. The relationship between proteins and their corresponding peptides can be represented by bipartite graphs. We conduct a comprehensive analysis of bipartite graphs using quantified peptides from measured data sets as well as theoretical peptides from an in silico digestion of the corresponding complete taxonomic protein sequence databases. The aim of this study is to characterize and structure the different types of graphs that occur and to compare them between data sets. We observed a large influence of the accepted minimum peptide length during in silico digestion. When changing from theoretical peptides to measured ones, the graph structures are subject to two opposite effects. On the one hand, the graphs based on measured peptides are on average smaller and less complex compared to graphs using theoretical peptides. On the other hand, the proportion of protein nodes without unique peptides, which are a complicated case for protein inference and quantification, is considerably larger for measured data. Additionally, the proportion of graphs containing at least one protein node without unique peptides rises when going from database to quantitative level. The fraction of shared peptides and proteins without unique peptides as well as the complexity and size of the graphs highly depends on the data set and organism. Large differences between the structures of bipartite peptide-protein graphs have been observed between database and quantitative level as well as between analyzed species. In the analyzed measured data sets, the proportion of protein nodes without unique peptides ranged from 6.4% to 55.0%. This highlights the need for novel methods that can quantify proteins without unique peptides. The knowledge about the structure of the bipartite peptide-protein graphs gained in this study will be useful for the development of such algorithms.

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Interactions and information: Exploring task allocation in ant colonies using network analysis In animal societies, individuals may take on different roles to fulfil their own needs and the needs of their groups. Ant colonies display high levels of organisational complexity, with ants fulfilling different roles at different timescales (what is known as task allocation). Factors affecting task allocation can be at the individual level (e.g., physiology), or at the group level (e.g., the network of interactions). We focus on group level processes by exploring the relationship between interaction networks, task allocation and task switching using a previously published dataset (Mersch et al., 2013) tracking the behaviour of six Camponotus fellah colonies over 41 days. In our new analyses, our goal was to better explain the noisy process of task switching beyond simple age polyethism. First, we investigated the architecture of interaction networks using node (individual) level network measures and their relation to the individuals task - foraging, cleaning or nursing - and whether or not the ant switched tasks. We then explored how noisy information propagation was among ants, as a function of the colony composition (how many ants carried out which tasks), through the information-theoretic metric of Effective Information. Our results show that interaction history was tied to task allocation: ants who switched to a task are more likely to have interacted with other ants carrying out that task. The degree to which interactions related to task allocation, as well as the noise in those interactions, depended on which groups of ants were interacting. Overall, we showed that colony cohesion was stable even as ant-level network measures varied more for ants when they switched functional groups; thus, ant colonies maintained a high level of information flow as determined by network analysis, and ant functional groups played different roles in maintaining colony cohesion through varied information flows.

HighlightsO_LIWe analysed the interaction networks of six Camponotus fellah colonies
C_LIO_LIWe tested how centrality and information flow were tied to task switching
C_LIO_LINode-level network metrics and the information theoretic measure of Effective Information explained differences among functional groups
C_LIO_LIInteractions were correlated with task switching, but the strength of the correlation differed across functional groups
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sentences: Multidimensional brain-age prediction reveals altered brain developmental trajectory in psychiatric disorders Neuroimaging-based brain-age prediction has emerged as an important new approach for studying brain development. However, brain regions change in different ways and at different rates. Unitary brain-age indices used in previous studies represent developmental status averaged across the whole brain and therefore do not capture the divergent developmental trajectories of various brain structures. Importantly, this staggered developmental unfolding, determined by genetics and postnatal experience, is implicated in the progression of psychiatric and neurological disorders. Here we propose an analytic method for computing a multidimensional brain-age index (MBAI) that provides regional age predictions. Using a database of 556 subjects (ages 8-21) that includes psychiatric and neurological patients as well as healthy controls, we conducted robust regression and cluster analyses to identify clusters of imaging features with distinct developmental trajectories. We then built machine-learning models to obtain brain-age predictions from each of the identified clusters to form the MBAI. Our results show that the MBAI provides a flexible analysis of region-specific brain-age changes that are invisible to unidimensional brain-age prediction methods. Importantly, brain ages computed from region-specific feature clusters contain complementary information and demonstrate differential ability to classify disorder groups (e.g., specific phobia, depression, ADHD) from healthy controls. Compared to unidimensional brain-age indices, we show that the MBAI is sensitive to alterations in brain structures and captures distinct regional change patterns which may serve as biomarkers that may contribute to our understanding of healthy and pathological brain development and to the characterization, diagnosis, and, potentially, treatment of various disorders.

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Predicting vertical ground reaction forces from 3D accelerometery using reservoir computers leads to accurate gait event detection Accelerometers are low-cost measurement devices that can readily be used outside the lab. However, determining isolated gait events from accelerometer signals, especially foot-off events during running, is an open problem. We outline a two-step approach where machine learning serves to predict vertical ground reaction forces from accelerometer signals, followed by force-based event detection.

We collected shank accelerometer signals and ground reaction forces from five adults during comfortable walking and running on an instrumented treadmill. We trained one common reservoir computer using segmented data using both walking and running data. Despite being trained on just a small number of strides, this reservoir computer predicted vertical ground reaction forces in continuous gait with excellent quality. The subsequent foot contact and foot off event detection proved highly accurate when compared to the golden standard based on co-registered ground reaction forces. Our proof-of-concept illustrates the capacity of combining accelerometery with machine learning for detecting isolated gait events irrespective of mode of locomotion.

sentences: Water stress and nitrogen supply affect floral traits and pollination in the white mustard, <Sinapis alba> (Brassicaceae) Changes in environmental conditions are likely to have a complex effect on the growth of plants, their phenology, plant-pollinator interactions, and reproductive success. The current world is facing an ongoing climate change along with other human-induced environmental changes. Most research has focused on the impact of increasing temperature as a major driving force for climate change, but other factors may have important impacts on plant traits and pollination too and these effects may vary between different seasons. In addition, it is likely that the effects of multiple environmental factors, such as increasing temperature, water stress, and nitrogen enrichment are not independent. Therefore, we tested the impact of three key factors - season, water, and nitrogen supply - on plant traits, pollination, and seed production in Sinapis alba (Brassicaceae). Plants were grown in three seasons with two levels of water and nitrogen supply in a factorial design. We collected data on multiple vegetative and floral traits and assessed the response of pollinators in the field. Additionally, we evaluated the effect of growing conditions on seed set in plants exposed to pollinators and in hand-pollinated plants. Our results show that water stress impaired vegetative growth, decreased flower production, reduced visitation by pollinators and seed set, while nitrogen availability played an important role in nectar production. Seasonality modulated the effect of water and nitrogen availability on vegetative and floral traits and strongly affected flowering phenology and flower production. We demonstrated that changes in water and nitrogen availability alter plant vegetative and floral traits, which impacts flower visitation and consequently plant reproduction. We conclude that ongoing environmental changes such as altered precipitation regimes and nitrogen enrichment may thus affect plant-pollinator interactions with negative consequences for the reproduction of wild plants and insect-pollinated crops.

sentences: Rapid carbon accumulation at a saltmarsh restored by managed realignment far exceeds carbon emitted in site construction Increasing attention is being paid to the carbon sequestration and storage services provided by coastal blue carbon ecosystems such as saltmarshes. Sites restored by managed realignment, where existing sea walls are breached to reinstate tidal inundation to the land behind, have considerable potential to accumulate carbon through deposition of sediment brought in by the tide and burial of vegetation in the site. While this potential has been recognised, it is not yet a common motivating factor for saltmarsh restoration, partly due to uncertainties about the rate of carbon accumulation and how this balances against the greenhouse gases emitted during site construction. We use a combination of field measurements over four years and remote sensing to quantify carbon accumulation at a large managed realignment site, Steart Marshes, UK. Sediment accumulated rapidly at Steart Marshes (mean of 75 mm yr-1) and had a high carbon content (4.4% total carbon, 2.2% total organic carbon), resulting in carbon accumulation of 36.6 t ha-1 yr-1 total carbon (19.4 t ha- 1 yr-1 total organic carbon). This rate of carbon accumulation is an order of magnitude higher than reported in many other restored saltmarshes, and is higher although more similar to values previously reported from another hypertidal system (Bay of Fundy, Canada). The estimated carbon emissions associated with the construction of the site were [~]2-4% of the observed carbon accumulation during the study period, supporting the view that managed realignment projects in such settings are likely to have significant carbon accumulation benefits. We outline further considerations that are needed to move towards a full carbon budget for saltmarsh restoration.

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An efficient coalescent epoch model for Bayesian phylogenetic inference We present a two headed approach called Bayesian Integrated Coalescent Epoch PlotS (BICEPS) for efficient inference of coalescent epoch models. Firstly, we integrate out population size parameters and secondly we introduce a set of more powerful Markov chain Monte Carlo (MCMC) proposals for flexing and stretching trees. Even though population sizes are integrated out and not explicitly sampled through MCMC, we are still able to generate samples from the population size posteriors. This allows demographic reconstruction through time and estimating the timing and magnitude of population bottlenecks and full population histories. Altogether, BICEPS can be considered a more muscular version of the popular Bayesian skyline model.

We demonstrate its power and correctness by a well calibrated simulation study. Furthermore, we demonstrate with an application to SARS-CoV-2 genomic data that some analyses that have trouble converging with the traditional Bayesian skyline prior and standard MCMC proposals can do well with the BICEPS approach.

BICEPS is available as open source package for BEAST 2 under GPL license and has a user friendly graphical user interface. Bayesian phylogenetics, coalescent model, BEAST 2, BICEPS

sentences: Modeling the stepwise extension of recombination suppression on sex chromosomes and other supergenes through deleterious mutation sheltering Many organisms have sex chromosomes with large non-recombining regions that have expanded stepwise, generating "evolutionary strata" of differentiation. The reasons for this remain poorly understood, but the principal hypotheses proposed to date are based on antagonistic selection due to differences between sexes. However, it has proved difficult to obtain empirical evidence of a role for sexually antagonistic selection in extending recombination suppression, and antagonistic selection has been shown to be unlikely to account for the evolutionary strata observed on fungal mating-type chromosomes. There may, therefore, be other mechanisms involved in the extension of non-recombining regions. We show here, by mathematical modeling and stochastic simulation, that recombination suppression on sex chromosomes and around supergenes can expand in a stepwise manner under a wide range of parameter values simply because it shelters recessive deleterious mutations, which are ubiquitous in genomes. Permanently heterozygous alleles, such as the maledetermining allele in XY systems, protect linked chromosomal inversions against the expression of their recessive mutation load, leading to the successive accumulation of inversions around these alleles without the need for antagonistic selection. Similar results were obtained with models assuming recombination-suppressing mechanisms other than chromosomal inversions, and for supergenes other than sex chromosomes, including those without XY-like asymmetry, such as fungal mating-type chromosomes. However, inversions capturing a permanently heterozygous allele were found to be less likely to spread when the mutation load was lower (e.g. under conditions of large effective population size, low mutation rates and high dominance coefficients). This may explain why sex chromosomes remain homomorphic in some organisms but are highly divergent in others. Here, we explicitly state and model a simple and testable hypothesis explaining the existence of stepwise extensions of recombination suppression on sex chromosomes, which can also be applied to mating-type chromosomes and supergenes in general.

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Absolute Scaling of Single-Cell Transcriptomes Reveals Pervasive Hypertranscription in Adult Stem and Progenitor Cells Hypertranscription facilitates biosynthetically demanding cellular state transitions through global upregulation of the nascent transcriptome. Despite its potential widespread relevance, documented examples of hypertranscription remain few and limited predominantly to early development. This limitation is in large part due to the fact that modern sequencing approaches, including single-cell RNA sequencing (scRNA-seq), generally assume similar levels of transcriptional output per cell. Here, we use molecule counting and spike-in normalization to develop absolute scaling of single-cell RNA sequencing data. Absolute scaling enables an estimation of total transcript abundances per cell, which we validate in embryonic stem cell (ESC) and germline data and apply to adult mouse organs at steady-state or during regeneration. The results reveal a remarkable dynamic range in transcriptional output among adult cell types. We find that many different multipotent stem and progenitor cell populations are in a state of hypertranscription, including in the hematopoietic system, intestine and skin. Hypertranscription marks cells with multilineage potential in adult organs, is redeployed in conditions of tissue injury, and can precede by 1-2 days bursts of proliferation during regeneration. In addition to the association between hypertranscription and the stem/progenitor cell state, we dissect the relationship between transcriptional output and cell cycle, ploidy and secretory behavior. Our analyses reveal a common set of molecular pathways associated with hypertranscription across adult organs, including chromatin remodeling, DNA repair, ribosome biogenesis and translation. Our findings introduce an approach towards maximizing single-cell RNA-seq profiling. By applying this methodology across a diverse collection of cell states and contexts, we put forth hypertranscription as a general and dynamic cellular program that is pervasively employed during development, organ maintenance and regeneration.

SUMMARY STATEMENTAbsolute scaling of single-cell transcriptomic data reveals highly dynamic global levels of transcription across adult cell lineages

sentences: Neural signature of everyday function in older adults at-risk of cognitive impairment Assessment of everyday activities are central to the diagnosis of pre-dementia and dementia. Yet, little is known about the brain substrates and processes that contribute to everyday functional impairment, particularly during early stages of cognitive decline. We investigated everyday function using a complex gait task in normal older adults stratified by risk of cognitive impairment. We applied a novel EEG approach, which combines electroencephalographic with 3D-body tracking technology to measure brain-gait dynamics with millisecond precision while participants are in motion. Twenty-six participants (mean age = 74.9 years) with cognitive and everyday functional profiles within the normal range for their age and sex were ranked for risk of cognitive impairment. We used the Montreal Cognitive Assessment battery, a global index of cognition with a range from 0 to 30, to classify individuals as being at higher (22-26) and lower risk (27+). Individuals walking on a treadmill were exposed to visual perturbation designed to destabilize gait. Assuming that brain changes precede behavioral decline, we predicted that older adults increase step width to gain stability, yet the underlying neural signatures would be different for lower versus higher risk individuals. When pooling across risk groups, we found that step width increased and fronto-parietal activation shifted from transient, during swing phases, to sustained across the gait cycle during visually perturbed input. As predicted, step width increased in both groups but underlying neural signatures were different. Fronto-medial theta (3-7Hz) power of gait-related brain oscillations were increased in higher risk individuals during both perturbed and unperturbed inputs. On the other hand, left central gyri beta (13-28Hz) power was decreased in lower risk individuals, specifically during visually perturbed input. Finally, relating MoCA scores to spectral power pooled across fronto-parietal regions, we found associations between increased theta power and worse MoCA scores and between decreased beta power and better MoCA scores.Able-bodied older adults at-risk of cognitive impairment are characterized by unique neural signatures of mobility. Stronger reliance on frontomedial theta activation in at-risk individuals may reflect higher-order compensatory responses for deterioration of basic sensorimotor processes. Region and spectral-specific signatures of mobility may provide brain targets for early intervention against everyday functional decline.

sentences: Genomic prediction with allele dosage information in highly polyploid species Several studies have shown how to leverage allele dosage information to improve the accuracy of genomic selection models in autotetraploids. In this study we expanded the methodology used for genomic selection in autotetraploids to higher (and mixed) ploidy levels. We adapted the models to build covariance matrices of both additive and digenic dominance effects that are subsequently used in genomic selection models. We applied these models using estimates of ploidy and allele dosage to sugarcane and sweet potato datasets and validated our results by also applying the models in simulated data. For the simulated datasets, including allele dosage information led up to 140% higher mean predictive abilities in comparison to using diploidized markers. Including dominance effects was highly advantageous when using diploidized markers, leading to mean predictive abilities which were up to 115% higher in comparison to only including additive effects. When the frequency of heterozygous genotypes in the population was low, such as in the sugarcane and sweet potato datasets, there was little advantage in including allele dosage information in the models. Overall, we show that including allele dosage can improve genomic selection in highly polyploid species under higher frequency of different heterozygous genotypic classes and high dominance degree levels.

sentences: Single cell transcriptomics and developmental trajectories of murine cranial neural crest cell fate determination and cell cycle progression Cranial neural crest (NC) cells migrate long distances to populate the future craniofacial regions and give rise to various tissues, including facial cartilage, bones, connective tissues, and cranial nerves. However, the mechanism that drives the fate determination of cranial NC cells remains unclear. Using single-cell RNA sequencing combined genetic fate mapping, we reconstructed developmental trajectories of cranial NC cells, and traced their differentiation in mouse embryos. We identified four major cranial NC cell lineages at different status: pre-epithelial-mesenchymal transition, early migration, NC-derived mesenchymal cells, and neural lineage cells from embryonic days 9.5 to 12.5. During migration, the first cell fate determination separates cranial sensory ganglia, the second generates mesenchymal progenitors, and the third separates other neural lineage cells. We then focused on the early facial prominences that appear to be built by undifferentiated, fast-dividing NC cells that possess similar transcriptomic landscapes, which could be the drive for the facial developmental robustness. The post-migratory cranial NC cells exit the cell cycle around embryonic day 11.5 after facial shaping is completed and initiates further fate determination and differentiation processes. Our results demonstrate the transcriptomic landscapes during dynamic cell fate determination and cell cycle progression of cranial NC lineage cells and also suggest that the transcriptomic regulation of the balance between proliferation and differentiation of the post-migratory cranial NC cells can be a key for building up unique facial structures in vertebrates.

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The co-stimulatory activity of Tim-3 requires Akt and MAPK signaling and immune synapse recruitment Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic T cell activation, including in chronic infection and solid tumors. We and others previously reported that Tim-3 exerts apparently paradoxical co-stimulatory activity in T cells (and other cells), including enhancement of ribosomal S6 protein phosphorylation (pS6). Here we examined the upstream signaling pathways that control Tim3-mediated increases in pS6 in T cells. We have also defined the localization of Tim-3 relative to the T cell immune synapse and impacts on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired Tim-3 co-stimulation of pS6. Strikingly, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in the context of a chimeric antigen receptor still allowed for robust T cell activation. Our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.

One Sentence SummaryHere we define elements of signaling and localization associated with Tim-3 co-stimulatory function in T cells.

sentences: Design Maps of TRPM8 as a Thermometer for Cold Detection The menthol sensor TRPM8 can be activated by cold and thus serves as a thermometer in a primary afferent sensory neuron for noxious cold detection. However, the underlying design principle is unknown. Here, a hairpin topological structural model and graph theory were prepared to test a role of the cold-dependent hairpin formation in the cold-evoked gating pathway of TRPM8. The results showed that the formation of a large lipid-dependent hairpin initiates a low temperature threshold in favor of TRPM8 activation. Furthermore, two smaller hairpins, which enhance the coupled interactions of the voltage-sensor-like domain with both the pore domain and the TRP domain, can stabilize the cold efficacy and work as a fuse to prevent cold denaturation. The cold-induced hairpin rearrangements along the gating pathway may be necessary for the high cold sensitivity. This hairpin model may provide a structural basis for activation of the thermo-gated TRP channels at low temperature.

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Recombination facilitates adaptive evolution in rhizobial soil bacteria Homologous recombination is expected to increase natural selection efficacy by decoupling the fate of beneficial and deleterious mutations and by readily creating new combinations of beneficial alleles. Here, we investigate how the proportion of amino acid substitutions fixed by adaptive evolution () depends on the recombination rate in bacteria. We analyze 3086 core protein-coding sequences from 196 genomes belonging to five closely-related species of the genus Rhizobium. These genes are found in all species and do not display any signs of introgression between species. We estimate  using the site frequency spectrum (SFS) and divergence data for all pairs of species. We evaluate the impact of recombination within each species by dividing genes into three equally sized recombination classes based on their average level of intragenic linkage disequilibrium. We find that  varies from 0.07 to 0.39 across species and is positively correlated with the level of recombination. This is both due to a higher estimated rate of adaptive evolution and a lower estimated rate of non-adaptive evolution, suggesting that recombination both increases the fixation probability of advantageous variants and decreases the probability of fixation of deleterious variants. Our results demonstrate that homologous recombination facilitates adaptive evolution measured by  in the core genome of prokaryote species in agreement with studies in eukaryotes.

Significance statementWhether intraspecific homologous recombination has a net beneficial or detrimental effect on adaptive evolution is largely unexplored in natural bacterial populations. We address this question by evaluating polymorphism and divergence data across the core genomes of 196 bacterial sequences--belonging to five closely related species of the genus Rhizobium. We show that the proportion of amino acid changes fixed due to adaptive evolution () increases with an increased recombination rate. This correlation is observed both in the interspecies and intraspecific comparisons. By using a population genetics approach our results demonstrate that homologous recombination directly impacts the efficacy of natural selection in the core genome of prokaryotes, as previously reported in eukaryotes.

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Orbitofrontal cortex and learning predictions of state transitions Learning the transition structure of the environment - the probabilities of transitioning from one environmental state to another - is a key prerequisite for goal-directed planning and model-based decision making. To investigate the role of the orbitofrontal cortex (OFC) in goal-directed planning and decision making, we used fMRI to assess univariate and multivariate activity in the OFC while humans experienced state transitions that varied in degree of surprise. Converging with recent evidence, we found that OFC activity was related to greater learning about transition structure. However, the observed relationship was inconsistent with a straightforward interpretation of OFC activity as representing a state prediction error that would facilitate learning of transitions via error-correcting mechanisms. The state prediction error hypothesis predicts that OFC activity at the time of observing an outcome should increase expectation of that same observed outcome on subsequent trials. Instead, our results showed that OFC activity was associated with increased expectation of the more probable outcome; that is, with more optimal predictions. These results suggest that the OFC is involved in updating or reinforcing a learned transition model on a trial-by-trial basis, specifically for the currently observed cue-outcome associations. Our findings add to the evidence of OFC involvement in learning state-to-state transition structure, while providing new constraints for algorithmic hypotheses regarding how these transitions are learned.

Significance StatementThe orbitofrontal cortex (OFC) has been implicated in model-based decision making--the kind of decisions that result from planning using an "environment model" of how current actions affect our future states. However, the widely suggested role of the OFC in representing expected values of future states is not sufficient to explain why the OFC would be critical for planning in particular. A new line of evidence implicates the OFC in learning about transition structure of the environment - a key component of the "environment model" used for planning. We investigate this function, adding to the growing literature on the role of the OFC in learning and decision making, while unveiling new questions about the algorithmic role of OFC in goal-directed planning.

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PhyloHerb: A phylogenomic pipeline for processing genome skimming data for plants O_LIPremise of the study: The application of high throughput sequencing, especially to herbarium specimens, is greatly accelerating biodiversity research. Among various techniques, low coverage Illumina sequencing of total genomic DNA (genome skimming) can simultaneously recover the plastid, mitochondrial, and nuclear ribosomal regions across hundreds of species. Here, we introduce PhyloHerb -- a bioinformatic pipeline to efficiently and effectively assemble phylogenomic datasets derived from genome skimming.
C_LIO_LIMethods and Results: PhyloHerb uses either a built-in database or user-specified references to extract orthologous sequences using BLAST search. It outputs FASTA files and offers a suite of utility functions to assist with alignment, data partitioning, concatenation, and phylogeny inference. The program is freely available at https://github.com/lmcai/PhyloHerb/.
C_LIO_LIConclusions: Using published data from Clusiaceae, we demonstrated that PhyloHerb can accurately identify genes using highly fragmented assemblies derived from sequencing older herbarium specimens. Our approach is effective at all taxonomic depths and is scalable to thousands of species.
C_LI

sentences: Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional Bayesian approaches Calling germline SNP variants from bisulfite-converted sequencing data poses a challenge for conventional software, which have no inherent capability to dissociate true polymorphisms from artificial mutations induced by the chemical treatment. Nevertheless, SNP data is desirable both for genotyping and to understand the DNA methylome in the context of the genetic background. The confounding effect of bisulfite conversion can be resolved by observing differences in allele counts on a per-strand basis, whereby artificial mutations are reflected by non-complementary base pairs. Herein, we present a computational pre-processing approach for adapting sequence alignment data, thus indirectly enabling downstream analysis in this manner using conventional variant calling software such as GATK or Freebayes. In comparison to specialised tools, the method represents a marked improvement in precision-sensitivity based on high-quality, published benchmark datasets for both human and model plant variants.

sentences: Superscan: Supervised Single-Cell Annotation Automated cell type annotation of single-cell RNA-seq data has the potential to significantly improve and streamline single cell data analysis, facilitating comparisons and meta-analyses. However, many of the current state-of-the-art techniques suffer from limitations, such as reliance on a single reference dataset or marker gene set, or excessive run times for large datasets. Acquiring high-quality labeled data to use as a reference can be challenging. With CITE-seq, surface protein expression of cells can be directly measured in addition to the RNA expression, facilitating cell type annotation. Here, we compiled and annotated a collection of 16 publicly available CITE-seq datasets. This data was then used as training data to develop Superscan, a supervised machine learning-based prediction model. Using our 16 reference datasets, we benchmarked Superscan and showed that it performs better in terms of both accuracy and speed when compared to other state-of-the-art cell annotation methods. Superscan is pre-trained on a collection of primarily PBMC immune datasets; however, additional data and cell types can be easily added to the training data for further improvement. Finally, we used Superscan to reanalyze a previously published dataset, demonstrating its applicability even when the dataset includes cell types that are missing from the training set.

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On the topochronic map of the human brain dynamics Two structurally connected brain regions are more likely to interact, with the lengths of the structural bundles, their widths, myelination, and the topology of the structural connectome influencing the timing of the interactions. We introduce an in vivo approach for measuring functional delays across the whole brain using magneto/electroencephalography and integrating them with the structural bundles. The resulting topochronic map of the functional delays/velocities shows that larger bundles have faster velocities. We estimated the topochronic map in multiple sclerosis patients, who have damaged myelin sheaths, and controls, demonstrating greater delays in patients across the network and that structurally lesioned tracks were slowed down more than unaffected ones. We provide a novel framework for estimating functional transmission delays in vivo at the single-subject and single-fiber level.

One-Sentence SummaryA non-invasive estimation of the individual deterministic spatio-temporal scaffold underlying the evolution of brain dynamics.

sentences: Spike-in normalization for single-cell RNA-seq reveals dynamic global transcriptional activity mediating anti-cancer drug response The transcriptional plasticity of cancer cells promotes intercellular heterogeneity in response to anti-cancer drugs and facilitates the generation of subpopulation surviving cells. Characterizing single-cell transcriptional heterogeneity after drug treatments can provide mechanistic insights into drug efficacy. Here we used single-cell RNA-seq to examine transcriptomic profiles of cancer cells treated with paclitaxel, celecoxib, and the combination of the two drugs. By normalizing the expression of endogenous genes to spike-in molecules, we found that celluar mRNA abundance shows dynamic regulation after drug treatment. Using a random forest model, we identified gene signatures classifying single cells into three states: transcriptional repression, amplification, and control-like. Treatment with paclitaxel or celecoxib alone generally repressed gene transcription across single cells. Interestingly, the drug combination resulted in transcriptional amplification and hyperactivation of mitochondrial oxidative phosphorylation pathway linking to enhanced cell killing efficiency. Finally, we identified a regulatory module enriched with metabolism and inflammation-related genes activated in a subpopulation of paclitaxel-treated cells, the expression of which predicted paclitaxel efficacy across cancer cell lines and in vivo patient samples. Our study highlights the dynamic global transcriptional activity driving single-cell heterogeneity during drug response and emphasizes the importance of adding spike-in molecules to study gene expression regulation using single-cell RNA-seq.

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The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.

Author SummarySARS-CoV-2 is the latest pathogenic coronavirus to emerge as a public health threat. We create a database of proximal host proteins to 17 SARS-CoV-2 viral proteins. We validate that NSP1 is proximal to the EIF3 translation initiation complex and is a potent inhibitor of translation. We also identify ORF6 antagonism of RNA-mediate innate immune signaling. We produce a database of potential host targets of the viral protease NSP5, and create a fluorescence-based assay to screen cleavage of peptide sequences. We believe that this data will be useful for identifying roles for many of the uncharacterized SARS-CoV-2 proteins and provide insights into the pathogenicity of new or emerging coronaviruses.

sentences: Early identification of cooperative fragments for protein-protein interaction stabilization Modulating protein-protein interactions (PPIs) is an effective approach to drug discovery, with several drugs in the clinic that inhibit PPIs. The orthogonal approach of PPI stabilization has developed slowly, a function of the complicated dynamics of multi-component protein complexes. In contrast to PPI inhibition, where ligand affinity is the driving parameter for efficacy, cooperativity is frequently the directing variable for PPI stabilization. Here we show how STD NMR allows for early-stage detection of cooperativity using the hub protein 14-3-3, a focused library of fragments and several 14-3-3 partner proteins. Further, we validate that the observed enhancement in STD signal is a function of cooperativity of the ternary 14-3-3 complex, using mutagenesis and X-ray crystallography. Additionally, we assess the differential cooperativity of three fragments in a panel of 14-3-3 interaction partners. Finally, we demonstrate how selective 14-3-3 complex formation is a function of cooperativity effects

sentences: Comparative Analysis of Genetically-Modified Crops: Conditional Equivalence Criteria The comparative assessment of genetically-modified (GM) crops relies on the principle of substantial equivalence, which states that such products should be compared to conventional counterparts that have an established history of safe use. In an effort to operationalize this principle, the GMO Panel of the European Food Safety Authority proposed an equivalence test that directly compares a GM test variety with a set of unrelated, conventionally-bred reference varieties with part of the difference as the known background of the test (the same as the given control). The criterion of the EFSA test, however, is defined solely by genotypic differences between the non-traited control and reference varieties (i.e. the background effect) while assuming the so-called GM trait effect as zero. As the outcome of an EFSA equivalence test is determined primarily by the similarity, or lack thereof, of the control and references, a conditional equivalence criterion is proposed in this investigation that focuses on "unintended" effects of a GM trait which is irrespective of the (random) genotypic value of a given control. The new criterion also includes a mean-scaled standard similar to the 80-125% rule for bioequivalence assessment practiced in the pharmaceutical industry as an alternative when the reference variation is zero or close to zero. In addition, optional criteria are proposed with a step-wise procedure to control the rate of false negatives (non-equivalence by chance) providing a comprehensive assessment under multiple comparisons. An application to maize grain composition data demonstrates that the conditional equivalence criterion provides effect-specific and more robust assessment of equivalence than the EFSA criterion did, especially for GM traits showing negligible or no unintended effects which are likely true for most traits in the current market.

sentences: JIP3 regulates bi-directional organelle transport in neurons through its interaction with dynein and kinesin-1 The conserved MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. Whether and how JIP3s interaction with dynein and kinesin-1 contributes to this critical organelle clearance function is unclear. Using purified recombinant human proteins, we show that dynein light intermediate chain (DLIC) binds to the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in a hydrophobic pocket of the RH1 domain, previously shown to abrogate RILPL2 binding to myosin Va, abrogates the binding of JIP3/4 and RILP to DLIC without perturbing the interaction between the JIP3 RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3s leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bi-directional organelle transport.

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Coverage-preserving sparsification of overlap graphs for long-read assembly Read-overlap-based graph data structures play a central role in computing de novo genome assembly using long reads. Many assembly tools use the string graph model [Myers, Bioinformatics 2005] to sparsify overlap graphs. Graph sparsification improves accuracy by removing spurious and redundant connections. However, a graph model must be coverage-preserving, i.e., it must ensure that each chromosome can be spelled as a walk in the graph, given sufficient sequencing coverage. This property becomes even more important for diploid genomes, polyploid genomes and metagenomes where there is a risk of losing haplotype-specific information.

We develop a novel theoretical framework under which the coverage-preserving properties of a graph model can be analysed. We first prove that de Bruijn graph and overlap graph models are guaranteed to be coverage-preserving. We also prove that the standard string graph model lacks this guarantee. The latter result is consistent with the observation made in [Hui et al., 2016; Nurk et al., 2021] that removal of contained reads during string graph construction can lead to coverage gaps. To remedy this, we propose practical heuristics that are well-supported by our theoretical results to sparsify overlap graphs. In our experiments using simulated long reads from HG002 human diploid genome, we find that 98 and 5 coverage gaps are introduced by ignoring contained reads from nanopore and HiFi long-read datasets respectively. We test the proposed heuristics for deciding which contained reads should be retained to avoid the coverage gaps. The proposed method retains a small fraction of contained reads (1 - 2%) and closes all coverage gaps. This compares favorably to other known solutions for this problem.

sentences: Waffle Method: A general and flexible approach for FIB-milling small and anisotropically oriented samples Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens that do not vitrify well, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and cellular specimens that distribute poorly across grid squares. Here we present a general approach we call the  Waffle Method which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation of the spores on the grid. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with both manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. Additionally, we describe applications of the Waffle Method which are currently being explored. We propose the Waffle Method as a way to achieve many of the advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

sentences: Optogenetic stimulation of medial amygdala GABA neurons with kinetically different channelrhodopsin variants yield opposite behavioral outcomes Optogenetic manipulation of genetically-specified neuron populations has become a key tool in circuit neuroscience. The medial amygdala (MeA) receives pheromone information about conspecifics and has crucial functions in social behaviors; interestingly, this amygdalar structure contains a majority of GABAergic projection neurons. A previous study showed that optogenetic activation of MeA GABA neurons with channelrhodopsin-2H134R (ChR2) strongly enhanced inter-male aggression (Hong et al. 2014, Cell). When we attempted to reproduce these findings, accidentally using a faster channelrhodopsin variant (channelrhodopsin-2H134R,E123T or ChETA), we found the opposite results. We therefore systematically compared the behavioral outcome of optogenetic stimulation of MeApd GABA neurons with ChETA versus ChR2, employing two widely used AAV serotypes. This revealed that optogenetic stimulation with ChETA suppressed aggression, whereas optogenetic stimulation with ChR2 increased aggression. Recordings of membrane potential changes following optogenetic stimulation with ChETA versus ChR2 revealed larger plateau depolarizations, smaller action potential amplitudes, and larger local inhibition of neighboring inhibitory neurons with ChR2 as compared to ChETA. Our study shows that channelrhodopsin variants have to be chosen with care for in-vivo optogenetic experiments. Furthermore, the role of MeApd GABA neurons in aggression control should be re- evaluated.

sentences: SARS-CoV-2 Permissive Glioblastoma Cell Line for High Throughput Antiviral Screening Despite the great success of the administered vaccines against SARS-CoV-2, the virus can still spread, as evidenced by the current circulation of the highly contagious Omicron variant. This emphasizes the additional need to develop effective antiviral countermeasures. In the context of early preclinical studies for antiviral assessment, robust cellular infection systems are required to screen drug libraries. In this study, we reported the implementation of a human glioblastoma cell line, stably expressing ACE2, in a SARS-CoV-2 cytopathic effect (CPE) reduction assay. These glioblastoma cells, designated as U87.ACE2+, expressed ACE2 and cathepsin B abundantly, but had low cellular levels of TMPRSS2 and cathepsin L. The U87.ACE2+ cells fused highly efficiently and quickly with SARS-CoV-2 spike expressing cells. Furthermore, upon infection with SARS-CoV-2 wild-type virus, the U87.ACE2+ cells displayed rapidly a clear CPE that resulted in complete cell lysis and destruction of the cell monolayer. By means of several readouts we showed that the U87.ACE2+ cells actively replicate SARS-CoV-2. Interestingly, the U87.ACE2+ cells could be successfully implemented in an MTS-based colorimetric CPE reduction assay, providing IC50 values for Remdesivir in the low nanomolar range. Lastly, the U87.ACE2+ cells were consistently permissive to all tested SARS-CoV-2 variants of concern, including the current Omicron variant. Thus, ACE2 expressing glioblastoma cells are highly permissive to SARS-CoV-2 with productive viral replication and with the induction of a strong CPE that can be utilized in high-throughput screening platforms.

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The impact of genetic risk for Alzheimers disease on the structural brain networks of young adults To facilitate pre-symptomatic diagnosis of late-onset Alzheimers disease, non-invasive imaging biomarkers could be combined with genetic risk information. In this work, we investigated the structural brain networks of young adults in relation to polygenic risk for Alzheimers disease, using magnetic resonance imaging (MRI) and genotype data for 562 19-year-old participants from the Avon Longitudinal Study of Parents and Children. Diffusion MRI data were acquired on a 3T scanner, and the data were used to perform whole-brain tractography. The resulting tractograms were used to generate structural brain networks, using the number of streamlines and the diffusion tensor fractional anisotropy as edge weights. This was done for the whole-brain connectome, and for the default mode, limbic and visual subnetworks. The mean clustering coefficient, mean betweenness centrality, characteristic path length, global efficiency and mean nodal strength were calculated for these networks, for each participant. The hubs of the networks were also identified, and the connectivity of the rich-club, feeder and local connections was also calculated. Polygenic risk scores (PRS), estimating the burden of genetic risk carried by an individual, were calculated both at genome-wide level and for nine specific disease pathways. The correlation coefficients were calculated between the PRS and a) the graph theoretical metrics of the structural networks and b) the rich-club, feeder and local connectivity of the whole-brain networks.

In the visual subnetwork, the mean nodal strength was negatively correlated with the genome-wide PRS including the APOE locus (r = -0.19, p = 1.3x10-5), the mean betweenness centrality was positively correlated with the pathway-specific PRS for plasma lipoprotein particle assembly including the APOE locus (r = 0.16, p = 9.2x10-4), and the mean clustering coefficient was negatively correlated with the tau protein binding PRS including the APOE locus (r = 0.16, p = 9.2x10-4). In the default mode network, the mean nodal strength was negatively correlated with the genome-wide PRS, including the APOE locus (r = -0.14, p=1.5x10-3). The rich-club and feeder connectivities were negatively correlated with the genome-wide PRS including the APOE locus (r = -0.16, p = 3.7x10-4; r = -0.15, p 8.8x10-4, respectively). Our results indicate small changes in the brain connectome of young adults at risk of developing Alzheimers disease in later life.

sentences: Beetle bioluminescence outshines aerial predators Bioluminescence is found across the tree of life and has many functions. Yet we understand very little about its timing and origins, particularly as a predator avoidance strategy. Understanding the timing between bioluminescence and predator origins has yet to be examined and can help elucidate the evolution of this ecologically important aposematic signal. Using the most prevalent bioluminescent group, fireflies, where bioluminescence primarily functions as aposematic and sexual signals, the timing for the origins of both potential predators of fireflies and bioluminescence is explored. Divergence time estimations were performed using genomic-scale phylogenetic reconstructions, and multiple fossil calibration points, allowing for a robust estimate for the origin of lampyrid bioluminescence as a terrestrial and as an aerial signal. Our results recover the origins of terrestrial beetle bioluminescence at 141.17 (122.63-161.17) mya and firefly aerial bioluminescence at 133.18 (117.86-152.47) mya with a large dataset focused on Lampyridae; and terrestrial bioluminescence as 148.03 (130.12-166.80) mya, with the age of aerial bioluminescence at 104.97 (99.00-120.90) mya with a complementary broad Elateroidea dataset. These ages predate the origins of all known extant aerial predators (i.e., bats and birds) and support the much older terrestrial predators (assassin bugs, frogs, ground beetles, lizards, snakes, hunting spiders, and harvestmen) as the most likely drivers of terrestrial bioluminescence in beetles, and sexual signaling likely being the original function in aerial fireflies.

sentences: Predicting cancer prognosis and drug response from the tumor microbiome Tumor gene expression is predictive of patient prognosis in some cancers. However, RNA- seq and whole genome sequencing data contain not only reads from host tumor and normal tissue, but also reads from the tumor microbiome, which can be used to infer the microbial abundances in each tumor. Here, we show that tumor microbial abundances, alone or in combination with tumor gene expression data, can predict cancer prognosis and drug response to some extent - microbial abundances are significantly less predictive of prognosis than gene expression, although remarkably, similarly as predictive of drug response, but in mostly different cancer-drug combinations. Thus, it appears possible to leverage existing sequencing technology, or develop new protocols, to obtain more non-redundant information about prognosis and drug response from RNA-seq and whole genome sequencing experiments than could be obtained from tumor gene expression or genomic data alone.

sentences: A global survey of specialized metabolic diversity encoded in bacterial genomes Bacterial specialized metabolites are a proven source of antibiotics and cancer therapies, but whether we have sampled all the secondary metabolite chemical diversity of cultivated bacteria is not known. We analysed ~ 170,000 bacterial genomes and ~ 47,000 metagenome assembled genomes (MAGs) using a modified BiG-SLiCE and the new clust-o-matic algorithm. We found that only 3% of the natural products potentially encoded in bacterial genomes have been experimentally characterized. We show that the variation of secondary metabolite biosynthetic diversity drops significantly at the genus level, identifying it as an appropriate taxonomic rank for comparison. Equal comparison of genera based on Relative Evolutionary Distance revealed that Streptomyces bacteria encode the largest biosynthetic diversity by far, with Amycolatopsis, Kutzneria and Micromonospora also encoding substantial diversity. Finally we find that several less-well-studied taxa, such as Weeksellaceae (Bacteroidota), Myxococcaceae (Myxococcota), Pleurocapsa and Nostocaceae (Cyanobacteria), have potential to produce highly diverse sets of secondary metabolites that warrant further investigation.

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Calcium current modulation by the γ1 subunit depends on alternative splicing of CaV1.1 The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming 1S subunit and the auxiliary 2{delta}-1 and {gamma}1 subunits. Particularly, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage-dependence of activation to positive potentials outside the physiological range. We generated a new HEK293-cell line stably expressing 2{delta}-1, {beta}3, and STAC3. When the adult (CaV1.1a) and the embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage-dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further co-expressed the {gamma}1 subunit, the current density of CaV1.1a, but not of CaV1.1e, was reduced by >50 %. In addition, {gamma}1 caused a shift of the voltage-dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of {gamma}1, but not its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between oppositely charged residues in the IVS3-S4 loop and the {gamma}1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the {gamma}1-dependent reduction of current density, suggesting that structural rearrangements of CaV1.1a induced by inclusion of exon 29 allosterically empower the {gamma}1 subunit to exert its inhibitory action on CaV1.1 calcium currents.

SummaryEl Ghaleb et al. analyzed the effects of the {gamma}1 subunit on current properties and expression of the adult (CaV1.1a) and embryonic (CaV1.1e) calcium channel splice variants, demonstrating that {gamma}1 reduces the current amplitude in a splicing-dependent manner.

sentences: Ligand-independent modulation of GIPR signaling by splice variants Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 7 reported (EMBL-EBI, 2022; NCBI, 2022a, 2022b) splice variants (SVs) with previously undefined functions. To explore their roles in endogenous peptide mediated GIPR signaling, we investigated the outcome of co-expressing each of the four SVs in question with GIPR in terms of ligand binding, cAMP accumulation, Gs activation, {beta}-arrestin recruitment and cell surface localization. The effects of these SVs on intracellular cAMP responses modulated by receptor activity-modifying proteins (RAMPs) were also studied. It was found that while SVs of GIPR neither bound to the hormone nor affected its signal transduction per se, they differentially regulated GIPR-mediated cAMP and {beta}-arrestin responses. Specifically, SV1 and SV4 were preferable to Gs signaling, SV3 was biased towards {beta}-arrestin recruitment, whereas SV2 was inactive on both pathways. In the presence of RAMPs, only SV1 and SV4 synergized the repressive action of RAMP3 on GIP-elicited cAMP production. The results suggest a new form of signal bias that is constitutive and ligand-independent, thereby expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-dependent (e.g., SV1 of the growth hormone-releasing hormone receptor).

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Filamentation in Candida albicans is modulated by adaptive translation of farnesol signalling genes The complex biology of the human pathogen Candida albicans is reflected in its remarkable ability to proliferate in numerous body sites, adapt to drastic changes in the environment, form various types of colonies and grow in yeast, pseudo-hyphal and hyphal forms. Much has been learnt in recent years about the relevance of this phenotypic plasticity, but the mechanisms that support it are still not fully understood. We have demonstrated that atypical translation of the CUG codon is a source of unexpected morphological diversity. The CUG codon is translated as both leucine (Leu) (~3%) and serine (Ser) (~97%) in normal growth conditions, but Ser/Leu levels change in response to stress. Remarkably, recombinant C. albicans strains incorporating between 20% and 99% of Leu at CUG sites display a diverse array of phenotypes and produce colonies of variable morphology containing a mixture of yeast, pseudohyphal and hyphal cells. In this work we investigate the role of the CUG codon in the yeast-hypha transition. Our data show that increasing incorporation levels of Leu at CUG sites trigger hyphal initiation under non-inducing conditions by reducing farnesol production, and increasing the degradation of the Nrg1 hyphal repressor. We propose that dual CUG Ser/Leu translation triggers filamentation via the Nrg1 pathway.

ImportanceThe unique translation of the CUG codon as both Ser (~97%) and Leu (~3%) plays a key role in the production of high genomic and phenotypic diversity in C. albicans. The molecular mechanisms that support such diversity are poorly understood. Here, we show that increased Leu incorporation at CUG sites induce hyphae formation in media where C. albicans normally grows in the yeast form. The data show that increasing Leu at CUG sites triggers the degradation of the hyphal repressor Nrg1, allowing for full expression of hyphal genes. Since filamentation is important for invasion of host tissues, this work shows how the atypical translation of a single codon may play a critical role in the virulence of all fungi of the CTG clade.

sentences: Population dynamics of epithelial-mesenchymal heterogeneity in cancer cells Phenotypic heterogeneity is a hallmark of aggressive cancer behaviour and a clinical challenge. Despite much characterisation of this heterogeneity at a multi-omics level in many cancers, we have a limited understanding of how this heterogeneity emerges spontaneously in an isogenic cell population. Some longitudinal observations of dynamics in epithelial-mesenchymal heterogeneity, a canonical example of phenotypic heterogeneity, have offered us opportunities to quantify the rates of phenotypic switching that may drive such heterogeneity. Here, we offer a mathematical modeling framework that explains the salient features of population dynamics noted in PMC42-LA cells: a) predominance of EpCAMhigh subpopulation, b) re-establishment of parental distributions from the EpCAMhigh and EpCAMlow subpopulations, and c) enhanced heterogeneity in clonal populations established from individual cells. Our framework proposes that fluctuations or noise in content duplication and partitioning of SNAIL - an EMT-inducing transcription factor - during cell division can explain spontaneous phenotypic switching and consequent dynamic heterogeneity in PMC42-LA cells observed experimentally at both single-cell and bulk level analysis. Together, we propose that asymmetric cell division can be a potential mechanism for phenotypic heterogeneity.

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Autism is associated with inter-individual variations of gray and white matter morphology BackgroundAlthough many studies have explored atypicalities in gray and white matter (GM, WM) morphology of autism, most of them rely on unimodal analyses that do not benefit from the likelihood that different imaging modalities may reflect common neurobiology. We aimed to establish multimodal brain patterns that differentiate between autism and typically developing (TD) controls and explore associations between these brain patterns and clinical measures.

MethodsWe studied 183 individuals with autism and 157 TD individuals (6-30 years) in a large deeply phenotyped autism dataset (EU-AIMS LEAP). Linked Independent Component Analysis was utilized to link all participants GM and WM images, and group comparisons of modality shared variances were examined. Subsequently, we performed a canonical correlation analysis to explore the aggregated effects between all multimodal GM-WM covariations and clinical profiles.

ResultsOne multimodal pattern was significantly related to autism. This pattern was primarily associated with GM in bilateral insula, frontal, pre- and post-central, cingulate, and caudate areas, and co-occurred with altered WM features in the superior longitudinal fasciculus. The canonical analysis showed a significant multivariate correlation primarily between multimodal brain patterns that involved variation of corpus callosum, and symptoms of social affect in the autism group.

ConclusionsOur findings demonstrate the assets of integrated analyses of GM and WM alterations to study the brain mechanisms that underpin autism, and show that the complex clinical autism phenotype can be interpreted by multimodal brain patterns that are spread across the brain involving both cortical and subcortical areas.

sentences: Cleavage of histone H2A during embryonic stem cell differentiation destabilizes nucleosomes to counteract gene activation Histone proteolysis is a poorly understood phenomenon in which the N-terminal tails of histones are irreversibly cleaved by intracellular proteases. During development, histone post-translational modifications are known to orchestrate gene expression patterns that ultimately drive cell fate decisions. Therefore, deciphering the mechanisms of histone proteolysis is necessary to enhance the understanding of cellular differentiation. Here we show that H2A is cleaved by the lysosomal protease Cathepsin L during ESCs differentiation. Using quantitative mass spectrometry (MS), we identified L23 to be the primary cleavage site that gives rise to the clipped form of H2A (cH2A), which reaches a maximum level of [~]1% of total H2A after four days of differentiation. Using ChIP-seq, we found that preventing proteolysis leads to an increase in acetylated H2A at promoter regions in differentiated ES cells. We also report the identification of novel readers of acetylated H2A in pluripotent ES cells, including members of the PBAF remodeling complex, which can recognize different acetylated forms of H2A. Finally, we show that H2A proteolysis abolishes this recognition. Altogether, our data suggests that proteolysis serves as an efficient mechanism to silence pluripotency genes and destabilize the nucleosome core particle.

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A ratiometric dual color luciferase reporter for fast characterization of transcriptional regulatory elements Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Therefore, transcriptional reporter systems are a key tool in understanding control of gene expression in biology.

In this work we construct and characterize a dual-color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is cheaper to perform than current dual-luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.

sentences: RNA splicing programs define tissue compartments and cell types at single cell resolution More than 95% of human genes are alternatively spliced. Yet, the extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach that is agnostic to transcript annotation, to detect cell-type-specific regulated splicing in > 110K carefully annotated single cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type specifically spliced. These results are validated with RNA FISH, single cell PCR, and in high throughput with Smart-seq2. Regulated splicing is found in ubiquitously expressed genes such as actin light chain subunit MYL6 and ribosomal protein RPS24, which has an epithelial-specific microexon. 13% of the statistically most variable splice sites in cell-type specifically regulated genes are also most variable in mouse lemur or mouse. SpliZ analysis further reveals 170 genes with regulated splicing during sperm development using, 10 of which are conserved in mouse and mouse lemur. The statistical properties of the SpliZ allow model-based identification of subpopulations within otherwise indistinguishable cells based on gene expression, illustrated by subpopulations of classical monocytes with stereotyped splicing, including an un-annotated exon, in SAT1, a Diamine acetyltransferase. Together, this unsupervised and annotation-free analysis of differential splicing in ultra high throughput droplet-based sequencing of human cells across multiple organs establishes splicing is regulated cell-type-specifically independent of gene expression.

sentences: Lung injury induces alveolar type 2 cell hypertrophy and polyploidy with implications for repair and regeneration Epithelial polyploidization post-injury is a conserved phenomenon, recently shown to improve barrier restoration during wound healing. Whether lung injury can induce alveolar epithelial polyploidy is not known. We show that bleomycin injury induces AT2 cell hypertrophy and polyploidy. AT2 polyploidization is also seen in short term ex vivo cultures, where AT2-to-AT1 trans-differentiation is associated with substantial binucleation due to failed cytokinesis. Both hypertrophic and polyploid features of AT2 cells can be attenuated by inhibiting the integrated stress response (ISR) using the small molecule ISRIB. These data suggest that AT2 hypertrophic growth and polyploidization may be a feature of alveolar epithelial injury. As AT2 cells serve as facultative progenitors for the distal lung epithelium, a propensity for injury-induced binucleation has implications for AT2 self-renewal and regenerative potential upon reinjury, which may benefit from targeting the ISR.

sentences: Structural insights into G protein activation by D1 dopamine receptor G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors and are the most important drug targets. An agonist-bound GPCR engages heterotrimeric G proteins and triggers the exchange of GDP with GTP to promote G proteins activation. A complete understanding of the molecular mechanisms of G proteins activation has been hindered by a lack of structural information of GPCR-G protein complex in nucleotide-bound states. Here, we present the cryoelectron microscopy (cryo-EM) structures of D1 dopamine receptor (D1R)-Gs in the nucleotide-free state, the GDP-bound state and the GTP-bound state with endogenous ligand dopamine. These structures reveal important conformational changes accounting for the release of GDP and the GTP-dependent dissociation of G from G{beta}{gamma} subunits. Combining mutagenesis functional studies, we also identified an important sequence motif in D1R that determines its G protein selectivity. Taken together, these results shed light into the molecular basis of G protein selectivity and the entire molecular signaling events of GPCR-mediated G protein activation.

sentences: Optical Tissue Clearing Enables Rapid, Precise and Comprehensive Assessment of Three-Dimensional Morphology in Experimental Nerve Regeneration Research Morphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections. Novel optical tissue clearing techniques enabling three-dimensional imaging of entire organs at a subcellular resolution have revolutionized morphological studies of the brain. To extend their applicability to experimental nerve repair studies we adapted these techniques to nerves and their motor and sensory targets in rats. The solvent-based protocols rendered harvested peripheral nerves and their target organs transparent within 24 h while preserving tissue architecture and fluorescence. Optical clearing is compatible with conventional laboratory techniques, including retrograde labelling studies, and computational image segmentation, providing fast and precise cell quantitation. Further, optically cleared organs enable three-dimensional morphometry at an unprecedented scale including dermatome-wide innervation studies, tracing of intramuscular nerve branches and mapping of neurovascular networks. Given their wide-ranging applicability, rapid processing times and low costs, tissue clearing techniques are likely to be a key technology for next-generation nerve repair studies.

sentences: Endomembrane targeting of human OAS1 p46 augments antiviral activity Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.

sentences: Modeling ex vivo tumor-infiltrating lymphocyte expansion from established solid malignancies Adoptive transfer of tumor-infiltrating lymphocytes (TIL) elicits the regression of metastatic malignancies, yet a low proportion of patients achieve complete durable responses. The high incidence of relapse in these patients highlights the need to better understand mechanisms of tumor escape from T cell control. While melanoma has provided the foundation for developing TIL therapy, much less is known about TIL efficacy and relapse in other malignancies. We sought to investigate TIL characteristics in mouse tumors which have not been studied in this setting. Here, we expanded murine TIL ex vivo in IL-2 from fragments of multiple tumor models, including oral cavity cancer models of varying immunogenicity. Additionally, TIL was expanded from pmel-1 mice bearing B16 melanoma, yielding an enriched population of tumor-infiltrating TCR transgenic T cells. Murine TILs are similar to human TIL in that they express high levels of inhibitory receptors (PD-1, Tim-3, etc.) and can be expanded ex vivo in IL-2 extensively. Of clinical relevance, we draw parallels between murine and patient TIL, evaluating relationships between PD-1, Lag-3, and Tim-3 on TILs from a cohort of oral cavity cancer patients. This platform can be used by labs even in the absence of clinical specimens or clean cell facilities and will be important to more broadly understand TIL phenotypes across many different malignancies.

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Opposing roles of blood-borne monocytes and tissue-resident macrophages in limbal stem cell damage after ocular injury Limbal stem cell (LSC) deficiency is a frequent and severe complication after chemical injury to the eye. Previous studies have assumed that this is mediated directly by the caustic agent. Here we show that LSC damage occurs through immune cell mediators, even without direct injury to LSCs. In particular, pH elevation in the anterior chamber (AC) causes acute uveal stress, release of inflammatory cytokines at the basal limbal tissue, and subsequent LSC loss. Peripheral CCR2+ CX3CR1- monocytes are the key mediators of LSC damage, through the upregulation of tumor necrosis factor alpha (TNF-) at the limbus. In contrast to peripherally-derived monocytes, CX3CR1+ CCR2- tissue-resident macrophages have a protective role and their depletion prior to injury exacerbates LSC loss and increases LSC vulnerability to TNF--mediated apoptosis independently of CCR2+ cell infiltration into the tissue. Consistently, repopulation of the tissue by new resident macrophages not only restores the protective M2-like phenotype of macrophages but also suppresses LSC loss after exposure to inflammatory signals. These findings may have clinical implication in patient with chemical burns.

SignificanceLimbal stem cell (LSC) loss after chemical burn is a significant clinical problem that has long been thought to be the result of direct chemical injury. This study demonstrates the existence of competing inflammatory mechanism responsible for LSC death, mediated by infiltrating peripheral monocytes that antagonize and overcome the protective role of tissue-resident macrophages. Thus, enhancing the role of tissue-resident macrophages while suppressing that of infiltrating monocytes could be a novel therapeutic strategy for LSC survival.

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Chemical ecology of an apex predator life cycle Microbial symbiotic interactions, mediated by small molecule signaling, drive physiological processes of higher order systems. Metabolic analytic technologies advancements provide new avenues to examine how chemical ecology, or conversion of existing biomass to new forms, changes over a symbiotic lifecycle. We examine such processes using the tripartite relationship between nematode host Steinernema carpocapsae, its obligate mutualist bacterium, Xenorhabdus nematophila, and the insects they infect together. We integrate trophic, metabolomics, and gene regulation analyses to understand insect biomass conversion to nematode or bacterium biomass. Trophic analysis established bacteria as the primary insect consumers, with nematodes at trophic position 4.37, indicating consumption of bacteria and likely other nematodes. Significant, discrete metabolic phases were distinguishable from each other, indicating the insect chemical environment changes reproducibly during bioconversion. Tricarboxylic acid cycle components and amino acids were significantly affected throughout infection. These findings contribute to an ongoing understanding of how symbiont associations shape chemical environments.

TeaserEntomopathogenic nematodes act as an apex predator in some ecosystems through altering chemical environments of their prey.

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utR.annotation: a tool for annotating genomic variants that could influence post-transcriptional regulation SummaryWhole genome sequencing of patient populations is identifying thousands of new variants in UnTranslated Regions(UTRs). While the consequences of UTR mutations are not as easily predicted from primary sequence as coding mutations are, there are some known features of UTRs modulate their function. utR.annotation is an R package that can be used to annotate potential deleterious variants in the UTR regions for both human and mouse species. Given a CSV or VCF format variant file, utR.annotation provides information of each variant on whether and how it alters known translational regulators including:upstream Open Reading Frames (uORFs), upstream Kozak sequences, polyA signals, the Kozak sequence at the annotated translation initiation site, start codon, and stop codon, conservation scores in the variant position, and whether and how it changes ribosome loading based on a model from empirical data.

Availability and implementationutR.annotation is freely available on Bitbucket (https://bitbucket.org/jdlabteam/utr.annotation/src/master/) and CRAN (to be updated)

Supplementary informationSupplementary data are available at https://wustl.box.com/s/yye99bryfin89nav45gv91l5k35fxo7z

sentences: In vivo prenylomic profiling in the brain of a transgenic mouse model of Alzheimer's disease reveals increased prenylation of a key set of proteins Dysregulation of protein prenylation has been implicated in many diseases, including Alzheimers disease (AD). Prenylomic analysis, the combination of metabolic incorporation of an isoprenoid analogue (C15AlkOPP) into prenylated proteins with a bottom-up proteomic analysis, has allowed identification of prenylated proteins in various cellular models. Here, transgenic AD mice were administered with C15AlkOPP through intracerebroventricular (ICV) infusion over 13 days. Using prenylomic analysis, 36 prenylated proteins were enriched in the brains of AD mice. Importantly, the prenylated forms of 15 proteins were consistently upregulated in AD mice compared to non-transgenic wild-type controls. These results highlight the power of this in vivo metabolic labeling approach to identify multiple post-translationally modified proteins that may serve as potential therapeutic targets for a disease that has proved refractory to treatment thus far. Moreover, this method should be applicable to many other types of protein modifications, significantly broadening its scope.

sentences: RBCeq: An Integrated Bioinformatics Algorithm Designed to Improve Blood Type Compatibility Testing While blood transfusion is an essential cornerstone of hematological care, patients that require repetitive transfusion remain at persistent risk of alloimmunization due to the diversity of human blood group polymorphisms. Next-generation sequencing (NGS) is an effective means of identifying genotypic and phenotypic variations among the blood groups, while the accurate interpretation of such NGS data is currently hampered by a lack of accessibility to bioinformatics support. To address this unmet need, we have developed the RBCeq (https://www.rbceq.org/) platform, which consists of a novel bioinformatics algorithm coupled with a user-friendly web server capable of comprehensively delineating different blood group variants from genomics data with advanced visualization of results. The software profiles genomic data for 36 blood group systems, including two transcription factors and can identify small genetic alterations, including small indels and copy number variants. The RBCeq algorithm was validated on 403 samples which include 58 complex serology cases from Australian Red Cross LifeBlood, 100 samples from The MedSeq Project (phs000958) and a further 245 from Indigenous Australian participants. The final blood typing data from RBCeq was 99.83% concordant for 403 samples (85 different antigens in 21 blood group systems) with that listed from the International Society for Blood Transfusion database.

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Quantitative multiplex immunohistochemistry reveals inter- and intra-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma PurposeQuantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma.

Experimental DesignWe established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naive, primary cutaneous melanomas.

ResultsHere we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naive, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates.

ConclusionsWe describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for tumor cell exit (metastasis) or leukocyte trafficking (immune response).

This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

TRANSLATIONAL RELEVANCEThis report describes a quantitative, image-based method to investigate the relationship between the tumor-associated lymphovasculature and immune landscape in treatment naive, primary human melanoma. The research shows that melanoma-associated blood and lymphatic vessels display context-dependent phenotypes that associate both with the risk of regional progression and immune infiltration. These findings indicate that stromal/vascular heterogeneity may underlie regional differences in immunogenicity and thus present opportunities for future biomarker development and therapeutic intervention.

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hei-tag: a highly efficient tag to boost targeted genome editing Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems1. While active in all species tested so far, editing efficiencies still leave room for improvement. To reach its target, the bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion of nuclear localization signals (NLSs) to the Cas9 protein2. Additional domains such as FLAG- or myc-tags are added for immediate detection or straight-forward purification3. To avoid steric hinderance impacting on activity, amino acid linkers are employed connecting Cas9 and additional domains.

We present the  hei-tag (high efficiency-tag), boosting the activity of the wide variety of CRISPR/Cas genome editing tools. The addition of the hei-tag to Cas9 or a C-to-T base editor dramatically enhances the respective targeting efficiency in model systems ranging from fish to mammals, including tissue culture applications. This allows to instantly upgrade existing and potentially highly adapted systems as well as establish novel highly efficient tools.

sentences: Fingerprinting of skin cells by live cell Raman spectroscopy reveals melanoma cell heterogeneity and cell-type specific responses to UVR Raman spectroscopy is an emerging dermatological technique with the potential to discriminate biochemically between cell types in a label free and non-invasive manner. Here we use live single cell Raman spectroscopy and principal component analysis (PCA) to fingerprint mouse melanoblasts, melanocytes, keratinocytes and melanoma cells. We show the differences in their spectra are attributable to biomarkers in the melanin biosynthesis pathway and that melanoma cells are a heterogeneous population that sit on a trajectory between undifferentiated melanoblasts and differentiated melanocytes. We demonstrate the utility of Raman spectroscopy as a highly sensitive tool to probe the melanin biosynthesis pathway and its immediate response to UV irradiation revealing previously undescribed opposing responses to UVA and UVB irradiation in melanocytes. Finally, we identify melanocyte specific accumulation of {beta}-carotene correlated with a stabilisation of the UVR response in lipids and proteins consistent with a {beta}-carotene mediated photoprotective mechanism. In summary our data show that Raman spectroscopy can be used to determine the differentiation status of cells of the melanocyte lineage and describe the immediate and temporal biochemical changes associated with UV exposure which differ depending on cell type, differentiation status and competence to synthesise melanin.

sentences: Self-consistent migration puts tight constraintson the spatio-temporal organization ofspecies-rich metacommunities Biodiversity is often attributed to a dynamic equilibrium between immigration of species and competition-driven extinction. This equilibrium forms a common basis for studying ecosystem assembly from a static reservoir of migrants-the mainland. Yet, natural ecosystems often consist of many coupled communities (i.e. metacommunities) and migration occurs between these communities. The pool of migrants then depends on what is sustained in the ecosystem, which in turn depends on the dynamic migrant pool. This chicken-and-egg problem of survival and migration is poorly understood in communities of many competing species, except for the neutral case - the "unified neutral theory of biodiversity". Employing spatio-temporal simulations and mean-field analyses, we show that self-consistent migration puts rather tight constraints on the dynamic migration-extinction equilibrium. When the number of species is large, even weak competitions push species to the edge of their global extinction, such that the overall diversity is highly sensitive to perturbations in demographic parameters, including growth and dispersal rates. When migration is short-range, the resulting spatiotemporal abundance patterns follow broad scale-free distributions that correspond to a directed percolation phase transition. The qualitative agreement of our results for short-range and long-range migration suggests that this self-organization process is a general property of species-rich metacommunities. Our study shows that self-sustaining metacommunities are highly sensitive to environmental change and provides insights into how biodiversity can be rescued and maintained.

sentences: Probing the stability of the SpCas9-DNA complex after cleavage CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 as well as its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different S. pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After target-strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability compared to the pre-cleavage state. This suggests that Cas9 has evolved to remain tightly bound to its cut target.

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Diversity and selection of SARS-CoV-2 minority variants in the early New York City outbreak High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. Here we analyzed minority variants within the SARS-CoV-2 data in 12 samples from the early outbreak in New York City, using replicate sequencing for reliable identification. While most minority variants were unique to a single sample, we found several instances of shared variants. We provide evidence that some higher-frequency minority variants may be transmitted between patients or across short transmission chains, while other lower-frequency, more widely shared variants arise independently. Further, our data indicate that even with a small transmission bottleneck, the heterogeneity of intra-host viral populations is enhanced by minority variants present in transmission samples. Our data suggest that analysis of shared minority variants could help identify regions of the SARS-CoV-2 genome that are under increased selective pressure, as well as inform transmission chains and give insight into variant strain emergence.

IMPORTANCEWhen viruses replicate inside a host, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus, nor strongly beneficial, can lead to minority variants. In this study, we analyzed the minority variants in SARS-CoV-2 patient samples from New York City during the early outbreak. We found common minority variants between samples that were closely related and showed that these minority variants may be transmitted from one patient to another. We show that in general, transmission events between individuals likely contain genetically diverse viral particles, and we find signatures of selection governing intra-host evolution. We conclude that the analysis of shared minority variants can help to identify transmission events and give insight into emergence of new viral variants.

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Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimised miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses are calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.

HighlightsO_LImiCLIP2 produces complex libraries to map m6A RNA modifications
C_LIO_LIMettl3 KO miCLIP2 allows to identify Mettl3-dependent RNA modification sites
C_LIO_LIMachine learning predicts genuine m6A sites from human and mouse miCLIP2 data without Mettl3 KO
C_LIO_LIm6A modifications occur outside of DRACH motifs and associate with alternative splicing
C_LI

sentences: The Mechanism of Replication Stalling and Recovery within Repetitive DNA Accurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstituted replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We found that structure-prone repeats are sufficient to impair replication. Whilst template unwinding was unaffected, leading strand synthesis was inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats relied on replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats required an extrinsic accessory helicase. DNA-induced fork stalling was mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress also applies to stalling induced by repetitive sequences.

sentences: Negligible impact of SARS-CoV-2 variants on CD4+ and CD8+ T cell reactivity in COVID-19 exposed donors and vaccinees. The emergence of SARS-CoV-2 variants highlighted the need to better understand adaptive immune responses to this virus. It is important to address whether also CD4+ and CD8+ T cell responses are affected, because of the role they play in disease resolution and modulation of COVID-19 disease severity. Here we performed a comprehensive analysis of SARS-CoV-2-specific CD4+ and CD8+ T cell responses from COVID-19 convalescent subjects recognizing the ancestral strain, compared to variant lineages B.1.1.7, B.1.351, P.1, and CAL.20C as well as recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. Similarly, we demonstrate that the sequences of the vast majority of SARS-CoV-2 T cell epitopes are not affected by the mutations found in the variants analyzed. Overall, the results demonstrate that CD4+ and CD8+ T cell responses in convalescent COVID-19 subjects or COVID-19 mRNA vaccinees are not substantially affected by mutations found in the SARS-CoV-2 variants.

sentences: Interplay between positive and negative regulation by B3-type transcription factors is critical for the accurate expression of the ABA INSENSITIVE 4 gene. The ABA-INSENSITIVE 4 transcription factor is key for the regulation of diverse aspects of plant development and environmental responses, including proper perception of hormonal and nutritional signals. ABI4 activity is highly regulated at the transcriptional and post-transcriptional levels leading to precise expression mainly in the developing seed and early seedling development. Based on genetic and molecular approaches in the current study we provide new insights into the central mechanism underpinning the transcriptional regulation of ABI4 during both seed and vegetative development. We identified a complex interplay between the LEC2 and ABI3 transcriptional activators and the HSI/VAL repressors that is critical for proper ABI4 expression. Interestingly, the regulation by these proteins relies on the two RY cis-acting motifs present two kb upstream of the ABI4 gene. Our analysis also shows that the chromatin landscape of the ABI4 loci is highly dependent on the LEC2 and HSI2/VAL proteins. LEC2 regulation extends to the vegetative development and the absence of this factor results in ABA- and sugar-insensitive signaling in the developing plant. This regulatory circuit functions as a major control module for the correct spatial-temporal expression of ABI4 and prevents its ectopic accumulation that is harmful to the plant.

sentences: A novel missense mutation in the proprotein convertase gene furinb causes hepatic cystogenesis during liver development in zebrafish Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that develops hepatic cysts during larval stages. Cyst formation in these mutants is not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time-lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed an excessive and disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole-genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene which encodes a proprotein convertase. The mutation alters Furinb localization and causes endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4-phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant livers also exhibited increased mTOR signaling and treatment with the mTOR inhibitor rapamycin partially blocked cyst formation by reducing ER stress. Our study has established a novel vertebrate model for studying hepatic cystogenesis and illustrated the role of ER stress in the disease pathogenesis.

sentences: Dicer-to-Argonaute switch controls biogenesis of oncogenic miRNA miRNAs are post-transcriptional regulators of gene expression, controlling biological processes from development to pathogenesis. We asked whether the reshaped functional miRNA landscape in cancers is driven by altered transcription of its precursors, or altered biogenesis and maturation of miRNAs. Integrated analysis of genomic and transcriptomic data in 9,111 samples across 10 cancer types and healthy tissues revealed a recurrent genomic switch from DICER-dependent to non-canonical Argonaute-mediated, DICER-independent, miRNA biogenesis. Experimental validation in AGO2-amplified clinical samples and cancer cell lines confirmed that canonical miRNAs can undergo maturation in a DICER-independent manner, and that elevated Argonaute levels promote selective maturation of the oncogenic miR-106b/25 cluster as shown by the altered ratio of mature miRNA to immature pri-miRNA levels. The preferential maturation of these oncogenic miRNAs, whose processing bypasses DICER1, promotes cancer progression and predicts poor prognosis. This highlights the evolution of non-canonical AGO2-dependent oncomiR processing as a novel driver pathway in cancer.

sentences: Photon-free (s)CMOS camera characterization for artifact reduction in high- and super-resolution microscopy Modern implementations of widefield fluorescence microscopy often rely on sCMOS cameras, but this camera architecture inherently features pixel-to-pixel variations. Such variations lead to image artifacts and render quantitative image interpretation difficult. Although a variety of algorithmic corrections exists, they require a thorough characterization of the camera, which typically is not easy to access or perform. Here, we developed a fully automated pipeline for camera characterization based solely on thermally generated signal, and implemented it in the popular open-source software Micro-Manager and ImageJ/Fiji. Besides supplying the conventional camera maps of noise, offset and gain, our pipeline also gives access to dark current and thermal noise as functions of the exposure time. This allowed us to avoid structural bias in single-molecule localization microscopy (SMLM), which without correction is substantial even for scientific-grade, cooled cameras. In addition, our approach enables high-quality 3D super-resolution as well as live-cell time-lapse microscopy with cheap, industry-grade cameras. As our approach for camera characterization does not require any user interventions or additional hardware implementations, numerous correction algorithms demanding camera characterization become easily applicable.

sentences: Pharmacological impacts on schizophrenia functional analysis: a postmortem proteome study. Schizophrenia (SCZ) is a severe and debilitating mental illness. Antipsychotic drugs (APDs) are used to treat both positive and negative SCZ symptoms, by influencing the cellular, subcellular-synaptic, and molecular processes. We posit that these effects influence our understanding of SCZ. To address this, we analyzed postmortem dorsolateral prefrontal cortex grey matter samples from control and SCZ subjects (n=10/group) using liquid-chromatography mass-spectrometry-based proteomics. We retrieved SCZ-altered and APD-influenced proteome-sets using linear and mixed linear models, respectively, and validated them experimentally using independent cohorts and insilico using published datasets. Functional analysis of proteome-sets was contrasted at the biological pathway, cell-type, subcellular-synaptic, and drug-target levels. The SCZ-altered proteome was conserved across several studies from DLPFC and other brain areas and was dependent on drug effect. At the pathway level, we observed an aberrant extracellular event and, except for homeostasis, signal-transduction, cytoskeleton, and dendrites associated downregulated changes, the APDs compensated for the majority of the SCZ-altered pathways. At the cell-type level, the up-and down-regulated SCZ-altered events were associated with two different subsets of striatum projecting layer-5 pyramidal-neurons regulating dopaminergic secretion. At the subcellular synaptic level, compensatory pre- and post-synaptic events were observed. At the drug target level, dopaminergic processes influence the SCZ-altered up-regulated proteome, whereas non-dopaminergic and a diverse array of non-neuromodulatory mechanisms influence the SCZ-altered down-regulated proteome. While these findings are dependent on pharmacological effects, they are also consistent with previous SCZ studies, implying the need to re-evaluate previous results. We discuss our findings in the context of cortico-striatal influence in SCZ-pathology.

sentences: Yin Yang 1 sets up the stage for cerebellar astrocyte maturation Diverse subpopulations of astrocytes tile different brain regions to accommodate local requirements of neurons and associated neuronal circuits. Nevertheless, molecular mechanisms governing astrocyte diversity remain mostly unknown. We explored the role of a zinc finger transcription factor Yin Yang 1 (YY1) that is expressed in astrocytes. We found that specific deletion of YY1 from astrocytes causes severe motor deficits in mice, induces Bergmann gliosis, and results in simultaneous loss of GFAP expression in velate and fibrous cerebellar astrocytes. Single cell RNA-seq analysis showed that YY1 exerts specific effects on gene expression in subpopulations of cerebellar astrocytes. We found that although YY1 is dispensable for the initial stages of astrocyte development, it regulates subtype-specific gene expression during astrocyte maturation. Moreover, YY1 is continuously needed to maintain mature astrocytes in the adult cerebellum. Our findings suggest that YY1 plays critical roles regulating cerebellar astrocyte maturation during development and maintaining a mature phenotype of astrocytes in the adult cerebellum.

sentences: Circuit motifs and graph properties of connectome development in C. elegans Network science is a powerful tool that can be used to better explore the complex structure of brain networks. Leveraging graph and motif analysis tools, we interrogate C. elegans connectomes across multiple developmental time points and compare the resulting graph characteristics and substructures over time. We show the evolution of the networks and highlight stable invariants and patterns as well as those that grow or decay unexpectedly, providing a substrate for additional analysis.

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Intestinal Gluconeogenesis Regulates Brown and White Adipose Tissues Functions in mice ObjectiveIntestinal gluconeogenesis, via the initiation of a gut-brain nervous circuit, accounts for the metabolic benefits linked to dietary proteins or fermentable fibre in rodents and has been positively correlated with the rapid amelioration of body weight after gastric bypass surgery in obese humans. In particular, the activation of intestinal gluconeogenesis moderates the development of hepatic steatosis accompanying obesity. In this study, we investigated the specific effects of intestinal gluconeogenesis on adipose tissue metabolism, independently of its induction by nutritional manipulation.

MethodsWe used two transgenic mouse models of suppression or overexpression of G6PC, the catalytic subunit of glucose-6 phosphatase, the key enzyme of endogenous glucose production, specifically in the intestine.

ResultsUnder a hypercaloric diet, mice with a genetic overexpression of intestinal gluconeogenesis showed a lower adiposity and higher thermogenic capacities than wild-type mice, featuring marked browning of white adipose tissue and prevention of the whitening of brown adipose tissue. Suppression of sympathetic nervous signalling in brown adipose tissue impairs the activation of thermogenesis. Conversely, mice with genetic suppression of intestinal gluconeogenesis exhibit an increase in adiposity under standard diet, associated with a decreased expression of markers of thermogenesis in both the brown and white adipose tissues.

ConclusionIntestinal gluconeogenesis is sufficient in itself to activate the sympathetic nervous system and prevent the expansion and the metabolic alterations of brown and white adipose tissues metabolism under high calorie diet, thus preventing the development of obesity. These data increase knowledge of the mechanisms of weight reduction in gastric bypass surgery and pave the way of new approaches to prevent or cure obesity.

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Layered Feedback Control Overcomes Performance Trade-off in Synthetic Biomolecular Networks Layered feedback is an optimization strategy in feedback control designs widely used in electrical and mechanical engineering. Layered control theory suggests that the performance of controllers is bound by the universal robustness-efficiency tradeoff limit, which could be overcome by layering two or more feedbacks together. In natural biological networks, genes are often regulated with redundancy and layering to adapt to environmental perturbations. Control theory hypothesizes that this layering architecture is also adopted by nature to overcome this performance trade-off. In this work, we validated this property of layered control with a synthetic network in living E. coli cells. We performed system analysis on a node-based design to confirm the tradeoff properties before proceeding to simulations with an effective mechanistic model, which guided us to the best performing design to engineer in cells. Finally, we interrogated its system dynamics experimentally with eight sets of perturbations on chemical signals, nutrient abundance, and growth temperature. For all cases, we consistently observed that the layered control overcomes the robustness-efficiency trade-off limit. This work experimentally confirmed that layered control could be adopted in synthetic biomolecular networks as a performance optimization strategy. It also provided insights in understanding genetic feedback control architectures in nature.

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sentences: Functional Annotation of Human Cognitive States using Deep Graph Convolution A key goal in neuroscience is to understand brain mechanisms of cognitive functions. An emerging approach is "brain decoding", which consists of inferring a set of experimental conditions performed by a participant, using pattern classification of brain activity. Few works so far have attempted to train a brain decoding model that would generalize across many different cognitive tasks drawn from multiple cognitive domains. To tackle this problem, we proposed a multidomain brain decoder that automatically learns the spatiotemporal dynamics of brain response within a short time window using a deep learning approach. We evaluated the decoding model on a large population of 1200 participants, under 21 different experimental conditions spanning six different cognitive domains, acquired from the Human Connectome Project task-fMRI database. Using a 10s window of fMRI response, the 21 cognitive states were identified with a test accuracy of 90% (chance level 4.8%). Performance remained good when using a 6s window (82%). It was even feasible to decode cognitive states from a single fMRI volume (720ms), with the performance following the shape of the hemodynamic response. Moreover, a saliency map analysis demonstrated that the high decoding performance was driven by the response of biologically meaningful brain regions. Together, we provide an automated tool to annotate human brain activity with fine temporal resolution and fine cognitive granularity. Our model shows potential applications as a reference model for domain adaptation, possibly making contributions in a variety of domains, including neurological and psychiatric disorders.

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High-Resolution Genomic Profiling of Carbapenem-Resistant Klebsiella pneumoniae Isolates: A Multicentric Retrospective Indian Study summaryWe report insights into genome sequences of Indian K. pneumoniae isolates, highlighting the presence of high-risk international clones and genetic pools different from those predominating in other regions. Identification of multidrug-resistant and hypervirulent K. pneumoniae elicits public health concerns.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a threat to public health in India due to its high dissemination, mortality, and limited treatment options. Its genomic variability is reflected in the diversity of sequence types, virulence factors, and antimicrobial resistance (AMR) mechanisms. This study aims to characterize the clonal relationships and genetic mechanisms of resistance and virulence in CRKP isolates in India.

Materials and MethodsWe characterized 344 retrospective K. pneumoniae clinical isolates collected from 8 centers across India collected in 2013-2019. Susceptibility to antibiotics was tested with VITEK 2. Capsular types, MLST, virulence genes, AMR determinants, plasmid replicon types, and a single-nucleotide polymorphism (SNP) phylogeny were inferred from their whole genome sequences.

ResultsPhylogenetic analysis of the 325 Klebsiella isolates that passed QC revealed 3 groups: K. pneumoniae sensu stricto (n=307), K. quasipneumoniae (n=17), and K. varicolla (n=1). Sequencing and capsular diversity analysis of the 307 K. pneumoniae sensu stricto isolates revealed 28 sequence types, 26 K-locus types, and 11 O-locus types, with ST231, KL51, and O1V2 being predominant. blaOXA-48-like and blaNDM-1/5 were present in 73.2% and 24.4% of isolates respectively. The major plasmid replicon types associated with carbapenase genes were IncF (51.0%), and Col group (35.0%).

ConclusionOur study documents for the first time the genetic diversity of K- and O-antigens circulating in India. The results demonstrate the practical applicability of genomic surveillance and its utility in tracking the population dynamics of CRKP. It alerts us to the urgency for longitudinal surveillance of these virulent and transmissible lineages.

FundingThis work was supported by Official Development Assistance (ODA) funding from the National Institute of Health Research [grant number 16_136_111].

This research was commissioned by the National Institute of Health Research using Official Development Assistance (ODA) funding. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.

Conflict of InterestThe authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

sentences: Cell type-specific meQTL extends melanoma GWAS annotation beyond eQTL and informs melanocyte gene regulatory mechanisms While expression quantitative trait loci (eQTL) have been powerful in identifying susceptibility genes from genome-wide association studies (GWAS) findings, most trait-associated loci are not explained by eQTL alone. Alternative QTLs including DNA methylation QTL (meQTL) are emerging, but cell-type-specific meQTL using cells of disease origin has been lacking. Here we established an meQTL dataset using primary melanocytes from 106 individuals and identified 1,497,502 significant cis-meQTLs. Multi-QTL colocalization using meQTL, eQTL, and mRNA splice-junction QTL from the same individuals together with imputed methylome-wide and transcriptome-wide association studies identified susceptibility genes at 63% of melanoma GWAS loci. Among three molecular QTLs, meQTLs were the single largest contributor. To compare melanocyte meQTLs with those from malignant melanomas, we performed meQTL analysis on skin cutaneous melanomas from The Cancer Genome Atlas (n = 444). A substantial proportion of meQTL probes (45.9%) in primary melanocytes are preserved in melanomas, while a smaller fraction of eQTL genes is preserved (12.7%). Integration of melanocyte multi-QTL and melanoma meQTL identified candidate susceptibility genes at 72% of melanoma GWAS loci. Beyond GWAS annotation, meQTL-eQTL colocalization in melanocytes suggested that 841 unique genes potentially share a causal variant with a nearby methylation probe in melanocytes. Finally, melanocyte trans-meQTL identified a hotspot for rs12203592, a cis-eQTL of a transcription factor, IRF4, with 131 candidate target CpGs. Motif enrichment and IRF4 ChIPseq analysis demonstrated that these target CpGs are enriched in IRF4 binding sites, suggesting an IRF4-mediated regulatory network. Our study highlights the utility of cell-type-specific meQTL.

sentences: Sensorimotor transformation underlying odor-modulated locomotion in walking Drosophila Most real-world behaviors are performed with incomplete information. Odor-guided locomotion, an ecologically important behavior essential to an animals survival, is an example of such a behavior. Different odors activate different patterns of olfactory receptor neuron (ORN) classes providing information about which odor is present but does not provide any navigational information. In this study, we investigate the sensorimotor transformation that relates ORN activation to locomotion changes in Drosophila by optogenetically activating different combinations of ORN classes and measuring the resulting changes in locomotion. Three features describe this sensorimotor transformation: First, locomotion depends on both the instantaneous firing frequency (f) and its change (df); the two together serve as short-term memory that allows the fly to automatically adapt its motor program to sensory context. Second, the mapping between f-df and locomotor parameters such as speed or curvature is distinct for each pattern of activated ORNs. Finally, the sensorimotor mapping changes with time after odor exposure allowing integration of information over a longer timescale.

sentences: Temporal pattern and synergy influence activity of ERK signaling pathways during L-LTP induction Long-lasting long-term potentiation (L-LTP) is a cellular mechanism of learning and memory storage. Studies have demonstrated a requirement for extracellular signal-regulated kinase (ERK) activation in L-LTP produced by a diversity of temporal stimulation patterns. Multiple signaling pathways converge to activate ERK, with different pathways being required for different stimulation patterns. To answer whether and how different temporal patterns select different signaling pathways for ERK activation, we developed a computational model of five signaling pathways (including two novel pathways) leading to ERK activation during L-LTP induction. We show that calcium and cAMP work synergistically to activate ERK and that stimuli given with large inter-trial intervals activate more ERK than shorter intervals. Furthermore, these pathways contribute to different dynamics of ERK activation. These results suggest that signaling pathways with different temporal sensitivity facilitate ERK activation to diversity of temporal patterns.

sentences: Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic stem cells. When human cord blood derived CD34+ cells are induced to differentiate in vitro, they undergo rapid and dynamic morphological and molecular transformations that are critical for fate commitment. Using ATAC-seq and single-cell RNA sequencing, we detected two phases in this process. In the first phase, we observed a rapid and global chromatin opening that makes most of the gene promoters in the genome accessible, followed by widespread upregulation of gene transcription and a concomitant increase in the cell-to-cell variability of gene expression. The second phase is marked by a slow chromatin closure and a subsequent overall downregulation of gene transcription and emergence of coherent expression profiles corresponding to distinct cell subpopulations. These observations are consistent with a model based on the spontaneous probabilistic organization of the cellular process of fate commitment.

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Competent immune responses to SARS-CoV-2 variants in older adults following mRNA vaccination Aging is associated with a reduced magnitude of primary immune responses to vaccination and constriction of immune receptor repertoire diversity. Clinical trials demonstrated high efficacy of mRNA based SARS-CoV-2 vaccines in older adults but concerns about virus variant escape have not been well addressed. We have conducted an in-depth analysis of humoral and cellular immunity against an early-pandemic viral isolate and compared that to the P.1. (Gamma) and B.1.617.2 (Delta) variants in <50 and >55 age cohorts of mRNA vaccine recipients. We have further measured neutralizing antibody titers for B.1.617.1 (Kappa) and B.1.595; a SARS-CoV-2 isolate bearing Spike mutation E484Q. As reported, robust immunity required the second dose of vaccine. Older vaccinees manifested robust cellular immunity against early-pandemic SARS-CoV-2 and more recent variants, which remained statistically comparable to the adult group. The older cohort had lower neutralizing capacity at the first time point following the second dose, but at later time points immunity was indistinguishable between them. While the duration of these immune responses remains to be determined over longer periods of time, these results provide reasons for optimism regarding vaccine protection of older adults against SARS-CoV-2 variants and inform thinking about boost vaccination with variant vaccines.

eTOC summaryVaccine responses are often diminished with aging, but we found strong responses to SARS-CoV-2 in older adults following mRNA vaccination. T cell responses were not diminished when confronted by SARS-CoV-2 variants. Neutralizing Ab were reduced but not more than those in adults.

Graphical Abstract

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sentences: Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remain poorly understood. Here, we probed the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that both the rates of microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, while a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of cytoplasm modulates the reactions underlying microtubule dynamic behaviors.

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Orally administered antigen can reduce or exacerbate pathology in an animal model of inflammatory arthritis dependent upon the timing of administration Currently, treatments for rheumatoid arthritis (RA) are focussed on treatment of disease symptoms rather than addressing the cause of disease, which could lead to remission and cure. Central to disease development is the induction of autoimmunity through a breach of self-tolerance. There is considerable research in RA focussed on antigens and approaches to re-establish antigen specific tolerance. A crucial step in this research is to employ appropriate animal models to test prospective antigen specific immunotherapies, preferably in the context of joint inflammation.

In this short communication, we use our previously developed model of antigen specific inflammatory arthritis in which OVA-specific TcR tg T cells drive breach of tolerance to endogenous antigens to determine the impact that the timing of therapy administration has upon disease progression. Using antigen feeding to induce tolerance we demonstrate that administration prior to articular challenge results in a reduced disease score as evidenced by pathology and serum antibody responses. By contrast, feeding antigen after articular challenge had the opposite effect and resulted in the exacerbation of pathology.

Although preliminary, these data suggest that the timing of antigen administration may be key to the success of tolerogenic immunotherapies. This has important implications for the timing of potential tolerogenic therapies in patients.

sentences: Tumor Microenvironment Alters Chemoresistance of Hepatocellular Carcinoma Through CYP3A4 Metabolic Activity Variations in tumor biology from patient to patient combined with the low overall survival rate of hepatocellular carcinoma (HCC) present significant clinical challenges. During the progression of chronic liver diseases from inflammation to the development of HCC, microenvironmental properties, including tissue stiffness and oxygen concentration, change over time. This can potentially impact drug metabolism and subsequent therapy response to commonly utilized therapeutics, such as doxorubicin, multi-kinase inhibitors (e.g., sorafenib), and other drugs, including immunotherapies. In this study, we utilized four common HCC cell lines embedded in 3D collagen type-I gels of varying stiffnesses to mimic normal and cirrhotic livers with environmental oxygen regulation to quantify the impact of these microenvironmental factors on HCC chemoresistance. In general, we found that HCC cells with higher baseline levels of cytochrome p450-3A4 (CYP3A4) enzyme expression, HepG2 and C3Asub28, exhibited a cirrhosis-dependent increase in doxorubicin chemoresistance. Under the same conditions, HCC cell lines with lower CYP3A4 expression, HuH-7 and Hep3B2, showed a decrease in doxorubicin chemoresistance in response to an increase in microenvironmental stiffness. This differential therapeutic response was correlated with the regulation of CYP3A4 expression levels under the influence of stiffness and oxygen variation. In all tested HCC cell lines, the addition of sorafenib lowered the required doxorubicin dose to induce significant levels of cell death, demonstrating its potential to help reduce systemic doxorubicin toxicity when used in combination. These results suggest that patient-specific tumor microenvironmental factors, including tissue stiffness, hypoxia, and CYP3A4 activity levels, may need to be considered for more effective use of chemotherapeutics in HCC patients.

sentences: Combined fluorescence, optical diffraction tomography and Brillouin microscopy Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples -- so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fuorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample -- a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

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ACORBA: Automated workflow to measure Arabidopsis thaliana root tip angle dynamic Plants respond to the surrounding environment in countless ways. One of these responses is their ability to sense and orient their root growth toward the gravity vector. Root gravitropism is studied in many laboratories as a hallmark of auxin-related phenotypes. However, manual analysis of images and microscopy data is known to be subjected to human bias. This is particularly the case for manual measurements of root bending as the selection lines to calculate the angle are set subjectively. Therefore, it is essential to develop and use automated or semi-automated image analysis to produce reproducible and unbiased data. Moreover, the increasing usage of vertical-stage microscopy in plant root biology yields gravitropic experiments with an unprecedented spatiotemporal resolution. To this day, there is no available solution to measure root bending angle over time for vertical-stage microscopy. To address these problems, we developed ACORBA (Automatic Calculation Of Root Bending Angles), a fully automated software to measure root bending angle over time from vertical-stage microscope and flatbed scanner images. Moreover, the software can be used semi-automated for camera, mobile phone or stereomicroscope images. ACORBA represents a flexible approach based on both traditional image processing and deep machine learning segmentation to measure root angle progression over time. By its automated nature, the workflow is limiting human interactions and has high reproducibility. ACORBA will support the plant biologist community by reducing time and labor and by producing quality results from various kinds of inputs.

Significance statementACORBA is implementing an automated and semi-automated workflow to quantify root bending and waving angles from images acquired with a microscope, a scanner, a stereomicroscope or a camera. It will support the plant biology community by reducing time and labor and by producing trustworthy and reproducible quantitative data.

sentences: Optimization of the TeraTox assay for preclinical teratogenicity assessment Current animal-free methods to assess teratogenicity of drugs under development still deliver high numbers of false negatives, and more sensitive approaches of toxicity prediction are required. To address this issue, we characterized the TeraTox test, a newly developed multi-lineage differentiation assay for human teratogenicity prediction using 3D human induced pluripotent stem cells. TeraTox produces as primary output concentration-dependent data sets for each test compound on cytotoxicity and altered gene expression. These data are then fed into a prediction model based on an interpretable machine-learning approach. The final information obtained relates to the concentration-dependent human teratogenicity potential of drug candidates. We applied TeraTox to profile 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. This way, it was possible to relate the test predictions to known human or animal toxicity. The TeraTox had an accuracy of 69% (specificity: 53%, sensitivity: 79%). It clearly performed better than two quantitative structure-activity relationship (QSAR) models and it had a higher sensitivity than the murine embryonic stem cell test (mEST) run in the same laboratory. By combining TeraTox and mEST data, the overall prediction accuracy was further improved. The knowledge on the pattern of altered gene expression may provide additional value in grouping toxicologically similar compounds and possibly deducing common modes of action. The assay will thus be a helpful additional tool in drug discovery, and the dataset provided here will be a valuable resource for the field of stem cell-based drug profiling.

sentences: Dopamine D2Rs Coordinate Cue-Evoked Changes in Striatal Acetylcholine Levels In the striatum, acetylcholine (ACh) neuron activity is modulated co-incident with dopamine (DA) release in response to unpredicted rewards and reward predicting cues and both neuromodulators are thought to regulate each other. While this co-regulation has been studied using stimulation studies, the existence of this mutual regulation in vivo during natural behavior is still largely unexplored. One long-standing controversy has been whether striatal DA is responsible for the induction of the cholinergic pause or whether D2R modulate a pause that is induced by other mechanisms. Here, we used genetically encoded sensors in combination with pharmacological and genetic inactivation of D2Rs from cholinergic interneurons (CINs) to simultaneously measure ACh and DA levels after CIN D2R inactivation. We found that CIN D2Rs are not necessary for the induction of cue induced dips in ACh levels but regulate dip lengths and rebound ACh levels. Importantly, D2R inactivation strongly decreased the temporal correlation between DA and Ach signals not only at cue presentation but also during the intertrial interval. This points to a general mechanism by which D2Rs coordinate both signals. At the behavioral level D2R antagonism increased the latency to lever press, which was not observed in CIN-selective D2R knock out mice. This latency correlated with the cue evoked dip length supporting a role of the ACh dip and its regulation by D2Rs in motivated behavior. Overall, our data indicate that striatal DA coordinate phasic ACh and DA signals via CIN D2Rs which is important for the regulation of motivated behavior.

sentences: Turning plants from passive to active material: FERONIA and microtubules independently contribute to mechanical feedback To survive, cells must constantly resist mechanical stress. In plants, this involves the reinforcement of cell walls, notably through microtubule-dependent cellulose deposition, and thus wall sensing. Several receptor-like kinases have been proposed to act as mechanosensors. Here we tested whether the microtubule response to stress acts downstream of known wall sensors. Using a multi-step screen with eleven mutant lines, we identify FERONIA as the primary candidate for controlling the microtubule response to stress. However, when performing mechanical perturbations, we show that the microtubule response to stress can be independent from FER. We reveal that the feronia phenotype can be partially rescued by reducing tensile stress levels. Conversely, in the absence of both microtubules and FER, cells swell and burst like soap bubbles. Altogether, this shows that the microtubule response to stress acts as an independent pathway to resist stress, in parallel to FER. We propose that both pathways are key components to turn plant cells from passive to active material.

sentences: The neuroinflammatory interleukin-12 signaling pathway drives Alzheimer's disease-like pathology by perturbing oligodendrocyte survival and neuronal homeostasis Alzheimers disease (AD) is characterized by deposition of pathological amyloid-{beta} (A{beta}) and tau protein aggregates and involves chronic neuroinflammation, ultimately leading to neurodegeneration and cognitive decline. Central in AD-related neuroinflammation is the proinflammatory interleukin-12 (IL-12)/IL-23 signaling pathway whose inhibition has been shown to attenuate pathology and cognitive defects in AD-like mice. In order to explore which cell types are involved in this neuroinflammatory cascade, we used single-nuclei RNA sequencing in AD-like APPPS1 mice lacking or harboring IL-12/IL-23 signaling. We found Il12b transcripts encoding the common p40 subunit of IL-12/IL-23 signaling to be expressed preferentially, but not exclusively, in microglia in an AD-specific manner. In contrast, transcripts for the other subunits of the IL-12 signaling pathway were expressed constitutively in neurons and oligodendrocytes irrespective of AD pathology, while transcripts for IL-23 were almost undetectable. Notably, genetic ablation of IL-12/IL-23 signaling did not affect the inflammatory gene expression profile of the AD-specific disease associated microglia (DAM), but reversed the loss of mature myelin-producing oligodendrocytes and alterations in neuronal homeostasis in APPPS1 mice. Taken together, our results reveal that IL-12, but not IL-23 is the main driver of AD-specific IL-12/IL-23 neuroinflammation, which alters neuronal and oligodendrocyte functions. Given that drugs targeting IL-12 already exist, our data may foster first clinical trials in AD subjects using this novel neuroimmune target.

sentences: A human embryonic limb cell atlas resolved in space and time Human limbs emerge during the fourth post-conception week as mesenchymal buds which develop into fully-formed limbs over the subsequent months. Limb development is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common. Decades of work with model organisms has outlined the fundamental processes underlying vertebrate limb development, but an in-depth characterisation of this process in humans has yet to be performed. Here we detail the development of the human embryonic limb across space and time, using both single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells, progressing from a restricted number of multipotent progenitors to myriad mature cell states, and identify several novel cell populations, including perineural fibroblasts and multiple distinct mesenchymal states. We uncover two waves of human muscle development, each characterised by different cell states regulated by separate gene expression programmes. We identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity and validate this by performing MSC knock down in human embryonic myoblasts, which results in significant upregulation of late myogenic genes. Spatially mapping the cell types of the limb across a range of gestational ages demonstrates a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncovers two transcriptionally and spatially distinct populations of the progress zone, which we term "outer" and "transitional" layers. The latter exhibits a transcriptomic profile similar to that of the chondrocyte lineage, but lacking the key chondrogenic transcription factors SOX5,6 & 9. Finally, we perform scRNA-seq on murine embryonic limbs to facilitate cross-species developmental comparison at single-cell resolution, finding substantial homology between the two species.

sentences: The phosphoproteome is a first responder in tiered cellular adaptation to chemical stress followed by proteomics and transcriptomics alteration Next-generation risk assessment for environmental chemicals and ingredients in consumer products involves a weight of evidence (WoE) framework integrating a suite of new approach methodologies (NAMs) based on points of departure (PoD) obtained from in vitro assays. Omics techniques provide broad coverages of the molecular toxicity pathway space. Transcriptomics assays especially play a leading role by providing relatively conservative PoDs in comparison with apical endpoints. However, it is unclear whether and how parameters measured using other omics technicquesparticipate in the cellular response to chemical perturbations, especially at exposure levels below the transcriptomically defined PoD. Multi-omics coverage may provide additional sensitive or confirmative biomarkers to complement and reduce the uncertainty in safety decisions made using targeted and transcriptomics assays. In the present study, we compared changes in transcriptomics, proteomics and phosphoproteomics with two prototype compounds, coumarin, as a main study and doxorubicin, as a complementary study to understand the sensitivity of the three omics techniques in response to chemically-induced changes in HepG2 and AC16 cells. With measurements obtained for multiple chemical concentrations and time points, we demonstrated that, compared with proteomics and transcriptomics, phosphoproteomics alterations occur not only earlier in time as expected, but also at much lower chemical concentrations and hence are proximal to the very early effects induced by chemical exposure. The phosphoproteomics changes appear to approach maximum when the transcriptomics alterations begin to be initiated. The results are consistent with a tiered framework of cellular response to chemical insults, where posttranslational modification of preexisting proteins is first seen before transcriptomics induction is engaged to launch a more energy-expensive defense that defines a useful PoD. We conclude that as the cost becomes more affordable, proteomics covering posttranslational modifications can be utilized to provide a more complete coverage of chemical-induced cellular alteration and supplement transcriptomics-based health safety decision making.

sentences: Ease of de novo gene birth through spontaneous mutations predicted in a parsimonious model Phylostratigraphy suggests that new genes are continually born de novo from non-genic sequences, and the genes that persist found new lineages, contributing to the adaptive evolution of organisms. While recent evidence supports the view that de novo gene birth is frequent and widespread, the mechanisms underlying this process are yet to be discovered. Here we hypothesize and examine a potential general mechanism of gene birth driven by the accumulation of beneficial mutations at non-genic loci. To demonstrate this possibility, we model this mechanism within the boundaries set by current knowledge on mutation effects. Estimates from this analysis are in line with observations of recurrent and extensive gene birth in genomics studies. Thus, we propose that, rather than being inactive and silent, non-genic regions are likely to be dynamic storehouses of potential genes.

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Ownership, Coverage, Utilisation and Maintenance of Long-lasting insecticidal nets in Three Health Districts in Cameroon: A Cross-Sectional Study IntroductionThe Bamenda, Santa and Tiko Health Districts are in the highest malaria transmission strata of Cameroon. The purpose of this study was to explore the indicators of ownership and utilisation as well as maintenance of long-lasting insecticidal nets (LLINs) in three health districts in Cameroon.

MethodsA cross-sectional household survey involving 1,251 households was conducted in the Bamenda, Santa and Tiko Health Districts in Cameroon. A structured questionnaire was used to collect data on LLINs ownership, utilisation, and maintenance as well as demographic characteristics.

ResultsThe average number of LLINs per household was higher in the Bamenda Health District (BHD) compared to the Tiko Health District (THD) (2.5{+/-}1.4 vs 2.4{+/-}1.6) as well as the household ownership at least one LLIN (93.3% vs. 88.9%). The proportion of the defacto population with universal utilisation was higher in BHD compared to THD (13.1% vs 0.2%). In multinomial regression analysis, households in the SHD (OR = 0.4, 95% = C.I; 0.2 - 0.8, p = 6.10x10-3), were less likely to own at least one LLIN, while those in the BHD (OR = 1.3, 95% = C.I; 0.8 - 2.1, p = 0.33) were more likely to maintain LLINs compared to those in THD.

ConclusionOwnership of LLINs was low in SHD and THD in comparison to the goal of one LLIN for every two household members. Overall, LLINs coverage and accessibility was still low after the free Mass Distribution Campaigns (MDCs), making it difficult for all household members to effectively use LLINs.

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Isolation and characterization of novel temperate virus Aeropyrum globular virus 1 infecting hyperthermophilic archaeon Aeropyrum We isolate a novel archaeal temperate virus named Aeropyrum globular virus 1 (AGV1) from the host Aeropyrum culture. Reproduction of AGV1 was induced by adding 20 mM tris-acetate buffer to exponentially growing host cells. Negatively stained virions showed spherical morphology (60 {+/-}2 nm in diameter) similar to Globuloviridae viruses. The double-stranded circular DNA genome of AGV1 contains 18,222 bp encoding 34 open-reading frames. No ORFs showed significant similarity with Globuloviridae viruses. AGV1 shares three genes, including an integrase gene, with reported spindle-shaped temperate viruses. However we couldnt detect its integration site in the host genome. Moreover AGV1 seemed not to replicate autonomously because there are no origin recognition boxes in the genome. qPCR results showed that the genome copy number of AGV1 was lower than that of the host genome (10-3 copies per host genome). Upon the addition of tris-acetate buffer, a steep increase in the AGV1 genome copy number (9.5-26 copies per host genome at 2 days post-treatment) was observed although clustered regularly interspaced short palindromic repeat (CRISPR) elements of the host genome showed significant matches with AGV1 protospacers. Our findings suggest that AGV1 is a novel globular virus exhibiting an unstable carrier state in the growing host and in that way AGV1 can escape from the host defense system and propagate under stressful host conditions.

ImportanceStudying archaeal viruses yields novel insights into the roles of virospheres and viruses in the evolutionary process of their hosts. Here, we isolated a novel spherical virus named Aeropyrum globular virus 1. AGV1 has integrase gene but its genome is not integrated into the host genome. AGV1 could not replicate autonomously due to the lack of origin recognition boxes and thus its copy number was too low (10-3 copies per host genome) without any inducing stimulus. However, upon the addition of tris-acetate buffer, the AGV1 genome copy number steeply increased instead of a perfect sequence match between the spacer of the host CRISPR/Cas system and the protospacer. Our findings suggest that AGV1 can escape from the host defense system and propagate under stressful conditions for the host by establishing an unstable carrier state. These results reveals a novel aspect of host-virus interactions in extreme environments.

sentences: Multilocus adaptation to vaccination Pathogen adaptation to public health interventions, such as vaccination, may take tortuous routes and involve multiple mutations at distinct locations in the pathogen genome, acting on distinct phenotypic traits. Despite its importance for public health, how these multilocus adaptations jointly evolve is poorly understood. Here we consider the joint evolution of two adaptations: the pathogens ability to escape the vaccine-induced immune response and adjustments to the pathogens virulence and transmissi-bility. We elucidate the role played by epistasis and recombination, with an emphasis on the different protective effects of vaccination. We show that vaccines reducing transmission and/or increasing clearance generate positive epistasis between the vaccine-escape and virulence alleles, favouring strains that carry both mutations, whereas vaccines reducing virulence mortality generate negative epistasis, favouring strains that carry either mutation, but not both. High rates of recombination can affect these predictions. If epistasis is positive, frequent recombination can lead to the sequential fixation of the two mutations and prevent the transient build-up of more virulent escape strains. If epistasis is negative, frequent recombination between loci can create an evolutionary bistability, such that whichever adaptation is more accessible tends to be favoured in the long-term. Our work provides a timely alternative to the variant-centered perspective on pathogen adaptation and captures the effect of different types of vaccines on the interference between multiple adaptive mutations.

sentences: Fate transitions in Drosophila neural lineages: a single cell road map to mature neurons. Neuron specification and maturation are essential for proper central nervous system development. However, the precise mechanisms that govern neuronal maturation remain poorly understood. Here, we use single-cell RNA sequencing, combined with a conditional genetic strategy to analyse neuronal lineages and their new born neurons in the Drosophila larval brain. A focused analysis on the transcriptional alterations that occur right after neuron generation revealed that neuron maturation starts shortly after neuronal birth, with transcription, but no translation, of mature neuronal features such as neurotransmitter (NT) genes. Using NT gene Choline acetyltransferase as an example, we show that the timings of translation initiation are not solely dependent on neuron age but are rather coordinated with the animal developmental stage. This study is the first characterization of the initial phases of neuron maturation, supporting a model where neuron maturation is coordinated with the animal developmental stage through post-transcriptional regulation of terminal effector genes.

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Orbitofrontal cortex contributes to the comparison of values underlying economic choices Economic choices between goods entail the computation and comparison of subjective values. Previous studies examined neuronal activity in the orbitofrontal cortex (OFC) of monkeys choosing between different types of juices. Three groups of neurons were identified: offer value cells encoding the value of individual offers, chosen juice cells encoding the identity of the chosen juice, and chosen value cells encoding the value of the chosen offer. The encoded variables capture both the input (offer value) and the output (chosen juice, chosen value) of the decision process, suggesting that values are compared within OFC. Recent work demonstrates that choices are causally linked to the activity of offer value cells. Conversely, the hypothesis that OFC contributes to value comparison has not been confirmed. Here we show that weak electrical stimulation of OFC specifically disrupts value comparison without altering offer values. This result implies that neuronal populations in OFC participate in the decision process.

SignificanceEconomic choices encompass two mental stages: values are assigned to the available offers, and a decision is made by comparing values. Previous work showed that choices are causally related to offer values encoded in the orbitofrontal cortex (OFC). Conversely, the neural underpinnings of value comparison remain poorly understood. Here we show that weak electrical stimulation of OFC selectively disrupts the decision process without affecting offer values. Hence, neurons in OFC contribute to value comparison.

sentences: Medial prefrontal-hippocampal concordance in child-parent dyads underlies children's psychological wellbeing Negative family environments can be harmful to childrens psychological wellbeing via altered child-parent interactions. Single-brain paradigms have uncovered changes in brain systems related to negative family environments, but how neurobiological reciprocity between child and parent brains contribute to childrens psychological wellbeing remains unknown. Across two studies examining 395 child-parent dyads, we show that children from negative family environments experienced more psychopathological symptoms. Dyad-based analysis of movie-watching functional magnetic resonance imaging data revealed higher inter-subject correlations in dorsal- and ventral-MPFC, and greater concordance of activity with regions critical for socioemotional skills in child-parent than child-stranger dyads. Negative family environment reduced inter-subject correlation of ventral-MPFC with the hippocampus in child-parent dyads, which further mediated childrens internalizing symptoms. The ventral-MPFC and hippocampus exhibited reciprocal activity, which helped child-parent dyads form shared meaning during movie watching. Our findings suggest child-parent brain-to-brain concordance may be a neurobiological mechanism for how family environments affect psychological wellbeing.

sentences: LRR protein RNH1 dampens the inflammasome activation and is associated with adverse clinical outcomes in COVID-19 patients Inflammasomes are cytosolic innate immune sensors of pathogen infection and cellular damage that induce caspase-1 mediated inflammation upon activation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and can be detrimental, such as in COVID-19. However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine rich repeat (LRR) protein Ribonuclease inhibitor (RNH1), which shares homology with LRRs of NLRP proteins, attenuates inflammasome activation. Deletion of RNH1 in macrophages increases IL-1{beta} production and caspase-1 activation for inflammasome stimuli. Mechanistically, RNH1 decreases pro-IL-1{beta} expression and induces proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and LPS-induced endotoxemia, which are dependent on caspase-1, respectively show increased neutrophil infiltration and lethality in Rnh1-/- mice compared to WT mice. Furthermore, RNH1 protein levels are negatively correlated with inflammation and disease severity in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity.

sentences: Wood ants express uncertainty when engaging with magnetic cues for directional guidance Wood ants were trained indoors to follow a route in a chosen magnetic direction from the centre of a small, circular arena to find a drop of sucrose at the edge. The arena, surrounded by a white cylindrical wall, was in the centre of a 3D coil system that generated an inclined Earth strength magnetic field in any horizontal direction. Between trials, the chosen magnetic training direction was rotated to a new orientation. Tests were given without food and with fresh or reversed paper on the floor of the arena. In a significant number of tests, ants left the centre facing the goal, or in the opposite direction, but they mostly failed to reach the goal. Tests given early in the day, before any training, show that ants remember the magnetic route direction overnight. On some training trials, the position of the sucrose was also indicated by a black stripe. Not uncommonly, ants first moved in the opposite direction to the stripe before switching to the correct direction. Travel away from the reward seems to express the ants uncertainty about the correct path to take. Tests show that this uncertainty may stem from competing directional cues linked to the room, suggesting that ants are reluctant to rely on magnetic information alone. We conclude that ants can remember a route direction defined by an Earth-strength magnetic field and that they express any uncertainty about the correct direction by moving for a stretch in the opposite direction. In a second experiment, an upright and an inverted triangle were fixed 90{degrees} from each other to the inside of the cylinder. Sucrose was placed beneath one of the triangles, dependent on the direction of the magnetic field. Ants failed to master this task and to approach the magnetically cued triangle. Instead, they preferred to approach the upright triangle. The ants were again uncertain of the correct direction and expressed this uncertainty through paths that had segments directed towards both the inverted and the upright triangles.

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Interpretable deep learning to uncover the molecular binding patterns determining TCR-epitope interactions BackgroundThe recognition of an epitope by a T-cell receptor (TCR) is crucial for eliminating pathogens and establishing immunological memory. Prediction of the binding of any TCR-epitope pair is still a challenging task, especially with novel epitopes, because the underlying patterns that drive the recognition are still largely unknown to both domain experts and machine learning models.

ResultsThe binding of a TCR and epitope sequence can only occur when amino acids from both sequences are in close contact with each other. We analyze the distance between interacting molecules of the TCR and epitope sequences and compare this to the amino acids that are important for TCR-epitope prediction models. Important residues are determined by using interpretable deep learning techniques or, more specifically, feature attribution extraction methods, on two state-of-the-art TCR-epitope prediction models: ImRex and TITAN. Highlighting feature attributions on the molecular complex reveals additional insights to the domain expert about why the prediction was made and can offer novel insights into the factors that determine TCR affinity on a molecular level. We also show which residues of the TCR and epitope sequences determine binding prediction for ImRex and TITAN and use those to explain model performance.

ConclusionsExtracting feature attributions is a useful way to verify your model and data for challenging problems where small hard-to-detect problems can accumulate to inaccurate results.

sentences: Tetrazoles as PPARγ ligands: Structural and Computational Investigation. Diabetes is an important chronic disease affecting about 10% of the adult population in the US and over 420 million people worldwide, resulting in 1.6 million deaths every year, according to the World Health Organization. The most common type of the disease, type 2 diabetes, can be pharmacologically managed using oral hypoglycemic agents or thiazolidinediones (TZDs), such as pioglitazone, which act by activating the Peroxisome Proliferated-Activated Receptor {gamma}. Despite their beneficial effects in diabetes treatment, TZDs like rosiglitazone and troglitazone were withdrawn due to safety reasons, creating a void in the pharmacological options for the treatment of this important disease. Here, we explored a structure-based approach in the screening for new chemical probes for a deeper investigation of the effects of PPAR{gamma} activation. A class of tetrazole compounds was identified and the compounds named T1, T2 and T3 were purchased and evaluated for their ability to interact with the PPAR{gamma} ligand binding domain (LBD). The compounds were binders with micromolar range affinity, as determined by their IC50 values. A Monte Carlo simulation of the compound T2 revealed that the tetrazole ring makes favorable interaction with the polar arm of the receptor binding pocket. Finally, the crystal structure of the PPAR{gamma}-LBD-T2 complex was solved at 2.3 [A], confirming the binding mode for this compound. The structure also revealed that, when the helix H12 is mispositioned, an alternative binding conformation is observed for the ligand suggesting an H12-dependent binding conformation for the tetrazole compound.

sentences: Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-{zeta} signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

sentences: Toward a More Accurate 3D Atlas of C. elegans Neurons Determining cell identity in volumetric images of tagged neuronal nuclei is an ongoing challenge in contemporary neuroscience. Frequently, cell identity is determined by aligning and matching tags to an "atlas" of labeled neuronal positions and other identifying characteristics. Previous analyses of such C. elegans datasets have been hampered by the limited accuracy of such atlases, especially for neurons present in the ventral nerve cord, and also by time-consuming manual elements of the alignment process. We present a novel automated alignment method for sparse and incomplete point clouds of the sort resulting from typical C. elegans fluorescence microscopy datasets. This method involves a tunable learning parameter and a kernel that enforces biologically realistic deformation. We also present a pipeline for creating alignment atlases from datasets of the recently developed NeuroPAL transgene. In combination, these advances allow us to label neurons in volumetric images with confidence much higher than previous methods. We release, to the best of our knowledge, the most complete C. elegans 3D positional neuron atlas, encapsulating positional variability derived from 7 animals, for the purposes of cell-type identity prediction for myriad applications (e.g., imaging neuronal activity, gene expression, and cell-fate).

sentences: Federated analysis of BRCA1 and BRCA2 variation in a Japanese cohort More than 40% of the germline variants in ClinVar today are variants of uncertain significance (VUS). These variants remain unclassified in part because the patient-level data needed for their interpretation is siloed. Federated analysis can overcome this problem by "bringing the code to the data": analyzing the sensitive patient-level data computationally within its secure home institution and providing researchers with valuable insights from data that would not otherwise be accessible. We tested this principle with a federated analysis of breast cancer clinical data at RIKEN, derived from the BioBank Japan repository. We were able to analyze these data within RIKENs secure computational framework, without the need to transfer the data, gathering evidence for the interpretation of several variants. This exercise represents an approach to help realize the core charter of the Global Alliance for Genomics and Health (GA4GH): to responsibly share genomic data for the benefit of human health.

sentences: Preferred synonymous codons are translated more accurately: Proteomic evidence, among-species variation, and mechanistic basis A commonly stated cause of the widespread phenomenon of unequal uses of synonymous codons is their differential translational accuracies. However, this long-standing translational accuracy hypothesis (TAH) of codon usage bias has had no direct evidence beyond anecdotes. Analyzing proteomic data from Escherichia coli, we observe higher translational accuracies of more frequently used synonymous codons, offering direct, global evidence for the TAH. The experimentally measured codon-specific translational accuracies validate a sequence-based proxy; this proxy provides support for the TAH from the vast majority of over 1000 taxa surveyed in all domains of life. We find that the relative translational accuracies of synonymous codons vary substantially among taxa and are strongly correlated with the amounts of cognate tRNAs relative to those of near-cognate tRNAs. These and other observations suggest a model in which selections for translational efficiency and accuracy drive codon usage bias and its coevolution with the tRNA pool.

sentences: Vesalius: Tissue anatomy from spatial transcriptomic data. Characterization of tissue architecture promises to deliver insights into development, cell communication and disease. In silico spatial domain retrieval methods have been developed for spatial transcriptomics (ST) data assuming transcriptional similarity of neighboring barcodes. However, domain retrieval approaches with this assumption cannot work in complex tissues composed of multiple cell types. This task becomes especially challenging in cellular resolution ST methods. We developed Vesalius to decipher tissue anatomy from ST data by applying image processing technology. Vesalius uniquely detected territories composed of multiple cell types and successfully recovered tissue structures in high-resolution ST data including in mouse brain, embryo, liver, and colon. Utilizing this tissue architecture, Vesalius identified tissue morphology specific gene expression and regional specific gene expression changes for astrocytes, interneuron, oligodendrocytes, and entorhinal cells in the mouse brain.

sentences: Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

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The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalyzed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well-studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for trimethylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted mass spectrometry analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi.

Data SummaryThe predicted proteomes used in this study are publicly available at the JGI (Joint Genome Institute) MycoCosm repository (1); the species names and abbreviations are listed in Table S1. To evaluate the completeness of the predicted proteomes and to obtain a species phylogeny, 758 fungal BUSCO (Benchmarking Universal Single-Copy Ortholog) genes were used, and their names are listed in Table S2. The fasta, trimmed alignment, and maximum-likelihood phylogenetic tree files can be found in Supplementary Data 1 and 2 deposited at Zenodo (doi:10.5281/zenodo.5907090), as well as the mass spectrometry results are mentioned in Table S4.

Impact StatementUncovering how fungi regulate gene expression and genome organization is an important step to understand how they control biological processes such as growth or biosynthesis of bioactive molecules. Despite the known importance of chromatin modifications in controlling a plethora of biological processes across eukaryotes, they remain particularly poorly understood in filamentous fungi, even in model organisms like Aspergilli. Our systematic evolutionary analysis provides a robust framework for the functional analysis of chromatin modifications in Aspergillus species and other fungi. Our results do not only implicate candidate enzymes to play a role in new chromatin modifications, but they also point at species that have experienced duplications or losses of genes encoding enzymes for specific chromatin modifications. We therefore expect that this work will set the stage for future research into the complexity of the histone code and its functional implications on gene regulation and genome organization in fungi.

sentences: Functional brain network community structure in childhood: Unfinished territories and fuzzy boundaries Adult cortex is organized into distributed functional communities. Yet, little is known about community architecture of childrens brains. Here, we uncovered the community structure of cortex in childhood using fMRI data from 670 children aged 9-11 years from the Adolescent Brain and Cognitive Development study. Children showed similar community structure to adults in early-developing sensory and motor communities, but differences emerged in transmodal areas. Children have more cortical territory in the limbic community, which is involved in emotion processing, than adults. Regions of association cortex interact more flexibly across communities, creating uncertainty for the model-based assignment algorithm, and perhaps reflecting cortical boundaries that are not yet solidified. Uncertainty was highest for cingulo-opercular areas involved in flexible deployment of cognitive control. Collectively, our findings suggest that community boundaries are not solidified by middle childhood, an instability that provides important context for childrens thoughts and behaviors.

sentences: Isolating perceptual biases caused by trial history during auditory categorization Just as most experiences have their origin in our perceptions, our perceptions can also be fundamentally shaped by our experiences. However, isolating which events in the recent past can impact perceptual judgments remains a difficult question, partly because post-perceptual processes can also introduce strong history dependencies. Two mechanisms have been hypothesized to specifically modulate perception: 1) the repulsive influence caused by previous stimuli and generally labeled as aftereffects, and 2) the modulation caused by stimulus predictions based on learned temporal regularities of the sensory environment, a key assumption in the predictive coding framework. Here, we ask whether these two mechanisms do indeed bias perception by training rats in an auditory task featuring serial correlations along the sequence of stimuli. We develop a detailed behavioral model that isolates the repulsive aftereffect generated by previous stimuli and shows that this repulsion cannot be explained from an interaction between past and current stimuli, and that it is still present in catch trials lacking the current stimulus. Moreover, the model describes that the bias caused by the animals expectation, as they leverage the predictability of the stimulus sequence, is present in a foraging task without the sensory component but with the same serial correlations in the sequence of rewards. These results indicate that the aftereffect and the prediction biases observed during an auditory task can all be revealed in the absence of a sensory stimulus, putting into question their perceptual nature.

sentences: Omicron Spike protein has a positive electrostatic surface that promotes ACE2 recognition and antibody escape High transmissibility is a hallmark of the Omicron variant of SARS-CoV-2. Understanding the molecular determinants of Omicrons transmissibility will impact development of intervention strategies. Here we map the electrostatic potential surface of the Spike protein to show that major SARS-CoV-2 variants have accumulated positive charges in solvent-exposed regions of the Spike protein, especially its ACE2-binding interface. Significantly, the Omicron Spike-ACE2 complex has complementary electrostatic surfaces. In contrast, interfaces between Omicron and neutralizing antibodies tend to have similar positively charged surfaces. Structural modeling demonstrates that the electrostatic property of Omicrons Spike receptor binding domain (S RBD) plays a role in enhancing ACE2 recognition and destabilizing Spike-antibody complexes. Collectively, our structural analysis implies that Omicron S RBD interaction interfaces have been optimized to simultaneously promote access to human ACE2 receptors and evade antibodies. These findings suggest that electrostatic interactions are a major contributing factor for increased Omicron transmissibility relative to other variants.

sentences: Transcriptional and cellular signatures of cortical morphometric similarity remodelling in chronic pain Chronic pain is a highly debilitating and poorly understood condition. Here, we attempt to advance our understanding of the brain mechanisms driving chronic pain by investigating alterations in morphometric similarity (MS) and corresponding transcriptomic and cellular signatures, in three cohorts of patients with distinct chronic pain syndromes (knee osteoarthritis, low back pain and fibromyalgia). We uncover a novel pattern of cortical MS remodelling involving mostly MS increases in the insula and limbic cortex, which cuts across the boundaries of specific pain syndromes. We show that cortical MS remodelling in chronic pain spatially correlates with the brain-wide expression of genes involved in the glial immune response and neuronal plasticity. Cortical remodelling in chronic pain might involve a disruption of multiple elements of the cellular architecture of the brain. Therefore, multi-target therapeutic approaches tackling both glial activation and neuronal hyperexcitability might better encompass the full neurobiology of chronic pain.

sentences: Single-Cell Atlas of Common Variable Immunodeficiency reveals germinal center-associated epigenetic dysregulation in B cell responses Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, is characterized by impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and a wide range of phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. We used single-cell epigenomics and transcriptomics to create a cell census of naive-to-memory B cell differentiation in a pair of CVID-discordant MZ twins. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B cells that mirror defective cell-cell communication defects following activation. These findings were validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naive-to-memory B-cell transition in CVID and reveal links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, paves the way for future diagnosis and treatments of CVID patients.

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HillTau: A fast, compact abstraction for model reduction in biochemical signaling networks Signaling networks mediate many aspects of cellular function. The conventional, mechanistically motivated approach to modeling such networks is through mass-action chemistry, which maps directly to biological entities and facilitates experimental tests and predictions. However such models are complex, need many parameters, and are computationally costly. Here we introduce the HillTau form for signaling models. HillTau retains the direct mapping to biological observables, but it uses far fewer parameters, and is 100 to over 1000 times faster than ODE-based methods. In the HillTau formalism, the steady-state concentration of signaling molecules is approximated by the Hill equation, and the dynamics by a time-course tau. We demonstrate its use in implementing several biochemical motifs, including association, inhibition, feedforward and feedback inhibition, bistability, oscillations, and a synaptic switch obeying the BCM rule. The major use-cases for HillTau are system abstraction, model reduction, scaffolds for data-driven optimization, and fast approximations to complex cellular signaling.

Author summaryChemical signals mediate many computations in cells, from housekeeping functions in all cells to memory and pattern selectivity in neurons. These signals form complex networks of interactions. Computer models are a powerful way to study how such networks behave, but it is hard to get all the chemical details for typical models, and it is slow to run them with standard numerical approaches to chemical kinetics. We introduce HillTau as a simplified way to model complex chemical networks. HillTau models condense multiple reaction steps into single steps defined by a small number of parameters for activation and settling time. As a result the models are simple, easy to find values for, and they run quickly. Remarkably, they fit the full chemical formulations rather well. We illustrate the utility of HillTau for modeling several signaling network functions, and for fitting complicated signaling networks.

sentences: Proteolytic activation of SARS-CoV-2 spike at the S1/S2 boundary: potential role of proteases beyond furin The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2 site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 pre-cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.

sentences: Recombinant annexin A6 promotes membrane repair in a stem cell derived-cardiomyocyte model of dystrophic cardiomyopathy Heart failure is a major source of mortality in Duchenne muscular dystrophy (DMD). DMD arises from mutations that ablate expression of the protein dystrophin, which render the plasma membrane unusually fragile and prone to disruption. In DMD patients, repeated mechanical stress leads to membrane damage and cardiomyocyte loss. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer the opportunity to study specific mutations in the context of a human cell, but these models can be improved by adding physiologic stressors. We modeled the primary defect underlying DMD by applying equibiaxial mechanical strain to DMD iPSC-CMs. DMD iPSC-CMs demonstrated an increased susceptibility to equibiaxial strain after 2 hours at 10% strain relative to healthy control cells, measured as increased lactate dehydrogenase (LDH) release. After 24 hours, both DMD and healthy control iPSC-CMs showed evidence of injury with release of LDH and cardiac troponin T. We exposed iPSC-CMs to recombinant annexin A6, a protein resealing agent, and found reduced LDH and troponin release in DMD and control iPSC-CMs that had been subjected to 24 hour strain at 10%. We used aptamer protein profiling of media collected from DMD and control iPSC-CMs and compared these results to serum protein profiling from DMD patients. We found a strong correlation between the proteins in DMD patient serum and media from DMD iPSC-CMs subjected to mechanical stress. By developing an injury assay that specifically targets an underlying mechanism of injury seen in DMD-related cardiomyopathy, we demonstrated the potential therapeutic efficacy of the protein membrane resealer, recombinant annexin A6, for the treatment of DMD-related cardiomyopathy and general cardiac injury.

sentences: Identification of SGLT2 inhibitor Ertugliflozin as a treatment for COVID-19 using computational and experimental paradigm Drug repurposing can expedite the process of drug development by identifying known drugs which are effective against SARS-CoV-2. The RBD domain of SARS-CoV-2 Spike protein is a promising drug target due to its pivotal role in viral-host attachment. These specific structural domains can be targeted with small molecules or drug to disrupt the viral attachment to the host proteins. In this study, FDA approved Drugbank database were screened using a virtual screening approach and computational chemistry methods. Five drugs were short listed for further profiling based on docking score and binding energies. Further these selected drugs were tested for their in vitro biological activity. There was significant correlation between the prediction from computational studies and the actual RBD-ACE2 binding inhibition by the drugs. Then, we performed a series of studies that mimic some of the biological events seen in COVID-19 patients such as secretion of IL1{beta}, presentation of a more thrombogenic endothelium by production of thrombomodulin and accumulation of inflammatory cells such as monocytes in the lungs. Of all the drugs, most promising drug was Ertugliflozin which is used for type-2 diabetes. This drug possesses several desired properties and may be a good candidate for immediate repurposing for treatment of COVID-19.

sentences: Iron Deficiency Induces Heart Failure with Ectopic Cardiac Calcification in Mice with Metabolic Syndrome Iron deficiency is linked to worse clinical status and outcomes in heart failure. Although metabolic syndrome contributes to the development of heart failure, the impact of iron deficiency in heart failure complicated with metabolic syndrome remains obscure. KKAy mice were used as a model of metabolic syndrome. Four-week-old male C57BL/6J and KKAy mice were fed either a normal diet or iron-restricted (IR) diet for 12 weeks. During the experiment, 40% of mice died due to pulmonary congestion in KKAy mice with IR diet (KKAy-IR), while no mice died in other groups. Necropsy showed the presence of multiple white lesions on the cardiac surface in those KKAy-IR mice. Echocardiography and histological analyses revealed that KKAy-IR mice exhibited cardiac hypertrophy and cardiac dysfunction with cardiac calcification. Cardiac mRNA of ectonucleotide pyrophosphatase/phosphodiesterase-1 (Enpp1), a key enzyme for bone mineralization, was highly abundant in KKAy-IR mice. Of note, iron restriction-induced cardiac calcification and dysfunction were attenuated by etidronate, an inhibitor of bone mineralization, with decreased cardiac Enpp1 mRNA abundance in KKAy-IR mice. In conclusion, iron deficiency leads to ectopic cardiac calcification and dysfunction with the increase of Enpp1 in metabolic syndrome model mice.

sentences: Magnesium Force Fields for OPC Water with Accurate Solvation, Ion-Binding, and Water-Exchange Properties: Successful Transfer from SPC/E Magnesium plays a vital role in a large variety of biological processes. To model such processes by molecular dynamics simulations, researchers rely on accurate force field parameters for Mg2+ and water. OPC is one of the most promising water models yielding an improved description of biomolecules in water. The aim of this work is to provide force field parameters for Mg2+ that lead to accurate simulation results in combination with OPC water. Using twelve different Mg2+ parameter sets, that were previously optimized with different water models, we systematically assess the transferability to OPC based on a large variety of experimental properties. The results show that the Mg2+ parameters for SPC/E are transferable to OPC and closely reproduce the experimental solvation free energy, radius of the first hydration shell, coordination number, activity derivative, and binding affinity toward the phosphate oxygens on RNA. Two optimal parameter sets are presented: MicroMg yields water exchange in OPC on the microsecond timescale in agreement with experiments. NanoMg yields accelerated exchange on the nanosecond timescale and facilitates the direct observation of ion binding events for enhanced sampling purposes.

sentences: A new insight into RecA filament regulation by RecX from the analysis of conformation-specific interactions RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on single-stranded DNA (ssDNA) in an ATP dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (E. coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

sentences: Understanding the Separation of Timescales in Rhodococcus erythropolis Proteasome Core Particle Assembly The 20S proteasome Core Particle (CP) is a molecular machine that is a key component of cellular protein degradation pathways. Like other molecular machines, it is not synthesized in an active form, but rather as a set of subunits that assemble into a functional complex. The CP is conserved across all domains of life and is composed of 28 subunits, 14  and 14 {beta}, arranged in four stacked 7-member rings (7{beta}7{beta}77). While details of CP assembly vary across species, the final step in the assembly process is universally conserved: two half proteasomes (HP: 7{beta}7) dimerize to form the CP. In the bacterium Rhodococcus erythropolis, experiments have shown that the formation of the HP is completed within minutes, while the dimerization process takes hours. The N-terminal propeptide of the {beta} subunit, which is autocatalytically cleaved off after CP formation, plays a key role in regulating this separation of time scales. However, the detailed molecular mechanism of how the propeptide achieves this regulation is unclear. In this work, we used Molecular Dynamics (MD) simulations to characterize HP conformations and found that the HP exists in two states: one where the propeptide interacts with key residues in the HP dimerization interface and likely blocks dimerization, and one where this interface is free. We found that a propeptide mutant that dimerizes extremely slowly is essentially always in the non-dimerizable state, while the WT rapidly transitions between the two. Based on these simulations, we designed a propeptide mutant that favored the dimerizable state in MD simulations. In vitro assembly experiments confirmed that this mutant dimerizes significantly faster than WT. Our work thus provides unprecedented insight into how this critical step in CP assembly is regulated, with implications both for efforts to inhibit proteasome assembly and for the evolution of hierarchical assembly pathways.

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Amyloid aggregation of Streptococcus mutans Cnm influences its collagen-binding activity The glycosylated collagen- and laminin-binding surface adhesin Cnm is present in approximately 20% of S. mutans clinical isolates and is associated with systemic infections and increased caries risk. Other surface-associated collagen-binding proteins of S. mutans such as P1 and WapA have been demonstrated to form an amyloid quaternary structure with functional implications within biofilms. In silico analysis predicted that the {beta}-sheet rich N-terminal collagen-binding domain (CBD) of Cnm has propensity for amyloid aggregation, whereas the threonine-rich C-terminal domain was predicted to be disorganized. In this study, thioflavin-T fluorescence and electron microscopy were used to show that Cnm forms amyloids either in its native glycosylated or recombinant non-glycosylated forms and that the CBD of Cnm is the main amyloidogenic unit of Cnm. We then performed a series of in vitro, ex vivo and in vivo assays to characterize the amylogenic properties of Cnm. In addition, Congo red birefringence indicated that Cnm is a major amyloidogenic protein of S. mutans biofilms. Competitive binding assays using collagen-coated microtiter plates and dental roots, a substrate rich in collagen, revealed that Cnm monomers inhibit S. mutans binding to collagenous substrates whereas Cnm amyloid aggregates lose this property. Thus, while Cnm contributes to recognition and initial binding of S. mutans to collagen-rich surfaces, Cnm amyloid aggregation appears to represent a mechanism to modulate this activity in mature biofilms.

IMPORTANCEStreptococcus mutans is a keystone pathogen that promotes caries by acidifying the dental biofilm milieu. The collagen- and laminin-binding glycoprotein Cnm is a virulence factor found in about 20% of the clinical isolates of S. mutans. Expression of Cnm by S. mutans is associated with niche expansion, allowing colonization of multiple sites in the body including collagen-rich surfaces such as dentin and heart valves. Here, we demonstrate for the first time that Cnm function appears to be modulated by its aggregation status. As a monomer, its primary function is to promote attachment to collagenous substrates via its collagen binding domain (CBD). However, in later stages of biofilm maturation, the same CBD of Cnm self-assembles into amyloid fibrils, losing the ability to bind to collagen and likely becoming a component of the biofilm matrix. Our findings shed light into the role of functional amyloids in S. mutans pathobiology and ecology.

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Mild SARS-CoV-2 infection in rhesus macaques is associated with viral control prior to antigen-specific T cell responses in tissues SARS-CoV-2 primarily replicates in mucosal sites, and more information is needed about immune responses in infected tissues. We used rhesus macaques to model protective primary immune responses in tissues during mild COVID-19. Viral RNA levels were highest on days 1-2 post-infection and fell precipitously thereafter. 18F-fluorodeoxyglucose (FDG)-avid lung abnormalities and interferon (IFN)-activated myeloid cells in the bronchoalveolar lavage (BAL) were found on days [~]3-4. Virus-specific effector CD8 and CD4 T cells were detectable in the BAL and lung tissue on days [~]7-10, after viral RNA, lung inflammation, and IFN-activated myeloid cells had declined. Notably, SARS-CoV-2-specific T cells were not detectable in the nasal turbinates, salivary glands, and tonsils on day 10 post-infection. Thus, SARS-CoV-2 replication wanes in the lungs prior to T cell responses, and in the nasal and oral mucosa despite the apparent lack of Ag-specific T cells, suggesting that innate immunity efficiently restricts viral replication during mild COVID-19.

ONE SENTENCE SUMMARYSARS-CoV-2 infection leads to mild, focal lung inflammation, and type I IFN activated myeloid cells that mostly resolve prior to the influx of virus-specific effector T cells or antibody responses in rhesus macaques.

sentences: Temporal and environmental factors interact with rootstock genotype to shape leaf elemental composition in grafted grapevines Plants take up elements through their roots and transport them to their shoot systems for use in numerous biochemical, physiological, and structural functions. Elemental composition of above-ground plant tissues, such as leaves, reflects both above- and below-ground activities of the plant genotype, as well the local environment. Perennial, grafted plants, where the root system of one individual is fused to the shoot system of a genetically distinct individual, offer a powerful experimental system in which to study the role of the root system in the elemental composition of the shoot system. We measured elemental composition of over 7000 leaves in the grapevine cultivar  Chambourcin growing ungrafted and grafted to three rootstock genotypes. Leaves were collected over multiple years and phenological stages (across the season) and along a developmental time series. Temporal components of this study had the largest effect on leaf elemental composition; and rootstock genotype interacted with year, phenological stage, and leaf age to differentially modulate leaf elemental composition. Further, the local, above-ground environment affected leaf elemental composition, an effect influenced by rootstock genotype. This work highlights the dynamic nature by which root systems interact with shoot systems to respond to temporal and environmental variation.

sentences: A genetic variant alters the secondary structure of the lncRNA H19 and is associated with Dilated Cardiomyopathy lncRNAs are at the core of many regulatory processes and have also been recognized to be involved in various complex diseases. They affect gene regulation through direct interactions with RNA, DNA or proteins. Accordingly, lncRNAs structure is likely to be essential for their regulatory function. Point mutations, which manifest as SNPs (single nucleotide polymorphisms) in genome screens, can substantially alter their function and, subsequently, the expression of their down-stream regulated genes. To test the effect of SNPs on structure, we investigated lncRNAs associated with dilated cardiomyopathy. Among 322 human candidate lncRNAs we demonstrate first the significant association of a SNP located in lncRNA H19 using data from 1084 diseased and 751 control patients. H19 is generally highly expressed in the heart, with a complex expression pattern during heart development. Next, we used MFE (minimum free energy) folding to demonstrate a significant refolding in the secondary structure of this 861 nt long lncRNA. Since MFE folding may overlook the importance of sub-optimal structures, we showed that this refolding also manifests in the overall Boltzmann structure ensemble. There, the composition of structures is tremendously affected in their thermodynamic probabilities through the genetic variant. Finally, we confirmed these results experimentally, using SHAPE-Seq, corroborating that SNPs affecting such structures may explain hidden genetic variance not accounted for through genome wide association studies. Our results suggest that structural changes in lncRNAs, and lncRNA H19 in particular, affect regulatory processes and represent optimal targets for further in-depth studies probing their molecular interactions.

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EGFR inhibition leads to enhanced desmosome assembly and cohesion in cardiomyocytes The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase, which is dysregulated in several kinds of cancer and heart diseases. Arrhythmogenic cardiomyopathy (AC) is a familial heart disease in part caused by impaired desmosome turnover. Thus, stabilization of desmosome integrity, may provide potential new treatment options. Desmosomes, apart from cellular cohesion, provide the structural framework of a signaling hub. Here, we investigated the role of EGFR inhibition on cardiomyocyte cohesion under physiological and pathophysiological conditions using the murine plakoglobin knockout AC model, in which EGFR was upregulated. EGFR inhibition led to enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR and desmoglein 2 (DSG2). Immunostaining and AFM revealed enhanced DSG2 localization and binding at cell borders upon EGFR inhibition. Enhanced area composita length and desmosome assembly were observed upon EGFR inhibition, confirmed by enhanced DSG2 and desmoplakin (DP) recruitment to cell borders. Thus, inhibiting EGFR, thereby stabilizing desmosome integrity, might provide new treatment options for AC.

SummaryShoykhet et al. show EGFR inhibition-enhanced cardiomyocyte cohesion. This positive adhesiotropy is achieved via an increase in desmosomal assembly and DP/DSG2 localization at cell borders and is the first step towards a novel therapeutic approach for arrhythmogenic cardiomyopathy.

sentences: Towards a mathematical understanding of colonization resistance in multispecies microbial communities Microbial community composition and dynamics are key to health and disease. Explaining the forces generating and shaping diversity in the microbial consortia making up our bodys defenses is a major aim of current research in microbiology. For this, tractable models are needed, that bridge the gap between observations of patterns and underlying mechanisms. While most microbial dynamics models are based on the Lotka-Volterra framework, we still do not have an analytic quantity for colonization resistance, by which a microbial systems fitness as a whole can be understood. Here, inspired by an epidemiological perspective, we propose a rather general modeling framework whereby colonization resistance can be clearly mathematically defined and studied. In our model, N similar species interact with each other through a co-colonization interaction network encompassing pairwise competition and cooperation, abstractly mirroring how organisms effectively modify their micro-scale environment in relation to others. This formulation relies on a generic notion of shared resources between members of a consortium, yielding explicit frequency-dependent dynamics among N species, in the form of a replicator equation, and offering a precise definition of colonization resistance. We demonstrate that colonization resistance arises and evolves naturally in a multispecies system as a collective quadratic term in a replicator equation, describing dynamic mean invasion fitness. Each pairwise invasion growth rate between two ecological partners, [Formula], is derived explicitly from species asymmetries and mean traits. This makes the systemic colonization resistance [Formula] also an emergent function of global mean-field parameters and trait variation architecture, weighted by the evolving relative abundances among species. In particular, if the underlying invasion fitness matrix {Lambda} displays species-specific  invasiveness or  invasibility structure, colonization resistance will be insensitive to mean micro-scale cooperation or competition. However, in general, colonization resistance depends on and may undergo critical transitions with changes in mean  environment, e.g. cooperation and growth level in a community. We illustrate several key links between our proposed measure of colonization resistance and invader success, including sensitivity to timing, and to the intrinsic pairwise invasion architecture of the resident community. Our simulations reveal that symmetric and invader-driven mutual invasion among resident species tend to maximize systemic colonization resistance to outsiders, when compared to resident-driven, anti-symmetric, almost anti-symmetric and random {Lambda} structures. We contend this modeling approach is a powerful new avenue to study, test and validate interaction networks and invasion topologies in diverse microbial consortia, and quantify analytically their role in colonization resistance, system function, and invasibility.

sentences: Chromosomal integration of blaCTX-M genes in diverse Escherichia coli isolates recovered from river water in Japan Occurrence of extended-spectrum {beta}-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in environmental waters is of great concern. However, unlike clinical ESBLEC, their genetic characteristics, in particular the genetic contexts of ESBL genes, are not well understood. In this study, we sequenced and analyzed the genomes of CTX-M-producing E. coli isolates recovered from river water to fully characterize the genetic contexts of blaCTX-M genes. Among the 14 isolates with completed genomes, blaCTX-M genes were detected on the chromosome in nine isolates. All but one chromosomal blaCTX-M genes were associated with ISEcp1 and were carried on different transposition units (TPU) ranging in size from 2,855 bp to 11,093 bp; the exception, blaCTX-M-2, was associated with ISCR1. In many cases, the TPU were directly inserted into chromosomes. However, in one case, the TPU was part of a 36-kbp multiresistance region bracketed by IS26, and the whole multiresistance region was inserted into the chromosome. We also found a case where a chromosomally-located TPU and an adjacent region had transposed into another chromosomal location, leading to duplication of blaCTX-M. The remaining five isolates carried blaCTX-M genes on epidemic IncI1 plasmids of different STs (ST3, ST16, ST113, and ST167) (n = 4) or on an IncB/O/K/Z plasmid (n = 1). This study revealed that environmental E. coli carry blaCTX-M genes in diverse genetic contexts. Apparent high prevalence of chromosomal blaCTX-M potentially indicates that some environmental E. coli can stably maintain blaCTX-M genes in the absence of antibiotic selection pressure, though further studies are needed to confirm this.

sentences: Sugar feeding enhances gut immunity and protects against arboviral infection in the mosquito vector Aedes aegypti As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, it has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been analysed. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families, highlighting a broad antiviral action of sugar. Sugar feeding blocks arbovirus initial infection and dissemination from the gut, lowers infection prevalence and intensity, thereby decreasing transmission potential of female mosquitoes. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity and vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this could increase the spread of mosquito-borne arboviral diseases.

sentences: Naturalized Dolichogenidea gelechiidivoris Marsh (Hymenoptera: Braconidae) complement the resident parasitoid complex of Tuta absoluta (Meyrick) (Lepidopera:Gelechiidae) in Spain Our study aimed to assess the contribution of natural parasitism due to Necremnus tutae Ribes & Bernardo (Hymenoptera: Eulophidae) to the biological control of Tuta absoluta (Meyrick) (Lepidopera:Gelechiidae) in commercial plots where an IPM program based on the use of predatory mirid bugs was implemented. During the samplings, the presence of another parasitoid was detected and, therefore, a second part of our study intended to identify this species and to evaluate the importance of its natural populations in the biological control of the pest. Leaflets with T. absoluta galleries were collected during 2017-2020 from commercial tomato plots in the horticultural production area of Catalonia (Northeast Spain), including greenhouses, open fields, and roof covered tunnels that lack side walls. In the laboratory, T. absoluta larvae were classified as ectoparasitized, alive, or dead. Reared parasitoids from ectoparasitized larvae were mostly morphologically identified as Necremnus sp. with parasitism rates that peaked in summer months with values between 9 and 15%. Some of these ectoparasitized larvae also yielded another parasitoid identified as Dolichogenidea gelechiidivoris Marsh (Hymenoptera: Braconidae) by both morphological and molecular-DNA barcoding methods. In 2020, parasitism rates due to D. gelechiidivoris that increased with season up to 22%. Our work reports for the first time in Europe the presence of the neotropical species D. gelechiidivoris adding this biocontrol agent to the resident parasitoid complex of T. absoluta in Spain.

sentences: HIV-1 Rev-RRE Functional Activity in Primary Isolates is Highly Dependent on Minimal Context-Dependent Changes in Rev During HIV infection, intron-containing viral mRNAs have to be exported efficiently from the host cell nucleus to the cytoplasm in order to complete the replication cycle. To overcome cellular restrictions to export incompletely spliced transcripts, HIV encodes a protein, Rev, that is constitutively expressed from a completely spliced transcript. Rev is then imported into the nucleus where it binds to an RNA structure on intron-containing viral mRNAs called the Rev Response Element (RRE). Bound Rev multimerizes and recruits cellular factors that permit the nuclear export of the resulting ribonucleoprotein complex. Primary HIV isolates display substantial variation in the functional activity of the Rev-RRE axis, which may permit viral adaptation to differing immune environments. We describe two subtype G primary isolates with disparate Rev activity. Rev activity was correlated with in vitro fitness of replication-competent viral constructs. Amino acid differences within the oligomerziation domain, but not within the arginine-rich motif or nuclear export signal, determined the different levels of Rev activity. Two specific amino acid substitutions were demonstrated to be able to alter the low-activity Rev to a high-activity phenotype. However, introducing the original amino acids from the the low activity Rev into high activity Rev in this position did not result in significant alterations in activity, highlighting the importance of the broader sequence context for functional activity. These results demonstrate that studies of Rev and RRE activity variation, which may have broader implications for HIV transmission and pathogenesis, should include sequences from primary isolates, as findings using only laboratory-adapted strains cannot be generalized.

sentences: A representative reference for MRI-based human axon radius assessment using light microscopy Non-invasive assessment of axon radii via MRI bears great potential for clinical and neuroscience research as it is a main determinant of the neuronal conduction velocity. However, there is a lack of representative histological reference data on the scale of the cross-section of MRI voxels for validating the MRI-visible, effective radius (reff). Because the current gold standard stems from neuroanatomical studies designed to estimate the frequency-weighted arithmetic mean radius (rarith) on small ensembles of axons, it is unsuited to estimate the tail-weighted reff. We propose CNN-based segmentation on high-resolution, large-scale light microscopy (lsLM) data to generate a representative reference for reff. In a human corpus callosum, we assessed estimation accuracy and bias of rarith and reff. Furthermore, we investigated whether mapping anatomy-related variation of rarith and reff is confounded by low-frequency variation of the image intensity, e.g., due to staining heterogeneity. Finally, we analyzed the potential error due to outstandingly large axons in reff. Compared to rarith, reff was estimated with higher accuracy (normalized-root-mean-square-error of reff: 7.2 %; rarith: 21.5 %) and lower bias (normalized-mean-bias-error of reff: -1.7 %; rarith: 16 %). While rarith was confounded by variation of the image intensity, variation of reff seemed anatomy-related. The largest axons contributed between 0.9 % and 3 % to reff. In conclusion, the proposed method accurately estimates reff at MRI voxel resolution across a human corpus callosum sample. Further investigations are required to assess generalization to brain areas with different axon radii ensembles.

sentences: A Ferroptosis-mediated regulation of the biogenesis of the oxidative phosphorylation system Several subunits in the matrix domain of mitochondrial complex I (CI) have been posited to be redox sensors for CI; but how elevated levels of reactive oxygen species (ROS) impinge on CI assembly is unknown. We report that when the mitochondrial NADPH-generating enzyme - Isocitrate Dehydrogenase 2 - is genetically disrupted, ROS levels are elevated and assembly of the oxidative phosphorylation system (OXPHOS) is impaired. Mechanistically, this begins with a ROS-mediated inhibition of biosynthesis of the matrix domain of CI, which progresses to a point where ferroptotic signals are induced, the mitochondrial unfolded protein response is activated and multiple OXPHOS complexes are impaired. Disruption of other enzymes that eliminate hydrogen peroxide, but not those that eliminate the superoxide radical, recapitulates the phenotype; implicating hydrogen peroxide as the signaling molecule involved. Thus, the redox status of the mitochondrion modulates the assembly of the matrix domain of CI and ultimately that of the entire OXPHOS.

sentences: Neuronal KGB-1 JNK MAPK signaling regulates the dauer developmental decision in response to environmental stress in C. elegans In response to stressful growth conditions of high population density, food scarcity and elevated temperature, young larvae of nematode Caenorhabditis elegans can enter a developmentally arrested stage called dauer that is characterized by dramatic anatomic and metabolic remodeling. Genetic analysis of dauer formation of C. elegans has served as an experimental paradigm for the identification and characterization of conserved neuroendocrine signaling pathways. Here, we report the identification and characterization of a conserved JNK-like mitogen-activated protein kinase (MAPK) pathway that is required for dauer formation in response to environmental stressors. We observed that loss-of-function mutations in the MLK-1-MEK-1-KGB-1 MAPK pathway suppress dauer entry. Loss-of-function mutation in the VHP-1 MAPK phosphatase, a known negative regulator of KGB-1 signaling, results in constitutive dauer formation which is dependent on the presence of dauer pheromone but independent of diminished food levels or elevated temperatures. Our data suggest that KGB-1 pathway acts in the sensory neurons, in parallel to established insulin and TGF-{beta} signaling pathways, to transduce the dauer-inducing environmental cues of diminished food levels and elevated temperature.

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CCR2 Regulates Vaccine-Induced Mucosal T cell Memory to Influenza A Virus Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T-cell based vaccines against respiratory viral pathogens such as influenza A virus (IAV). Chemokine receptor 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs, to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendrtitic cells (DCs) and monocyte-derived DCs internalized and processed vaccine antigen in lungs. We also found that Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF-3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127HI/KLRG-1LO, OX40+veCD62L+ve and mucosally imprinted CD69+veCD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs, induced by CCR2 deficiency was linked to dampened expression of T-bet, but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens including IAV and SARS-CoV-2.

ImportanceWhile antibody-based immunity to influenza A virus (IAV) is type and sub-type specific, lung and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T-cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of anti-viral lung-resident memory T cells, following intranasal vaccination. These findings suggested that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses such as IAV and SARS-CoV-2.

sentences: Characterization of mouse Bmp5 regulatory injury element in zebrafish wound models Many key signaling molecules used to build tissues during embryonic development are re-activated at injury sites to stimulate tissue regeneration and repair. Bone morphogenetic proteins provide a classic example, but the mechanisms that lead to reactivation of BMPs following injury are still unknown. Previous studies have mapped a large "injury response element" (IRE) in the mouse Bmp5 gene that drives gene expression following bone fractures and other types of injury. Here we show that the large mouse IRE region is also activated in both zebrafish tail resection and mechanosensory hair cell injury models. Using the ability to test multiple constructs and image temporal and spatial dynamics following injury responses, we have narrowed the original size of the mouse IRE region by over 100 fold and identified a small 142 bp minimal enhancer that is rapidly induced in both mesenchymal and epithelial tissues after injury. These studies identify a small sequence that responds to evolutionarily conserved local signals in wounded tissues and suggest candidate pathways that contribute to BMP reactivation after injury.

sentences: Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions led to two outcomes -- removal of cytoplasmic material and stem cell division, or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality.

sentences: Joint effect of changing selection and demography on the site frequency spectrum The site frequency spectrum (SFS) is an important statistic that summarizes the molecular variation in a population, and used to estimate population-genetic parameters and detect natural selection. While the equilibrium SFS in a constant environment is quite well studied, recent research has focused on nonequilibrium SFS to elucidate the role of demography when selection is constant in time and of fluctuating selection in a population of constant size. However, the joint effect of time-dependent selection and population size has not been investigated so far. Here, we study the SFS in a randomly mating, diploid population in which both the population size and selection coefficient vary periodically with time using a diffusion theory approach, and derive simple analytical expressions for the time-averaged SFS in slowly and rapidly changing environments. We show that for strong selection and in slowly changing environments, the time-averaged SFS differs significantly from the equilibrium SFS when the population experiences both positive and negative cycles of the selection coefficient. The deviation depends on the time spent by the population in the deleterious part of the selection cycle and the phase difference between the selection coefficient and population size. In particular, we find that the time-averaged SFS in slowly to moderately fast varying, on-average neutral environment has the same qualitative shape as the equilibrium SFS for positively selected mutant but differs quantitatively from it which can be captured by an effective population size.

sentences: Long-term ketogenic diet increases expression of SOX2-dependent oligodendrocyte- and myelination-associated genes in the aged mouse brain. Aging is associated with multiple neurodegenerative conditions that severely limit quality of life and shorten lifespan. Studies in rodents indicate that in addition to extending lifespan, the ketogenic diet improves cognitive function in aged animals, thus improving healthspan. To broadly investigate what mechanisms might be activated in the brain in response to ketogenic diet, we conducted transcriptome wide analysis on whole brain samples from 13-month-old mice, 26-month-old mice, and 26-month-old mice fed a ketogenic diet. We observed clear activation of inflammation and complement system pathways in the 26-month-old mice relative to the younger animals. Interestingly, ketogenic diet caused a modest but significant increase in the expression of SOX2-dependent oligodendrocyte/myelination markers.

sentences: A nesprin-4/kinesin-1 cargo model for nucleus positioning in cochlear outer hair cells Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in-vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

sentences: Antagonism of GluK1-containing Kainate Receptors Reduces Ethanol Consumption by Modulating Ethanol Reward and Withdrawal Alcohol use disorder (AUD) is a neuropsychiatric condition affecting millions of people worldwide. Topiramate (TPM) is an antiepileptic drug that has been shown to reduce ethanol drinking in humans. However, TPM is associated with a variety of adverse effects due to its interaction with many receptor systems and intracellular pathways. Thus, a better understanding of the role of TPMs main molecular targets in AUD could yield better therapeutic tools. GluK1-containing kainate receptors (GluK1*KARs) are non-selectively inhibited by TPM, and genetic association studies suggest that this receptor system could be targeted to reduce drinking in AUD patients. We examined the efficacy of LY466195, a selective inhibitor of GluK1*KAR, in reducing ethanol consumption in the intermittent two-bottle choice paradigm in mice. The effect of LY466195 on various ethanol-related phenotypes was investigated by quantification of alcohol intake, physical signs of withdrawal, conditioned place preference (CPP) and in vivo microdialysis in the nucleus accumbens. Selective GluK1*KAR inhibition reduced ethanol intake and preference in a dose-dependent manner. LY466195 treatment attenuated the physical manifestations of ethanol withdrawal and influenced the rewarding properties of ethanol. Interestingly, LY466195 injection also normalized changes in dopamine levels in response to acute ethanol in ethanol-dependent mice, but had no effect in ethanol-naive mice, suggesting ethanol state-dependent effects. The data point to GluK1*KARs as an attractive pharmacological target for the treatment of AUD.

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GwEEP - A comprehensive approach for genome-wide efficiency profiling of DNA modifying enzymes A precise understanding of DNA methylation dynamics on a genome wide scale is of great importance for the comprehensive investigation of a variety of biological processes such as reprogramming of somatic cells to iPSCs, cell differentiation and also cancer development. To date, a complex integration of multiple and distinct genome wide data sets is required to derive the global activity of DNA modifying enzymes. We present GwEEP - Genome-wide Epigenetic Efficiency Profiling as a versatile approach to infer dynamic efficiency changes of DNA modifying enzymes at base pair resolution on a genome wide scale. GwEEP relies on genome wide oxidative Hairpin Bisulfite sequencing (HPoxBS) data sets, which are translated by a sophisticated hidden Markov model into quantitative enzyme efficiencies with reported confidence around the estimates. GwEEP in its present form predicts de novo and maintenance methylation efficiencies of Dnmts, as well as the hydroxylation efficiency of Tets but its purposefully flexible design allows to capture further oxidation processes such as formylation and carboxylation given available data in the future. Applied to a well characterized ES cell model, GwEEP precisely predicts the complex epigenetic changes following a Serum-to-2i shift i.e., (i) instant reduction in maintenance efficiency (ii) gradually decreasing de novo methylation efficiency and (iii) increasing Tet efficiencies. In addition, a complementary analysis of Tet triple knock-out ES cells confirms the previous hypothesized mutual interference of Dnmts and Tets. GwEEP is applicable to a wide range of biological samples including cell lines, but also tissues and primary cell types.

MOTIVATIONDynamic changes of DNA methylation patterns are a common phenomenon in epigenetics. Although a stable DNA methylation profile is essential for cell identity, developmental processes require the rearrangement of 5-methylcytosine in the genome. Stable methylation patterns are the result of balanced Dnmts and Tets activities, while methylome transformation results from a coordinated change in Dnmt and Tet efficiencies. Such transformations occur on a global scale, for example during the reprogramming of maternal and paternal methylation patterns and the establishment of novel cell type specific methylomes during embryonic development in vivo, but also in vitro during (re)programming of induced pluripotent stem cells, as well as somatic cells. In addition, local (de)methylation events are key for gene regulation during cell differentiation. A detailed understanding of Dnmt and Tet cooperation is essential for understanding natural epigenetic adaptation as well as optimization of in vitro (re)programming protocols. For this purpose, we developed a pipeline for quantitative and precise estimation of Dnmt and Tet activity. Using only double strand methylation information, GwEEP infers accurate maintenance and de novo methylation efficiency of Dnmts, as well as hydroxylation efficiency of Tets at single base resolution. Thus, we believe GwEEP provides a powerful tool for the investigation of methylome rearrangements in various systems.

sentences: Spatiotemporal patterns of neuronal subtype genesis suggest hierarchical development of retinal diversity How do neuronal subtypes emerge during development? Recent molecular studies have expanded our knowledge of existing neuronal diversity. However, the genesis of neuronal subtypes remains elusive and previous studies have been limited by a lack of quantitative methods for simultaneous detection of subtype diversity in situ. The bipolar interneurons of the mammalian retina represent a diverse neuronal class, characterized by distinct functions, morphologies, and recently discovered transcriptional profiles. Here, we developed a comprehensive spatiotemporal map of bipolar subtype genesis in the retina. Combining multiplexed detection of 16 RNA markers with timed delivery of EdU and BrdU, we analyzed more than 30,000 single cells in full retinal sections to classify all bipolar subtypes and their birthdates. We found that bipolar subtype birthdates are ordered and follow a centrifugal developmental axis. Spatial analysis revealed a striking oscillatory wave pattern of bipolar subtype birthdates, and lineage analyses suggest clonal restriction on homotypic subtype production. These results inspired a hierarchical model of neuronal subtype genesis in the mammalian retina, with the wave pattern of subtype birthdates arising from early asymmetric cell divisions among founding retinal progenitor cells. Our results provide an outline of the developmental logic that generates diverse neuronal subtypes, and establishes a framework for studying subtype diversification.

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Meta-transcriptomic analysis reveals the gene expression and novel conserved sub-genomic RNAs in SARS-CoV-2 and MERS-CoV. BackgroundFundamental to viral biology is identification and annotation of viral genes and their function. Determining the level of coronavirus gene expression is inherently difficult due to the positive stranded RNA genome and the identification of sub-genomic RNAs (sgRNAs) that are required for expression of most viral genes. In the COVID-19 epidemic so far, few genomic studies have looked at viral sgRNAs and none have systematically examined the sgRNA profiles of large numbers of SARS-CoV2 datasets in conjuction with data for other coronaviruses.

ResultsWe developed a bioinformatic pipeline to analyze the sgRNA profiles of coronaviruses and applied it to 588 individual samples from 20 independent studies, covering more than 10 coronavirus species. Our result showed that SARS-CoV, SARS-CoV-2 and MERS-CoV each had a core sgRNA repertoire generated via a canonical mechanism. Novel sgRNAs that encode peptides with evolutionarily conserved structures were identified in several coronaviruses and were expressed in vitro and in vivo. Two novel peptides may have direct functional relevance to disease, by alluding interferon responses and disrupting IL17E (IL25) signaling. Relevant to coronavirus infectivity and transmission, we also observed that the level of Spike sgRNAs were significantly higher in-vivo than in-vitro, while the opposite held true for the Nucleocapside protein.

ConclusionsOur results greatly expanded the predicted number of coronaviruses proteins and identified potential viral peptide suggested to be involved in viral virulence. These methods and findings shed new light on coronavirus biology and provides a valuable resource for future genomic studies of coronaviruses.

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Integration of immunome with disease-gene network reveals common cellular mechanisms between IMIDs and drug repurposing strategies ObjectiveDevelopment and progression of immune-mediated inflammatory diseases (IMIDs) involve intricate dysregulation of the disease associated genes (DAGs) and their expressing immune cells. Due to the complex molecular mechanism, identifying the top disease associated cells (DACs) in IMIDs has been challenging. Here, we aim to identify the top DACs and DAGs to help understand the cellular mechanism involved in IMIDs and further explore therapeutic strategies.

MethodUsing transcriptome profiles of 40 different immune cells, unsupervised machine learning, and disease-gene networks, we constructed the Disease-gene IMmune cell Expression (DIME) network, and identified top DACs and DAGs of 12 phenotypically different IMIDs. We compared the DIME networks of IMIDs to identify common pathways between them. We used the common pathways and publicly available drug-gene network to identify promising drug repurposing targets.

ResultWe found CD4+Treg, CD4+Th1, and NK cells as top DACs in the inflammatory arthritis such as ankylosing spondylitis (AS), psoriatic arthritis, and rheumatoid arthritis (RA); neutrophils, granulocytes and BDCA1+CD14+ cells in systemic lupus erythematosus and systemic scleroderma; ILC2, CD4+Th1, CD4+Treg, and NK cells in the inflammatory bowel diseases (IBDs). We identified lymphoid cells (CD4+Th1, CD4+Treg, and NK) and their associated pathways to be important in HLA-B27 type diseases (psoriasis, AS, and IBDs) and in primary-joint-inflammation-based inflammatory arthritis (AS and RA). Based on the common cellular mechanisms, we identified lifitegrast as potential drug repurposing candidate for Crohns disease, and other IMIDs.

ConclusionOur method identified top DACs, DAGs, common pathways, and proposed potential drug repurposing targets between IMIDs. To extend our method to other diseases, we built the DIME tool. Thus paving way for future (pre-)clinical research.

sentences: Structuring time: The hippocampus constructs sequence memories that generalize temporal relations across experiences The hippocampal-entorhinal region supports memory for episodic details, such as temporal relations of sequential events, and mnemonic constructions combining experiences for inferential reasoning. However, it is unclear whether hippocampal event memories reflect temporal relations derived from mnemonic constructions, event order, or elapsing time, and whether these sequence representations generalize temporal relations across similar sequences. Here, participants mnemonically constructed times of events from multiple sequences using infrequent cues and their experience of passing time. After learning, event representations in the anterior hippocampus reflected temporal relations based on constructed times. Temporal relations were generalized across sequences, revealing distinct representational formats for events from the same or different sequences. Structural knowledge about time patterns, abstracted from different sequences, biased the construction of specific event times. These findings demonstrate that mnemonic construction and the generalization of relational knowledge combine in the hippocampus, consistent with the simulation of scenarios from episodic details and structural knowledge.

sentences: baredSC: Bayesian Approach to Retrieve Expression Distribution of Single-Cell The number of studies using single-cell RNA sequencing (scRNA-seq) is constantly growing. This powerful technique provides a sampling of the whole transcriptome of a cell. However, the commonly used droplet-based method often produces very sparse samples. Sparsity can be a major hurdle when studying the distribution of the expression of a specific gene or the correlation between the expressions of two genes. We show that the main technical noise associated with these scRNA-seq experiments is due to the sampling (i.e. Poisson noise). We developed a new tool named baredSC, for Bayesian Approach to Retrieve Expression Distribution of Single-Cell, which infers the intrinsic expression distribution in single-cell data using a Gaussian mixture model (GMM). baredSC can be used to obtain the distribution in one dimension for individual genes and in two dimensions for pairs of genes, in particular to estimate the correlation in the two genes expressions. We apply baredSC to simulated scRNA-seq data and show that the algorithm is able to uncover the expression distribution used to simulate the data, even in multi-modal cases with very sparse data. We also apply baredSC to two real biological data sets. First, we use it to measure the anti-correlation between Hoxd13 and Hoxa11, two genes with known genetic interaction in embryonic limb. Then, we study the expression of Pitx1 in embryonic hindlimb, for which a trimodal distribution has been identified through flow cytometry. While other methods to analyze scRNA-seq are too sensitive to sampling noise, baredSC reveals this trimodal distribution.

sentences: Facilitating the design of combination therapy in cancer using multipartite network models: Emphasis on acute myeloid leukemia From the drug discovery perspective, combination therapy is recommended in cancer due to efficiency and safety compared to the common cytotoxic and single-targeted monotherapies. However, identifying effective drug combinations is time- and cost-consuming. Here, we offer a novel strategy of predicting potential drug combinations and patient subclasses by constructing multipartite networks using drug response data on patient samples. In the present study, we used Beat AML and GDSC, two comprehensive datasets based on patient-derived and cell line-based samples, to show the potential of multipartite network modeling in cancer combinatorial therapy. We used the median values of cell viability to compare drug potency and reconstruct a weighted bipartite network, which models the interaction of drugs and biological samples. Then, clusters of network communities were identified in two projected networks based on the topological structure of networks. Chemical structures, drug-target networks, protein-protein interactions, and signaling networks were used to corroborate the intra-cluster homogeneity. We further leveraged the community structures within the drug-based multipartite networks to discover effective multi-targeted drug combinations, and the synergy levels which were supported with more evidence using the DrugComb and the ALMANAC databases. Furthermore, we confirmed the potency of selective combinations of drugs against monotherapy in vitro experiment using three acute myeloid leukemia (AML) cell lines. Taken together, this study presents an innovative data-driven strategy based on multipartite networks to suggest potential drug combinations to improve treatment of AML.

sentences: Pkd1 and Wnt5a genetically interact to control lymphatic vascular morphogenesis in mice Lymphatic vascular development is regulated by well-characterised signalling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity and and morphogenesis. Canonical and non-canonical WNT signalling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Here we find that loss of non-canonical WNT signalling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1/Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1-/- mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a-/- mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild-type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signalling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signalling during the formation of lymphatic vascular networks.

sentences: Identifying C. elegans Lifespan Mutants by Screening for Early-Onset Protein Aggregation Genetic screens have been widely used to identify genetic pathways that control specific biological functions. In C. elegans, forward genetic screens rely on the isolation of reproductively active mutants that can self-propagate clonal populations. Since aged individuals are unable to generate clonal populations, screens that target post-reproductive phenotypes, such as longevity, are challenging. In this work, we developed an approach that combines microfluidic technologies and image processing to perform a high-throughput, automated screen for mutants with shortened lifespan using protein aggregation as a marker for aging. We take advantage of microfluidics for maintaining a reproductively-active adult mutagenized population and for performing serial high-throughput analysis and sorting of animals with increased protein aggregation, using fluorescently labeled PAB-1 as a readout. We identified five mutants with increased aggregation levels, of which two exhibited a reduced lifespan. We demonstrate that lifespan mutants can be identified by screening for accelerated protein aggregation through quantitative analysis of fluorescently-labeled aggregates in populations that do not require conditional sterilization or manual separation of parental and progeny populations. We further analyzed the morphology of protein aggregates and reveal that patterns of aggregation in naturally-aging animals differ from mutants with increased aggregation, suggesting aggregate growth is time-dependent. This screening approach can be customized to other non-developmental phenotypes that appear during adulthood, as well as to other aging markers to identify additional longevity-regulating genetic pathways.

sentences: Emergence of integrated information at macro timescales in real neural recordings How a system generates conscious experience remains an elusive question. One approach towards answering this is to consider the information available in the system from the perspective of the system itself. Integrated information theory (IIT) proposes a measure to capture this, integrated information ({Phi}). While {Phi} can be computed at any spatiotemporal scale, IIT posits that it be applied at the scale at which the measure is maximised. Importantly, {Phi} should emerge to be maximal not at the smallest spatiotemporal scale, but at some macro scale where system elements or timesteps are grouped into larger elements or timesteps. Emergence in this sense has been demonstrated in artificial binary systems, but it remains unclear whether it occurs in real neural recordings which are generally continuous and noisy. Here we first utilise a computational model to confirm that {Phi} becomes maximal at the temporal scales underlying its generative mechanisms. Second, we search for emergence in local field potentials from the fly brain recorded during wakefulness and anaesthesia, finding that normalised {Phi} (wake/anaesthesia), but not raw {Phi} values, peaks at 5 ms. Lastly, we extend our model to investigate why raw {Phi} values themselves did not peak. This work extends the application of {Phi} to simple artificial systems consisting of logic gates towards searching for emergence of a macro spatiotemporal scale in real neural systems.

sentences: A neural network account of memory replay and knowledge consolidation Replay can consolidate memories through offline neural reactivation related to past experiences. Category knowledge is learned across multiple experiences, and its subsequent generalisation is promoted by consolidation and replay during rest and sleep. However, aspects of replay are difficult to determine from neuroimaging studies. We provided insights into category knowledge replay by simulating these processes in a neural network which approximated the roles of the human ventral visual stream and hippocampus. Generative replay, akin to imagining new category instances, facilitated generalisation to new experiences. Consolidation-related replay may therefore help to prepare us for the future as much as remember the past. Generative replay was more effective in later network layers functionally similar to the lateral occipital cortex than layers corresponding to early visual cortex, drawing a distinction between neural replay and its relevance to consolidation. Category replay was most beneficial for newly acquired knowledge, suggesting replay helps us adapt to changes in our environment. Finally, we present a novel mechanism for the observation that the brain selectively consolidates weaker information; a reinforcement learning process in which categories were replayed according to their contribution to network performance. This reinforces the idea of consolidation-related replay as an active rather than passive process.

sentences: A remote lecture series roadmap to equity, diversity, and inclusion in STEM Within a year of the shutdowns caused by the COVID-19 pandemic, virtual meetings transformed from an auxiliary service to an essential work platform for hundreds of millions of people worldwide. Universities rapidly accelerated adoption of virtual platforms for remote conferences, classes, and seminars amidst a second crisis that started in May 2020 and has tested institutional commitment to the principles of equity, diversity, and inclusion. To address these concerns, we began the Diversity and Science Lecture series (DASL), a cross-institutional national platform where junior life scientists present personal stories, professional progress, and advice for their peers. We first demonstrate benchmarking of speaker diversity using public data and review scientific talk content by text mining of talk titles from DASL. We then review insight into equity, diversity, and inclusion in STEM presented by DASL speakers. Notably, nearly every speaker highlighted the importance of social or interpersonal support to their career progression. Three-fifths of speakers commented on race or ethnicity. Other identities received considerable attention from a minority of speakers: immigrant identity, gender identity, mental illness, sexual minorities, disability, and rural or agricultural background. In addition, we report that ancillary small group sessions with speakers achieve greater engagement when held before formal seminars versus after. To facilitate future programming, we provide exhaustive guides that frame the goals of virtual programming in terms of benefits for executive team members, speakers, and attendees. In total, we have curated over 75 resources for personal and professional development cited by trainee life scientists, attracted over 4,000 unique website visitors, and exceeded 3,500 hours of engagement from attendees at weekly seminars. Our success with DASL confirms that community building can flourish under a remote lecture framework.

sentences: Describing Chemical Kinetics in the Time Dimension with Mean Reaction Time The chemical kinetics is such a fundamental topic in chemistry. By analyzing how the experimental conditions and parameters influence the reaction rate, chemists have deciphered the molecular details on how the chemical reactions occur. The quantitative analysis of the reaction rate requires the formulation of the rate equation, which describes the dependence of the reaction rate on the concentrations of the reactants and also the rate constants of the elementary steps. Though the methods to derive the rate equation from the kinetic model have been known for a century, it is still mathematically challenging to derive the rate equation for complex reactions with multiple steps, which requires a solution for simultaneous differential equations. Therefore, chemists frequently resort to the steady-state approximation or the numerical simulation. One way to avoid the mathematical difficulty is to describe the chemical kinetics in the time dimension. Describing the mean reaction time, the average of the time required for the completion of the chemical reaction, using the elementary rate constants of the kinetic model is much simpler mathematically than deriving the rate equation for the kinetic model. Here, we describe the basic rules for the derivation of the formula of the mean reaction time, derive the generalized equation for the mean reaction time, and analyze the components of the formula to determine how the individual steps in the complex reaction contribute to the mean reaction time. Being the ensemble-averaged value, the mean reaction time does not provide the information on the actual distribution of the reaction time of individual chemical entity. However, the formula of the mean reaction time reveals invaluable insights on how the energy levels of the ground state and the transition states affect the kinetics of the complex reaction and offers a way to identify the most time-consuming process of the complex reaction in a straightforward manner. We also apply the mean reaction time to enzyme kinetics and demonstrate that one can derive the expressions of the kinetic parameters (kcat/KM and kcat) in a surprisingly simple way even without resorting to the steady-state application.

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Three-dimensional models of the cervicovaginal epithelia to study host-microbiome interactions and sexually transmitted infections Two-dimensional (2D) cell culture systems have provided controlled, reproducible means to analyze host-pathogen interactions. Although inexpensive, straightforward, and requiring very short time commitment, these models recapitulate neither the functionality of multi-layered cell types nor the microbial diversity of an infected human. Animal models have commonly been used to recreate the complexity of human infections. However, extensive modifications are commonly required to recreate interactions that resemble those in the human reproductive tract microbiologically and physiologically. Three-dimensional (3D) cell culture models have emerged as alternative means of reproducing key elements of human infections at a fraction of the cost of animal models and on a scale that allows for replicative experiments to be readily performed. Here we describe a new 3D model that utilizes transwells with epithelial cells seeded apically and a basolateral extra cellular matrix (ECM)-like layer containing collagen and fibroblasts. In this system, basal feeding creates a liquid/air interface on the apical side. The model produced tissues with close morphologic and physiological resemblance to human cervical and vaginal epithelia, including observable levels of mucus produced by cervical cells. Infection by both Chlamydia trachomatis and Neisseria gonorrhoeae was demonstrated as well as the growth of bacterial species observed in the human vaginal microbiota, enabling controlled mechanistic analyses of the interactions between host cells, vaginal microbiota and STI pathogens. Future experiments may include immune cells to mimic more closely the genital environment. Finally, the modular set up of the model makes it fully applicable to the analysis of non-genital host-microbiome-pathogen interactions.

IMPORTANCEInfected sites in humans are a complex mix of host and microbial cell types interacting with each other to perform specific and necessary functions. The ability to understand the mechanism(s) that facilitate these interactions, and interactions with external factors is paramount to being able to develop preventative therapies. Models that attempt to faithfully replicate the complexity of these interactions are time intensive, costly, and not conducive to high throughput analysis. Two-dimensional (2D) models that have been used as a platform to understand these interactions, while cost effective, are generally limiting in experimental flexibility and structural/physiological relevance. Our three-dimensional (3D) models of the cervicovaginal epithelium can facilitate analysis of interactions between the host epithelium, sexually transmitted pathogens and bacteria present in the vaginal microbiota. Due to the modular design, additional cell types and environmental modulators can be introduced to the system to provide added complexity, approaching conditions in the infected human host.

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Dissolved oxygen minimally affects magnetic susceptibility in biologically relevant conditions PurposeTo investigate the potential of quantitative susceptibility mapping (QSM) with MRI as a biomarker for tissue oxygenation in fat-water mixture. Oxygen molecules (O2) are paramagnetic. This suggests that dissolved O2 in tissue should affect the measured magnetic susceptibility. However, direct measurements of dissolved O2 in tissues is challenging with QSM as the induced change in susceptibility is below the sensitivity of existing algorithms. QSM in regions that contain fat could be sensitive enough to be used as a marker of tissue oxygenation as oxygen has a larger solubility in fat than in water.

MethodsThe relationship between dissolved O2 concentration and magnetic susceptibility was investigated based on MRI measurements using phantoms made of fat-water emulsions. Dairy cream was used to approximate fat-containing biological tissues. Phantoms based on dairy cream with 35 % fat were designed with controlled concentrations of dissolved O2. O2 was bubbled into the dairy cream to reach O2 concentrations above the concentration at atmospheric pressure, while nitrogen was bubbled in cream to obtain O2 concentrations below atmospheric pressure. Magnetic susceptibility was expected to increase, becoming more paramagnetic, as O2 concentration was increased.

ResultsMagnetic susceptibility from MRI-based QSM measurements did not reveal a dependence on O2 concentration in fat-water mixture phantoms. The relationship between susceptibility and O2 was weak and inconsistent among the various phantom experiments.

ConclusionQSM in fat-water mixture appears to be minimally sensitive to dissolved O2 based on phantom experiments. This suggests that QSM is not likely to be sensitive enough to be proposed as a marker for tissue oxygenation, as the change in magnetic susceptibility induced by the change in dissolved O2 concentration is below the current detection limit, even in the presence of fat.

sentences: Dengue emergence in the temperate Argentinian province of Santa Fe, 2009-2020. Dengue virus (DENV) transmission occurs primarily in tropical and subtropical climates, but within the last decade it has extended to temperate regions. Santa Fe, a temperate province in Argentina, has experienced an increase in dengue cases and virus circulation since 2009, with the recent 2020 outbreak being the largest in the province to date. The aim of this work is to describe spatio-temporal fluctuations of dengue cases from 2009 to 2020 in Santa Fe Province. The data presented in this work provide a detailed description of DENV transmission for Santa Fe Province by department. These data are useful to assist in investigating drivers of dengue emergence in Santa Fe Province and for developing a better understanding of the drivers and the impacts of ongoing dengue emergence in temperate regions across the world. This work provides data useful for future studies including those investigating socio-ecological, climate, and environmental factors associated with DENV transmission, as well as those investigating other variables related to the biology and the ecology of vector-borne diseases.

sentences: Lifelong musical activity is associated with multi-domain cognitive and brain benefits in older adults 1AO_SCPLOWBSTRACTC_SCPLOWRegular musical activity as a highly-stimulating lifestyle activity is proposed to be protective against age-related cognitive decline and Alzheimers disease (AD). This study investigated associations between lifelong regular musical instrument playing, late-life cognitive abilities and brain morphology in older adults. We show that musical activity over the life course is associated with better global cognition, working memory, executive functions, language, and visuospatial abilities accounting for reserve proxies. Playing music is not significantly associated with gray matter volume in regions most affected by aging and AD. Selectively in the musically active participants, multi-domain cognitive abilities were enhanced with preserved gray matter volume in frontal and temporal regions. Our correlational findings suggest that playing a musical instrument may improve the recruitment of existing brain resources to facilitate late-life cognitive capacities. We propose that engaging in regular musical activity could serve as a low-threshold multimodal enrichment strategy that may promote cognitive resilience in advanced age.

sentences: Inflammatory signals are sufficient to elicit TOX expression in mouse and human CD8 T cells T cell receptor (TCR) stimulation leads to expression of the transcription factor TOX. Prolonged TCR signaling, such as encountered during chronic infections or in tumors, leads to sustained TOX expression, which induces a state of exhaustion or dysfunction. While CD8 memory T cells (Tmem) in specific pathogen-free laboratory mice typically do not express TOX, functional human Tmem show heterogeneous TOX expression levels. Whether TCR-independent mechanisms can alter TOX expression in human and murine Tmem has not been defined. We report that human and mouse Tmem increase TOX expression following stimulation with inflammatory cytokines IL-12, IL-15, and IL-18. TOX and PD-1 expression patterns often appear to be directly correlated, however, we found that TOX is not necessary for cytokine-driven expression of PD-1. Together, these observations highlight that inflammation is sufficient to alter TOX and PD-1 expression and that the signals regulating TOX expression appear well conserved in human and murine Tmem.

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Contributions of the four essential entry glycoproteins to HSV-1 tropism and the selection of entry routes. Herpes Simplex viruses (HSV-1 and HSV-2) encode up to 16 envelope proteins, four of which are essential for entry. However, whether these four proteins alone are sufficient to dictate the broad cellular tropism of HSV-1 and the selection of different cell-type dependent entry routes is unknown. To begin addressing this, we previously pseudotyped VSV, lacking its native glycoprotein G, with only the four essential entry glycoproteins of HSV-1: gB, gH, gL, and gD. This novel VSV{Delta}G-BHLD pseudotype recapitulated several important features of HSV-1 entry: the requirement for gB, gH, gL, gD, a cellular receptor, and sensitivity to anti-gB and anti-gH/gL neutralizing antibodies. However, due to the use of a single cell type in that study, the tropism of the VSV{Delta}G-BHLD pseudotype was not investigated. Here, we show that the cellular tropism of the pseudotype is severely limited compared to wild-type HSV-1 and that its entry pathways differ from the native HSV-1 entry pathways. To test the hypothesis that other HSV-1 envelope proteins may contribute to HSV-1 tropism, we generated a derivative pseudotype containing the HSV-1 glycoprotein gC (VSV{Delta}G-BHLD-gC) and observed a gC-dependent increase in entry efficiency in two cell types. We propose that the pseudotyping platform developed here has the potential to uncover functional contributions of HSV-1 envelope proteins to entry in a gain-of-function manner.

ImportanceHerpes simplex viruses (HSV-1 and HSV-2) contain up to 16 different proteins in their envelopes. Four of these, glycoproteins gB, gD, gH, and gL, are termed essential with regard to entry whereas the rest are typically referred to as non-essential based on the entry phenotypes of the respective single genetic deletions. However, the single-gene deletion approach, which relies on robust loss-of-function phenotypes, may be confounded by functional redundancies among the many HSV-1 envelope proteins. We have developed a pseudotyping platform, in which the essential four entry glycoproteins are isolated from the rest, which can be added back individually for systematic gain-of-function entry experiments. Here, we show the utility of this platform for dissecting the contributions of HSV envelope proteins, both the essential four and the remaining dozen (using gC as an example), to HSV entry.

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Arousal state affects perceptual decision-making by modulating hierarchical sensory processing in a large-scale visual system model Arousal levels strongly affect task performance. Yet, what arousal level is optimal for a task depends on its difficulty. Easy task performance peaks at higher arousal levels, whereas performance on difficult tasks displays an inverted U-shape relationship with arousal, peaking at medium arousal levels, an observation first made by Yerkes and Dodson in 1908. It is commonly proposed that the noradrenergic locus coeruleus system regulates these effects on performance through a widespread release of noradrenaline resulting in changes of cortical gain. This account, however, does not explain why performance decays with high arousal levels only in difficult, but not in simple tasks. Here, we present a mechanistic model that revisits the Yerkes-Dodson effect from a sensory perspective: a deep convolutional neural network augmented with a global gain mechanism reproduced the same interaction between arousal state and task difficulty in its performance. Investigating this model revealed that global gain states differentially modulated sensory information encoding across the processing hierarchy, which explained their differential effects on performance on simple versus difficult tasks. These findings offer a novel hierarchical sensory processing account of how, and why, arousal state affects task performance.

Authors summaryOver a hundred years ago, it was first observed that the effect of arousal on performance depends on task difficulty: the Yerkes-Dodson effect. Difficult tasks are best solved at intermediate arousal levels, whereas easy tasks benefit from a high arousal state. Current theories on how arousal affects neural processing cannot explain this effect of task difficulty. Here, we implement a key effect of arousal on cortical processing, a change in neuronal gain, in a computational model of visual processing capable of object recognition. Across a series of experiments, we find that our model can reproduce the Yerkes-Dodson effect behaviorally and that this effect can be explained by where in the processing hierarchy different arousal states optimize sensory information encoding.

sentences: Multiple causal DNA variants in a single gene affect gene expression in trans Identifying the specific causal DNA differences in a genome that contribute to variation in phenotypic traits is a key goal of genetic research. Trans-acting DNA variants that alter gene expression are important sources of genetic variation. Several genes are known to carry single causal variants that affect the expression of numerous genes in trans. Whether these single variants are representative of the architecture of trans-acting variation is unknown. Here, we studied the gene IRA2, which carries variants with broad, trans-acting effects on gene expression in two strains of the yeast Saccharomyces cerevisiae. We found that IRA2 contains at least seven causal nonsynonymous variants. The causal variants were located throughout the gene body and included a pair of neighboring variants with opposing effects that largely canceled each other out. The causal variants showed evidence for non-additive epistatic interactions, in particular among variants at the 5 end of the gene. These results show that the molecular basis of trans-acting variation can involve considerable complexity even within a single gene.

sentences: Dynein acts to cluster glutamate receptors and traffic the PIP5 kinase, Skittles, to regulate postsynaptic membrane organization at the neuromuscular junction Cytoplasmic dynein is essential in motoneurons for retrograde cargo transport that sustains neuronal connectivity. Little, however, is known about dyneins function on the postsynaptic side of the circuit. Here we report distinct postsynaptic roles for dynein at neuromuscular junctions (NMJs). Intriguingly, we show that dynein punctae accumulate postsynaptically at glutamatergic synaptic terminals. Moreover, Skittles, a phosphatidylinositol 4-phosphate 5-kinase that produces PI(4,5)P2 to organize the spectrin cytoskeleton, also localizes specifically to glutamatergic synaptic terminals. Depletion of postsynaptic dynein disrupts the accumulation of Skittles, PI(4,5)P2 phospholipid, and organization of the spectrin cytoskeleton at the postsynaptic membrane. Coincidental with dynein depletion, we observe an increase in the clusters size of ionotropic glutamate receptor (iGluR), and an increase in the amplitude and frequency of mEJPs. However, PI(4,5)P2 levels do not affect iGluR clustering and dynein does not affect the protein levels of iGluR subunits at the NMJ, suggesting a separate, transport independent function for dynein in iGluR cluster organization. As dynein punctae closely associate with iGluR clusters, we propose that dynein physically tethers iGluR clusters at the postsynaptic membrane to ensure proper synaptic transmission.

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Multi-scale Inference of Genetic Trait Architecture using Biologically Annotated Neural Networks In this article, we present Biologically Annotated Neural Networks (BANNs), a nonlinear probabilistic framework for association mapping in genome-wide association (GWA) studies. BANNs are feedforward models with partially connected architectures that are based on biological annotations. This setup yields a fully interpretable neural network where the input layer encodes SNP-level effects, and the hidden layer models the aggregated effects among SNP-sets. We treat the weights and connections of the network as random variables with prior distributions that reflect how genetic effects manifest at different genomic scales. The BANNs software uses variational inference to provide posterior summaries which allow researchers to simultaneously perform (i) mapping with SNPs and (ii) enrichment analyses with SNP-sets on complex traits. Through simulations, we show that our method improves upon state-of-the-art association mapping and enrichment approaches across a wide range of genetic architectures. We then further illustrate the benefits of BANNs by analyzing real GWA data assayed in approximately 2,000 heterogenous stock of mice from the Wellcome Trust Centre for Human Genetics and approximately 7,000 individuals from the Framingham Heart Study. Lastly, using a random subset of individuals of European ancestry from the UK Biobank, we show that BANNs is able to replicate known associations in high and low-density lipoprotein cholesterol content.

Author SummaryA common goal in genome-wide association (GWA) studies is to characterize the relationship between genotypic and phenotypic variation. Linear models are widely used tools in GWA analyses, in part, because they provide significance measures which detail how individual single nucleotide polymorphisms (SNPs) are statistically associated with a trait or disease of interest. However, traditional linear regression largely ignores non-additive genetic variation, and the univariate SNP-level mapping approach has been shown to be underpowered and challenging to interpret for certain trait architectures. While nonlinear methods such as neural networks are well known to account for complex data structures, these same algorithms have also been criticized as "black box" since they do not naturally carry out statistical hypothesis testing like classic linear models. This limitation has prevented nonlinear regression approaches from being used for association mapping tasks in GWA applications. Here, we present Biologically Annotated Neural Networks (BANNs): a flexible class of feedforward models with partially connected architectures that are based on biological annotations. The BANN framework uses approximate Bayesian inference to provide interpretable probabilistic summaries which can be used for simultaneous (i) mapping with SNPs and (ii) enrichment analyses with SNP-sets (e.g., genes or signaling pathways). We illustrate the benefits of our method over state-of-the-art approaches using extensive simulations. We also demonstrate the ability of BANNs to recover novel and previously discovered genomic associations using quantitative traits from the Wellcome Trust Centre for Human Genetics, the Framingham Heart Study, and the UK Biobank.

sentences: Transcriptional regulatory features associated with Coccidioides immitis phase transition Coccidioidomycosis (Valley Fever) is an emerging endemic fungal infection with a rising incidence and an expanding geographic range. It is caused by Coccidiodes, which are thermally dimorphic fungi that grow as mycelia in soil but transition in the lung to form pathogenic spherules. The regulatory mechanisms underlying this transition are not understood. Exploiting capped small (cs)RNA-seq, which identifies actively initiated stable and unstable transcripts and thereby detects acute changes in gene regulation with remarkable sensitivity, here we report the changes in architectural organization and key sequence features underlying phase transition of this highly pathogenic fungus. Spherule transition was accompanied by large-scale transcriptional reprogramming, functional changes in transcript isoforms, and a massive increase in promoter-distal transcription of ncRNAs. Analysis of spherule-activated regulatory elements revealed a motif predicted to recruit a WOPR family transcription factor, which are known regulators of virulence in other fungi. We identify CIMG_02671 as a C. immitis WOPR homologue and show that it activates transcription in a WOPR motif-dependent manner, suggesting it is an important regulator of pathogenic phase transition. Collectively, this also highlights csRNA-seq as a powerful means to identify transcriptional mechanisms that control pathogenesis.

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Comprehensive machine-learning-based analysis of microRNA-target interactions reveals variable transferability of interaction rules across species BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally via base-pairing with complementary sequences on messenger RNAs (mRNAs). Due to the technical challenges involved in the application of high-throughput experimental methods, datasets of direct bona-fide miRNA targets exist only for a few model organisms. Machine learning (ML) based target prediction methods were successfully trained and tested on some of these datasets. There is a need to further apply the trained models to organisms where experimental training data is unavailable. However, it is largely unknown how the features of miRNA-target interactions evolve and whether there are features that have been fixed during evolution, questioning the general applicability of these ML methods across species.

ResultsIn this paper, we examined the evolution of miRNA-target interaction rules and used data science and ML approaches to investigate whether these rules are transferable between species. We analyzed eight datasets of direct miRNA-target interactions in four organisms (human, mouse, worm, cattle). Using ML classifiers, we achieved high accuracy for intra-dataset classification and found that the most influential features of all datasets significantly overlap. To explore the relationships between datasets we measured the divergence of their miRNA seed sequences and evaluated the performance of cross-datasets classification. We showed that both measures coincide with the evolutionary distance of the compared organisms.

ConclusionsOur results indicate that the transferability of miRNA-targeting rules between organisms depends on several factors, the most associated factors being the composition of seed families and evolutionary distance. Furthermore, our feature importance results suggest that some miRNA-target features have been evolving while some have been fixed during evolution. Our study lays the foundation for the future developments of target prediction tools that could be applied to "non-model" organisms for which minimal experimental data is available.

Availability and implementation The code is freely available at https://github.com/gbenor/TPVOD

sentences: Neuromuscular denervation and deafferentation but not motor neuron death are disease features in the Smn 2B/- mouse model of SMA Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of motor neurons and skeletal muscle atrophy which is caused by ubiquitous deficiency in the survival motor neuron (SMN) protein. Several cellular defects contribute to sensory-motor circuit pathology in SMA mice, but the underlying mechanisms have often been studied in one mouse model without validation in other available models. Here, we used Smn2B/- mice to investigate specific behavioral, morphological, and functional aspects of SMA pathology that we previously characterized in the SMN{Delta}7 model. Smn2B/- SMA mice on a pure FVB/N background display deficits in body weight gain and muscle strength with onset in the second postnatal week and median survival of 19 days. Morphological analysis revealed severe loss of proprioceptive synapses on the soma of motor neurons and prominent denervation of neuromuscular junctions (NMJs) in axial but not distal muscles. In contrast, no evidence of cell death emerged from analysis of several distinct pools of lumbar motor neurons known to be lost in the disease. Moreover, SMA motor neurons from Smn2B/- mice showed robust nuclear accumulation of p53 but lack of phosphorylation of serine 18 at its amino-terminal, which selectively marks degenerating motor neurons in the SMN{Delta}7 mouse model. These results indicate that NMJ denervation and deafferentation, but not motor neuron death, are conserved features of SMA pathology in Smn2B/- mice.

sentences: Lipid nanodiscs facilitate the identification of a fragment compound inhibiting the enzymatic activity of the bacterial membrane protein MsbA. Membrane proteins are critical elements of numerous therapeutic approaches ranging from cancer to bacterial infections. MsbA is a bacterial membrane protein that has received increasing attention as an antibacterial target for its role in the processing of Lipid A, a key precursor of lipopolysaccharide that is essential for bacterial growth. When employing nanodiscs it is possible to stabilize MsbA by providing a membrane-like environment that enhances its enzymatic activity. Taking advantage of this property we have carried out a fragment screening using the biophysical method of surface plasmon resonance. This approach identified several compounds that bind specifically to MsbA. In particular, one of these fragment molecules not only binds to the target, but also inhibits the ATPase activity of the MsbA protein. The similarity of this fragment to the adenine moiety of ATP points at a route to generate stronger and more potent inhibitors for MsbA and even other proteins of its family of ABC transporters. Collectively, our study reveals biophysical approaches that facilitate the identification of fragment candidates inhibiting the activity of membrane proteins.

sentences: Pupil diameter tracked during motor adaptation in humans Pupil diameter, under constant illumination, is known to reflect individuals internal states, such as surprise about observation and environmental uncertainty. Despite the growing use of pupillometry in cognitive learning studies as an additional measure for examining internal states, few studies have used pupillometry in human motor learning studies. Here we provide the first detailed characterization of pupil diameter changes in a short-term reach adaptation paradigm. We measured pupil changes in 91 human participants while they adapted to abrupt, gradual, or switching force field conditions. Sudden increases in movement error caused by the introduction/reversal of the force field resulted in strong phasic pupil dilation during movement accompanied by a transient increase in tonic pre-movement baseline pupil diameter in subsequent trials. In contrast, clear changes in pupil responses were absent when the force field was gradually introduced, indicating that error drove the changes in pupil responses. Nevertheless, we found an association between baseline pupil diameter and awareness of the gradually-introduced perturbation assessed post-experimentally. In all experiments, we also found a strong co-occurrence of larger baseline pupil diameter and slower reaction and movement time after each set break. Interestingly, error-induced pupil responses gradually became insensitive after experiencing multiple reversals. Collectively, these results suggest that tonic baseline pupil diameter reflects ones belief about environmental uncertainty, whereas phasic pupil dilation during movement reflects surprise about a sensory outcome (i.e., movement error), and both effects are modulated by novelty. Our results provide a new approach for non-verbally assessing participants internal states during motor learning.

sentences: Age-dependent formation of TMEM106B amyloid filaments in human brain Many age-dependent neurodegenerative diseases, like Alzheimers and Parkinsons, are characterised by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-{beta} (A{beta}), -synuclein and TDP-43 are the most common. Here, we used electron cryo-microscopy (cryo-EM) structure determination to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in the human brain. We solved cryo-EM structures of TMEM106B filaments from the brains of 22 individuals with neurodegenerative conditions, including sporadic and inherited tauopathies, A{beta}-amyloidoses, synucleinopathies and TDP-43opathies, as well as from the brains of two neurologically normal individuals. We observed three different TMEM106B folds, with no clear relationship between folds and diseases. The presence of TMEM106B filaments correlated with that of a 29 kDa sarkosyl-insoluble fragment of the protein on Western blots. The presence of TMEM106B filaments in the brains of older, but not younger, neurologically normal individuals indicates that they form in an age-dependent manner.

sentences: Porcine pancreatic ductal epithelial cells transformed with KRASG12D and SV40T are tumorigenic We describe our initial studies in the development of an orthotopic, genetically-defined, large animal model of pancreatic cancer. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs. A transformed cell line was generated from these primary cells with oncogenic KRAS and SV40T. The transformed cell lines outperformed the primary and SV40T immortalized cells in terms of proliferation, population doubling time, soft agar growth, transwell migration and invasion. The transformed cell line grew tumors when injected subcutaneously in nude mice, forming glandular structures and staining for epithelial markers. Future work will include implantation studies of these tumorigenic porcine pancreatic cell lines into the pancreas of allogeneic and autologous pigs. The resultant large animal model of pancreatic cancer could be utilized for preclinical research on diagnostic, interventional, and therapeutic technologies.

sentences: Calibration-free 3D reconstruction of firefly trajectories from 360-degree cameras Over the past few decades, progress in animal tracking techniques, from large migrating mammals to swarming insects, has facilitated significant advances in ecology, behavioural biology, and conservation science. Recently, we developed a technique to record and track flashing fireflies in their natural habitat using pairs of 360-degree cameras. The method, which has the potential to help identify and monitor firefly populations worldwide, was successfully implemented in various natural swarms. However, camera calibration remained tedious and time-consuming. Here, we propose and implement an algorithm that calibrates the cameras directly from the data, requiring minimal user input. We explain the principles of the calibration-free algorithm, and demonstrate the ease and efficiency of its implementation. This method is relatively inexpensive, versatile, and well-suited for automatic processing and the collection of a large dataset of firefly trajectories across species and populations. This calibration-free method paves the way to citizen science efforts for monitoring and conservation of firefly populations.

sentences: Underlying environmental factors and geomagnetic fields linked to 45 pilot whales Globicephala macrorhynchus stranding in Modung white beach, Indonesian coast on 19th February 2021 The reason whale and dolphin stranding is not fully understood and it is not linked to a standalone variable. Theories assume intertwined factors including sickness, underwater noise, navigational error, geographical features, the presence of predators, poisoning from pollution or algal blooms, geomagnetic field, and extreme weather are responsible to whale stranding. On 19th February 2021, a pod consists of 45 pilot whales Globicephala macrorhynchus was stranded in a remote 7050 m2 Modung white beach of Indonesian coast. This paper aims to assess the environmental factors that may be can explain and link to this stranding cases. Those factors include bathymetry, plankton cell density measured using MODIS, water sediment load measured using Sentinel 2 Bands 4,3,1, vessel traffic, precipitation (inch) and thunder (CAPE index J/kg), water salinity and temperature, and geomagnetic field (nT). The results show the water near stranding sites were shallow, has sediment load, high plankton density, warmer, receiving torrential rain prior stranding, having weak geomagnetic field and high total magnetic field change/year. The combination of those environmental covariates may influence the behavior, navigation, and echolocation of the said stranded pilot whale.

sentences: A novel effective live-attenuated human metapneumovirus vaccine candidate produced in the serum-free suspension DuckCelt-T17 cell platform Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen for which there is currently no specific treatment or licensed vaccine. Different strategies have been evaluated to prevent this infection, including the use of live-attenuated vaccines (LAVs). However, further development of LAV approaches is often hampered by the lack of highly efficient and scalable cell-based production systems that support worldwide vaccine production. In this context, avian cell lines cultivated in suspension are currently competing with traditional cell platforms used for viral vaccine manufacturing. We investigated whether the DuckCelt(R)-T17 avian cell line (Vaxxel) we previously described as an efficient production system for several influenza strains could also be used to produce a new HMPV LAV candidate (Metavac(R)), an engineered SH gene-deleted mutant of the A1/C-85473 strain of HMPV. To that end, we characterized the operational parameters of multiplicity of infection (MOI), cell density, and trypsin addition to achieve optimal production of the LAV Metavac(R) in the DuckCelt(R)-T17 cell line platform. We demonstrated that the DuckCelt(R)-T17 cell line is permissive and is well adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac(R) vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt(R)-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, as well as its capacity to induce efficient neutralizing antibodies in a mouse model. Our results suggest that the DuckCelt(R)-T17 avian cell line is a very promising platform for scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac(R).

sentences: An anaphase switch in astral microtubule dynamics specifically requires the APC/C-Cdc20-dependent degradation of the mitotic cyclin Clb4 Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their dynamics accordingly. Spindle microtubules are conventionally divided into three classes: kinetochore, interpolar and astral microtubules (kMTs, iMTs and aMTs, respectively), among all aMT regulation remains elusive. Here, we show that aMT dynamics are tightly regulated. aMTs remain unstable up to metaphase and are stabilized at anaphase onset. This switch in aMT dynamics, crucial for proper spindle orientation, specifically requires the degradation of the mitotic cyclin Clb4 by the Anaphase Promoting Complex bound to its activator subunit Cdc20 (APC/CCdc20). These data highlight a unique role for mitotic cyclin Clb4, provide a framework to understand aMT regulation in vertebrates and uncover mechanistic principles of how the APC/CCdc20 choreographs the timing of late mitotic events by sequentially impacting on the three classes of spindle microtubules.

sentences: A field-wide assessment of differential high throughput sequencing reveals widespread bias Here we assess reproducibility and inferential quality in the field of differential HT-seq, based on analysis of datasets submitted 2008-2019 to the NCBI GEO data repository. Analysis of GEO submission file structures places an overall 56% upper limit to reproducibility without querying other sources. We further show that only 23% of experiments resulted in theoretically expected p value histogram shapes, although both reproducibility and p value distributions show marked improvement over time. Uniform p value histogram shapes, indicative of <100 true effects, were extremely few. Our calculations of {pi}0, the fraction of true nulls, showed that 36% of experiments have {pi}0 <0.5, meaning that in over a third of experiments most RNA-s were estimated to change their expression level upon experimental treatment. Both the fraction of different p value histogram types and {pi}0 values are strongly associated with the software used for calculating these p values by the original authors, indicating widespread bias.

sentences: Beyond Spike: Identification of nine highly prevalent SARS-CoV-2-specific CD8 T-cell epitopes in a large Norwegian cohort T-cell epitopes with broad population coverage may form the basis for a new generation of SARS-CoV-2 vaccines. However, published studies on immunoprevalence are limited by small test cohorts, low frequencies of antigen-specific cells and lack of data correlating eluted HLA ligands with T-cell responsiveness. As the protective role of pre-existing cross-reactivity to homologous peptides is unclear, we aimed to identify SARS-CoV-2-specific minimal epitopes recognized by CD8 T-cells among 48 peptides eluted from prevalent HLA alleles, and an additional 84 predicted binders, in a large cohort of convalescents (n=83) and pre-pandemic control samples (n=19). We identified nine conserved SARS-CoV-2-specific epitopes restricted by four of the most prevalent HLA class I alleles in the Norwegian study cohort, to which responding CD8 T cells were detected in 70-100% of convalescents expressing the relevant HLA allele. Only two of these were derived from the Spike protein, included in current vaccines. We found a strong correlation between immunoprevalence and immunodominance. Thus, the CD8 T-cell response to SARS-CoV-2 is more focused than previously believed. Using a new algorithm, we predict that a vaccine including these epitopes could induce a T-cell response in 83% of Caucasians.

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GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNAs for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for atypical projects with 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens.

FindingsGuideMaker can rapidly design gRNAs for gene targets across the genome from a degenerate protospacer adjacent motif (PAM) and a GenBank file. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs. This allows the user to design gRNAs targeting all genes in a typical bacterial genome in about 1-2 minutes.

ConclusionsGuidemaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process smaller eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.

sentences: Scalable Adaptive Protein Ensemble Refinement Integrating Flexible Fitting Recent advances in cryo-electron microscopy (cryo-EM) has enabled modeling macro-molecular complexes that are essential components of life. The density maps obtained from cryo-EM experiments is often integrated with ab - initio, knowledge-driven or first principles-based computational methods to build, fit and refine protein structures inside the electron density maps. Going beyond a single stationary-structure determination scheme, it is becoming more common to interpret the experimental data with a set of multiple physical models all of which contributes to the average observation seen by the experiment. Hence, there is a need to decide on the quality of an ensemble of protein structures on-the-fly, while refining them against the density maps. In this work, we demonstrate such adaptive decision making capabilities during flexible fitting of biomolecules. Our solution uses RADICAL tools (RCT) and we test this new implementation in exascale high performance computing environment for two proteins, Adenylate Kinase (ADK) and Carbon Monoxide Dehydrogenase (CODH). Our results indicate that using multiple replicas in flexible fitting with adaptive decision making improves the overall quality of fit and model by 40 % improvement when compared against the traditional flexible fitting approach. These advances are agnostic to system-size and computing environments.

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Sigh breathing rhythm depends on intracellular calcium oscillations in a population of inspiratory rhythmogenic preBötzinger complex neurons in mice The preBotzinger Complex (preBotC) of the lower brainstem generates two breathing-related rhythms: one for inspiration on a timescale of seconds and another that produces larger amplitude sighs on the order of minutes. Their underlying mechanisms and cellular origins remain incompletely understood. We resolve these problems via a joint experiment and modeling approach. Blocking purinergic gliotransmission does not perturb either rhythm and imaging experiments show that both rhythms emanate from the same glutamatergic neuron population. We hypothesized that these two disparate rhythms emerge in tandem wherein recurrent excitation gives rise to inspiratory rhythm while a calcium oscillator generates sighs; there is no obligatory role for gliotransmission, hyperpolarization activated mixed cationic current (Ih) in neurons, or synaptic inhibition-mediated coupling of separate populations. We developed a mathematical model that instantiates our working hypothesis. Tests of model predictions validate the single-population rhythmogenic framework, reproducing disparate breathing-related frequencies and the ability for inspiratory and sigh rhythms to be separately regulated in support of respiration under a wide array of conditions. Here we show how a single neuron population exploits two cellular tool-kits: one involving voltage-dependent membrane properties and synaptic excitation for inspiratory breathing (eupnea) and an intracellular biochemical oscillator for sighs, which ventilate and maintain optimal function in the compliant mammalian lung.

SIGNIFICANCE STATEMENTBreathing consists of two vital rhythms: one for eupnea that serves periodic physiological gas exchange and the other for sighs, which are larger breaths that occur minutes apart and serve to optimize pulmonary function. These rhythms with disparate frequencies emerge via a mechanism that is simpler than previously envisaged: it results from one neuron population (not two as previously thought) without need for gliotransmission or synaptic inhibition-mediated coupling of neuronal populations. We show that a low-frequency intracellular calcium oscillation underlies sighs and functions in parallel with the higher-frequency voltage-dependent network oscillation that drives eupnea. Exploiting two separate cellular tool kits enables quasi-independent breathing rhythms, which are unique features of breathing in mammals with compliant lungs.

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Competitive coordination of the dual roles of the Hedgehog co-receptor in homophilic adhesion and signal reception Hedgehog (Hh) signaling patterns embryonic tissues and contributes to homeostasis in adults. In Drosophila, Hh transport and signaling are thought to occur along a specialized class of actin-rich filopodia, termed cytonemes. Here, we report that Interference hedgehog (Ihog) not only forms a Hh receptor complex with Patched to mediate intracellular signaling, but Ihog also engages in trans-homophilic binding leading to cytoneme stabilization in a manner independent of its role as the Hh receptor. Both functions of Ihog (trans-homophilic binding for cytoneme stabilization and Hh binding for ligand sensing) involve a region of the first fibronectin repeat of the extracellular domain. Thus, the Ihog-Ihog interaction and the Hh-Ihog interaction cannot occur simultaneously for a single Ihog molecule. By combining experimental data and mathematical modeling, we determined that Hh-Ihog heterophilic interaction dominates and Hh can disrupt and displace Ihog molecules involved in trans-homophilic binding. Consequently, we proposed that the weaker Ihog-Ihog trans interaction promotes and stabilizes direct membrane contacts along cytonemes and that, as the cytoneme encounters secreted Hh ligands, the ligands trigger release of Ihog from trans Ihog-Ihog complex enabling transport or internalization of the Hh ligand-Ihog-Patched -receptor complex. Thus, the seemingly incompatible functions of Ihog in homophilic adhesion and ligand binding cooperate to assist Hh transport and reception along the cytonemes.

One-sentence summaryApparently incompatible functions of Ihog in homophilic adhesion and ligand binding cooperate in Hh transport and reception.

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Investigating the chemolithoautotrophic and formate metabolism of Nitrospira moscoviensis by constraint-based metabolic modeling and 13C-tracer analysis Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used constraint-based analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and 13C-tracer experiments with bicarbonate and formate coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings support that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO2 fixation. We also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospiras in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes.

ImportanceNitrospira are globally abundant nitrifying bacteria in soil and aquatic ecosystems and wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of N. moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite- oxidizing bacteria.

sentences: Comparative viral metagenomics from chicken feces and farm dust in the Netherlands Farm animals may harbor many viral pathogens, some with zoonotic potential which can possibly cause severe clinical outcomes in animals and humans. Documenting viral content of dust may provide information of potential sources and movement of viruses. Here, we describe a dust sequencing strategy that provides detailed viral sequence characterization from farm dust samples and use this method to document the virus communities from chicken farm dust samples and paired feces collected at multiple time points during the production cycle in broiler farms in the Netherlands. From the sequencing data, Parvoviridae and Picornaviridae were the most prevalent families, detected in 85-100% of all feces and dust samples. Surprisingly large genomic diversity was identified in Picornaviridae and viruses from the Caliciviridae and Astroviridae were also obtained. This study provides a unique characterization of virus communities in farmed chickens and paired farm dust samples and our sequencing methodology enabled the recovery of viral genome sequences from farm dust, providing important tracking details for virus movement between livestock animals and their farm environment. This study serves as a proof of concept supporting that dust sampling could potentially be incorporated as part of viral metagenomic surveillance.

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TAPIOCA: Topological Attention and Predictive Inference of Chromatin Arrangement Using Epigenetic Features Chromatin conformation is an important characteristic of the genome which has been repeatedly demonstrated to play vital roles in many biological processes. Chromatin can be characterized by the presence or absence of structural motifs called topologically associated domains. The de facto strategy for determination of topologically associated domains within a cell line is the use of Hi-C sequencing data. However Hi-C sequencing data can be expensive or otherwise unavailable. Various epigenetic features have been hypothesized to contribute to the determination of chromatin conformation. Here we present TAPIOCA, a self-attention based deep learning transformer algorithm for the prediction of chromatin topology which circumvents the need for labeled Hi-C data and makes effective predictions of chromatin conformation organization using only epigenetic features. TAPIOCA outperforms prior art in established metrics of TAD prediction, while generalizing across cell lines beyond those used in training.

Availabilitythe source code of TAPIOCA and training and test datasets are available at https://github.com/Max-Highsmith/TAPIOCA

Author SummaryIn this paper we outline a machine learning approach for predicting the topological organization of chromosomes using epigenetic track data as features. By utilizing an architecture inspired by the sequence transduction transformer network we are able to effectively predict multiple metrics used to characterize topologically associated domains. Our experimental results demonstrate that once trained our algorithm can effectively predict topological organization on novel cell lines all without any exposure to original Hi-C data in test datasets.

sentences: Deciphering the genetic basis of wheat seminal root anatomy uncovers ancestral axial conductance alleles Root axial conductance which describes the ability of water to pass throw the xylem, contributes to the rate of water uptake from the soil throughout the whole plant lifecycle. In a rainfed wheat agro-system, grain-filling is typically occurring during declining water availability (i.e. terminal drought). Therefore, preserving soil water moisture during grain filling could serve as a key adaptive trait. We hypothesized that lower wheat root axial conductance can promote higher yields under terminal drought. A segregating population derived from a cross between durum wheat and its direct progenitor wild emmer wheat was used to underpin the genetic basis of seminal root architectural and functional traits. We detected 75 QTL associated with seminal roots morphological, anatomical, and physiological traits, with several hotspots harboring co-localized QTL. We further validated the axial conductance and central metaxylem QTL using wild introgression lines. Field-based characterization of genotypes with contrasting axial conductance suggested the contribution of low axial conductance as a mechanism for water conservation during grain filling and consequent increase in grain size and yield. Our findings underscore the potential of introducing wild alleles to reshape the wheat root system architecture for greater adaptability under changing climate.

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Tropical-temperate dichotomy falls apart in the Asian Palmate Group of Araliaceae PREMISEThe use of climatic data on phylogenetic studies has greatly increased in the last decades. High-quality spatial data and accurate climatic information are essential to minimize errors in the climatic reconstructions to the past. However, despite the huge amount of already available biodiversity digital information, the process of compiling, cleaning, and comparing spatial data from different open data sources is a time-consuming task that sometimes ends up with low-quality geographical information. For this reason, researchers often resort qualitative approximations among which World bioclimatic classification systems or the experts criteria are the most used. Our aim is to evaluate the climatic characterization of the genera of the Asian Palmate Group (AsPG) of the ginseng family (Araliaceae), one of the classical examples of tropical-temperate plant families.

METHODSWe compiled a curated worldwide spatial database of the AsPG genera. We then created five raster layers representing bioclimatic regionalizations of the World. Finally, we crossed the database with the layers to characterize the AsPG genera.

RESULTSWe found large disagreement in the climatic characterization of genera among regionalizations and little support for the tropical-temperate dichotomy. Both results are attributed to the complexity of delimiting tropical, subtropical and temperate climates in the World and to the distribution of the study group in regions with transitional climatic conditions.

CONCLUSIONSThe complexity in the climatic classification of this classical example tropical-temperate dichotomy, calls for a general revision in other families. In fact, we claim that to properly evaluate tropical-temperate transitions we cannot ignore the complexity of distribution ranges.

sentences: Conformational transitions of the mitotic adaptor Spindly underlie its interaction with Dynein and Dynactin Cytoplasmic Dynein 1, or Dynein, is a microtubule minus-end directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. This mechanism may be paradigmatic for Dynein activation by other adaptors at various cellular locales.

sentences: Preventing ovariectomy-induced weight gain decreases tumor burden in rodent models of obesity and postmenopausal breast cancer. Obesity and adult weight gain are linked to increased breast cancer risk and poorer clinical outcomes in postmenopausal women, particularly for hormone-dependent tumors. Menopause is a time when significant weight gain occurs in many women, and clinical and preclinical studies have identified menopause (or ovariectomy) as a period of vulnerability for breast cancer development and promotion. We hypothesized that preventing weight gain after ovariectomy (OVX) may be sufficient to prevent the formation of new tumors and decrease growth of existing mammary tumors. Here, we tested this hypothesis in a rat model of obesity and carcinogen-induced postmenopausal mammary cancer and validated our findings in a murine xenograft model with implanted human tumors. In both models, preventing weight gain after OVX significantly decreased obesity-associated tumor development and growth. Importantly, we did not induce weight loss in these animals, but simply prevented weight gain. In both lean and obese rats, preventing weight gain reduced visceral fat accumulation and associated insulin resistance. Similarly, the intervention decreased circulating tumor-promoting growth factors and inflammatory cytokines (ie, BNDF, TNF, FGF2), with greater effects in obese compared to lean rats. In obese rats, preventing weight gain decreased adipocyte size, adipose tissue macrophage infiltration, reduced expression of the tumor-promoting growth factor FGF-1, and reduced phosphorylated FGFR in tumors. Together, these findings suggest that the underlying mechanisms associated with the anti-tumor effects of weight maintenance are multifactorial, and that weight maintenance during the peri-/post-menopausal period may be a viable strategy for reducing obesity-associated breast cancer risk and progression in women.

sentences: Extracting Phylogenetic Information of Human Mitochondrial DNA by Linear Autoencoder We used a linear autoencoder (LAE) and its learning dynamics to analyze the high-order structure of human mitochondrial DNA (mtDNA). A total of 360 complete human mtDNA sequences were collected from the MITOMAP database and transformed into 1024-dimensional vectors of pentanucleotide frequencies. We compressed those into a three-dimensional (3D) coordinates by an LAE at each step of training by gradient descent with respect to the quadratic error function. Along the time axis of training epochs, the compressed 3D coordinates were gradually clustered and separated in accordance with the order of the genetic distance in the phylogenetic tree of human mtDNA haplogroups. This suggests that there is an association between the learning dynamics of LAE and the high-dimensional structure of human mtDNA sequences, similar to that of phylogenetic analysis and evolutionary pathways: the five clusters eventually contained only a single haplogroup of L0, M, N, R, and U, while the L3 cluster contained a small number of M members and The packing was comparable to that realized in learning dynamics similar to genetic classification and evolutionary pathways by LAE in principal component analysis (PCA), but somewhat denser than PCA.

sentences: Egg-adaptation pathway of human influenza H3N2 virus is contingent on natural evolution Egg-adaptive mutations in influenza hemagglutinin (HA) often emerge during the production of egg-based seasonal influenza vaccines, which contribute to the largest share in the global influenza vaccine market. While some egg-adaptive mutations have minimal impact on the HA antigenicity (e.g. G186V), others can alter it (e.g. L194P). Here, we show that the preference of egg-adaptation pathway in human H3N2 HA is strain-dependent. In particular, Thr160 and Asn190, which are found in many recent H3N2 strains, restrict the emergence of L194P but not G186V. Our results further suggest that natural amino acid variants at other HA residues also play a role in determining the egg-adaptation pathway. Consistently, recent human H3N2 strains from different clades acquire different mutations during egg passaging. Overall, these results demonstrate that natural mutations in human H3N2 HA can influence the egg-adaption pathway, which has important implications in seed strain selection for egg-based influenza vaccine.

sentences: EHMT2 Controls Transcriptional Noise and the Developmental Switch after Gastrulation in the Mouse Embryo Embryos that carry zygotic or parental mutations in Ehmt2, the gene encoding the main euchromatic histone H3K9 methyltransferase, EHMT2, exhibit variable developmental delay. We asked the question whether the delayed embryo is different transcriptionally from the normally developing embryo when they reach the same developmental stage. We collected embryos carrying a series of genetic deficiencies in the Ehmt2 gene and performed total RNA sequencing of somite stage-matched individual embryos. We applied novel four-way comparisons to detect differences between normal versus deficient embryos, and between 12-somite and 6-somite embryos. Importantly, we also identified developmental changes in transcription that only occur during the development of the normal embryo. We found that at the 6-somite stage, gastrulation-specific genes were not precisely turned off in the Ehmt2-/- embryos, and genes involved in organ growth, connective tissue development, striated muscle development, muscle differentiation, and cartilage development were not precisely switched on in the Ehmt2-/- embryos. Zygotic EHMT2 reduced transcriptional variation of developmental switch genes and at some repeat elements at the six-somite stage embryos. Maternal EHMT2-mutant embryos also displayed great transcriptional variation consistent with their variable survival, but transcription was normal in developmentally delayed parental haploinsufficient embryos, consistent with their good prospects. Global profiling of transposable elements in the embryo revealed that specific repeat classes responded to EHMT2. DNA methylation was specifically targeted by EHMT2 to LTR repeats, mostly ERVKs. Long noncoding transcripts initiated from those misregulated  driver repeats in Ehmt2-/- embryos, and extended to several hundred kilobases, encompassing a multitude of additional, similarly misexpressed  passenger repeats. These findings establish EHMT2 as an important regulator of the transition between gastrulation programs and organ specification programs and of variability.

sentences: Development and validation of a miniaturized host range screening assay for bacteriophages Antimicrobial resistance is a global health crisis, partly contributed by inappropriate use of antibiotics. The increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Current procedures assessing susceptibility and breadth of host range to bacteriophage are conducted using large-scale manual processes that are labor-intensive. The aim here was to establish and validate a scaled down methodology for high-throughput screening in order to reduce procedural footprint. Bacteriophages were isolated from wastewater samples and screened for specificity against 29 clinical Pseudomonas aeruginosa isolates and PA01 using a spot test (2 L/ drop). Host range assessment was performed on four representative P. aeruginosa isolates using both double agar overlay assay on petri dishes and 24-well culture plates. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates were corroborated between the current standard practice of whole plate phage assay and 24-well phage assay. The high correlation achieved in this study confirms miniaturization as the first step in future automation that could test phage diversity and efficacy as antimicrobials.

sentences: MiR-1253 Potentiates Cisplatin Response in Pediatric Group 3 Medulloblastoma by Regulating Ferroptosis Medulloblastoma (MB), the most common malignant pediatric brain tumor and a leading cause of childhood mortality, is stratified into four primary subgroups, WNT (wingless), SHH (sonic hedgehog), group 3, and group 4. Patients with group 3 tumors have the poorest prognosis. Loss of 17p13.3, which houses the tumor suppressor gene miR-1253, is a frequent high-risk feature of group 3 tumors.. In this study, we show that miR-1253 levels can disrupt iron homeostasis, induce oxidative stress and lipid peroxidation, triggering an iron-mediated form of cell death called ferroptosis. In silico and in vitro analyses of group 3 tumors revealed deregulation of ABCB7, a mitochondrial iron transporter and target of miR-1253, and GPX4, a critical regulator of ferroptosis. Restoration of miR-1253 levels in group 3 cell lines resulted in downregulation of ABCB7 and GPX4, consequently increasing cytosolic and mitochondrial labile iron pools, reducing glutathione levels, in turn, resulting in mitochondrial oxidative stress and lipid peroxidation. Together, these events accelerated cancer cell death. Treating miR-1253-expressing cancer cells with cisplatin potentiated cell death by further elevating oxidative stress, depleting glutathione levels, and augmenting lipid peroxidation, with added inhibitory effects on cell viability and colony formation. Treatment with a ferroptosis inhibitor (ferrostatin-1) lead to recovery from the cytotoxic effects of this combination therapy. Together, these findings reveal a novel role for miR-1253 in enhancing ferroptosis to attenuate group 3 tumor cell growth. Our studies provide a proof-of-concept for using miR-based therapeutics to augment current chemotherapeutics in high-risk tumors. Leveraging the tumor-suppressive properties of miRNAs as adjuncts to chemotherapy may provide a promising alternative to current therapeutic strategies.

sentences: A theory of cortical map formation in the visual brain The cerebral cortex receives multiple afferents from the thalamus that segregate by stimulus modality forming cortical maps for each sense. In vision, the primary visual cortex maps the multiple dimensions of the visual stimulus in patterns that vary across species for reasons unknown. Here we introduce a general theory of cortical map formation, which proposes that map diversity emerges from species variations in the thalamic afferent density sampling sensory space. In the theory, increasing afferent sampling density enlarges the cortical domains representing the same visual point, allowing the segregation of afferents and cortical targets by multiple stimulus dimensions. We illustrate the theory with an afferent-density model that accurately replicates the maps of different species through afferent segregation followed by thalamocortical convergence pruned by visual experience. Because thalamocortical pathways use similar mechanisms for axon segregation and pruning, the theory may extend to other sensory areas of the mammalian brain.

sentences: Native structure of the RhopH complex, a key determinant of malaria parasite nutrient acquisition The RhopH complex is implicated in malaria parasites ability to invade and create new permeability pathways in host erythrocytes, but its mechanisms remain poorly understood. Here we enrich the endogenous RhopH complex in a native soluble form, comprising RhopH2, CLAG3.1 and RhopH3, directly from parasite cell lysates and determine its atomic structure using cryo electron microscopy, mass spectrometry, and the cryoID program. This first direct observation of an exported P. falciparum transmembrane protein--in a soluble, trafficking state and with atomic details of buried putative membrane-insertion helices--offers insights into assembly and trafficking of RhopH and other parasite-derived complexes to the erythrocyte membrane. Our study demonstrates the potential endogenous structural proteomics approach holds for elucidating the molecular mechanisms of hard-to-isolate complexes in their native, functional forms.

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Analytical Performance of a Standardized Kit for Mass Spectrometry-based Measurements of Human Glycosaminoglycans Glycosaminoglycans (GAGs) are long linear sulfated polysaccharides implicated in processes linked to disease development such as mucopolysaccharidosis, respiratory failure, cancer, and viral infections, thereby serving as potential biomarkers. A successful clinical translation of GAGs as biomarkers depends on the availability of standardized GAG measurements. However, owing to the analytical complexity associated with the quantification of GAG concentration and structural composition, a standardized method to simultaneously measure multiple GAGs is missing. In this study, we sought to characterize the analytical performance of a ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS)-based kit for the quantification of 17 GAG disaccharides. The kit showed acceptable linearity, selectivity and specificity, accuracy and precision, and analyte stability in the absolute quantification of 15 GAG disaccharides. In native human samples, here using urine as a reference matrix, the analytical performance of the kit was acceptable for the quantification of CS disaccharides. Intra- and inter-laboratory tests performed in an external laboratory demonstrated robust reproducibility of GAG measurements showing that the kit was acceptably standardized. In conclusion, these results indicated that the UHPLC-MS/MS kit was standardized for the simultaneous measurement of GAG disaccharides allowing for comparability of measurements and enabling translational research.

SummaryAnalytical performance of a kit for standardized GAG measurements, based on an established UHPLC-MS/MS method

sentences: Close-kin methods to estimate census size and effective population size The last two decades have witnessed rapid developments and increasing interest in use of (1) genetic methods to estimate effective population size (Ne) and (2) close-kin mark-recapture (CKMR) methods to estimate abundance based on the incidence of close relatives. Whereas Ne estimation methods have been applied to a wide range of taxa, all CKMR applications to date have been for aquatic species. These two fields of inquiry have developed largely independently, and this is unfortunate because deeper insights can be gained by joint evaluation of eco-evolutionary processes. In this synthesis, we use simple analytical models and simulated pedigree data to illustrate how various factors (life-history traits; patterns of variation in individual reproductive success; experimental design; stochasticity; marker type) can affect performance of the estimators. We show that the Ne/N ratio and the probability of a close-kin match both depend on a vector of parental weights that specify relative probabilities that different individuals will produce offspring. Although age-specific vital rates are central to both methodologies, for CKMR they can potentially bias abundance estimates unless properly accounted for, whereas they represent the signals of genetic drift that Ne estimation methods depend upon. Coordinating Ne and CKMR estimation methods using the same or overlapping datasets would facilitate joint evaluation of both the ecological and evolutionary consequences of abundance.

sentences: Transcription factor RFX7 governs a tumor suppressor network in response to p53 and stress Despite its prominence, the mechanisms through which the tumor suppressor p53 regulates most genes remain unclear. Recently, the regulatory factor X 7 (RFX7) emerged as a suppressor of lymphoid neoplasms, but its regulation and target genes mediating tumor suppression remain unknown. Here, we identify a novel p53-RFX7 signaling axis. Integrative analysis of the RFX7 DNA binding landscape and the RFX7-regulated transcriptome in three distinct cell systems reveals that RFX7 directly controls multiple established tumor suppressors, including PDCD4, PIK3IP1, MXD4, and PNRC1, across cell types and is the missing link for their activation in response to p53 and stress. RFX7 target gene expression correlates with cell differentiation and better prognosis in numerous cancer types. Interestingly, we find that RFX7 sensitizes cells to Doxorubicin by promoting apoptosis. Together, our work establishes RFX7s role as a ubiquitous regulator of cell growth and fate determination and a key node in the p53 transcriptional program.

sentences: Constitutive 5-HT2C receptor knock-out facilitates fear extinction through altered activity of a dorsal raphe-bed nucleus of the stria terminalis pathway Serotonin 2C receptors (5-HT2CRs) are widely distributed throughout the brain and are strongly implicated in the pathophysiology of anxiety disorders such as post-traumatic stress disorder (PTSD). Although in recent years, a considerable amount of evidence supports 5-HT2CRs facilitating effect on anxiety behavior, the involvement in learned fear responses and fear extinction is rather unexplored. Here we used a 5-HT2CR knock-out mouse line (2CKO) to gain new insights into the involvement of 5-HT2CRs in the neuronal fear circuitry. Using a cued fear conditioning paradigm, our results revealed that global loss of 5-HT2CRs exclusively accelerates fear extinction, without affecting fear acquisition and fear expression. To investigate the neuronal substrates underlying the extinction enhancing effect, we mapped the immediate-early gene product cFos, a marker for neuronal activity, in the dorsal raphe nucleus (DRN), amygdala and bed nucleus of the stria terminalis (BNST). Surprisingly, besides extinction-associated changes, our results revealed alterations in neuronal activity even under basal home cage conditions in specific subregions of the DRN and the BNST in 2CKO mice. Neuronal activity in the dorsal BNST was shifted in an extinction-supporting direction due to 5-HT2CR knock-out. Finally, the assessment of DRN-BNST connectivity using antero- and retrograde tracing techniques uncovered a discrete serotonergic DRC-BNSTad pathway showing increased activity in 2CKO mice. Thus, our results provide new insights for the fear extinction network by revealing a specific serotonergic DRC-BNSTad pathway underlying a 5-HT2CR-sensitive mechanism with high significance in the treatment of PTSD.

sentences: Predicting the evolution of Lassa Virus endemic area and population at risk over the next decades Lassa fever is listed among the diseases that pose the greatest risks to public health by the World Health Organization. This severe viral hemorrhagic fever is caused by Lassa virus, a zoonotic pathogen that repeatedly spills over to humans from its rodent reservoirs. It is currently not known how climate change, transformations in land use, and human population growth could affect the endemic area of this virus, currently limited to parts of West Africa. By exploring the environmental data associated with virus occurrence, we show how temperature, precipitation and the presence of pastures determine ecological suitability for virus circulation. We project that regions in Central and East Africa will likely become suitable for Lassa virus over the next decades and estimate that the total population living in areas suitable for Lassa virus may grow from about 100 million to 700 million by 2070. By analysing geotagged viral genomes, we find that in the event of Lassa virus being introduced into a new suitable region, its spread might remain spatially limited over the first decades. Our results highlight how the endemic area of Lassa virus may expand well beyond West Africa in the next decades due to human impact on the environment, putting hundreds of million more people at risk of infection.

sentences: Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding--analysis of extra-organismal DNA released into the environment--allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set "Amph16S" had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

sentences: PISCES: A pipeline for the Systematic, Protein Activity-based Analysis of Single Cell RNA Sequencing Data While single-cell RNA sequencing provides a new window on physiologic and pathologic tissue biology and heterogeneity, it suffers from low signal-to-noise ratio and a high dropout rate at the individual gene level, thus challenging quantitative analyses. To address this problem, we introduce PISCES (Protein-activity Inference for Single Cell Studies), an integrated analytical framework for the protein activity-based analysis of single cell subpopulations. PISCES leverages the assembly of lineage-specific gene regulatory networks, to accurately measure activity of each protein based on the expression its transcriptional targets (regulon), using the ARACNe and metaVIPER algorithms, respectively. It implements novel analytical and visualization functions, including activity-based cluster analysis, identification of cell state repertoires, and elucidation of master regulators of cell state and cell state transitions, with full interoperability with Seurats single-cell data format. Accuracy and reproducibility assessment, via technical and biological validation assays and by assessing concordance with antibody and CITE-Seq-based measurements, show dramatic improvement in the ability to identify rare subpopulations and to assess activity of key lineage markers, compared to gene expression analysis.

sentences: Comprehensive O-GlcNAc glycoproteomics on NOTCH1 EGF repeats implicated unique Lewis X epitopes in mammals The O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor-like (EGF) repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the glycan structures, the Lexis X antigen was detected on O-fucose glycans. Our results refined the putative consensus sequence for the EOGT-dependent extracellular O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans.

sentences: Computational Simulation of 4-D Micro-Circular Network in Zebrafish Tail Amputation and Regeneration Wall shear stress (WSS) in the micro-vasculature contributes to biomechanical cues that regulate mechanotransduction underlying vascular development, regeneration, and homeostasis. We hereby elucidate the interplay between hemodynamic shear forces and luminal remodeling in response to vascular injury and regeneration in the zebrafish model of tail amputation. Using the transgenic Tg(fli1:eGFP; Gata1:ds-red) line, we were able to track the enhanced green-fluorescent protein (eGFP)-labeled endothelial lining of the 3-D microvasculature for post-image segmentation and reconstruction of fluid domain for computational fluid dynamics (CFD) simulation. At 1 day post amputation (dpa), dorsal aorta (DA) and posterior cardinal vein (PCV) were severed, and vasoconstriction developed in the dorsal longitudinal anastomotic vessel (DLAV) with a concomitant increase in WSS in the segmental vessels (SV) proximal to the amputation site and a decrease in WSS in SVs distal to amputation. Simultaneously, we observed angiogenesis commencing at the tips of the amputated DLAV and PCV where WSS was minimal in the absence of blood flow. At 2 dpa, vasodilation occurred in a pair of SVs proximal to amputation, resulting in increased flow rate and WSS, whereas in the SVs distal to amputation, WSS normalized to the baseline. At 3 dpa, the flow rate in the arterial SV proximal to amputation continued to rise and merged with DLAV that formed a new loop with PCV. Thus, our CFD modeling uncovered a well-coordinated micro-vascular adaptation process following tail amputation, accompanied by the rise and fall of WSS and dynamic changes in flow rate during vascular regeneration.

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GEM-DeCan: Improving tumor immune microenvironment profiling by the integration of novel gene expression and DNA methylation deconvolution signatures Quantifying the proportion of the different cell types present in tumor biopsies remains a priority in cancer research. So far, a number of deconvolution methods have emerged for estimating cell composition using reference signatures, either based on gene expression or on DNA methylation from purified cells. These two deconvolution approaches could be complementary to each other, leading to even more performant signatures, in cases where both data types are available. However, the potential relationship between signatures based on gene expression and those based on DNA methylation remains underexplored.

Here we present five new deconvolution signature matrices, based on DNA methylation or RNAseq data, which can estimate the proportion of immune cells and cancer cells in a tumour sample. We test these signature matrices on available datasets for in-silico and in-vitro mixtures, peripheral blood, cancer samples from TCGA, bone marrow from multiple myeloma patients and a single-cell melanoma dataset. Cell proportions estimates based on deconvolution performed using our signature matrices, implemented within the EpiDISH framework, show comparable or better correlation with FACS measurements of immune cell-type abundance and with various estimates of cancer sample purity and composition than existing methods.

Using publicly available data of 3D chromatin structure in haematopoietic cells, we expanded the list of genes to be included in the RNAseq signature matrices by considering the presence of methylated CpGs in gene promoters or in genomic regions which are in 3D contact with these promoters. Our expanded signature matrices have improved performance compared to our initial RNAseq signature matrix. Finally, we show the value of our signatures in predicting patient response to immune checkpoint inhibitors in three melanoma and one bladder cancer cohorts, based on bulk tumour sample gene expression.

We also provide GEM-DeCan: a snakemake pipeline, able to run an analysis from raw sequencing data to deconvolution based on various gene expression signature matrices, both for bulk RNASeq and DNA methylation data. The code for producing the signature matrices and reproducing all the figures of this paper is available on the GEM-DeCan repository.

sentences: Epigenetic Control of Temperature-Dependent Female Reproductive Life History Trade-Offs in Seed Beetles, Callosobruchus maculatus Many species are threatened by climate change and must rapidly respond to survive changing environments. Epigenetic modifications, such as DNA methylation, can facilitate plastic responses by regulating gene expression in response to environmental cues. Understanding epigenetic responses is therefore essential for predicting species ability to rapidly adapt in the context of global environmental change. Here, we investigated the functional significance of DNA methylation on temperature-dependent life history in seed beetles, Callosobruchus maculatus. We assessed changes in DNA methyltransferase (Dnmt1 and Dnmt2) expression levels under ambient conditions and thermal stress, and reproductive performance following artificially-induced epimutation via 3-aminobenzamide (3AB) and Zebularine (Zeb), at a range of ambient and warmer temperatures over two generations. We found that Dnmt1 and Dnmt2 were greatly expressed in females, throughout the body, and exhibited temperature-dependence; in contrast, Dnmt expression was minimal in males. Epimutation led to shifts in female reproductive life history trade-off allocation, and differentially altered thermal optima of fecundity and offspring viability. This study revealed the optimal allocation strategy among these fitness components is temperature-dependent, and trade-offs become increasingly difficult to resolve epigenetically under more extreme warming. Results suggest that epigenetic mechanisms are strongly implicated in, and perhaps limiting of, invertebrate life history responses to temperature change. Further investigation will reveal targeted DNA methylation patterns and specific loci associated with temperature-dependent life history trade-offs in seed beetles and other invertebrates.

sentences: Nascent ribosomal RNA acts as surfactant that suppresses growth of fibrillar centers in nucleolus Liquid-liquid phase separation (LLPS) has been thought to be the assembly mechanism of the multiphase structure of nucleolus, the site of ribosomal biogenesis. Condensates assembled by LLPS increase their size to minimize the surface energy as far as their components are available. However, multiple microphases, fibrillar centers (FCs), dispersed in a nucleolus are stable and their size does not grow unless the transcription of pre-ribosomal RNA (pre-rRNA) is inhibited. To understand the mechanism of the suppression of the growth of FCs, we here construct a minimal theoretical model by taking into account the nascent pre-rRNA transcripts tethered to the surfaces of FCs by RNA polymerase I. Our theory predicts that nascent pre-rRNA transcripts generate the lateral osmotic pressure that counteracts the surface tension of the microphases and this suppresses the growth of the microphases over the optimal size. The optimal size of the microphases decreases with increasing the transcription rate and decreasing the rate of RNA processing. This prediction is supported by our experiments showing that the size of FCs increased with increasing the dose of transcription inhibitor. This theory may provide insight in the general mechanism of the size control of nuclear bodies.

sentences: Pink Cedar (Acrocarpus fraxinifolius): its prophylactic role against APAP induced organs toxicity in rats and its antiviral activity against Herpes simplex virus type 1 The possible protective effects of methanolic extract Acrocarpus fraxinifolius leaves (MEAFL) were assessed against the APAP-induced organ toxicity in male rats. Also, the content of polyphenols extracted from AFL was studied, and their relationship with antioxidant activity was investigated. MEAFL was tested for cytotoxicity on Vero cell line, with reference to IC50, and other non-toxic concentrations of all the extracts. The antiviral activity against HSV1 for all non-toxic concentrations of the extract was determined using plaque reduction assay. It was found that MEAFL showed a reduction of serum hepatic and renal cellular toxicity and cellular lipid peroxidation, as well as enhanced cellular antioxidant. Also, our results revealed that the inhibitory activity of the virus was dose dependent on the polyphenol content of the examined extract. The MIC for the MEAFL extract was determined as well as the EC50 and SI. Calculated SI showed promising value for the MEAFL, and hence can be used as therapeutic medication for HSV1. To study other possible mode of action, Vero cells were treated with the examined extracts before, during, and after virus infection to give an insight on the interference of the extract in each step in the virus life cycle. In conclusion, MEAFL showed a remarkable antioxidant effect against APAP induced organs toxicity. Also, examined extracts exhibited the antiviral activity against HSV1.

sentences: A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

sentences: Zebrafish airinemes optimize their shape between ballistic and diffusive search In addition to diffusive signals, cells in tissue also communicate via long, thin cellular protrusions, such as airinemes in zebrafish. Before establishing communication, cellular protrusions must find their target cell. Here we demonstrate that the shape of airinemes in zebrafish are consistent with a finite persistent random walk model. The probability of contacting the target cell is maximized for a balance between ballistic search (straight) and diffusive search (highly curved, random). We find that the curvature of airinemes in zebrafish, extracted from live cell microscopy, is approximately the same value as the optimum in the simple persistent random walk model. We also explore the ability of the target cell to infer direction of the airinemes source, finding that there is a theoretical trade-off between search optimality and directional information. This provides a framework to characterize the shape, and performance objectives, of non-canonical cellular protrusions in general.

sentences: Mutability of mononucleotide repeats, not oxidative stress, explains the discrepancy between laboratory-accumulated mutations and the natural allele-frequency spectrum in C. elegans Important clues about natural selection can be gleaned from discrepancies between the properties of segregating genetic variants and of mutations accumulated experimentally under minimal selection, provided the mutational process is the same in the lab as in nature. The ratio of transitions to transversions (Ts/Tv) is consistently lower in C. elegans mutation accumulation (MA) experiments than in nature, which has been argued to be in part due to increased oxidative stress in the lab environment. Using whole-genome sequence data from a set of C. elegans MA lines carrying a mutation (mev-1) that increases the cellular titer of reactive oxygen species (ROS), leading to increased endogenous oxidative stress, we find that the base-substitution spectrum is similar between mev-1 lines, its wild-type progenitor (N2), and another set of MA lines derived from a different wild strain (PB306). By contrast, the rate of short insertions is greater in the mev-1 lines, consistent with studies in other organisms in which environmental stress led to an increase in the rate of insertion-deletion mutations. Further, the mutational properties of mononucleotide repeats in all strains are qualitatively different from those of non-mononucleotide sequence, both for indels and base-substitutions, and whereas the non-mononucleotide spectra are fairly similar between MA lines and wild isolates, the mononucleotide spectra are very different. The discrepancy in mutational spectra between lab MA experiments and natural variation is likely due to a consistent (but unknown) effect of the lab environment that manifests itself via different modes of mutability and/or repair at mononucleotide loci.

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Multi-omics Topic Modeling for Breast Cancer Classification The integration of transcriptional data with other layers of information, such as the post-transcriptional regulation mediated by microRNAs, can be crucial to identify the driver genes and the subtypes of complex and heterogeneous diseases such as cancer. This paper presents an approach based on topic modeling to accomplish this integration task. More specifically, we show how an algorithm based on a hierarchical version of stochastic block modeling can be naturally extended to integrate any combination of  omics data. We test this approach on breast cancer samples from the TCGA database, integrating data on messenger RNA, microRNAs and copy number variations. We show that the inclusion of the microRNA layer significantly improves the accuracy of subtype classification. Moreover, some of the hidden structures or "topics" that the algorithm extracts actually correspond to genes and microRNAs involved in breast cancer development, and are associated to the survival probability.

Simple SummaryTopic models are algorithms introduced for discovering hidden topics or latent variables in large unstructured text corpora. Leveraging on analogies between texts and gene expression profiles, these algorithms can be used to find structures in expression data. This work presents an application of topic modeling techniques to the identification of breast cancer subtypes. In particular, we extended a specific class of topic models to allow a multi-omics approach. As an illustrative example, considering both messenger RNA and microRNA expression levels, we were able to clearly distinguish healthy from tumor samples, as well as the different breast cancer subtypes. The integration of different layers of information is crucial for the observed classification accuracy. Our approach naturally provides the genes and the microRNAs associated to the specific topics that are used for the sample organization. We show that indeed these topics often contain genes involved in breast cancer development, and are associated to different survival probabilities.

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BrainEXP-NPD: a database of transcriptomic profiles of human brains of six neuropsychiatric disorders BackgroundSpatio-temporal gene expression has been widely used to study gene functions and biological mechanisms in diseases. Numerous microarray and RNA sequencing data focusing on brain transcriptomes in neuropsychiatric disorders have accumulated. However, their consistency, reproducibility has not been properly evaluated. Except for a few psychiatric disorders, like schizophrenia, bipolar disorder and autism, most have not been compared to each other for cross-disorder comparisons.

MethodsWe organized 48 human brain transcriptome datasets from six sources. The original brain donors include patients with schizophrenia (SCZ, N=427), bipolar disorder (BD, N=312), major depressive disorder (MDD, N=219), autism spectrum disorder (ASD, N=53), Alzheimers disease (AD, N=765), Parkinsons disease (PD, N=163) as well as controls as unaffected by such disorders (CTRL, N=6,378), making it a total of 8,317 samples. Raw data included multiple brain regions of both sexes, with ages ranging from embryonic to seniors. After standardization, quality control, filtering and removal of known and unknown covariates, we performed comprehensive meta- and mega-analyses, including gene differential expression and gene co-expression network.

ResultsA total of 6922, 3011, 2703, 4389, 3507, 4279 significantly differentially expressed genes (FDR q < 0.05) were detected in the comparisons of 6 brain regions of SCZ-CTRL, 5 brain regions of BD-CTRL, 6 brain regions of MDD-CTRL, 4 brain regions of ASD-CTRL, 7 brain regions of AD-CTRL, and 6 brain regions of PD-CTRL, respectively. Most differentially expressed genes were brain region-specific and disease-specific. SCZ and BD have a maximal transcriptome similarity in striatum ({rho}=0.42) among the four brain regions, as measured by Spearmans correlation of differential expression log2 FC values. SCZ and MDD have a maximal transcriptome similarity in hippocampus ({rho}=0.30) among the five brain regions. BD and MDD have a maximal transcriptome similarity in frontal cortex ({rho}=0.45) among the five brain regions. Other disease pairs have a less transcriptome similarity ({rho}<0.1) in all brain regions. PD is negatively correlated with SCZ, BD, and MDD in cerebellum and striatum. We also performed coexpression network analyses for different disorders and controls separately. We developed a database named BrainEXP-NPD (http://brainexpnpd.org:8088/BrainEXPNPD/), to provide a userfriendly web interface for accessing the data, and analytical results of meta- and mega-analyses, including gene differential expression and gene co-expression networks between cases and controls on different brain regions, sexes and age groups. Discussion: BrainEXP-NPD compiled the largest collection of brain transcriptomic data of major neuropsychiatric disorders and presented lists of differentially expressed genes and coexpression modules in multiple brain regions of six major disorders.

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Ankylosis homologue mediates cellular efflux of ATP, not pyrophosphate The plasma membrane protein Ankylosis Homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints, but we recently showed that ANKH releases large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. This ATP is converted extracellularly into PPi and AMP by the ectoenzyme Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANK. We now addressed this question by determining the effect of a complete absence of ENPP1 on ANKH-dependent extracellular PPi concentrations. Introduction of ANKH in ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi seen in ENPP1-proficient cells.

Ank-activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1-/- mice contained < 2.5% of the PPi found in bones of wild type mice, showing that Enpp1-activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60-70 % of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification, therefore do so by extruding NTPs rather than PPi itself.

sentences: Transfer Learning for Inference of Metastatic Origin from Whole Slide Histology Accurate diagnosis of metastatic cancer is essential for prescribing optimal control strategies to halt further spread of metastasizing disease. While pathological inspection aided by immunohistochemistry staining provides a valuable gold standard for clinical diagnostics, deep learning methods have emerged as powerful tools for identifying clinically relevant features of whole slide histology relevant to a tumors metastatic origin. Although deep learning models require significant training data to learn effectively, transfer learning paradigms provide mechanisms to circumvent limited training data by first training a model on related data prior to fine-tuning on smaller data sets of interest. In this work we propose a transfer learning approach that trains a convolutional neural network to infer the metastatic origin of tumor tissue from whole slide images of hematoxylin and eosin (H&E) stained tissue sections and illustrate the advantages of pre-training network on whole slide images of primary tumor morphology. We further characterize statistical dissimilarity between primary and metastatic tumors of various indications on patch-level images to highlight limitations of our indication-specific transfer learning approach. Using a primary-to-metastatic transfer learning approach, we achieved mean class-specific areas under receiver operator characteristics curve (AUROC) of 0.779, which outperformed comparable models trained on only images of primary tumor (mean AUROC of 0.691) or trained on only images of metastatic tumor (mean AUROC of 0.675), supporting the use of large scale primary tumor imaging data in developing computer vision models to characterize metastatic origin of tumor lesions.

sentences: Programming bacteria to sense environmental DNA for multiplexed pathogen detection DNA is a universal and programmable signal of living organisms. Here we developed Cell-based DNA sensors (Cell-Sens) by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity and sensitivity. Multiplexed detection of combinations of different species genomic DNA was achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We demonstrate that the DNA sensors can detect presence of species in a microbial community without requiring DNA purification of the donor species. In sum, the modularity of the living cell-based DNA sensing mechanism and simple measurement procedure could enable programmable DNA sensing for broad applications.

sentences: Inertia triggers nonergodicity of fractional Brownian motion How related are the ergodic properties of the over- and underdamped Langevin equations driven by fractional Gaussian noise? We here find that for massive particles performing fractional Brownian motion (FBM) inertial effects not only destroy the stylized fact of the equivalence of the ensemble-averaged mean-squared displacement (MSD) to the time-averaged MSD (TAMSD) of overdamped or massless FBM, but also concurrently dramatically alter the values of the ergodicity breaking parameter (EB). Our theoretical results for the behavior of EB for underdamped ot massive FBM for varying particle mass m, Hurst exponent H, and trace length T are in excellent agreement with the findings of extensive stochastic computer simulations. The current results can be of interest for the experimental community employing various single-particle-tracking techniques and aiming at assessing the degree of nonergodicity for the recorded time series (studying e.g. the behavior of EB versus lag time). To infer FBM as a realizable model of anomalous diffusion for a set single-particle-tracking data when massive particles are being tracked, the EBs from the data should be compared to EBs of massive (rather than massless) FBM.

sentences: Mucins form a nanoscale material barrier against immune cell attack The cancer cell glycocalyx serves as a major line of defense against immune surveillance. However, how specific physical properties of the glycocalyx contribute to immune evasion and how these properties are regulated are not well understood. Here, we uncover how the surface density, glycosylation, and crosslinking of cancer-associated mucins contribute to the nanoscale material thickness of the glycocalyx, and further analyze the effect of the glycocalyx thickness on resistance to effector cell attack. Natural Killer (NK) cell-mediated cytotoxicity exhibits a near perfect inverse correlation with the glycocalyx thickness of target cells regardless of the specific glycan structures present. NK cells expressing a chimeric antigen receptor (CAR) have an enhanced ability to breach the glycocalyx and kill target cells. Equipping the NK cell surface with a mucin-digesting enzyme also improves killing with a performance enhancement that rivals or exceeds CARs in some cases. Together, our results provide new considerations for improving cancer immunotherapies.

sentences: Time-dependent memory and individual variation in Arctic brown bears (Ursus arctos) 1Animal movement modelling provides unique insight about how animals perceive their landscape and how this perception may influence space use. When coupled with data describing an animals environment, ecologists can fit statistical models to location data to describe how spatial memory informs movement. We performed such an analysis on a population of brown bears (Ursus arctos) in the Canadian Arctic using a model incorporating time-dependent spatial memory patterns. Brown bear populations in the Arctic lie on the periphery of the species range, and as a result endure harsh environmental conditions. In this kind of environment, effective use of memory to inform movement strategies could spell the difference between survival and mortality. The model we fit tests four alternate hypotheses (some incorporating memory; some not) against each other, and we found a high degree of individual variation in how brown bears used memory. We found that 52% (11 of 21) of the bears used complex, time-dependent spatial memory to inform their movement decisions. These results, coupled with existing knowledge on individual variation in the population, highlight the diversity of foraging strategies for Arctic brown bears while also displaying the inference that can be drawn from this innovative movement model.

sentences: An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2 Monoclonal antibodies targeting the SARS-CoV-2 spike (S) glycoprotein neutralize infection and are efficacious for the treatment of mild-to-moderate COVID-19. However, SARS-CoV-2 variants have emerged that partially or fully escape monoclonal antibodies in clinical use. Notably, the BA.2 sublineage of B.1.1.529/omicron escapes nearly all monoclonal antibodies currently authorized for therapeutic treatment of COVID-19. Decoy receptors, which are based on soluble forms of the host entry receptor ACE2, are an alternative strategy that broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22.v2.4-IgG1 was previously shown to be effective in vivo against SARS-CoV-2 variants when administered intravenously. Here, the inhalation of sACE22.v2.4-IgG1 is found to increase survival and ameliorate lung injury in K18-hACE2 transgenic mice inoculated with a lethal dose of the virulent P.1/gamma virus. Loss of catalytic activity reduced the decoys therapeutic efficacy supporting dual mechanisms of action: direct blocking of viral S and turnover of ACE2 substrates associated with lung injury and inflammation. Binding of sACE22.v2.4-IgG1 remained tight to S of BA.1 omicron, despite BA.1 omicron having extensive mutations, and binding exceeded that of four monoclonal antibodies approved for clinical use. BA.1 pseudovirus and authentic virus were neutralized at picomolar concentrations. Finally, tight binding was maintained against S from the BA.2 omicron sublineage, which differs from S of BA.1 by 26 mutations. Overall, the therapeutic potential of sACE22.v2.4-IgG1 is further confirmed by inhalation route and broad neutralization potency persists against increasingly divergent SARS-CoV-2 variants.

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In vitro screening of herbal medicinal products for their supportive curing potential in the context of SARS-CoV-2 BackgroundHerbal medicinal products have a long-standing history of use in the therapy of common respiratory infections. In the COVID-19 pandemic, they may have the potential for symptom relief in non-severe or moderate disease cases. Here we describe the results derived by in vitro screening of five herbal medicinal products with regard to their potential to i) interfere with the binding of the human Angiotensin-converting enzyme 2 (ACE2) receptor with the SARS-CoV-2 Spike S1 protein, ii) modulate the release of the human defensin HBD1 and cathelicidin LL-37 from human A549 lung cells upon Spike S1 protein stimulation and iii) modulate the release of IFN-{gamma} from activated human peripheral blood mononuclear cells (PBMC). The investigated extracts were: Sinupret extract (SINx), Bronchipret thyme-ivy (BRO TE), Bronchipret thyme-primrose (BRO TP), Imupret (IMU), and Tonsipret (TOP).

MethodsThe inhibitory effect of the herbal medicinal products on the binding interaction of Spike S1 protein and the human ACE2 receptor was measured by ELISA. The effects on intracellular IFN-{gamma} expression in stimulated human PBMCs were measured by flow cytometry. Regulation on HBD1 and LL-37 expression and secretion was assessed in 25d long-term cultured human lung A549 epithelial cells by RT-PCR and ELISA.

ResultsIMU and BRO TE concentration-dependently inhibited the interaction between spike protein and the ACE2 Receptor. However, this effect was only observed in the cell-free assay at a concentration range which was later on determined as cytotoxic to human PBMC. SINx, TOP and BRO TP significantly upregulated the intracellular expression of antiviral IFN{gamma} from stimulated PBMC. Co-treatment of A549 cells with IMU or BRO TP together with SARS-CoV-2 spike protein significantly upregulated mRNA expression (IMU) and release (IMU and BRO TP) of HBD1 and LL-37 (BRO TP).

ConclusionsThe in vitro screening results provide first evidence for an immune activating potential of some of the tested herbal medicinal extracts in the context of SARS-CoV-2. Whether these could be helpful in prevention of SARS-CoV-2 invasion or supportive in recovery from SARS-CoV-2 infection needs deeper understanding of the observations.

sentences: Deep learning-based segmentation and quantification of podocyte foot process morphology The kidneys constantly filter enormous amounts of fluid, with almost complete retention of albumin and other macromolecules in the plasma. Diseases of podocytes at the kidney filtration barrier reduce the intrinsic permeability of the capillary wall resulting in albuminuria. However, direct quantitative assessment of the underlying morphological changes has previously not been possible. Here we developed a deep learning-based approach for segmentation of foot processes in images acquired with optical microscopy. Our method - Automatic Morphological Analysis of Podocytes (AMAP) - accurately segments foot processes and robustly quantifies their morphology. It also robustly determined morphometric parameters, at a Pearson correlation of r > 0.71 with a previously published semi-automated approach, across a large set of mouse tissue samples. The artificial intelligence algorithm wasWe applied the analysis to a set of human kidney disease conditions allowing comprehensive quantification of various underlying morphometric parameters. These results confirmed that when podocytes are injured, they take on a more simplified architecture and the slit diaphragm length is significantly shortened, resulting in a reduction in the filtration slit area and a loss of the buttress force of podocytes which increases the permeability of the glomerular basement membrane to albumin.

sentences: Viruses inhibit TIR gcADPR signaling to overcome bacterial defense The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants, and animals. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signaling molecule whose molecular structure remained elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function. We identified a large family of phage-encoded proteins, denoted here Thoeris anti-defense 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are "sponges" that bind and sequester the immune signaling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. A high-resolution crystal structure of Tad1 bound to the signaling molecule revealed that its chemical structure is 1'-2' glycocyclic ADPR (gcADPR), a unique molecule not previously described in other biological systems. Our results define the chemical structure of a central immune signaling molecule, and reveal a new mode of action by which pathogens can suppress host immunity.

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Zika virus infection drives epigenetic modulation of immunity by the histone acetyltransferase CBP of Aedes aegypti Epigenetic mechanisms are responsible for a wide range of biological phenomena in insects, controlling embryonic development, growth, aging and nutrition. Despite this, the role of epigenetics in shaping insect-pathogen interactions has received little attention. Gene expression in eukaryotes is regulated by histone acetylation/deacetylation, an epigenetic process mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we explored the role of the Aedes aegypti histone acetyltransferase CBP (AaCBP) after infection with Zika virus (ZIKV), focusing on the two main immune tissues, the midgut and fat body. We showed that the expression and activity of AaCBP could be positively modulated by blood meal and ZIKV infection. Nevertheless, Zika-infected mosquitoes that were silenced for AaCBP revealed a significant reduction in the acetylation of H3K27 (CBP target marker), followed by downmodulation of the expression of immune genes, higher titers of ZIKV and lower survival rates. Importantly, in Zika-infected mosquitoes that were treated with sodium butyrate, a histone deacetylase inhibitor, their capacity to fight virus infection was rescued. Our data point to a direct correlation among histone hyperacetylation by AaCBP, upregulation of antimicrobial peptide genes and increased survival of Zika-infected-A. aegypti.

Author summaryPathogens have coevolved with mosquitoes to optimize transmission to hosts. As natural vectors, mosquitoes are permissive to and allow systemic and persistent arbovirus infection, which intriguingly does not result in dramatic pathological sequelae that affect their lifespan. In this regard, mosquitoes have evolved mechanisms to tolerate persistent infection and develop efficient antiviral strategies to restrict viral replication to nonpathogenic levels. There is a great deal of evidence supporting the implication of epigenetics in the modulation of the biological interaction between hosts and pathogens. This study reveals that Zika virus infection positively modulates the expression and activity of A. aegypti histone acetyltransferase CBP (AaCBP). This study shows that AaCBP plays a role in the activation of immune-responsive genes to limit Zika virus replication. This first description that Zika virus infection has epigenomic consequences in the regulation of A. aegypti immunity opens a new avenue for research on mosquito factors that can drive vector competence.

sentences: Contrasting processing tomato cultivars unlink yield and pollen viability under heat stress The occurring climate change is causing temperature increment in crop production areas worldwide, generating conditions of heat stress that negatively affect crop productivity. Tomato (Solanum lycopersicum), a major vegetable crop, is highly susceptible to conditions of heat stress. When tomato plants are exposed to ambient day/night temperatures that exceed 32{degrees}C/20{degrees}C respectively during the reproductive phase, fruit set and fruit weight are reduced, leading to a significant decrease in yield. Processing tomato cultivars are cultivated in open fields, where environmental conditions are not controlled, therefore plants are exposed to multiple abiotic stresses, including heat stress. Understanding the physiological response of modern processing tomato cultivars to heat stress may facilitate the development of thermotolerant cultivars. Here, we compared two tomato processing cultivars, H4107 and H9780, that we found to be constantly differing in yield performance. Using field and temperature-controlled greenhouse experiments, we show that the observed difference in yield is attributed to the occurrence of heat stress conditions. In addition, fruit-set and seed production were significantly improved in the thermotolerant cultivar H4107, compared with H9780. Despite the general acceptance of pollen viability as a measure of thermotolerance, there was no difference in the percentage of viable pollen between H4107 and H9780 under either of the conditions tested. Therefore, processing tomato cultivars may present a particular case, in which other factors are central for heat stress tolerance. Our results also demonstrate the value of combining controlled with uncontrolled experimental settings, in order to identify heat stress related responses and facilitate the development of thermotolerant processing tomato cultivars.

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Effect of imputation on gene network reconstruction from single-cell RNA-seq data Despite the advances in single-cell transcriptomics the reconstruction of gene regulatory networks remains challenging. Both the large amount of zero counts in experimental data and the lack of a consensus preprocessing pipeline for single-cell RNA-seq data make it hard to infer networks from transcriptome data. Data imputation can be applied in order to enhance gene-gene correlations and facilitate downstream data analysis. However, it is unclear what consequences imputation methods have on the reconstruction of gene regulatory networks.

To study this question, we evaluate the effect of imputation methods on the performance and structure of the reconstructed networks in different experimental single-cell RNA-seq data sets. We use state-of-the-art algorithms for both imputation and network reconstruction and evaluate the difference in results before and after imputation. We observe an inflation of gene-gene correlations that affects the predicted network structures and may decrease the performance of network reconstruction in general. Yet, within the modest limits of achievable results, we also make a recommendation as to an advisable combination of algorithms, while warning against the indiscriminate use of imputation before network reconstruction in general.

sentences: Antimicrobial resistance determinants in silage Animal products may play a role in developing and spreading antimicrobial resistance in several ways. On the one hand, residues of antibiotics not adequately used in animal farming can enter the human body via food. But resistant bacteria may also be present in animal products, which can transfer the antimicrobial resistance genes (ARG) to the bacteria in the consumers body by horizontal gene transfer. As previous studies have shown that fermented foods have a meaningful ARG content, it is indicated that such genes may also be present in silage used as mass feed in the cattle sector. In our study, we aspired to answer what ARGs occur in silage and what mobility characteristics they have? For this purpose, we have analyzed bioinformatically 52 freely available deep sequenced silage samples from shotgun metagenome next-generation sequencing. A total of 17 perfect matched ARGs occurred 55 times in the samples. More than half of these ARGs are mobile because they can be linked to integrative mobile genetic elements, prophages or plasmids. Our results point to a neglected but substantial ARG source in the food chain.

sentences: The architecture of phenotypic flexibility within a complex trait: an empirical case study using avian thermogenic performance Reversible modifications to trait values can allow individuals to match their phenotypes to changing environmental conditions, a phenomenon known as phenotypic flexibility. A systems capacity for flexibility may be determined by its underlying architecture, and these relationships can have important implications for both organismal adaptation and the evolvability of acclimatization responses. Theory provides two possible alternatives to explain the ways in which lower-level traits respond to environmental challenges and contribute to phenotypic flexibility in complex, whole-organism traits: symmorphosis predicts correspondence between structure and demand across all levels of a physiological system, while the alternative predicts that influence is concentrated in select elements of a physiological network. Here we provide a rich dataset -- composed of 20 sub-organismal, physiological traits paired with whole-organism metabolic rates for 106 adult Dark-eyed Juncos (Junco hyemalis) -- to explore the mechanistic basis of phenotypic flexibility in complex traits. When exposed to synthetic temperature cues, these individuals have previously been shown to increase their thermogenic capacity (Msum) and enhance their ability to maintain their body temperature in the cold. We show that the relationships among a number of the traits that contribute to Msum varied as the environmental context changed. Moreover, variation in Msum in response to temperature acclimation was correlated with only a handful of subordinate phenotypes. As a result, avian thermogenic flexibility does not appear to be a symmorphotic response. If this is generally true of complex traits, it suggests that simple and reversible modifications can significantly impact whole-organism performance, and thus that the evolution of phenotypic flexibility in a single component part could impart flexibility for the entire system.

sentences: Curvelet Transform-based Sparsity Promoting Algorithm for Fast Ultrasound Localization Microscopy Ultrasound localization microscopy (ULM) based on microbubble (MB) localization was recently introduced to overcome the resolution limit of conventional ultrasound. However, ULM is currently challenged by the requirement for long data acquisition times to accumulate adequate MB events to fully reconstruct vasculature. In this study, we present a curvelet transform-based sparsity promoting (CTSP) algorithm that improves ULM imaging speed by recovering missing MB localization signal from data with very short acquisition times. CTSP was first validated in a simulated microvessel model, followed by the chicken embryo chorioallantoic membrane (CAM), and finally, in the mouse brain. In the simulated microvessel study, CTSP robustly recovered the vessel model to achieve an 86.94% vessel filling percentage from a corrupted image with only 4.78% of the true vessel pixels. In the chicken embryo CAM study, CTSP effectively recovered the missing MB signal within the vasculature, leading to marked improvement in ULM imaging quality with a very short data acquisition. Taking the optical image as reference, the vessel filling percentage increased from 2.7% to 42.2% using 50ms of data acquisition after applying CTSP. CTSP used 80% less time to achieve the same 90% maximum saturation level as compared with conventional MB localization. We also applied CTSP on the microvessel flow speed maps and found that CTSP was able to use only 1.6s of microbubble data to recover flow speed images that have similar qualities as those constructed using 33.6s of data. In the mouse brain study, CTSP was able to reconstruct the majority of the cerebral vasculature using 1-2s of data acquisition. Additionally, CTSP only needed 3.2s of microbubble data to generate flow velocity maps that are comparable to those using 129.6s of data. These results suggest that CTSP can facilitate fast and robust ULM imaging especially under the circumstances of inadequate microbubble localizations.

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Noise source importance in linear stochastic models of biological systems that grow, shrink, wander, or persist While noise is an important factor in biology, biological processes often involve multiple noise sources, whose relative importance can be unclear. Here we develop tools that quantify the importance of noise sources in a network based on their contributions to variability in a quantity of interest. We generalize the edge importance measures proposed by Schmidt and Thomas [1] for first-order reaction networks whose steady-state variance is a linear combination of variance produced by each directed edge. We show that the same additive property extends to a general family of stochastic processes subject to a set of linearity assumptions, whether in discrete or continuous state or time. Our analysis applies to both expanding and contracting populations, as well as populations obeying a martingale ("wandering") at long times. We show that the original Schmidt-Thomas edge importance measure is a special case of our more general measure, and is recovered when the model satisfies a conservation constraint ("persists"). In the growing and wandering cases we show that the choice of observables (measurements) used to monitor the process does not influence which noise sources are important at long times. In contrast, in the shrinking or persisting case, which noise sources are important depends on what is measured. We also generalize our measures to admit models with affine moment update equations, which admit additional limiting scenarios, and arise naturally after linearization. We illustrate our results using examples from cell biology and ecology: (i) a model for the dynamics of the inositol trisphospate receptor, (ii) a model for an endangered population of white-tailed eagles, and (iii) a model for wood frog dispersal.

Author summaryBiological processes are frequently subject to an ensemble of independent noise sources. Noise sources produce fluctuations that propagate through the system, driving fluctuations in quantities of interest such as population size or ion channel configuration. We introduce a measure that quantifies how much variability each noise source contributes to any given quantity of interest. Using these methods, we identify which binding events contribute significantly to fluctuations in the state of a molecular signalling channel, which life history events contribute the most variability to an eagle population before and after a successful conservation effort rescued the population from the brink of extinction, and which dispersal events, at what times, matter most to variability in the recolonization of a series of ponds by wood frogs after a drought.

sentences: Behaviorally emergent hippocampal place maps remain stable during memory recall The hippocampus is critical for the formation and recall of episodic memories1, 2 which store past experience of events ( what) occurring at particular locations ( where) in time ( when). Hippocampal place cells, pyramidal neurons which show location-specific modulation of firing rates during navigation3, 4, together form a spatial representation of the environment. It has long been hypothesized that place cells serve as the neural substrate for long-term episodic memory of space5, 6. However, recent studies call to question this tenet of the field by demonstrating unexpected levels of representational drift in hippocampal place cells with respect to the duration of episodic memories in mice7, 8. In the present study, we examined behaviorally driven long-term organization of the place map, to resolve the relationship between memory and place cells. Leveraging the stability of two-photon calcium imaging, we tracked activity of the same set of CA1 pyramidal neurons during learning and memory recall in an operant, head-fixed, odorcued spatial navigation task. We found that place cells are rapidly recruited into task-dependent spatial maps, resulting in emergence of orthogonal as well as overlapping representations of space. Further, task-selective place cells used a diverse set of remapping strategies to represent changing task demands that accompany learning. We found behavioral performance dependent divergence of spatial maps between trial types occurs during learning. Finally, imaging during remote recall spanning up to 30 days revealed increased stabilization of learnt place cell maps following memory consolidation. Our findings suggest that a subset of place cells is recruited by rule based spatial learning, actively reconfigured to represent task-relevant spatial relationships, and stabilized following successful learning and consolidation.

sentences: Cumulative growth and stress responses to the 2018-2019 drought in a European floodplain forest Droughts increasingly threaten the worlds forests and their potential to mitigate climate change. In 2018-2019, Central European forests were hit by two consecutive hotter drought years, an unprecedented phenomenon that is likely to occur more frequently with climate change. Here, we examine tree growth and physiological stress responses (increase in carbon isotope composition; {Delta}{delta}13C) to this consecutive drought based on tree-rings of dominant tree species in a Central European floodplain forest. Tree growth was not reduced for most species in 2018, indicating that water supply in floodplain forests can partly buffer meteorological water deficits. Drought stress responses in 2018 were comparable to former single drought years but the hotter drought in 2018 induced drought legacies in tree growth while former droughts did not. We observed strong decreases in tree growth and increases in {Delta}{delta}13C across all tree species in 2019, which are likely driven by the cumulative stress both consecutive hotter droughts exerted. Our results show that consecutive hotter droughts pose a novel threat to forests under climate change, even in forest ecosystems with comparably high levels of water supply.

sentences: Characterization of the consensus mucosal microbiome of colorectal cancer Recent evidence suggests that dysbiosis, an imbalance of microbiota, is associated with increased risk of colorectal cancer. Diverse microbial organisms are physically associated with the cells found in tumor biopsies. Characterizing this mucosa-associated microbiome through genome sequencing has advantages compared to culture-based profiling. However, there are notable challenges in accurately characterizing the features of tumor microbiomes with methods like transcriptome sequencing. Most sequence reads originate from the host. Moreover, there is a high likelihood of bacterial contaminants being introduced. Another major challenge is the microbiome diversity among different studies. Colorectal tumors demonstrate a significant extent of microbiome variation among individuals from different geographic and ethnic origins. To address these challenges, we identified a consensus microbiome for colorectal cancer through analyzing 924 tumors from eight independent RNA-Seq data sets. A standardized meta-transcriptomic analysis pipeline was established and applied to the complete CRC cohort. Common contaminants were filtered out. Our study involved taxonomic investigation of non-human sequences, linked microbial signatures to phenotypes and the association of microbiome with tumor microenvironment components. Microbiome profiles across different CRC cohorts were compared, and recurrently altered microbial shifts specific to CRC were determined. We identified cancer-specific set of 114 microbial species associated with tumors that were found among all investigated studies. Validating our approach, we found that Fusobacterium nucleatum was one of the most enriched bacterial species in CRC. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were among the four most abundant phyla for CRC microbiome. Signficant associations between the consensus species and specific immune cell types were noted. Our results are available as a web data resource for other researchers to explore (https://crc-microbiome.stanford.edu).

sentences: Delineating antibody escape from Omicron variants SARS-CoV-2-neutralizing antibodies play a critical role for protection and treatment of COVID-19. Viral antibody evasion therefore threatens essential prophylactic and therapeutic measures. The high number of mutations in the Omicron BA.1 sublineage results in markedly reduced neutralization susceptibility. Consistently, Omicron is associated with lower vaccine effectiveness and a high re-infection rate. Notably, newly emerging Omicron sublineages (BA.1.1, BA.2) have rapidly become dominant. Here, we determine polyclonal serum activity against BA.1, BA.1.1 and BA.2 in 50 convalescent or vaccinated individuals as well as delineate antibody sensitivities on a monoclonal level using 163 antibodies. Our study reveals a significant but comparable reduction of serum activity against Omicron sublineages which markedly increases after booster immunization. However, notable differences in sensitivity to individual antibodies demonstrate distinct escape patterns of BA.1 and BA.2 that also affect antibodies in clinical use. The results have strong implications for vaccination strategies and antibody use in prophylaxis and therapy.

sentences: Antibody Display of cell surface receptor Tetraspanin12 and SARS-CoV-2 spike protein In previous work, Hsieh and Higgins presented a novel structure of antibodies identified from malaria-exposed individuals, in which the extracellular immunoglobulin (Ig)-like domain of leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) is presented on the third complementarity determining regions (CDR3) of the Ig heavy chain. Here we develop an Antibody Display technology based on this LAIR1-containing antibody, by grafting proteins of interest (POI) onto the heavy chain CDR3 while retaining the biological properties of the POI. As a proof of principle, we displayed the second extracellular domain of Tetraspanin12 (Tspan12EC2) and the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the heavy chain CDR3. Our data revealed that Antibody Display Tspan12EC2 bound to Norrie Disease Protein (Norrin) and Antibody Display SARS-CoV-2 RBD bound to angiotensin-converting enzyme 2 (ACE2) and neutralizing nanobodies. Collectively, Antibody Display technology offers the general strategy of designing novel antibodies by grafting POI onto the CDR3.

sentences: Association of RACK1 protein with ribosomes in Plasmodium falciparum The receptor for activated C-kinase 1 (RACK1), a highly conserved eukaryotic protein, is known to have many, varying biological roles and functions. Previous work has established RACK1 as a ribosomal protein, with defined regions important for binding ribosomes in both human and yeast cells. In Plasmodium falciparum, RACK1 has been shown to be required for parasite growth, however conflicting evidence has been presented about the RACK1 ribosome binding and its role in mRNA translation. Given the importance of RACK1 as a regulatory component of mRNA translation and ribosome quality control, the case could be made in parasites for either of the binary options: bound or unbound to the ribosome. Here we used bioinformatics and transcription analyses to describe P. falciparum RACK1 protein. Based on homology modeling and structural analyses, we generated a model of P. falciparum RACK1 protein. We created and explored mutant and chimeric human and P. falciparum RACK1 proteins binding properties to the human and P. falciparum ribosome. Wild type, chimeric and mutant RACK1 proteins suggest different binding characteristics for P. falciparum and human RACK1 proteins and different regions being involved in their ribosome association. The ribosomal binding of RACK1 variants in human and parasite cells shown here demonstrates that although RACK1 proteins have highly conserved sequences and structures across species, ribosomal binding is affected by species specific alterations to the protein. In conclusion, we show that in the case of P. falciparum, contrary to the structural data, RACK1 is found to be bound to ribosomes and in actively translating polysomes of P. falciparum cells.

sentences: Facultative variation across a shallow to deep torpor spectrum in hummingbirds Many small endotherms use torpor, saving energy by a controlled reduction of their body temperature and metabolic rate. Some species (e.g. arctic ground squirrels, hummingbirds) enter deep torpor, dropping their body temperatures by 23-37 {degrees}C, while others can only enter shallow torpor (e.g., pigeons, 3-10 {degrees}C reductions). However, deep torpor in mammals can increase predation risk (unless animals are in burrows or caves), inhibit immune function, and result in sleep deprivation, so even for species that can enter deep torpor, facultative shallow torpor might help balance energy savings with these potential costs. Deep torpor occurs in three avian orders. Although the literature hints that some bird species can use both shallow and deep torpor, little empirical evidence of such an avian torpor spectrum exists. We infrared imaged three hummingbird species that are known to use deep torpor, under natural temperature and light cycles, to test if they were also capable of shallow torpor. All three species used both deep and shallow torpor, often on the same night. Depending on the species, they used shallow torpor for 5-35% of the night. The presence of a bird torpor spectrum indicates a capacity for fine-scale physiological and genetic regulation of avian torpid metabolism.

sentences: Species Tree Inference on Data with Paralogs is Accurate Using Methods Intended to Deal with Incomplete Lineage Sorting AO_SCPLOWBSTRACTC_SCPLOWMany recent phylogenetic methods have focused on accurately inferring species trees when there is gene tree discordance due to incomplete lineage sorting (ILS). For almost all of these methods, and for phylogenetic methods in general, the data for each locus is assumed to consist of orthologous, single-copy sequences. Loci that are present in more than a single copy in any of the studied genomes are excluded from the data. These steps greatly reduce the number of loci available for analysis. The question we seek to answer in this study is: What happens if one runs such species tree inference methods on data where paralogy is present, in addition to or without ILS being present? Through simulation studies and analyses of two large biological data sets, we show that running such methods on data with paralogs can still provide accurate results. We use multiple different methods, some of which are based directly on the multispecies coalescent (MSC) model, and some of which have been proven to be statistically consistent under it. We also treat the paralogous loci in multiple ways: from explicitly denoting them as paralogs, to randomly selecting one copy per species. In all cases the inferred species trees are as accurate as equivalent analyses using single-copy orthologs. Our results have significant implications for the use of ILS-aware phylogenomic analyses, demonstrating that they do not have to be restricted to single-copy loci. This will greatly increase the amount of data that can be used for phylogenetic inference.

sentences: Pictorial balance is a bottom-up aesthetic property mediated by eye movements This paper identifies a specific pattern of luminance in pictures that creates a low level non-subjective neuro-aesthetic effect and provides a theoretical explanation for how it occurs. Pictures evoke both a top-down and a bottom-up visual percept of balance. Through its effect on eye movements, balance is a bottom-up conveyor of aesthetic feelings of unity and harmony in pictures. These movements are influenced by the large effects of saliency so that it is difficult to separate out the much smaller effect of balance. Given that balance is associated with a unified, harmonious picture and that there is a pictorial effect known to painters and historically documented that does just that, it was thought that such pictures are perfectly balanced. Computer models of these pictures were found to have bilateral quadrant luminance symmetry with a lower half lighter by a factor of ~1.07 +/- ~0.03. A top weighted center of quadrant luminance calculation is proposed to measure imbalance. A study was done comparing identical pictures in two different frames with respect to whether they appeared different given that the sole difference is balance. Results show that with observers, mostly painters, there was a significant correlation between average pair imbalance and observations that two identical pictures appeared different indicating at a minimum that the equation for calculating balance was correct. For those who can disregard saliency the effect is the result of the absence of forces on eye movements created by imbalance. This unaccustomed force of imbalance causes fatigue when viewing pictures carefully. A model is presented of how an equation of balance could be derived from any luminous object so that its approximate size, location and motion can be followed. Using this model the center of balance in non-rectangular pictures was determined.

sentences: Relocation of rDNA repeats for repair is dependent on SUMO-mediated nucleolar release by the Cdc48/p97 segregase Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to active transcription and their repetitive nature. Compartmentalization of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be released to the nucleoplasm to allow repair by homologous recombination. The process of rDNA relocation is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation targets CLIP-cohibin for disassembly mediated by the Cdc48/p97 chaperone, which recognizes SUMOylated CLIP-cohibin through its cofactor, Ufd1. Consistent with a conserved mechanism, UFD1L depletion impairs rDNA release in human cells. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.

sentences: Allele expression biases in mixed-ploid sugarcane accessions Allele-specific expression (ASE) represents differences in the magnitude of expression between alleles of the same gene. This is not straightforward for polyploids, especially autopolyploids, as knowledge about the dose of each allele is required for accurate estimation of ASE. This is the case for the genomically complex Saccharum species, characterized by high levels of ploidy and aneuploidy. We used a Beta-Binomial model to test for allelic imbalance in Saccharum, with adaptations for mixed-ploid organisms. The hierarchical Beta-Binomial model was used to test if allele expression followed the expectation based on genomic allele dosage. The highest frequencies of ASE occurred in sugarcane hybrids, suggesting a possible influence of interspecific hybridization in these genotypes. For all accessions, ASEGs were less frequent than those with balanced allelic expression. These genes were related to a broad range of processes, mostly associated with general metabolism, organelles, responses to stress and responses to stimuli. In addition, the frequency of ASEGs in high-level functional terms was similar among the genotypes, with a few genes associated with more specific biological processes. We hypothesize that ASE in Saccharum is largely a genotype-specific phenomenon, as a large number of ASEGs were exclusive to individual accessions.

sentences: Cis-Regulatory Element Hijacking by Structural Variants Overshadows Higher-Order Topological Changes in Primary Prostate Cancer Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However the impact of this instability on the three-dimensional chromatin organization and how this drives progression is unclear. Using primary benign and tumour tissue, we find a high concordance in the higher-order three-dimensional genome organization across normal and prostate cancer cells. This concordance argues for constraints to the topology of prostate tumour genomes. Nonetheless, we identify changes to focal chromatin interactions and show how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events result in opposing differential expression on genes found at antipodes of rearrangements. Collectively, our results argue that cis-regulatory element hijacking from structural variant-induced altered focal chromatin interactions overshadows higher-order topological changes in the development of primary prostate cancer.

sentences: Control of nuclear size by osmotic forces in Schizosaccharomyces pombe The size of the nucleus scales robustly with cell size so that the nuclear-to-cell volume ratio (N/C ratio) is maintained during cell growth in many cell types. The mechanism responsible for this scaling remains mysterious. Previous studies have established that the N/C ratio is not determined by DNA amount but is instead influenced by factors such as nuclear envelope mechanics and nuclear transport. Here, we developed a quantitative model for nuclear size control based upon colloid osmotic pressure and tested key predictions in the fission yeast Schizosaccharomyces pombe. This model posits that the N/C ratio is determined by the numbers of macromolecules in the nucleoplasm and cytoplasm. Osmotic shift experiments showed that the fission yeast nucleus behaves as an ideal osmometer whose volume is primarily dictated by osmotic forces. Inhibition of nuclear export caused accumulation of macromolecules and an increase in crowding in the nucleoplasm, leading to nuclear swelling. We further demonstrated that the N/C ratio is maintained by a homeostasis mechanism based upon synthesis of macromolecules during growth. These studies demonstrate the functions of colloid osmotic pressure in intracellular organization and size control.

sentences: Mapping the organization and dynamics of the posterior medial network during movie watching Brain regions within a posterior medial network (PMN) are characterized by sensitivity to episodic tasks, and they also demonstrate strong functional connectivity as part of the default network. Despite its cohesive structure, delineating the intranetwork organization and functional diversity of the PMN is crucial for understanding its contributions to multidimensional event cognition. Here, we probed functional connectivity of the PMN during movie watching to identify its pattern of connections and subnetwork functions in a split-sample replication of 136 participants. Consistent with prior findings of default network fractionation, we identified distinct PMN subsystems: a Ventral PM subsystem (retrosplenial cortex, parahippocampal cortex, posterior angular gyrus) and a Dorsal PM subsystem (medial prefrontal cortex, hippocampus, precuneus, posterior cingulate cortex, anterior angular gyrus). Ventral and Dorsal PM subsystems were differentiated by functional connectivity with parahippocampal cortex and precuneus and integrated by retrosplenial cortex and posterior cingulate cortex, respectively. Finally, the distinction between PMN subsystems is functionally relevant: whereas both Dorsal and Ventral PM connectivity tracked the movie content, only Ventral PM connections increased in strength at event transitions and appeared sensitive to episodic memory. Overall, these findings reveal PMN functional pathways and the distinct functional roles of intranetwork subsystems during event cognition.

sentences: Molecular basis for redox control by the human cystine/glutamate antiporter System xc-. Cysteine plays an essential role in cellular redox homeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc- is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc- is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc- in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.

sentences: Control of division and microtubule dynamics in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ( CDK) CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen (EB1-NG) allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1-NG signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by  comets of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC mitosis modulate microtubule dynamics while regulating mitotic progression.

sentences: Sensory neuron-associated macrophages proliferate in the sensory ganglia after peripheral nerve injury in a CX3CR1 signaling dependent manner Resident macrophages are distributed across all tissues and are highly heterogeneous as a consequence of adaptation to different tissue-specific environments. The resident macrophages of the sensory ganglia (sensory neuron-associated macrophages, sNAMs) are in close contact with the cell body of primary sensory neurons and might play physiological and pathophysiological roles. After peripheral nerve injury, there is an increase in the population of macrophages in the sensory ganglia which have been involved in different conditions, especially in neuropathic pain development and nerve regeneration. However, it is still under debate whether macrophages accumulation in the sensor ganglia after peripheral nerve injury is due to the local proliferation of resident macrophages or as a result of blood monocytes infiltration. Here, we confirmed that the number of macrophages increased in the DRGs after spared nerve injury (SNI) model in mice. By using different approaches, we found that the increase in the number of macrophages in the DRGs after SNI is mainly in consequence of the proliferation of resident CX3CR1+ macrophages but not due to infiltration of CCR2+ blood monocytes. These proliferating macrophages are the source of the production of pro-inflammatory cytokines such as TNF and IL-1b. In addition, we found that CX3CR1 signaling is involved in the sNAMs proliferation after peripheral nerve injury. In summary, these results indicated that peripheral nerve injury leads to sNAMs proliferation in the sensory ganglia in a CX3CR1-dependent manner. In conclusion, sNAMs proliferation could be modulated to change pathophysiological conditions such as chronic neuropathic pain and/or nerve regeneration.

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Identification of tissue-specific and common methylation quantitative trait loci in healthy individuals using MAGAR BackgroundUnderstanding the influence of genetic variants on DNA methylation is fundamental for the interpretation of epigenomic data in the context of disease. There is a need for systematic approaches not only for determining methylation quantitative trait loci (methQTL) but also for discriminating general from cell-type-specific effects.

ResultsHere, we present a two-step computational framework MAGAR, which fully supports identification of methQTLs from matched genotyping and DNA methylation data, and additionally the identification of quantitative cell-type-specific methQTL effects. In a pilot analysis, we apply MAGAR on data in four tissues (ileum, rectum, T-cells, B-cells) from healthy individuals and demonstrate the discrimination of common from cell-type-specific methQTLs. We experimentally validate both types of methQTLs in an independent dataset comprising additional cell types and tissues. Finally, we validate selected methQTLs (PON1, ZNF155, NRG2) by ultra-deep local sequencing. In line with previous reports, we find cell-type-specific methQTLs to be preferentially located in enhancer elements.

ConclusionsOur analysis demonstrates that a systematic analysis of methQTLs provides important new insights on the influences of genetic variants to cell-type-specific epigenomic variation.

sentences: Evaluating the reliability of human brain white matter tractometry The validity of research results depends on the reliability of analysis methods. In recent years, there have been concerns about the validity of research that uses diffusion-weighted MRI (dMRI) to understand human brain white matter connections in vivo, in part based on reliability of the analysis methods used in this field. We defined and assessed three dimensions of reliability in dMRI-based tractometry, an analysis technique that assesses the physical properties of white matter pathways: (1) reproducibility, (2) test-retest reliability and (3) robustness. To facilitate reproducibility, we provide software that automates tractometry (https://yeatmanlab.github.io/pyAFQ). In measurements from the Human Connectome Project, as well as clinical-grade measurements, we find that tractometry has high test-retest reliability that is comparable to most standardized clinical assessment tools. We find that tractometry is also robust: showing high reliability with different choices of analysis algorithms. Taken together, our results suggest that tractometry is a reliable approach to analysis of white matter connections. The overall approach taken here both demonstrates the specific trustworthiness of tractometry analysis and outlines what researchers can do to demonstrate the reliability of computational analysis pipelines in neuroimaging.

sentences: Individualized discovery of rare cancer drivers in global network context Late advances in genome sequencing expanded the space of known cancer driver genes several-fold. However, most of this surge was based on computational analysis of somatic mutation frequencies and/or their impact on the protein function. On the contrary, experimental research necessarily accounted for functional context of mutations interacting with other genes and conferring cancer phenotypes. Eventually, just such results become "hard currency" of cancer biology. The new method, NEAdriver employs knowledge accumulated thus far in the form of gene interaction networks and functionally annotated pathways in order to recover known and predict novel driver genes. The driver discovery was individualized by accounting for mutations co-occurrence in tumour genomes. For each somatic genome change, probabilistic estimates from two lanes of network analysis were combined into joint likelihoods of being a driver. Thus, ability to detect previously unnoticed candidate driver events emerged from combining individual genomic context with network perspective. The procedure was applied to ten largest cancer cohorts followed by evaluating error rates against previous cancer gene sets. The discovered driver combinations were shown to be informative on cancer outcome. We demonstrate that the individualized discovery revealed driver events which were individually rare, not detectable by other computational approaches, and related to cancer biology domains poorly covered by previous analyses. Considering the novel driver candidates and their constellations in individual tumor genomes opens a novel avenue for personalized cancer medicine.

sentences: Fatty acid-binding proteins and fatty acid synthase influence glial reactivity and promote the formation of Müller glia-derived progenitor cells in the avian retina The capacity for retinal regeneration varies greatly across vertebrates species. A recent comparative epigenetic and transcriptomic investigation of Muller glial (MG) in the retinas of fish, birds and mice revealed that Fatty Acid Binding Proteins (FABPs) are among the most highly up-regulated genes in activated chick MG (Hoang et al., 2020). Herein we provide an in-depth follow-up investigation to describe patterns of expression and how FABPs and fatty acid synthase (FASN) influence glial cells in the chick retina. During development, FABP7 is highly expressed by embryonic retinal progenitor cells (eRPCs) and maturing MG, whereas FABP5 is gradually up-regulated in maturing MG and remains elevated in mature glial cells. PMP2 (FABP8) is expressed by oligodendrocytes and FABP5 is expressed by non-astrocytic inner retinal glial cells, and both of these FABPs are significantly up-regulated in activated MG in damaged or growth factor-treated retinas. In addition to suppressing the formation of MGPCs, we find that FABP-inhibition suppressed the accumulation of proliferating microglia, although the microglia appeared highly reactive. scRNA-seq analyses of cells treated with FABP-inhibitor revealed distinct changes in patterns of expression suggesting that FABPs are involved in the transitions of MG from a resting state to a reactive state and conversion from reactive MG to MGPCs. Inhibition of FABPs in undamaged retinas had a significant impact upon the transcriptomic profiles of MG, with up-regulation of genes associated with gliogenesis, decreases in genes associated with neurogenesis, and suppression of the ability of MG to become MGPCs. scRNA-seq analyses of microglia indicated that FABP inhibition enhances gene modules related to reactivity, proliferation and cytokine signaling. We find that the proliferation of retinal progenitors in the circumferential marginal zone (CMZ) is unaffected by FABP-inhibitor. Upstream of FABP activity, we inhibited FASN in damaged retinas, which reduced numbers of dying cells, increased the proliferation of microglia, and potently suppressed the formation MGPCs in damaged retinas. We conclude that the activity of FASN and FABPs are required early during the formation of proliferating MGPCs. Fatty acid metabolism and cell signaling involving fatty acids are important in regulating glial homeostasis in the retina, and the dedifferentiation and proliferation of microglia and MGPCs.

sentences: Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) requires immediate investigation. Here, we used high-throughput yeast display screening1,2 to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs and showed that the NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications3-5. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlap with ACE2-binding motif, are largely escaped by K417N, G446S, E484A, and Q493R. Group E (S309 site)6 and F (CR3022 site)7 NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, N440K, and S371L. Furthermore, Omicron pseudovirus neutralization showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, the neutralization potency of LY-CoV016/LY-CoV555, REGN10933/REGN10987, AZD1061/AZD8895, and BRII-196 were greatly reduced by Omicron, while VIR-7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron would cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.

sentences: Cortical processing of reference in language revealed by computational models Human language processing involves not only combining word meanings in accordance with semantic and syntactic constraints, but also figuring out who and what is being referred to. Here we present a first study towards a mechanistic understanding of the neural basis for referential processing. Using both functional MRI and magnetoencephalography (MEG), we identified a consistent increase of activity in a network spanning the anterior and posterior left middle temporal gyrus and the angular gyrus for pronoun processing during naturalistic listening for both English and Chinese speakers. We then adopted a "reverse-engineering" approach to examine the cognitive processes underlying pronoun resolution. We evaluated the neural fit of three symbolic models that each formalizes a different strand of explanation for pronoun resolution in the cognitive and linguistic literature, as well as two deep neural network models with an LSTM or a Transformer architecture. Our results favor the memory-based symbolic model, suggesting a domain-general mechanism of pronoun resolution that resembles memory retrieval.

sentences: Thalamic control of sensory enhancement and sleep spindle properties in a biophysical model of thalamoreticular microcircuitry Thalamoreticular circuitry is known to play a key role in attention, cognition and the generation of sleep spindles, and is implicated in numerous brain disorders, but the cellular and synaptic mechanisms remain intractable. Therefore, we developed the first detailed computational model of mouse thalamus and thalamic reticular nucleus microcircuitry that captures morphological and biophysical properties of [~]14,000 neurons connected via [~]6M synapses, and recreates biological synaptic and gap junction connectivity. Simulations recapitulate multiple independent network-level experimental findings across different brain states, providing a novel unifying cellular and synaptic account of spontaneous and evoked activity in both wakefulness and sleep. Furthermore, we found that: 1.) inhibitory rebound produces frequency-selective enhancement of thalamic responses during wakefulness, in addition to its role in spindle generation; 2.) thalamic interactions generate the characteristic waxing and waning of spindle oscillations; and 3.) changes in thalamic excitability (e.g. due to neuromodulation) control spindle frequency and occurrence. The model is openly available and provides a new tool to interpret spindle oscillations and test hypotheses of thalamoreticular circuit function and dysfunction across different network states in health and disease.

sentences: Protein family content uncovers lineage relationships and bacterial pathway maintenance mechanisms in DPANN archaea DPANN are small-celled archaea that are generally predicted to be symbionts, and in some cases are known episymbionts of other archaea. As the monophyly of the DPANN remains uncertain, we hypothesized that proteome content could reveal relationships among DPANN lineages, constrain genetic overlap with bacteria, and illustrate how organisms with hybrid bacterial and archaeal protein sets might function. We tested this hypothesis using protein family content that was defined in part using 569 newly reconstructed genomes. Protein family content clearly separates DPANN from other archaea, paralleling the separation of Candidate Phyla Radiation (CPR) bacteria from all other bacteria. This separation is partly driven by hypothetical proteins, some of which may be symbiosis-related. Pacearchaeota with the most limited predicted metabolic capacities have Form II/III and III-like Rubisco, suggesting metabolisms based on scavenged nucleotides. Intriguingly, the Pacearchaeota and Woesearchaeota with the smallest genomes also tend to encode large extracellular murein-like lytic transglycosylase domain proteins that may bind and degrade components of bacterial cell walls, indicating that some might be episymbionts of bacteria. The pathway for biosynthesis of bacterial isoprenoids is widespread in Woesearchaeota genomes and is encoded in proximity to genes involved in bacterial fatty acids synthesis. Surprisingly, in some DPANN genomes we identified a pathway for synthesis of queuosine, an unusual nucleotide in tRNAs of bacteria. Other bacterial systems are predicted to be involved in protein refolding. For example, many DPANN have the complete bacterial DnaK-DnaJ-GrpE system and many Woesearchaeota and Pacearchaeota possess bacterial group I chaperones. Thus, many DPANN appear to have mechanisms to ensure efficient protein folding of both archaeal and laterally acquired bacterial proteins.

sentences: SOAR: a spatial transcriptomics analysis resource to model spatial variability and cell type interactions Spatial transcriptomics is an emerging technology to profile spatially resolved gene expression. Its preserved capture locations allow researchers to investigate transcriptomes in the tissue context. Large volumes of spatial transcriptomics data under different study designs have been generated, but the lack of a public database with systematically collected and processed data makes data reuse challenging. We present Spatial transcriptOmics Analysis Resource (SOAR), a database with analysis capability and an extensive collection of spatial transcriptomics data. We systematically curated, reviewed, annotated, and pre-processed 132 datasets containing 1,633 samples across 22 tissue types from 6 species. SOAR provides interactive web interfaces for users to visualize spatial gene expression, evaluate gene spatial variability across cell types, and assess cell-cell interactions. Besides data access and download, SOAR can aid researchers in investigating whether a specific gene expression is associated with distinct spatial patterns or cell-cell communications. SOAR is publicly available at https://soar.fsm.northwestern.edu/.

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Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV There is an urgent need to understand the nature of immune responses generated against SARS-CoV-2, to better inform risk-mitigation strategies for people living with HIV (PLWH). Although not all PLWH are considered immunosuppressed, residual cellular immune deficiency and ongoing inflammation could influence COVID-19 disease severity, the evolution and durability of protective memory responses. Here, we performed an integrated analysis, characterizing the nature, breadth and magnitude of SARS-CoV-2-specific immune responses in PLWH, controlled on ART, and HIV negative subjects. Both groups were in the convalescent phase of predominately mild COVID-19 disease. The majority of PLWH mounted SARS-CoV-2 Spike- and Nucleoprotein-specific antibodies with neutralizing activity and SARS-CoV-2-specific T cell responses, as measured by ELISpot, at levels comparable to HIV negative subjects. T cell responses against Spike, Membrane and Nucleocapsid were the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. Notably, the overall magnitude of SARS-CoV-2-specific T cell responses related to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH, in whom disparate antibody and T cell responses were observed. Both humoral and cellular responses to SARS-CoV-2 were detected at 5-7 months post-infection, providing evidence of medium-term durability of responses irrespective of HIV serostatus. Incomplete immune reconstitution on ART and a low CD4:CD8 ratio could, however, hamper the development of immunity to SARS-CoV-2 and serve as a useful tool for risk stratification of PLWH. These findings have implications for the individual management and potential effectiveness of vaccination against SARS-CoV-2 in PLWH.

One Sentence SummaryAdaptive immune responses to SARS-CoV-2 in the setting of HIV infection

sentences: Interpretable Drug Target Predictions using Self-Expressiveness The identification of missing drug targets is critical for the development of treatments and for the molecular elucidation of drug side effects. Drug targets have been predicted by exploiting molecular, biological or pharmacological features of drugs and protein targets. Yet, developing integrative and interpretable machine learning models for predicting drug targets remains a challenging task. We present Inception, an integrative and interpretable matrix completion model for predicting drug targets. Inception is a self-expressive model that learns two similarity matrices: one for drugs and another for protein targets. These learned similarity matrices are key for our models interpretability: they can explain how a predicted drug-target interaction can be explain in terms of a linear combination of chemical, biological and pharmacological similarities. We develop a novel objective function with efficient closed-form solution. To demonstrate the ability of Inception at recovering missing drug-target interactions (DTIs), we perform cross-validation experiments with stringent controls of data imbalance, chemical similarities between drugs and sequence similarities between targets. We also assess the performance of our model using a simulated prospective approach. Having trained our model with DTIs from a snapshot 2011 of the DrugBank database, we test whether we could predict DTIs from a 2020 snapshot of DrugBank. Inception outperforms two state-of-the-art drug target prediction models in all the scenarios. This suggests that Inception could be useful for predicting missing drug target interactions while providing interpretable predictions.

sentences: Pathways and signatures of mutagenesis at targeted DNA nicks Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at targeted nicks. BRCA2 inhibits all categories of mutational events at nicks, including indels, SNVs and HDR. DNA2 and RPA promote 5 resection. Most insertions at nicks consist of a single C incorporated opposite the nick by the translesion polymerase REV1. DNA2 and REV3 inhibit these 1 bp insertions; and DNA2 also inhibits 1 bp deletions. Longer deletions are stimulated by DNA2, REV7 and POLQ. Strikingly, POLQ generates most SNVs at both nicks and double-strand breaks. These results identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ at nicks and highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells.

sentences: Functional characterization of RELN missense mutations involved in recessive and dominant forms of Neuronal Migration Disorders RELN is a large secreted glycoprotein that acts at multiple steps of cerebral cortex development, including neuronal migration. Only recessive mutations of the Reelin gene (RELN) have been associated with human cortical malformations and none has been functionally characterized. We identified novel missense RELN mutations in both compound and de novo heterozygous patients exhibiting an array of neuronal migration disorders (NMDs) as diverse as pachygyria, polymicrogyria and heterotopia. Most mutations caused defective RELN secretion in vitro and, when ectopically expressed in the embryonic mouse cortex, affected neuronal aggregation and/or migration in vivo. We determined the de novo heterozygous mutations acted as dominant negative and demonstrated that RELN mutations mediate not only recessive, but also dominant NMDs. This work assesses for the first time the pathogenicity of RELN mutations showing a strong genotype-phenotype correlation. In particular, the behavior of the mutant proteins in vitro and in vivo predicts the severity of cortical malformations and provides valuable insight into the pathogenesis of these disorders.

sentences: Identification of the Molecular Targets of Disulfide Bond Disrupting Agents Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

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Metabolic breakdown of non-small cell lung cancers by mitochondrial HSPD1 targeting The identification of novel targets is of paramount importance to develop more effective drugs and improve the treatment of non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide. Since cells alter their metabolic rewiring during tumorigenesis and along cancer progression, targeting key metabolic players and metabolism-associated proteins represents a valuable approach with a high therapeutic potential. Metabolic fitness relies on the functionality of heat shock proteins (HSPs), molecular chaperones that facilitate the correct folding of metabolism enzymes and their assembly in macromolecular structures. Here, we show HSPD1 (HSP60) as a survival gene ubiquitously expressed in NSCLC and associated with poor patients prognosis. HSPD1 knockdown or its chemical disruption by the small molecule KHS101 induces a drastic breakdown of oxidative phosphorylation, and suppresses cell proliferation both in vitro and in vivo. By combining drug profiling with transcriptomics and through a whole-genome CRISPR/Cas9 screen, we demonstrate that HSPD1-targeted anti-cancer effects are dependent on OXPHOS and validated molecular determinants of KHS101 sensitivity, in particular, the creatine-transporter SLC6A8 and the subunit of the cytochrome c oxidase complex COX5B. These results highlight mitochondrial metabolism as an attractive target and HSPD1 as a potential theranostic marker for developing therapies to combat NCSLC.

SignificanceHSPD1 elimination or disruption interferes with NSCLC metabolic activity causing a strong OXPHOS-dependent energetic breakdown, which the cancer cells fail to overcome, highlighting HSPD1 as a potential theranostic marker for improving lung cancer therapy.

sentences: Got Milk? Maternal immune activation during the mid-lactational period affects nutritional milk quality and adolescent offspring sensory processing in male and female rats The neonatal environment requires a high level of maternal demand in terms of both breastfeeding and other forms of maternal care. Previous studies have underscored the importance of these maternal factors on offspring development and behavior. However, their contribution as dynamic variables in animal models of early life stress are often overlooked. In the present study, we show that lipopolysaccharide (LPS)-induced maternal immune activation (MIA) on postnatal day (P)10 elevated milk corticosterone concentrations, which recovered by P11. In contrast, both milk triglyceride and percent creamatocrit values demonstrated a prolonged decrease following MIA. Sustained changes to the nutritional quality of milk were also evidenced by an increase of microbial communities associated with inefficient energy and lipid metabolism. Nutritional deficits in early development have been associated with metabolic dysfunction later in life. Indeed, these nutritional milk changes were reflected by increased adolescent offspring bodyweights. While MIA did not decrease maternal care quality, there was a significant compensatory increase in maternal licking and grooming the day that followed the inflammatory challenge. However, this did not protect against disrupted neonatal huddling or later-life alterations in sensorimotor gating, mechanical allodynia, or hippocampal parvalbumin interneuron expression in MIA offspring. Since milk concentrations of LPS binding protein and interleukin-6 were not different between MIA and control dams, these offspring effects were likely driven by nutritional factors and not by a direct exposure to LPS or inflammatory molecules. Animal models of early life stress can impact both parents and their progeny; our data show that inflammatory stress affects maternal lactation quality which acts as a mechanism to confer multifaceted and compounding effects on offspring.

sentences: Structure of the 5' untranslated region in SARS-CoV-2 genome and its specific recognition by innate immune system via the human oligoadenylate synthase 1 2-5-Oligoadenylate synthetase 1 (OAS1) is one of the key enzymes driving the innate immune system response to SARS-CoV-2 infection whose activity has been related to COVID-19 severity. In particular, OAS1 is a sensor of endogenous RNA that triggers the 2-5 oligoadenylate/RNase L pathway in response to viral infections, ultimately activating the RNA-Lyase which cleaves endogenous and exogenous RNA impeding the viral maturation. Upon SARS-CoV-2 infection, OAS1 is responsible for the recognition of viral RNA and has been shown to possess a particularly high sensitivity for the 5-untranslated (5-UTR) RNA region, which is organized in a double-strand stem loop motif (SLA). Since the structure of the nucleic acid/protein complex has not been resolved, here we report its structure obtained by molecular modeling, including enhanced sampling approaches. We also pinpoint that the SL1 region enhances the interaction network with the enzyme, promoting specific hydrogen bonds, absent in normal double strand RNA fragments, hence rationalizing the high affinity for OAS1.

sentences: Grasp aperture corrections in reach-to-grasp movements do not alter size perception When grasping an object, the opening between the fingertips (grip aperture) scales with the size of the object. If an object changes in size, the grip aperture has to be corrected. In this study, it was investigated whether such corrections would influence the perceived size of objects. The grasping plan was manipulated with a preview of the object, after which participants initiated their reaching movement without vision. In a minority of the grasps, the object changed in size after the preview and participants had to adjust their grasping movement. Visual feedback was manipulated in two experiments. In experiment 1, vision was restored during reach and both visual and haptic information was available to correct the grasp and lift the object. In experiment 2, no visual information was provided during the movement and grasps could only be corrected using haptic information. Participants made reach-to-grasp movements towards two objects and compared these in size. Results showed that participants adjusted their grasp to a change in object size from preview to grasped object in both experiments. However, a change in object size did not bias the perception of object size or alter discrimination performance. In experiment 2, a small perceptual bias was found when objects changed from large to small. However, this bias was much smaller than the difference that could be discriminated and could not be considered meaningful. Therefore, it can be concluded that the planning and execution of reach-to-grasp movements do not affect the perception of object size.

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An Autoantigen Atlas from Human Lung HFL1 Cells Offers Clues to Neurological and Diverse Autoimmune Manifestations of COVID-19 COVID-19 is accompanied by a myriad of both transient and long-lasting autoimmune responses. Dermatan sulfate (DS), a glycosaminoglycan crucial for wound healing, has unique affinity for autoantigens (autoAgs) from apoptotic cells. DS-autoAg complexes are capable of stimulating autoreactive B cells and autoantibody production. Using DS affinity, we identified an autoantigenome of 408 proteins from human fetal lung fibroblast HFL11 cells, at least 231 of which are known autoAgs. Comparing with available COVID data, 352 proteins of the autoantigenome have thus far been found to be altered at protein or RNA levels in SARS-Cov-2 infection, 210 of which are known autoAgs. The COVID-altered proteins are significantly associated with RNA metabolism, translation, vesicles and vesicle transport, cell death, supramolecular fibrils, cytoskeleton, extracellular matrix, and interleukin signaling. They offer clues to neurological problems, fibrosis, smooth muscle dysfunction, and thrombosis. In particular, 150 altered proteins are related to the nervous system, including axon, myelin sheath, neuron projection, neuronal cell body, and olfactory bulb. An association with the melanosome is also identified. The findings from our study illustrate a strong connection between viral infection and autoimmunity. The vast number of COVID-altered proteins with propensity to become autoAgs offers an explanation for the diverse autoimmune complications in COVID patients. The variety of autoAgs related to mRNA metabolism, translation, and vesicles raises concerns about potential adverse effects of mRNA vaccines. The COVID autoantigen atlas we are establishing provides a detailed molecular map for further investigation of autoimmune sequelae of the pandemic.

Summary sentenceAn autoantigenome by dermatan sulfate affinity from human lung HFL1 cells may explain neurological and autoimmune manifestations of COVID-19

sentences: Optimal protein production by a synthetic microbial consortium: Coexistence, distribution of labor, and syntrophy The bacterium E. coli is widely used to produce recombinant proteins such as growth hormone and insulin. One inconvenience with E. coli cultures is the secretion of acetate through overflow metabolism. Acetate inhibits cell growth and represents a carbon diversion, which results in several negative effects on protein production. One way to over-come this problem is the use of a synthetic consortium of two different E. coli strains, one producing recombinant proteins and one reducing the acetate concentration. In this paper, we study a chemostat model of such a synthetic community where both strains are allowed to produce recombinant proteins. We give necessary and sufficient conditions for the existence of a coexistence equilibrium and show that it is unique. Based on this equilibrium, we define a multi-objective optimization problem for the maximization of two important bioprocess performance metrics, process yield and productivity. Solving numerically this problem, we find the best available trade-offs between the metrics. Under optimal operation of the mixed community, both strains must produce the protein of interest, and not only one (distribution instead of division of labor). Moreover, in this regime acetate secretion by one strain is necessary for the survival of the other (syntrophy). The results thus illustrate how complex multi-level dynamics shape the optimal production of recombinant proteins by synthetic microbial consortia.

sentences: Modulation of nuclear and cytoplasmic mRNA fluctuations by time-dependent stimuli: analytical distributions Temporal variation of environmental stimuli leads to changes in gene expression. Since the latter is noisy and since many reaction events occur between the birth and death of an mRNA molecule, it is of interest to understand how a stimulus affects the transcript numbers measured at various sub-cellular locations. Here, we construct a stochastic model describing the dynamics of signal-dependent gene expression and its propagation downstream of transcription. For any time-dependent stimulus and assuming bursty gene expression, we devise a procedure which allows us to obtain time-dependent distributions of mRNA numbers at various stages of its life-cycle, e.g. in its nascent form at the transcription site, post-splicing in the nucleus, and after it is exported to the cytoplasm. We also derive an expression for the error in the approximation whose accuracy is verified via stochastic simulation. We find that, depending on the frequency of oscillation and the time of measurement, a stimulus can lead to cytoplasmic amplification or attenuation of transcriptional noise.

sentences: Nuclear mRNA metabolism drives selective basket assembly on a subset of nuclear pores in budding yeast To determine which transcripts should reach the cytoplasm for translation, eukaryotic cells have established mechanisms to regulate selective mRNA export through the nuclear pore complex (NPC). The nuclear basket, a substructure of the NPC protruding into the nucleoplasm, is thought to function as a stable platform where mRNA-protein complexes (mRNPs) are rearranged and undergo quality control (QC) prior to export, ensuring that only mature mRNAs reach the cytoplasm. Here, we use proteomic, genetic, live-cell, and single-molecule resolution microscopy approaches in budding yeast to demonstrate that baskets assemble only on a subset of NPCs and that basket formation is dependent on RNA polymerase II (Pol II) transcription and subsequent mRNP processing. Specifically, we observe that the cleavage and polyadenylation machinery, the poly(A)-binding protein Pab1, and pre-mRNA-leakage factor Pml39 are required for basket assembly. We further show that while all nuclear pores can bind Mlp1, baskets assemble only on a subset of nucleoplasmic NPCs, and these basket-containing pores associate a distinct protein and RNA interactome. Taken together, our data points towards nuclear pore heterogeneity and an RNA-dependent mechanism for functionalization of nuclear pores in budding yeast through nuclear basket assembly.

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GlucoCEST MRI for the early evaluation response to chemotherapeutic and metabolic treatments in a murine triple negative breast cancer: a comparison with 18F-FDG-PET. PurposeTriple-negative breast cancer (TNBC) patients have usually poor outcome after chemotherapy and early prediction of therapeutic response would be helpful. [18F]F-FDG-PET/CT acquisitions are often carried out to monitor variation in metabolic activity associated to response to the therapy, despite moderate accuracy and radiation exposure limit its application. The glucoCEST technique relies on the use of unlabelled D-glucose to assess glucose uptake with conventional MRI scanners and is currently under active investigations at clinical level. This work aims at validating the potential of MRI-glucoCEST in monitoring early therapeutic responses in a TNBC tumor murine model.

ProceduresBreast tumor (4T1) bearing mice were treated with doxorubicin or dichloroacetate for one week. PET/CT with [18F]F-FDG and MRI-glucoCEST were performed at baseline and after 3 cycles of treatment. Metabolic changes measured with [18F]F-FDG-PET and glucoCEST were compared and evaluated with changes in tumor volumes.

ResultsDoxorubicin treated mice showed a significant decrease in tumor growth when compared to the control group. GlucoCEST imaging provided early metabolic response after three cycles of treatment, conversely, no variations were detect by in [18F]F-FDG uptake. Dichloroacetate treated mice did not show any decrease either in tumor volume or in tumor metabolic activity as assessed by both glucoCEST and [18F]F-FDG-PET.

ConclusionsEarly metabolic changes during doxorubicin treatment can be predicted by glucoCEST imaging that appears more sensitive than [18F]F-FDG-PET in reporting on early therapeutic response. These findings support the view that glucoCEST may be a sensitive technique for monitoring metabolic response, but future studies are needed to explore the accuracy of this approach in other tumor types and treatments.

sentences: Structural insights into an evolutionary turning-point of photosystem I from prokaryotes to eukaryotes Photosystem I (PSI) contributes to light-conversion reactions; however, its oligomerization state is variable among photosynthetic organisms. Herein we present a 3.8-[A] resolution cryo-electron microscopic structure of tetrameric PSI isolated from a glaucophyte alga Cyanophora paradoxa. The PSI tetramer is organized in a dimer of dimers form with a C2 symmetry. Different from cyanobacterial PSI tetramer, two of the four monomers are rotated around 90{degrees}, resulting in a totally different pattern of monomer-monomer interactions. Excitation-energy transfer among chlorophylls differs significantly between Cyanophora and cyanobacterial PSI tetramers. These structural and spectroscopic features reveal characteristic interactions and energy transfer in the Cyanophora PSI tetramer, thus offering an attractive idea for the changes of PSI from prokaryotes to eukaryotes.

sentences: Hierarchical Bayesian Model for Estimating Migratory Bird Harvest in Canada The Canadian Wildlife Service (CWS) requires reliable estimates of the harvest of migratory game birds, including waterfowl, to effectively manage populations of these hunted species. The National Harvest Survey is an annual survey of hunters who purchase Canadas mandatory migratory game bird hunting permit, integrating information from a survey of hunting activity with information from a separate survey of species composition in the harvest. We use these survey data to estimate the number of birds harvested for each species, as well as hunting activity metrics such as the number of active hunters and days spent hunting. The analytical methods used to generate these estimates have not changed since the survey was first designed in the early 1970s. Here we describe a new hierarchical Bayesian integrated model, which replaces the series of ratio estimators that comprised the old model. We are now using this new model to generate estimates for migratory bird harvests as of the 2019-2020 hunting season, and to generate updated estimates for all earlier years. The hierarchical Bayesian model uses over-dispersed Poisson distributions to model mean hunter activity and harvest (zero inflated Poisson and zero truncated Poisson, respectively). It also includes multinomial distributions to model some key components including, variation in total harvest across periods of the hunting season, the species composition of the harvest within each of those periods, and the age and sex composition in the harvests of a given species. We estimated the parameters of the Poisson and the multinomial distributions for each year as random effects using first-difference time-series. This time-series component allows the model to share information across years and reduces the sensitivity of the estimates to annual sampling noise. The new model estimates are generally very similar to those from the old model, particularly for the species that occur most commonly in the harvest, and so the results do not suggest any major changes to harvest management decisions and regulations. However, estimates for all species from the new model are more precise and less susceptible to annual sampling error, particularly for species that occur less commonly in the harvest (e.g., sea ducks and other species of conservation concern). This new model, with its hierarchical Bayesian framework, will also facilitate future improvements and elaborations, allowing the incorporation of prior information from the rich literature and knowledge in game bird management and biology.

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Not all smokers are alike: The hidden cost of sustained attention during nicotine abstinence BackgroundNicotine Withdrawal Syndrome (NWS)-associated cognitive deficits are heterogeneous, suggesting underlying endophenotypic subgroups. We identified smoker subgroups based on response accuracy during a cognitively demanding Parametric Flanker Task (PFT) and characterized their distinct neuroimaging endophenotypes using a nicotine state manipulation (sated, abstinent).

MethodsForty-five smokers completed the 25-min PFT in two fMRI sessions (nicotine sated, abstinent). Task-evoked NWS-associated errors of omission (EOm), brain activity, underlying functional connectivity (FC), and brain-behavior correlations between subgroups were assessed.

ResultsBased on their response accuracy in the high demand PFT condition, smokers split into high (HTP, n=21) and low task performer (LTP, n=24) subgroups. Behaviorally, HTPs showed greater response accuracy independent of nicotine state and greater vulnerability to abstinence-induced EOm. HTPs showed greater BOLD responses in attentional control brain regions for the [correct responses (-) errors of commission] PFT contrast across states. A whole-brain FC analysis with these subgroup-derived regions as seeds revealed two circuits: L Precentral : R Insula and L Insula : R Occipital, with abstinence-induced FC strength increases only in HTPs. Finally, abstinence-induced brain (FC) and behavior (EOm) differences were positively correlated for HTPs in a L Precentral : R Orbitofrontal cortical circuit.

ConclusionWe used a cognitive stressor (PFT) to fractionate smokers into two subgroups (HTP/LTP). Only the HTPs demonstrated sustained attention deficits during nicotine abstinence, a stressor in dependent smokers. Unpacking underlying smoker heterogeneity with this  dual stressor approach revealed distinct smoker subgroups with differential attention deficit responses to withdrawal that could be novel targets for therapeutic interventions to improve cessation outcomes.

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A distinctive neural nexus in blind individuals supports Braille reading Natural Braille reading, a demanding cognitive skill, poses a huge challenge for the brain network of the blind. Here, with behavioral measurement and functional MRI imaging data, we pinpointed the neural pathway and investigated the neural mechanisms of individual differences in Braille reading in late blindness. Using resting state fMRI, we identified a distinct neural link between the higher-tier  visual cortex--the lateral occipital cortex (LOC), and the inferior frontal cortex (IFC) in the late blind brain, which is significantly stronger than sighted controls. Individual Braille reading proficiency positively correlated with the left-lateralized LOC-IFC functional connectivity. In a natural Braille reading task, we found an enhanced bidirectional information flow with a stronger top-down modulation of the IFC-to-LOC effective connectivity. Greater top-down modulation contributed to higher Braille reading proficiency via a broader area of task-engaged LOC. Together, we established a model to predict Braille reading proficiency, considering both functional and effective connectivity of the LOC-IFC pathway. This  two-tale model suggests that developing the underpinning neural circuit and the top-down cognitive strategy contributes uniquely to superior Braille reading performance.

SIGNIFICANCE STATEMENTFor late blind humans, one of the most challenging cognitive skills is natural Braille reading. However, little is known about the neural mechanisms of significant differences in individual Braille reading performance. Using functional imaging data, we identified a distinct neural link between the left lateral occipital cortex (LOC) and the left inferior frontal cortex (IFC) for natural Braille reading in the late blind brain. To better predict individual Braille reading proficiency, we proposed a linear model with two variables of the LOC-IFC link: the resting-state functional connectivity and the task-engaged top-down effective connectivity. These findings suggest that developing the underpinning neural pathway and the top-down cognitive strategy contributes uniquely to superior Braille reading performance.

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SSAM-lite: a light-weight web app for rapid analysis of spatially resolved transcriptomics data 1The combination of a cells transcriptional profile and location defines its function in a spatial context. Spatially resolved transcriptomics (SRT) has emerged as the assay of choice for characterizing cells in situ. SRT methods can resolve gene expression up to single-molecule resolution. A particular computational problem with single-molecule SRT methods is the correct aggregation of mRNA molecules into cells. Traditionally, aggregating mRNA molecules into cell-based features begins with the identification of cells via segmentation of the nucleus or the cell membrane. However, recently a number of cell-segmentation-free approaches have emerged. While these methods have been demonstrated to be more performant than segmentation-based approaches, they are still not easily accessible since they require specialized knowledge of programming languages and access to large computational resources. Here we present SSAM-lite, a tool that provides an easy-to-use graphical interface to perform rapid and segmentation-free cell-typing of SRT data in a web browser. SSAM-lite runs locally and does not require computational experts or specialized hardware. Analysis of a tissue slice of the mouse somatosensory cortex took less than a minute on a laptop with modest hardware. Parameters can interactively be optimized on small portions of the data before the entire tissue image is analyzed. A server version of SSAM-lite can be run completely offline using local infrastructure. Overall, SSAM-lite is portable, lightweight, and easy to use, thus enabling a broad audience to investigate and analyze single-molecule SRT data.

Availability and ImplementationSSAM-lite is an open-source browser-based web application with source code freely available on Github via https://github.com/HiDiHlabs/ssam-lite. Stable releases can be accessed via https://ssam-lite.bihealth.org and https://ssam-lite.netlify.app, and developmental releases can be accessed via https://dev--ssam-lite.netlify.app. The source code for a locally deployable server version, SSAM-lite-server, is available on GitHub via https://github.com/HiDiHlabs/ssam-lite-server. Both versions require a modern browser with JavaScript and WebGL support. Detailed user guides and documentation can be found at https://ssam-lite.readthedocs.io.

sentences: Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag) Individual analysis of the epigenome of preimplantation embryos is useful for characterizing each embryo and for investigating the effects of environmental factors on their epigenome. However, it is difficult to analyze genome-wide epigenetic modifications, especially histone modifications, in a large number of single embryos due to the small number of cells and the complexity of the analysis methods. To solve this problem, we further modified the CUT&Tag method, which can analyze histone modifications in a small number of cells, such that the embryo is handled as a cell mass in the reaction solutions in the absence of the solid-phase magnetic beads that are used for antibody and enzyme reactions in the original method (NON-TiE-UP CUT&Tag; NTU-CAT). By using bovine blastocysts as a model, we showed that genome-wide profiles of representative histone modifications, H3K4me3 and H3K27me3, could be obtained by NTU-CAT that are in overall agreement with the conventional chromatin immunoprecipitation-sequencing (ChIP-seq) method, even from single embryos. However, this new approach has limitations that require attention, including false positive peaks and lower resolution for broad modifications. Despite these limitations, we conclude that NTU-CAT is a promising replacement for ChIP-seq with the great advantage of being able to analyze individual embryos.

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Mycobacteria tuberculosis PPE36 modulates host inflammation by promoting E3 ligase Smurf1-mediated MyD88 degradation Mycobacterium tuberculosis (Mtb) PPE36, a cell-wall associated protein is highly specific and conserved for the Mtb complex group. Although it has been proven essential for iron utilization, little is known about the role of PPE36 in regulating host immune responses. Here we exhibited that PPE36 preferentially enriched in Mtb virulent strains, and could efficiently inhibit host inflammatory responses and increase bacterial loads both in mycobacterium-infected macrophages and mice. In exploring the underlying mechanisms, we found that PPE36 could robustly inhibit the activation of inflammatory NF-{kappa}B and MAPK (ERK, p38 and JNK) pathways by promoting E3 ligase Smurf1-mediated ubiquitination and proteasomal degradation of MyD88 protein. Our research revealed a previously unknown function of PPE36 on modulating host immune responses, and provided some clues to the development of novel tuberculosis treatment strategies based on immune regulation.

Author SummaryMycobacterium tuberculosis (Mtb) has developed diverse immune evasion strategies to successfully establish infection in host. Identifying the important Mtb immune regulatory proteins and elucidating the underlying mechanisms are critical for tuberculosis control. Here we demonstrated that PPE36, a Mtb cell-wall associated protein, was predominantly enriched in virulent mycobacterial strains, and obviously inhibited inflammatory responses and facilitated bacterial survival in infected macrophages. Compared with the wild-type BCG, BCG lacking PPE36 (BCG{Delta}PPE36) induced more inflammation, lower bacterial loads as well as the improved histopathological changes in the lungs of infected mice. We further found that PPE36 significantly reduced host MyD88 abundance, and inhibited the activation of subsequunt inflammatory NF-{kappa}B and MAPK pathways. In addition, this direct inhibition effect of PPE36 on MyD88 was mediated by the promoted E3 ligase Smurf1 ubiquitin -protesome pathway. This study identified PPE36 as a immune regulatory protein of Mtb, and showed it played an important role in the Mtb immune evasion.

sentences: What happened when? Brain and behavioral responses to violated expectations about the structure and content of episodic memories Episodic memories are not static but can be modified on the basis of new experiences, potentially allowing us to make valid predictions in the face of an ever-changing environment. Recent research has identified prediction errors during retrieval as a possible trigger for such changes. In the present study, we used modified episodic cues to investigate whether different types of mnemonic prediction errors modulate brain activity and subsequent memory performance. Participants encoded different episodes which consisted of short toy stories. During a subsequent functional magnetic resonance imaging (fMRI) session, participants were then presented videos showing the original episodes, or modified versions thereof. In modified videos, either the order of two subsequent action steps was changed or an object was exchanged for another. Content modifications recruited inferior frontal, parietal, and temporo-occipital areas reflecting the processing of the new object information, while brain responses to structure modifications in right dorsal premotor and inferior frontal areas remained subthreshold. In a post-fMRI memory test, the participants tendency to accept modified episodes as originally encoded increased significantly when they had been presented modified versions already during the fMRI session. Our study provides valuable initial insights into the neural processing of different types of episodic prediction errors and their influence on subsequent memory.

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A Pan-Cancer Census of Dominant Tumor Immune Archetypes Cancers display significant heterogeneity with respect to tissue of origin, driver mutations and other features of the surrounding tissue. It is likely that persistent tumors differentially engage inherent patterns-here  Archetypes-of the immune system, to both benefit from a tumor immune microenvironment (TIME) and to disengage tumor-targeting. To discover dominant immune system archetypes, the Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub compartments uncovered archetypes that exist across indications. These Immune composition-based archetypes differentiate tumors based upon unique immune and tumor gene-expression patterns. Archetypes discovered this way also tie closely to well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune infiltration and provides a rational path forward to learn how to modulate these patterns to improve therapy.

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sentences: Rapid inactivation in vitro of SARS-CoV-2 in saliva by black tea and green tea Saliva plays major roles in human-to-human transmission of the SARS-CoV-2. Recently we reported that black, green and oolong tea significantly inactivated SARS-CoV-2 within 1 min. Theaflavin-3,3-di-gallate (TFDG), theasinensin A (TSA) and (-) epigallocatechin gallate (EGCG) were involved in the anti-viral activities. Here we examined how long period is required for the compounds to inactivate the virus. We also assessed whether tea inactivates SARS-CoV-2 diluted in human saliva. Treatment of SARS-CoV-2 with 500 M TFDG or TSA for 10 sec reduced the virus titer to undetectable levels (less than 1/1,000). Black and green tea decreased virus titer to less than 1/100 within 10 sec even in saliva. These findings suggest a possibility that intake of, or gargling with, tea may inactivate SARS-CoV-2 in saliva in infected individuals, which may eventually attenuate spread of COVID-19 within a population, although clinical studies are required to test this hypothesis by determining the intensity and duration of the anti-viral effect of tea in saliva in humans.

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SARS-CoV-2 transmission via apical syncytia release from primary bronchial epithelia and infectivity restriction in children epithelia The beta-coronavirus SARS-CoV-2 is at the origin of a persistent worldwide pandemic. SARS-CoV-2 infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract eventually causing acute severe respiratory syndrome with a high degree of mortality in the elderly. Here we use reconstituted primary bronchial epithelia from adult and children donors to follow the infection dynamic following infection with SARS-CoV-2. We show that in bronchial epithelia derived from adult donors, infections initiate in multi-ciliated cells. Then, infection rapidly spread within 24-48h throughout the whole epithelia. Within 3-4 days, large apical syncytia form between multi-ciliated cells and basal cells, which dissipate into the apical lumen. We show that these syncytia are a significant source of the released infectious dose. In stark contrast to these findings, bronchial epithelia reconstituted from children donors are intrinsically more resistant to virus infection and show active restriction of virus spread. This restriction is paired with accelerated release of IFN compared to adult donors. Taken together our findings reveal apical syncytia formation as an underappreciated source of infectious virus for either local dissemination or release into the environment. Furthermore, we provide direct evidence that children bronchial epithelia are more resistant to infection with SARS-CoV-2 providing experimental support for epidemiological observations that SARS-CoV-2 cases fatality is linked to age.

Significance StatementBronchial epithelia are the primary target for SARS-CoV-2 infections. Our work uses reconstituted bronchial epithelia from adults and children. We show that infection of adult epithelia with SARS-CoV-2 is rapid and results in the synchronized release of large clusters of infected cells and syncytia into the apical lumen contributing to the released infectious virus dose. Infection of children derived bronchial epithelia revealed an intrinsic resistance to infection and virus spread, probably as a result of a faster onset of interferon secretion. Thus, our data provide direct evidence for the epidemiological observation that children are less susceptible to SARS-CoV-2.

sentences: Long-Term Imaging of Living Adult Zebrafish The zebrafish has become a widely used animal model due in large part to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior, and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels.

sentences: Impact of fungal hyphae on growth and dispersal of obligate anaerobic bacteria in aerated habitats Anoxic microsites arising in fungal biofilms may foster the presence of obligate anaerobes even in well-areated environments. Here, we analyzed whether and to which degree fractal hyphae of Coprinopsis cinerea thriving in oxic habitats enable the germination, growth, and dispersal of obligate anaerobic soil bacterium Clostridium acetobutylicum. Time-resolved optical oxygen mapping, microscopy and metabolite analysis revealed the formation and persistence of anoxic circum hyphal niches allowing for spore germination, growth and fermentative activity of the obligate anaerobe in an otherwise oxic environment. Hypoxic liquid films containing 80 {+/-} 10% of atmospheric oxygen saturation around single air-exposed hyphae thereby allowed for efficient clostridial dispersal amid spatially separated (>0.5 cm) anoxic sites. Our results suggest that fungal biomass typical in soil (<550 g g-1soil) may create anoxic microniches and enable activity as well as dispersal of obligate anaerobes near hyphae in an otherwise inhabitable environment.

sentences: Sparse Representation Learning Derives Biological Features with Explicit Gene Weights from the Allen Mouse Brain Atlas We have developed representation learning methods, specifically to address the constraints and advantages of complex spatial data. Sparse filtering (SFt), uses principles of sparsity and mutual information to build representations from both global and local features from a minimal list of samples. Critically, the samples that comprise each representation are listed and ranked by informativeness. We used the Allen Mouse Brain Atlas gene expression data for prototyping and established performance metrics based on representation accuracy to labeled anatomy. SFt, implemented with the PyTorch machine learning libraries for Python, returned the most accurate reconstruction of anatomical ground truth of any method tested. SFt generated gene lists could be further compressed, retaining 95% of informativeness with only 580 genes. Finally, we build classifiers capable of parsing anatomy with >95% accuracy using only 10 derived genes. Sparse learning is a powerful, but underexplored means to derive biologically meaningful representations from complex datasets and a quantitative basis for compressed sensing of classifiable phenomena. SFt should be considered as an alternative to PCA or manifold learning for any high dimensional dataset and the basis for future spatial learning algorithms.

sentences: Expectation violations produce error signals in mouse V1 Repeated exposure to visual sequences changes the form of evoked activity in the primary visual cortex (V1). Predictive coding theory provides a potential explanation for this, namely that plasticity shapes cortical circuits to encode spatiotemporal predictions and that subsequent responses are modulated by the degree to which actual inputs match these expectations. Here we use a recently developed statistical modeling technique called Model-Based Targeted Dimensionality Reduction (MbTDR) to study visually-evoked dynamics in mouse V1 in context of a previously described experimental paradigm called "sequence learning". We report that evoked spiking activity changed significantly with training, in a manner generally consistent with the predictive coding framework. Neural responses to expected stimuli were suppressed in a late window (100-150ms) after stimulus onset following training, while responses to novel stimuli were not. Omitting predictable stimuli led to increased firing at the expected time of stimulus onset, but only in trained mice. Substituting a novel stimulus for a familiar one led to changes in firing that persisted for at least 300ms. In addition, we show that spiking data can be used to accurately decode time within the sequence. Our findings are consistent with the idea that plasticity in early visual circuits is involved in coding spatiotemporal information.

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Developmental exposure to domoic acid disrupts startle response behavior and circuitry Harmful algal blooms produce potent neurotoxins that accumulate in seafood and are hazardous to human health. Developmental exposure to the harmful algal bloom toxin, domoic acid (DomA), has behavioral consequences well into adulthood, but the cellular and molecular mechanisms are largely unknown. To assess these, we exposed zebrafish embryos to DomA during the previously identified window of susceptibility (2 days post-fertilization) and used the well-known startle response circuit as a tool to identify specific neuronal components that are targeted by exposure to DomA. Exposure to DomA reduced the probability of eliciting a startle after auditory/vibrational or electrical stimuli and led to the dramatic reduction of one type of startle, short latency c-start (SLC) responses. Furthermore, DomA-exposed larvae had altered kinematics of both SLC and long latency c-start (LLC) startle responses, exhibiting shallower bend angles and slower maximal angular velocities. Using vital dye staining, immunolabelling, and live imaging of transgenic lines, we determined that while the sensory inputs were intact, the reticulospinal neurons required for SLC responses were absent in most DomA-exposed larvae. Furthermore, axon tracing revealed that DomA-treated larvae also showed significantly reduced primary motor neuron axon collaterals. Overall, these results show that developmental exposure to DomA leads to startle deficits by targeting specific subsets of neurons. These findings provide mechanistic insights into the neurodevelopmental effects of excess glutamatergic signaling caused by exposure to DomA. It further provides a model for using the startle response circuit to identify neuronal populations targeted by toxin or toxicant exposures.

Summary statementWe used the zebrafish startle response as a tool to identify sensory-motor deficits and the loss of specific neural populations after developmental exposure to the harmful algal bloom toxin domoic acid.

GRAPHICAL ABSTRACT

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Genetic interaction analysis reveals that Cryptococcus neoformans utilizes multiple acetyl-CoA-generating pathways during infection Cryptococcus neoformans is an important human fungal pathogen for which the external environment is its primary niche. To cause infection and disease, C. neoformans must adapt to a plethora of conditions and stresses inherent to the host environment. Of these stresses, the role of central carbon metabolism has been of interest not only at the level of fundamental pathobiology but also as a potential target for new antifungal drug therapy. Previous work has shown that two non-essential acetyl-CoA metabolism enzymes, ATP-citrate lyase (ACL1) and acetyl-CoA synthetase (ACS1), play important roles in C. neoformans infection. Here, we took a genetic interaction approach to studying the interplay between these two enzymes along with an enzyme initially called ACS2 but which we have found is an acetoacetyl-CoA synthetase; we have renamed the gene 2-ketobutyryl CoA synthetase 1 (KBC1) based on its biochemical activity and the systematic name of its substrate. ACL1 and ACS1 represent a synthetic lethal pair of genes based on our genetic interaction studies. Double mutants of KBC1 with either ACS1 or ACL1 do not have significant synthetic phenotypes in vitro, although we find that deletion of any one of these enzymes reduces fitness within macrophages. Importantly, the acs1{Delta} kbc1{Delta} double mutant has significantly reduced fitness in the CNS relative to either single mutant as well as WT (~2 log10 CFU reduction in fungal burden), indicating the important role these enzymes play during infection. The expression of both ACS1 and KBC1 is increased in vivo relative to in vitro conditions. The acs1{Delta} mutant is hypersusceptible to fluconazole in vivo despite its minimal in vitro phenotypes. These data not only provide insights into the in vivo mechanism of action for a new class of antifungal Acs inhibitors but also into metabolic adaptations of C. neoformans to the host environment.

Author SummaryThe adaptation of environmental fungal pathogens to the mammalian host is critical to pathogenesis. Of these adaptations, the remodeling of carbon metabolism is particularly important. Here, we generated a focused set of double mutants of non-essential genes (ACL1, ACS1, KBC1) involved in acetyl-CoA metabolism and examined their fitness in vitro and during CNS infection. From these studies, we found that all three enzymes play important roles during infection but that the role of ACS1/KBC1 was minimal in vitro. Consistent with these observations, the expression of ACS1 and KBC1 was increased in vivo relative to standard in vitro conditions. Furthermore, strains lacking both ACL1 and ACS1 were not viable. These data clearly show that C. neoformans employs multiple carbon metabolism pathways to adapt to the host environment. They also provide insights into the potential mechanism of action for anti-cryptococcal Acs inhibitors.

sentences: STEPS 4.0: Fast and memory-efficient molecular simulations of neurons at the nanoscale AO_SCPLOWBSTRACTC_SCPLOWRecent advances in computational neuroscience have demonstrated the usefulness and importance of stochastic, spatial reaction-diffusion simulations. However, ever increasing model complexity renders traditional serial solvers, as well as naive parallel implementations, inadequate. This paper introduces a new generation of the STochastic Engine for Pathway Simulation (STEPS) project, denominated STEPS 4.0, and its core components which have been designed for improved scalability, performance, and memory efficiency. STEPS 4.0 aims to enable novel scientific studies of macroscopic systems such as whole cells while capturing their nanoscale details. This class of models is out of reach for serial solvers due to the vast quantity of computation in such detailed models, and also out of reach for naive parallel solvers due to the large memory footprint. Based on a distributed mesh solution, we introduce a new parallel stochastic reaction-diffusion solver and a deterministic membrane potential solver in STEPS 4.0. The distributed mesh, together with improved data layout and algorithm designs, significantly reduces the memory footprint of parallel simulations in STEPS 4.0. This enables massively parallel simulations on modern HPC clusters and overcomes the limitations of the previous parallel STEPS implementation. Current and future improvements to the solver are not sustainable without proper software engineering practices. For this reason, we also give an overview of how the STEPS codebase and the development environment have been updated to follow modern software development practices. We benchmark performance improvement and memory footprint on three published models with different complexities, from a simple spatial stochastic reaction-diffusion model, to a more complex one that is coupled to a deterministic membrane potential solver to simulate the calcium burst activity of a Purkinje neuron. Simulation results of these models suggest that the new solution dramatically reduces the per-core memory consumption by more than a factor of 30, while maintaining similar or better performance and scalability.

sentences: Human iPSC-derived renal cells change their immunogenic properties during maturation: Implications for regenerative therapies Therapeutic success of human induced pluripotent stem cell (hiPSC)-based therapies critically depends on immunological compatibility of the hiPSC-derived transplant. As grafted hiPSC-derived cells are often immature, we hypothesized that their immunologic properties may change due to post-grafting maturation. Subsequently, this will affect their interaction with the host immune system and may compromise graft tolerance. In the present study allogeneic and autologous cellular immunity of primary cells, therof reprogrammed hiPSC, hiPSC-derived progenitor and terminally differentiated cells was investigated in vitro by using renal cells as a model system. In contrast to allogeneic primary cells, hiPSC-derived early renal progenitors and mature renal epithelial cells were both tolerated not only by autologous but also by allogeneic T cells. These immune-privileged properties resulted from active immune-modulation and low immune visibility, which declined during the process of cell maturation. However, autologous and allogeneic natural killer (NK) cell responses were not suppressed by hiPSC-derived renal cells and efficiently changed NK cell activation status. These findings clearly show a dynamic stage-specific dependency of autologous and allogeneic T- and NK cell responses to the hiPSC-derived renal cell lineage with consequences for effective cell therapies. The study suggests that hiPSC-derived early progenitors may provide advantageous immune suppressive properties when applied in cell therapy. The data furthermore indicate a need to suppress NK cell activation in allogeneic as well as autologous settings.

sentences: Tideglusib mitigates dystrophic pathology in skeletal muscle and restores diastolic function in young D2 mdx mice Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder that leads to early mortality. We examined the pathogenic contribution of glycogen synthase kinase 3 (GSK3) to DMD using the mdx model. GSK3 is a serine/threonine kinase that has been implicated in other muscular dystrophies and our initial results showed that overactivation of GSK3 may contribute to increased disease severity found in DBA/2J (D2) mdx mice vs C57BL/10 mdx mice. In support of this, treating D2 mdx mice with the GSK3 inhibitor, tideglusib (10 mg/kg/day), increased muscle mass, strength, and fatigue resistance. We also found elevated proportions of oxidative fibers and increased utrophin mRNA, while muscle necrosis and oxidative stress were reduced. Finally, young D2 mdx mice displayed early diastolic dysfunction, and this was blunted with tideglusib treatment - an effect attributed to lowered oxidative stress and fibrosis. This study highlights the therapeutic potential of tideglusib and GSK3 inhibition for DMD.

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Relapse to cotinine seeking in rats: Differential effects of sex Relapse is a defining feature of smoking and a significant challenge in cessation management. Elucidation of novel mechanisms underlying relapse may inform future treatments. Cotinine, the major metabolite of nicotine, has been shown to support intravenous self-administration in rats, suggesting it as one potential mechanism contributing to nicotine reinforcement. However, it remains unknown whether cotinine would induce relapse-like behaviors. The current study investigated relapse to cotinine seeking in two relapse models, the reinstatement of drug seeking and incubation of drug craving models. In the reinstatement model, rats were trained to self-administer cotinine, extinguished cotinine-associated responses, and underwent cue-, drug-, or stress-induced reinstatement. Conditioned cues associated with cotinine self-administration, cotinine (1-2 mg/kg), or the pharmacological stressor yohimbine (1.25-2.5 mg/kg) reinstated cotinine seeking. Female rats displayed more pronounced cue-induced, but not drug- or stress-induced reinstatement than male rats. In addition, an overall analysis revealed that female rats exhibited greater cotinine self-administration, but less extinction than male rats. In the incubation model, rats were trained to self-administer cotinine, and underwent forced withdrawal in home cages. Rats were tested for cue-induced cotinine seeking on both withdrawal day 1 and withdrawal day 18. Rats exhibited greater cotinine-seeking on withdrawal day 18 compared to withdrawal day 1, with no difference between male and female rats. These findings indicate that cotinine induces sex-dependent relapse to cotinine seeking in rats, suggesting that cotinine may be a novel mechanism contributing to relapse. These rat models are valuable preclinical tools for interrogation of neurobiological underpinnings of relapse to cotinine seeking.

Significance StatementRelapse is a defining feature of smoking and a significant challenge in cessation management. Elucidation of novel mechanisms underlying relapse may inform future treatment. Cotinine, the major metabolite of nicotine, has previously been shown as a potential mechanism underlying the development of nicotine reinforcement because it supports intravenous self-administration in rats. The current study found that conditioned cues, priming injection of cotinine, and acute exposure to the pharmacological stressor yohimbine induced robust cotinine-seeking behavior in rats with a history of cotinine self-administration. In addition, significant sex effects were revealed in that female rats exhibited greater cotinine self-administration, less extinction, and more robust cue-induced reinstatement of cotinine seeking than male rats. These findings suggest that cotinine may be a novel mechanism contributing to relapse to nicotine seeking. In addition, these rat models are valuable preclinical tools for interrogation of behavioral and neurobiological underpinnings to relapse to cotinine seeking, thus forming basis for developing effective therapeutic strategy to aid in smoking cessation.

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

sentences: "Two-story building" of a ctenophore comb plate provides structural and functional integrity for motility of giant multicilia Comb plates (CPs) are large compound cilia uniquely seen in a basal metazoan group of ctenophores.1-3 Tens of thousands of cilia are bundled in a CP via structures connecting adjacent cilia, called compartmenting lamella (CL), which are the basis for the structural iridescent color and the coordination of ciliary movement of the CP.4-6 We previously reported the first component of CL, CTENO64, and found that it was convergently acquired in ctenophores and was essential for determination of ciliary orientation.3 However, CTENO64 is localized only in the proximal region of the CL; therefore, the molecular architecture of CL over the entire length has not been elucidated. Here, we identified a second CL component, CTENO189. This ctenophore-specific protein was present in the distal region of the CL, with a localization clearly segregated from CTENO64. Knockdown of the CTENO189 gene with morpholino antisense oligonucleotides resulted in complete loss of CLs in the distal region, but did not affect either the formation of CP or the orientation of each cilium. However, the hexagonal distribution of cilia was disarranged, and the metachronal coordination of CP along a comb row was lost in the CTENO189 morphants. The morphant CP showed asymmetric ciliary-type movement in normal seawater, and in a high-viscosity solution, it could not maintain the normal waveforms, becoming a symmetric flagellar-type. Our findings demonstrate a "two-story building" of CP, comprising the proximal CL, as the building foundation that rigidly fixes the ciliary orientation. The distal CL would reinforce the elastic connection among cilia to overcome the hydrodynamic drag of giant multiciliary plates.

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Interneuronal network model of theta-nested fast oscillations predicts differential effects of heterogeneity, gap junctions and short term depression for hyperpolarizing versus shunting inhibition Theta and gamma oscillations in the hippocampus have been hypothesized to play a role in the encoding and retrieval of memories. Recently, it was shown that an intrinsic fast gamma mechanism in medial entorhinal cortex can be recruited by optogenetic stimulation at theta frequencies, which can persist with fast excitatory synaptic transmission blocked, suggesting a contribution of interneuronal network gamma (ING). We calibrated the passive and active properties of a 100-neuron model network to capture the range of passive properties and frequency/current relationships of experimentally recorded PV+ neurons in the medial entorhinal cortex (mEC). The strength and probabilities of chemical and electrical synapses were also calibrated using paired recordings, as were the kinetics and short-term depression (STD) of the chemical synapses. Gap junctions that contribute a noticeable fraction of the input resistance were required for synchrony with hyperpolarizing inhibition; these networks exhibited theta-nested high frequencies ([~]200 Hz) similar to the putative ING observed experimentally in the optogenetically-driven PV-ChR2 mice. With STD included in the model, fast oscillations were only observed before the peak of the theta drive, whereas without STD, they were observed symmetrically before and after the peak. Because hyperpolarizing synapses provide a synchronizing drive that contributes to robustness in the presence of heterogeneity, synchronization decreases as the hyperpolarizing inhibition becomes weaker. In contrast, networks with shunting inhibition required non-physiological levels of gap junctions to synchronize using conduction delays within the measured range; synaptic depression of shunting synapses facilitated fast oscillations, suggesting that shunting inhibition in mEC is desynchronizing.

Author SummaryFast oscillations nested within slower oscillations have been hypothesized to play a role in the encoding and retrieval of memories by chunking information within each fast cycle; networks of parvalbumin positive inhibitory interneurons contribute to the generation of fast oscillations. We show that, in the entorhinal cortex, the intrinsic dynamical properties of these neurons are sufficiently heterogeneous that electrical synapses are likely required to synchronize fast oscillations. Moreover, synchrony likely requires the chemical synapses to have a reversal potential that is negative relative to the action potential threshold of individual neurons during these oscillations. We show that the range of slow phases that support a fast oscillation is controlled by short term synaptic depression. The precise phase locking of the fast oscillation within the slow oscillations is hypothesized to allow for multiplexing of information.

sentences: An energy-dissipative state of the major antenna complex of plants Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as non-photochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants survival under abiotic stress. Here, we tested whether we could isolate photosynthetic subcomplexes (from plants in which qH was induced) that would remain in an energy-dissipative state. Interestingly chlorophyll fluorescence lifetimes were decreased by qH in isolated major trimeric antenna complexes, providing a natively quenched complex with physiological relevance to natural conditions. Next, we monitored the changes in thylakoid pigment, protein or lipid content of antenna with active or inactive qH, and no evident differences were detected. Finally, we investigated whether specific antenna subunits of the major antenna were required for qH but found it insensitive to trimer composition. Because qH can occur in the absence of specific xanthophylls, and no changes in pigments were detected, we propose that the energy-dissipative state reported here may stem from chlorophyll-chlorophyll excitonic interaction.

sentences: An Efficient and Cost-effective Purification Methodology for Cas9 Nuclease With an ever-increasing demand for laboratory-grade Cas9 proteins by many groups advancing the use of CRISPR technology, a more efficient and scalable process for generating the proteins, coupled with rapid purification methods is in urgent demand. Here, we introduce a modified methodology for rapid purification of active SaCas9 protein within 24 hours. The product has over 90% protein purity. The simplicity and cost-effectiveness of such methodology will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating the advancement of CRISPR/Cas9-based research.

sentences: Evolutionary dynamics of pseudoautosomal region 1 in humans and great apes The pseudoautosomal region 1 (PAR1) is a 2.7 Mb telomeric region of human sex chromosomes. As the largest point of contact between the X and Y, PAR1 has a crucial role in ensuring proper segregation of sex chromosomes during male meiosis, exposing it to extreme recombination and associated mutational processes. We investigate PAR1 evolution using population genomic datasets of extant humans, eight populations of great apes and two archaic human genome sequences. We find that the PAR1 sequence is closer to nucleotide equilibrium than autosomal telomeric sequences. We detect a difference between long-term substitution patterns and extant diversity in PAR1 that is mainly driven by the conflict between strong mutation and recombination-associated fixation bias at CpG sites. Additionally, we detect excess C[->]G mutations in PAR1 of all great ape species, specific to the mutagenic effect of male recombination. Analysis of differences between frequencies of alleles segregating in females and males provided no evidence for sexually antagonistic selection in this region. Furthermore, despite recent evidence for Y chromosome introgression from humans into Neanderthals, we find that the Neanderthal PAR1 retained similarity to the Denisovan sequence, as is the case for the X chromosome and the autosomes. Lastly, we study repeat content and double-strand break hotspot regions in PAR1 and find that they may play roles in ensuring the obligate X-Y recombination event during male meiosis. Our study provides an unprecedented quantification of population genetic forces and insight into evolutionary processes governing PAR1 biology.

sentences: Altered Cortical Activation Associated with Mirror Overflow Driven by Non-Dominant Hand Movement in Attention-Deficit/Hyperactivity Disorder Mirror overflow is involuntary movement that accompanies unilateral voluntary movement on the opposite side of the body, and is commonly seen in Attention-Deficit/Hyperactivity Disorder (ADHD). Children with ADHD show asymmetry in mirror overflow between dominant and non-dominant hand, yet there are competing mechanistic accounts of why this occurs. Using EEG during a sequential, unimanual finger-tapping task, we found that children with ADHD exhibited significantly more mirror overflow than typically developing (TD) controls, especially during the tapping of the non-dominant hand. Furthermore, source-level EEG oscillation analysis revealed that children with ADHD showed decreased alpha (8-12 Hz) event-related desynchronization (ERD) compared with controls in both hemispheres, but only during tapping of the non-dominant hand. Moreover, only the ERD ipsilateral to the mirror overflow during non-dominant hand movement correlated with both magnitude of overflow movements and higher ADHD symptom severity (Conners ADHD Hyperactivity/Impulsiveness scale) in children with ADHD. TD controls did not show these relationships. Our findings suggest that EEG differences in finger-tapping in ADHD are related primarily to voluntary movement in the non-dominant hand. Our results are also consistent with the Ipsilateral Corticospinal Tract (CST) Hypothesis, which posits that the atypical persistence of mirror overflow in ADHD may originate in the sensorimotor areas ipsilateral to mirror overflow and be transmitted via non-decussating CST fibers.

sentences: Emergence of a recurrent insertion in the N-terminal domain of the SARS-CoV-2 spike glycoprotein Tracking the evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through genomic surveillance programs is undoubtedly one of the key priorities in the current pandemic situation. Although the genome of SARS-CoV-2 acquires mutations at a slower rate compared with other RNA viruses, evolutionary pressures derived from the widespread circulation of SARS-CoV-2 in the human population have progressively favored the global emergence, though natural selection, of several variants of concern that carry multiple non-synonymous mutations in the spike glycoprotein. These are often placed in key sites within major antibody epitopes and may therefore confer resistance to neutralizing antibodies, leading to partial immune escape, or otherwise compensate infectivity deficits associated with other non-synonymous substitutions. As previously shown by other authors, several emerging variants carry recurrent deletion regions (RDRs) that display a partial overlap with antibody epitopes located in the spike N-terminal domain (NTD). Comparatively, very little attention has been directed towards spike insertion mutations prior to the emergence of the B.1.1.529 (omicron) lineage. This manuscript describes a single recurrent insertion region (RIR1) in the N-terminal domain of SARS-CoV-2 spike protein, characterized by at least 41 independent acquisitions of 1-8 additional codons between Val213 and Leu216 in different viral lineages. Even though RIR1 is unlikely to confer antibody escape, its association with two distinct formerly widespread lineages (A.2.5 and B.1.214.2), with the quickly spreading omicron and with other VOCs and VOIs warrants further investigation concerning its effects on spike structure and viral infectivity.

sentences: GEF Cytohesin-2/ARNO: a novel bridge between cell migration and immunoregulation in synovial fibroblasts. The guanine nucleotide exchange factor cytohesin-2 (ARNO) is a major activator of the small GTPase ARF6, and has been shown to play an important role(s) in cell adhesion, migration and cytoskeleton reorganization in various cell types and models of disease. Interestingly, dysregulated cell migration, in tandem with hyper-inflammatory responses, is one of the hallmarks associated with activated synovial fibroblasts (SFs) during chronic inflammatory joint diseases, like rheumatoid arthritis. The role of ARNO in this process was unknown but we hypothesized that the pro-inflammatory milieu of inflamed joints induces local activation of ARNO-mediated pathways in SFs, promoting an invasive cell phenotype that ultimately leads to bone and cartilage damage. Thus, we used small interference RNA to investigate the impact of ARNO on the pathological migration and inflammatory responses of murine SFs, revealing a fully functional ARNO-ARF6 pathway in SFs, which can be rapidly activated by IL-1{beta}. Such activation promotes cell migration and formation of focal adhesions. Unexpectedly, ARNO was also shown to modulate SF-inflammatory responses, dictating the precise cytokine and chemokine expression profile. Our results uncover a novel role for ARNO in SF-dependent inflammation, that potentially links pathogenic migration with initiation of local joint inflammation, offering new approaches for targeting the fibroblast compartment in chronic arthritis and joint disease.

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Cortical-subcortical structural connections support transcranial magnetic stimulation engagement of the amygdala The amygdala processes valenced stimuli, influences affective states, and exhibits aberrant activity across anxiety disorders, depression, and PTSD. Interventions that modulate amygdala activity hold promise for treating transdiagnostic affective symptoms. We investigated (N=45) whether transcranial magnetic stimulation (TMS) elicits indirect changes in amygdala activity when applied to ventrolateral prefrontal cortex (vlPFC), a region important for affect regulation. Harnessing in-scanner interleaved TMS/functional MRI (fMRI), we reveal that vlPFC neurostimulation evoked acute, dose-dependent modulations of amygdala fMRI BOLD signal. Larger TMS-evoked changes in amygdala fMRI signal were associated with higher fiber density in a vlPFC-amygdala white matter pathway, suggesting this pathway facilitated stimulation-induced communication between cortex and subcortex. This work provides evidence of amygdala engagement by TMS, highlighting stimulation of vlPFC-amygdala circuits as a candidate treatment for affective psychopathology. More broadly, it indicates that targeting cortical-subcortical connections may enhance the impact of TMS on subcortical neural activity and, by extension, subcortex-subserved behaviors.

TeaserIndividualized, connectivity-guided transcranial magnetic stimulation modulates the amygdala, demonstrating therapeutic potential.

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Meiosis in the human pathogen Aspergillus fumigatus has the highest known number of crossovers Evidence from both population genetics and a laboratory sexual cycle indicate that sex is common in the fungus Aspergillus fumigatus. However, the impact of sexual reproduction has remained unclear. Here, we show that meiosis in A. fumigatus involves the highest known recombination rate, producing ~29 crossovers per chromosome. This represents the highest known crossover rate for any Eukaryotic species. We validate this recombination rate by mapping resistance to acriflavine, a common genetic marker. We further show that this recombination rate can produce the commonly encountered TR34/L98H azole-resistant cyp51A haplotype in each sexual event, facilitating its rapid and global spread. Understanding the consequences of this unparalleled crossover rate will not only enrich our genetic understanding of this emergent human pathogen, but of meiosis in general.

One-Sentence SummaryGenetic exchange between chromosomes during sex in Aspergillus fumigatus is higher than in any other known organism.

sentences: Lactate production can function to increase human epithelial cell iron concentration Iron is an essential micronutrient required by every cell, inclusive of both prokaryotes and eukaryotes. Under conditions of limited iron availability, plants and microbes evolved mechanisms to acquire iron which include carbon metabolism reprogramming, with the activity of several enzymes involved in the Krebs cycle and the glycolytic pathway being stimulated by metal deficiency. Following release, resultant carboxylates/hydroxycarboxylates can function as ligands to complex iron and facilitate its solubilization and uptake, reversing the deficiency. Human epithelial tissue may produce lactate, a hydroxycarboxylate, during absolute and functional iron deficiency in an attempt to import metal to reverse limited availability. Here we investigate 1) if lactate can increase cell metal import, 2) if lactic dehydrogenase (LDH) activity in and lactate production by cells correspond to metal availability, and 3) if blood concentrations of LDH in a human cohort correlate with indices of iron homeostasis. Exposures of Caco-2 cells to both Na lactate and ferric ammonium citrate (FAC) increased metal import relative to FAC alone. Fumaric, isocitric, malic, and succinic acid exposure revealed that FAC co-incubation similarly increased iron import relative to FAC alone. Increased iron import following exposures to Na lactate and FAC elevated both ferritin and metal associated with mitochondria. LDH in Caco-2 cell scrapings did not change after exposure to deferoxamine but decreased with 24 hr exposure to FAC. Lactate levels in both the supernatants and cell scrapings revealed decreased levels at 4, 8, and 24 hr with FAC. In the National Health and Nutrition Examination Survey (NHANES 2005-2010), Spearman correlations demonstrated significant negative relationships between LDH concentrations and serum iron. We conclude that iron import in human cells can involve lactate, LDH activity can reflect the availability of this metal, and blood LDH concentrations can correlate with indices of iron homeostasis.

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Bacterial, phytoplankton, and viral dynamics of meromictic Lake Cadagno offer insights into the Proterozoic ocean microbial loop Lake Cadagno, a permanently stratified high-alpine lake with a persistent microbial bloom in its anoxic chemocline, has long been considered a model for the low-oxygen, high-sulfide Proterozoic ocean where early microbial life gave rise to Earths oxygenated atmosphere. Although the lake has been studied for over 25 years, the absence of concerted study of the bacteria, phytoplankton, and viruses, together with primary and secondary production, has hindered a comprehensive understanding of its microbial food web. Here, the identities, abundances, and productivity of microbes were evaluated in the context of Lake Cadagno biogeochemistry. Photo-synthetic pigments and chloroplast 16S rRNA gene phylogenies suggested high abundances of eukaryotic phytoplankton, primarily Chlorophyta, through the water column. Of these, a close relative of Ankyra judayi, a high-alpine adapted chlorophyte, peaked with oxygen in the mixolimnion, while Closteriopsis-related chlorophytes peaked in the chemocline and monimolimnion. Anoxygenic phototrophic sulfur bacteria, Chromatium, dominated the chemocline along with Lentimicrobium, a newly observed genus of known fermenters. Secondary production peaked in the chemocline, suggesting anoxygenic primary producers depended on heterotrophic nutrient remineralization. Virus-to-microbe ratios spanned an order of magnitude, peaking with high phytoplankton abundances and at a minimum at the peak of Chromatium, dynamic trends that suggest viruses may play a role in the modulation of oxygenic and anoxygenic photo- and chemosynthesis in Lake Cadagno. Through the combined analysis of bacterial, eukaryotic, viral, and biogeochemical dynamics of Lake Cadagno, this study provides a new perspective on the biological and geochemical connections that comprised the food webs of the Proterozoic ocean.

IMPORTANCEAs a window to the past, the study offers insights into the role of microbial guilds of Proterozoic ocean chemoclines in the production and recycling of organic matter of sulfur- and ammonia-containing ancient oceans. The new observations described here suggest that eukaryotic algae were persistent in the low oxygen upper-chemocline in association with purple and green sulfur bacteria in the lower half of the chemocline. Further, this study provides the first insights into Lake Cadagno viral ecology. High viral abundances suggested viruses may be essential components of the chemocline where their activity may result in the release and recycling of organic matter. The framework developed in this study through the integration of diverse geochemical and biological data types lays the foundation for future studies to quantitatively resolve the processes performed by discrete populations comprising the microbial loop in this early anoxic ocean analogue.

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Human metapneumovirus P protein independently drives phase separation and recruits N protein to liquid-like inclusion bodies Human metapneumovirus (HMPV) inclusion bodies (IBs) are dynamic structures required for efficient viral replication and transcription. The minimum components needed to form IB-like structures in cells are the nucleoprotein (N) and the tetrameric phosphoprotein (P). HMPV P binds to two versions of N protein in infected cells: C-terminal P residues interact with oligomeric, RNA-bound N (N-RNA), and N-terminal P residues interact with monomeric N (N0) to maintain a pool of protein to encapsidate new RNA. Recent work on other negative-strand viruses has suggested that IBs are liquid-like organelles formed via liquid-liquid phase separation (LLPS). Here, HMPV IBs in infected or transfected cells were shown to possess liquid organelle properties, such as fusion and fission. Recombinant versions of HMPV N and P proteins were purified to analyze the interactions required to drive LLPS in vitro. Purified HMPV P was shown to form liquid droplets in the absence of other protein binding partners, a novel finding compared to other viral systems. Removal of nucleic acid from purified P altered phase separation dynamics, suggesting that nucleic acid interactions also play a role in IB formation. HMPV P also recruits monomeric N (N0-P) and N-RNA to IBs in vitro. These findings suggest that, in contrast to what has been reported for other viral systems, HMPV P acts as a scaffold protein to mediate multivalent interactions with monomeric and oligomeric HMPV N to promote phase separation of IBs.

IMPORTANCEHuman metapneumovirus (HMPV) is a leading cause of respiratory disease among children, immunocompromised individuals, and the elderly. Currently, no vaccines or antivirals are available for treatment of HMPV infections. Cytoplasmic inclusion bodies (IBs), where HMPV replication and transcription occur, represent a promising target for the development of novel antivirals. The HMPV nucleoprotein (N) and phosphoprotein (P) are the minimal components needed for IB formation in eukaryotic cells. However, interactions that regulate the formation of these dynamic structures are poorly understood. Here, we showed that HMPV IBs possess the properties of liquid organelles and that purified HMPV P phase separates independently in vitro. Our work suggests that HMPV P phase separation dynamics are altered by nucleic acid. We provide strong evidence that, unlike results reported from other viral systems, HMPV P alone serves as a scaffold for multivalent interactions with monomeric (N0) and oligomeric (N-RNA) HMPV N for IB formation.

sentences: The Enemy of my Enemy is my Friend: Immune-Mediated Facilitation Contributes to Fitness of Co-Infecting Helminths Our conceptual understanding of immune-mediated interactions between parasites is rooted in the theory of community ecology. One of the limitations of this approach is that most of the theory and empirical evidence has focused on resource or immune-mediated parasite competition and yet, there is ample evidence of positive interactions between species that could be generated by immune-mediated facilitation. Here, we develop an immuno-epidemiological framework and apply it to longitudinal infection data of two gastrointestinal helminths that infect a population of free-living rabbits to investigate, through model testing, the mechanisms of immune-mediated facilitation in dual infections. Simulations show that weakened, species-specific IgA antibody responses and unequal, albeit low, IgA cross-reactions explain higher parasite intensities in dual compared to single infections, for both helminths. Simulations also show that rabbits with dual infections shed more free-living stages that survive fort longer in the environment, implying greater onward transmission than hosts with single infections. These findings support the hypothesis that the two helminths interact through immune-mediated facilitation which contributes to greater fitness and the long-term co-circulation of both species in the host population.

sentences: Adipocyte lipolysis abrogates skin fibrosis in a Wnt/DPP4-dependent manner Tissue fibrosis in many organs results from altered and excessive extracellular matrix (ECM) protein deposition 1. Concomitant with ECM expansion, resident lipid-filled cells including mature adipocytes are lost in human and mouse fibrosis2-5, yet the mechanisms that drive mature adipocyte lipid loss and their contribution to tissue fibrosis are unknown. Here, we identify an early, fibro-protective role of mature adipocyte lipolysis driven by Wnt signaling during fibrosis onset. Using chemical and genetic mouse models of skin fibrosis, we show that fibrotic stimuli induce and maintain lipolysis in mature dermal adipocytes. Loss of the lipolytic rate-limiting enzyme adipocyte triglyceride lipase (ATGL)6,7 in murine dermal adipocytes exacerbates bleomycin-induced fibrosis development. Adipocyte lipolysis is stimulated in the early stages of Wnt signaling-induced skin fibrosis and by Wnt agonists in vitro. Furthermore, deletion or inhibition of the Wnt target gene, CD26/Dipeptidyl peptidase 4 (DPP4) prevented Wnt-induced lipolysis and skin fibrosis in mice. Notably, DPP4 expression correlates with skin fibrosis severity in human patients. Thus, we propose that adipocyte-derived fatty acids and the Wnt-DPP4 axis act as essential regulators of ECM homeostasis within tissues and provide a therapeutic avenue to manipulate fibrosis.

sentences: The Flowering Repressor SVP recruits the TOPLESS co-repressor to control flowering in chrysanthemum and Arabidopsis Plant flowering time is a consequence of the perception of environmental and endogenous signals. The MCM1-AGAMOUSDEFICIENS-SRF-box (MADS-box) gene SHORT VEGETATIVE PHASE (SVP) is a pivotal repressor that negatively regulates the floral transition during the vegetative phase. The transcriptional corepressor TOPLESS (TPL) plays critical roles in many aspects of plant life. An interaction first identified between the second LXLXLX motif (LRLGLP) of CmSVP with CmTPL1-2, which can repress the expression of a key flowering factor CmFTL3 by binding its promotor CArG element in chrysanthemum. Genetic analysis suggested that the CmSVP-CmTPL1-2 transcriptional complex is a prerequisite for SVP to act as a floral repressor, which reduces CmFTL3 transcriptional activity. CmSVP rescued the phenotype of the svp-31 mutant in Arabidopsis, and overexpression of AtSVP or CmSVP in the Arabidopsis dominant negative mutation tpl-1 led to a loss-of-function in late flowering, which confirmed the highly conserved function of SVP in the two completely different species. Thus, we have validated a conserved machinery wherein SVP relies on TPL to inhibit flowering through the direct regulation of FT, which is more meaningful for the evolution of species and could be translated to high-quality cultivation and breeding of crops.

sentences: Analysis of Mouse Vocal Communication (AMVOC): A deep, unsupervised method for rapid detection, analysis, and classification of ultrasonic vocalizations Some aspects of the neural mechanisms underlying mouse ultrasonic vocalizations (USVs) are a useful model for the neurobiology of human speech and speech-related disorders. Much of the research on vocalizations and USVs is limited to offline methods and supervised classification of USVs, hindering the discovery of new types of vocalizations and the study of real-time free behavior. To address these issues, we developed AMVOC (Analysis of Mouse VOcal Communication) as a free, open-source software to analyze and detect USVs in both online and offline modes. When compared to hand-annotated ground-truth USV data, AMVOCs detection functionality (both offline and online) has high accuracy, and outperforms leading methods in noisy conditions, thus allowing for broader experimental use. AMVOC also includes the implementation of an unsupervised deep learning approach that facilitates discovery and analysis of USV data by clustering USVs using latent features extracted by a convolutional autoencoder and isimplemented in a graphical user interface (GUI), also enabling users evaluation. These results can be used to explore the vocal repertoire space of the analyzed vocalizations. In this way, AMVOC will facilitate vocal analyses in a broader range of experimental conditions and allow users to develop previously inaccessible experimental designs for the study of mouse vocal behavior.

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Pulmonary mesenchymal stem cells are engaged in distinct steps of host response to respiratory syncytial virus infection Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications.

AUTHOR SUMMARYThis work identifies a novel function of lung-resident MSCs during virus-induced acute lung injury. These findings contribute to the understanding of host response and lung repair mechanisms during a highly prevalent clinical situation and may have therapeutic implications.

sentences: Population genomics of Pocillopora corals: insights from RAD-sequencing Scleractinian corals are of great ecological interest as ecosystem engineer species. Accordingly, there is a wealth of studies on their adaptive abilities facing climate change. Such studies should rely on precise species and population delimitation. Nevertheless species delimitation in corals can be hindered by the lack of adequate genetic markers, by hybridization, and by morphological plasticity. Here we applied RAD sequencing to the study of species delimitation and genetic structure in populations of Pocillopora spp. from Oman and French Polynesia with the objectives to test primary species hypotheses based on mitochondrial DNA sequencing, and to study the genetic structure among sampling sites inside species. Regarding the varying levels of missing data observed among samples we tested different filtering strategy. The main genetic differentiation was observed between samples from Oman and French Polynesia, which also corresponded to different mitochondrial lineages and species hypotheses. In Oman, we did not observe any clear differentiation according to the main mitochondrial lineages considered here, nor between sampling sites. In French Polynesia where a single mitochondrial lineage was studied, we did not evidence any differentiation according to sampling sites. These results provide an additional example of the importance of using independent nuclear markers for the study of species delimitation. Our analyses also allowed the identification of clonal lineages in our samples, and to take them into account in our interpretations. We used simulations to study the impact of clonal reproduction on the distribution of statistics of genetic diversity and genetic structure among loci.

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Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces robust neutralizing salivary IgA Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable  sterilizing immunity at the mucosal level. Our study uncovers, strong neutralizing mucosal component (NT50 [≤] 50pM), emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the Receptor-Binding-Domain (RBD) of SARS-CoV-2 spike protein and demonstrate that these IgAs are key mediators of potent neutralization. RBD-targeting IgAs were found to associate with the Secretory Component, indicating their bona-fide transcytotic origin and their dimeric tetravalent nature. The mechanistic understanding of the exceptionally high neutralizing activity provided by mucosal IgA, acting at the first line of defence, will advance vaccination design and surveillance principles, pointing to novel treatment approaches, and to new routes of vaccine administration and boosting.

Significance statementWe unveiled powerful mucosal neutralization upon BNT162b2 vaccination, mediated by temporary polymeric IgA and explored its longitudinal properties. We present a model, whereby the molecular architecture of polymeric mucosal IgA and its spatial properties are responsible for the outstanding SARS-CoV-2 neutralization potential. We established a methodology for quantitative comparison of immunoreactivity and neutralization for IgG and IgAs in serum and saliva in molar equivalents for standardization in diagnostics, surveillance of protection and for vaccine evaluations.

sentences: Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD deletion in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

sentences: Increasing triacylglycerides and lipid storage by unsaturated lipids protects renal proximal tubules In diabetic patients, dyslipidemia frequently contributes to organ damage such as diabetic kidney disease (DKD). DKD is associated with excessive renal deposition of triacylglycerol (TAG) in lipid droplets (LD). Yet, it is unclear whether LDs play a protective or damaging role and how this might be influenced by dietary patterns. By using a diabetes mouse model, we find here that high fat diet enriched in the unsaturated oleic acid (OA) caused more lipid storage in LDs in renal proximal tubular cells (PTC) but less tubular damage than a corresponding butter diet with the saturated palmitic acid (PA). Mechanistically, we identify endoplasmic reticulum (ER) stress as the main cause of PA-induced PTC injury. ER stress is caused by elevated cellular levels of saturated TAG precursors and to higher membrane order in the ER. The resulting cell death is preceded by a transcriptional rewiring of phospholipid metabolism. Simultaneous addition of OA rescues the cytotoxic effects by normalizing membrane order and by increasing the total TAG amount. The latter also stimulates the formation of LDs that in turn can release unsaturated lipids upon demand by lipolysis. Our study thus clarifies mechanisms underlying PA-induced cell stress in PTCs and emphasizes the importance of olive oil for the prevention of DKD.

sentences: A method to predict 3D structural ensembles of chromatins from pairwise distances: Applications to Interphase Chromosomes and Structural Variants The principles that govern the organization of genomes, which are needed for a deeper understanding of how chromosomes are packaged and function in eukaryotic cells, could be deciphered if the three dimensional (3D) structures are known. Recently, single-cell imaging experiments have determined the 3D coordinates of a number of loci in a chromosome. Here, we introduce a computational method (Distance Matrix to Ensemble of Structures, DIMES), based on the maximum entropy principle, with experimental pair-wise distances between loci as constraints, to generate a unique ensemble of 3D chromatin structures. Using the ensemble of structures, we quantitatively account for the distribution of pair-wise distances, three-body co-localization and higher-order interactions. We demonstrate that the DIMES method can be applied to both small length-scale and chromosome-scale imaging data to quantify the extent of heterogeneity and fluctuations in the shapes on various length scales. We develop a perturbation method that is used in conjunction with DIMES to predict the changes in 3D structures from structural variations. Our method also reveals quantitative differences between the 3D structures inferred from Hi-C and the ones measured in imaging experiments. Finally, the physical interpretation of the parameters extracted from DIMES provides insights into the origin of phase separation between euchromatin and heterochromatin domains.

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Functional ultrasound imaging of recent and remote memory recall in the associative fear neural network in mice Scientific AbstractEmotional learning and memory are affected in numerous psychiatric disorders. At a systems level, however, the underlying neural circuitry is not well defined. Rodent fear conditioning (FC) provides a translational model to study the networks underlying associative memory retrieval. In the current study, functional connectivity among regions related to the cue associative fear network were investigated using functional ultrasound (fUS), a novel imaging technique with great potential for detecting regional neural activity through cerebral blood flow. Behavioral fear expression and fUS imaging were performed one and thirty-one days after FC to assess recent and remote memory recall. Cue-evoked increases in functional connectivity were detected throughout the amygdala, with the lateral (LA) and central (CeA) amygdalar nuclei emerging as major hubs of connectivity, though CeA connectivity was reduced during remote recall. The hippocampus and sensory cortical regions displayed heightened connectivity with the LA during remote recall, whereas interconnectivity between the primary auditory cortex and temporal association areas was reduced. Subregions of the prefrontal cortex exhibited variable connectivity changes, where prelimbic connectivity with the amygdala was refined while specific connections between the infralimbic cortex and amygdalar subregions emerged during remote memory retrieval. Moreover, freezing behavior positively correlated with functional connectivity between hubs of the associative fear network, suggesting that emotional response intensity reflected the strength of the cue-evoked functional network. Overall, our data provide evidence of the functionality of fUS imaging to investigate the neural dynamics of memory encoding and retrieval, applicable in the development of innovative treatments for affective disorders.

HighlightsFunctional ultrasound imaging can elucidate fear associated neural networks

Freezing behavior correlates with cue-evoked functional connectivity changes

The lateral and central amygdalar nuclei are major hubs in the fear network

The hippocampus is active during recent and remote cued fear memory retrieval

Connectivity profiles of the prelimbic and infralimbic areas vary in remote recall

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Cholesterol Oxidation Modulates the Formation of Liquid-Ordered Domains in Model Membranes 7-ketocholesterol (KChol) is one of the most cytotoxic oxysterols found in the plasma membrane, and increased levels of KChol are associated with numerous pathologies. It is thought to induce apoptosis via inactivation of the phosphatidylinositol 3-kinase/Akt signaling pathway -- a pathway that depends on lipid-rafts as signaling platforms. By means of coarse-grained molecular dynamics simulations, we demonstrate that KChol disrupts the liquid-liquid phase separation seen in an equimolar mixture of (dipalmitoylphosphatidylcholine) DPPC, (dioleoylphosphatidylcholine) DOPC, and Cholesterol (Chol). This disruption occurs via two mechanisms: i) KChol adopts a wider range of orientations with the membrane, which disrupts the packing of neigh-boring lipids and ii) KChol has no preference for DPPC over DOPC, which is the main driving force for lateral demixing in DPPC/DOPC/Chol membranes. This provides a molecular description of the means by which KChol induces apoptosis, and illustrates that a single chemical substitution to cholesterol can have a profound impact on the lateral organization of lipid membranes.

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sentences: Intranasal oxytocin drives coordinated social approach Coordinated responses to challenge are essential to survival for bonded monogamous animals and may depend on behavioral compatibility. Oxytocin (OT) context-dependently regulates social affiliation and vocal communication, but its role in pair members decision to jointly respond to challenge is unclear. To test for OT effects, California mouse females received an intranasal dose of OT (IN-OT) or saline after bonding with males either matched or in their approach response to an aggressive vocal challenge. Pair mates were re-tested jointly for approach response, time spent together, and vocalizations. Females and males converged in their approach after pairing, but mismatched pairs with females given a single dose of IN-OT displayed a greater convergence that resulted from behavioral changes by both pair members. Unpaired females given IN-OT did not change their approach, indicating a social partner was necessary for effects to emerge. Moreover, IN-OT increased time spent approaching together, suggesting behavioral coordination beyond a further increase in bonding. This OT-induced increase in joint approach was associated with a decrease in the proportion of sustained vocalizations, a type of vocalization that can be associated with intra-pair conflict. Our results expand OTs effects on behavioral coordination and underscore the importance of emergent social context.

sentences: Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 [A]) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic, as well as a microtubule-unbound CaKip3-ADP crystal structure (2.0 [A]). Together with biochemical analyses of CaKip3 and kinesin-1 mutants, we define a model that explains the kinesin-8 mechanism. The microtubule depolymerization activity originates in conformational changes of the kinesin-8 motor core that are amplified by its dynamic loop-2. On curved microtubule ends, loop-1 assists depolymerization by inserting into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin. On straight tubulin protofilaments in the microtubule lattice, extended loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. These tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

sentences: Social determinants of health and disease in companion dogs: A cohort study from the Dog Aging Project Exposure to social environmental adversity is associated with health and survival in many social species, including humans. However, little is known about if and how these health and mortality effects vary across the lifespan, largely due to the difficulty of studying long-lived organisms across much of their lifespan. Here, we leveraged a relatively new and powerful model for human aging, the companion dog, to investigate which components of the social environment are associated with dog health and how these associations vary across the lifespan. We drew on comprehensive survey data collected on 21,410 dogs from the Dog Aging Project and identified five factors that together explained 33.7% of the variation in a dogs social environment. Factors capturing financial and household adversity were associated with poorer health and lower physical mobility in companion dogs, while factors that captured social support, such as living with other dogs, were associated with better health when controlling for dog age and weight. Some of these associations differed across a dogs lifespan, including a stronger relationship between owner age and health in younger (as compared to older) dogs. Taken together, these findings suggest the importance of income, stability, and owner age on owner-reported health outcomes in companion dogs and point to potential behavioral and/or environmental modifiers that can be used to promote healthy aging across species.

sentences: Elucidating Microscopic Events Driven by GTP Hydrolysis Reaction in Ras-GAP System with Semi-reactive Molecular Dynamics Simulation: Alternative Role of Phosphate Binding Loop as Mechanical Energy Storage ATPase and GTPase have been widely found as chemical energy-mechanical work transducers, whereas the physicochemical mechanisms are not satisfactorily understood. We addressed the problem by examining John Ross conjecture that repulsive Coulomb interaction between ADP/GDP and inorganic phosphate (Pi) does the mechanical work upon the system. We effectively simulated the consequence of GTP hydrolysis reaction in a complex system of Rat sarcoma (Ras) and GTPase activation protein (GAP) in the framework of classical molecular dynamics by switching force field parameters between the reactant and product systems. We then observed ca. 5 kcal/mol raise of potential energy about the phosphate-binding loop (P-loop) in Ras protein, indicating that the mechanical work generated via the GTP hydrolysis is converted into the local interaction energy and stored in the P-loop. Interestingly, this local energy storage in the P-loop depends on neither impulsive nor consecutive collisions of GDP and Pi with P-loop. Instead, GTP-GDP conversion itself does work on the Ras system, elevating the potential energy. These observations encourage us to challenge a conjecture previously given by Ross. We assert that triphosphate nucleotide hydrolyses do mechanical work by producing emergent steric interaction accompanied with relaxation, namely, a shift of biomolecular system to non-equilibrium state on the reshaped potential energy landscape. Recalling the universality of the P-loop motif among GTPases and ATPases, the observations that we obtained through this study would progress physicochemical understanding for the operating principles of GTP/ATP hydrolysis-driven biological nano-machines.

sentences: Provenance Attestation of Human Cells Using Physical Unclonable Functions We introduce a novel methodology, namely CRISPR-Engineered Attestation of Mammalian Cells using Physical Unclonable Functions (CREAM-PUFs), which can serve as the cornerstone for formally verifying transactions in human cell line distribution networks. A PUF is a physical entity which provides a measurable output that can be used as a unique and irreproducible identifier for the artifact wherein it is embedded. Popularized by the electronics industry, silicon PUFs leverage the inherent physical variations of semiconductor manufacturing to establish intrinsic security primitives for attesting integrated circuits. Owing to the stochastic nature of these variations and the multitude of steps involved, photo-lithographically manufactured silicon PUFs are impossible to reproduce (thus unclonable). Inspired by the success of silicon PUFs, we sought to exploit a combination of sequence-restricted barcodes and the inherent stochasticity of CRISPR-induced non-homologous end joining DNA error repair to create the first generation of genetic physical unclonable functions in three distinct human cells (HEK293, HCT116, and HeLa). We demonstrate that these CREAM-PUFs are robust (i.e., they repeatedly produce the same output), unique (i.e., they do not coincide with any other identically produced PUF), and unclonable (i.e., they are virtually impossible to replicate). Accordingly, CREAM-PUFs can serve as a foundational principle for establishing provenance attestation protocols for protecting intellectual property and confirming authenticity of engineered cell lines.

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Direct reprogramming of human embryonic to trophoblast stem cells During the first week of development, human embryos form a blastocyst comprised of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles and directed differentiation potential similar to existing human TSCs. Systematic omission of each or combinations of factors, revealed the critical importance of GATA2 and GATA3 for successful iTSC reprogramming. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.

Summary statementTranscriptional analysis of human blastocysts reveals transcription factors sufficient to derive induced trophoblast stem cells from primed human embryonic stem cells.

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Architecture of the cytoplasmic face of the nuclear pore The nuclear pore complex (NPC) is the sole bidirectional gateway for nucleocytoplasmic transport. Despite recent progress in elucidating the NPC symmetric core architecture, the asymmetrically decorated cytoplasmic face, essential for mRNA export and a hotspot for nucleoporin-associated diseases, has remained elusive. Here, we report a composite structure of the entire human cytoplasmic face obtained by combining biochemical reconstitution, crystal structure determination, docking into cryo-electron tomographic reconstructions, and physiological validation, accounting for a third of the NPCs mass. Whereas an evolutionarily conserved [~]540 kDa hetero-hexameric cytoplasmic filament nucleoporin complex is anchored by species-specific motifs above the central transport channel, attachment of the pentameric NUP358 bundles depends on the double-ring arrangement of the coat nucleoporin complex. Our results and the predictive power of our composite structure provide a rich foundation for elucidating the molecular basis of mRNA export and nucleoporin diseases.

One sentence summaryAn interdisciplinary analysis established the near-atomic molecular architecture of the cytoplasmic face of the human nuclear pore complex.

sentences: Aβ promotes amyloidogenic processing of APP through a Go/Gβγ signaling Alzheimers disease (AD) is characterized by a cognitive impairment associated to amyloid beta (A{beta}) aggregation and deposition in the brain. A{beta} is generated by sequential cleavage of the amyloid precursor protein (APP) by {beta}-site APP cleaving enzyme 1 (BACE1) and {gamma}-secretase complex. The mechanisms that underlie exacerbated production of A{beta}, favoring its deposition in the brain, is largely unknown. In vitro studies have shown that A{beta} aggregates trigger enhanced production of A{beta} by a yet non described mechanism. Here, we show that in different cell types, including human neurons derived from induced pluripotent stem cells (iPSC), oligomers and fibrils of A{beta} enhance the convergence and interaction of APP and BACE1 in endosomal compartments. We demonstrated a key role of A{beta}-APP/Go/G{beta}{gamma} signaling on the amyloidogenic processing of APP. We show that APP mutants with impaired capacity to bind A{beta} or to activate Go protein, are unable to exacerbate APP and BACE1 colocalization in the presence of A{beta}. Moreover, pharmacological inhibition of G{beta}{gamma} subunits signaling with gallein, abrogate A{beta}-dependent interaction of APP and BACE1 in endosomes preventing {beta}-processing of APP. Collectively, these findings uncover a feed-forward mechanism of amyloidogenesis that might contribute to A{beta} pathology in early stages of AD and suggest that gallein might have clinical relevance.

sentences: ReFeaFi: Genome-wide prediction of regulatory elements driving transcription initiation Regulatory elements control gene expression through transcription initiation (promoters) and by enhancing transcription at distant regions (enhancers). Accurate identification of regulatory elements is fundamental for annotating genomes and understanding gene expression patterns. While there are many attempts to develop computational promoter and enhancer identification methods, reliable tools to analyze long genomic sequences are still lacking. Prediction methods often perform poorly on the genome-wide scale because the number of negatives is much higher than that in the training sets. To address this issue, we propose a dynamic negative set updating scheme with a two-model approach, using one model for scanning the genome and the other one for testing candidate positions. The developed method achieves good genome-level performance and maintains robust performance when applied to other species, without re-training. Moreover, the unannotated predicted regulatory regions made on the human genome are enriched for disease-associated variants, suggesting them to be potentially true regulatory elements rather than false positives. We validated high scoring "false positive" predictions using reporter assay and all tested candidates were successfully validated, demonstrating the ability of our method to discover novel human regulatory regions.

sentences: Characterizing white matter alterations in drug-naïve de novo Parkinsons disease with diffusion MRI Parkinsons disease (PD) is a progressive neurodegenerative disorder that is characterized by a range of motor and non-motor symptoms, often with the motor dysfunction initiated unilaterally. Knowledge regarding disease-related alterations in white matter pathways can effectively help improve the understanding of the disease and propose targeted treatment strategies. Microstructural imaging techniques, including diffusion tensor imaging (DTI), allows inspection of white matter integrity to study the pathogenesis of various neurological conditions. Previous voxel-based analyses with DTI measures, such as fractional anisotropy and mean diffusivity have uncovered changes in brain regions that are associated with PD, but the conclusions were inconsistent, partially due to small patient cohorts and the lack of consideration for clinical laterality onset, particularly in early PD. Fixel-based analysis (FBA) is a recent framework that offers tract-specific insights regarding white matter health, but very few FBA studies on PD exist. We present a study that reveals strengthened and weakened white matter integrity that is subject to symptom laterality in a large drug-naive de novo PD cohort using complementary DTI and FBA measures. The findings suggest that the disease gives rise to both functional degeneration and the creation of compensatory networks in the early stage.

sentences: Mammary ductal epithelium controls cold-induced adipocyte thermogenesis Sympathetic activation during cold exposure increases adipocyte thermogenesis via expression of mitochondrial protein uncoupling protein 1 (UCP1)1. The propensity of adipocytes to express UCP1 is under a critical influence of the adipose microenvironment and varies among various fat depots2-7. Here we report that cold-induced adipocyte UCP1 expression in female mouse subcutaneous white adipose tissue (scWAT) is regulated by mammary gland ductal epithelial cells in the adipose niche. Single cell RNA-sequencing (scRNA-seq) show that under cold condition glandular alveolar and hormone-sensing luminal epithelium subtypes express transcripts that encode secretory factors involved in regulating adipocyte UCP1 expression. We term mammary duct secretory factors as "mammokines". Using whole-tissue immunofluorescence 3D visualization, we reveal previously undescribed sympathetic nerve-ductal points of contact and show that sympathetic nerve-activated mammary ducts limit adipocyte UCP1 expression via cold-induced mammokine production. Both in vivo and ex vivo ablation of mammary ductal epithelium enhances cold-induced scWAT adipocyte thermogenic gene program. The mammary duct network extends throughout most scWATs in female mice, which under cold exposure show markedly less UCP1 expression, fat oxidation, energy expenditure, and subcutaneous fat mass loss compared to male mice. These results show a previously uncharacterized role of sympathetic nerve-activated glandular epithelium in adipocyte thermogenesis. Overall, our findings suggest an evolutionary role of mammary duct luminal cells in defending glandular adiposity during cold exposure, highlight mammary gland epithelium as a highly active metabolic cell type, and implicate a broader role of mammokines in mammary gland physiology and systemic metabolism.

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Identification of residue inversions in large phylogenies of duplicated proteins Connecting protein sequence to function is becoming increasingly relevant since high-throughput sequencing studies accumulate large amounts of genomic data. In order to go beyond the existing database annotation, it is fundamental to understand the mechanisms underlying functional inheritance and divergence. If the homology relationship between proteins is known, can we determine whether the function diverged?

In this work, we analyze different possibilities of protein sequence evolution after gene duplication and identify "residue inversions", i.e., sites where the relationship between the ancestry and the functional signal is decoupled. Residues in these sites are masked from being recognized by other prediction tools. Still, they play a role in functional divergence and could indicate a shift in protein function. We develop a method to specifically recognize residue inversions in a phylogeny and test it on real and simulated datasets. In a dataset built from the Epidermal Growth Factor Receptor (EGFR) sequences found in 88 fish species, we identify 19 positions that went through inversion after gene duplication, mostly located at the ligand-binding extracellular domain.

Our work uncovers a rare event of protein divergence that has direct implications in protein functional annotation and sequence evolution as a whole. The developed method is optimized to work with large protein datasets and can be readily included in a targeted protein analysis pipeline.

sentences: Transcriptional Pathology Evolves Over Time in Rat Hippocampus Following Lateral Fluid Percussion Traumatic Brain Injury Traumatic brain injury (TBI) causes acute and lasting impacts on the brain, driving pathology along anatomical, cellular, and behavioral dimensions. Rodent models offer the opportunity to study TBI in a controlled setting, and enable analysis of the temporal progression that occurs from injury to recovery. We applied transcriptomic and epigenomic analysis, characterize gene expression and in ipsilateral hippocampus at 1 and 14 days following moderate lateral fluid percussion (LFP) injury. This approach enabled us to identify differential gene expression (DEG) modules with distinct expression trajectories across the two time points. The major DEG modules represented genes that were up- or downregulated acutely, but largely recovered by 14 days. As expected, DEG modules with acute upregulation were associated with cell death and astrocytosis. Interestingly, acutely downregulated DEGs related to neurotransmission mostly recovered by two weeks. Upregulated DEG modules related to inflammation were not necessarily elevated acutely, but were strongly upregulated after two weeks. We identified a smaller DEG module with delayed downregulation at 14 days including genes related to cholesterol metabolism and amyloid beta clearance. Finally, differential expression was paralleled by changes in H3K4me3 at the promoters of differentially expressed genes at one day following TBI. Following TBI, changes in cell viability, function and ultimately behavior are dynamic processes. Our results show how transcriptomics in the preclinical setting has the potential to identify biomarkers for injury severity and/or recovery, to identify potential therapeutic targets, and, in the future, to evaluate efficacy of an intervention beyond measures of cell death or spatial learning.

sentences: Power dynamics of theta oscillations during goal-directed navigation in freely moving humans: A mobile EEG-virtual reality T-maze study Theta oscillations ([~]4-12 Hz) are dynamically modulated by speed and direction in freely moving animals. However, due to the paucity of electrophysiological recordings of freely moving humans, this mechanism remains poorly understood. Here, we combined mobile-EEG with fully immersive virtual-reality to investigate theta dynamics in twenty-two healthy adults (aged 18-29 years old) freely navigating a T-maze to find rewards. Our results revealed three dynamic periods of theta modulation: 1) theta power increases coincided with the participants decision-making period; 2) theta power increased for fast and leftward trials as subjects approached the goal location; and 3) feedback onset evoked two phase-locked theta bursts over the right temporal and frontal-midline channels. These results suggest that recording scalp EEG in freely moving humans navigating a simple virtual T-maze can be utilized as a powerful translational model by which to map theta dynamics during "real-life" goal-directed behavior in both health and disease.

sentences: An averaging model for analysis and interpretations of high-order genetic interactions While combinatorial genetic data collection from biological systems in which quantitative phenotypes are controlled by functional and non-functional alleles in each of multiple genes (multi-gene systems) is becoming common, a standard analysis method for such data has not been established. A common additive model of the non-functional allele effects contrasted against the functional alleles, based on ANOVA with interaction, has three issues. First, although it is a long tradition of genetics, modeling the effect of the non-functional allele (a null mutant allele) contrasted against that of the functional allele (the wild-type allele) is not suitable for mechanistic understanding of multi-gene systems. Second, an additive model is highly problematic when the system has more than two genes and a limited phenotypic range: errors propagate toward higher order interactions. Third, interpretations of higher-order interactions defined by an additive model are not intuitive. I propose an averaging model, which is suitable for mechanistic understanding of multi-gene systems. The effect of the functional allele is contrasted against the effect of the non-functional allele for easier mechanistic interpretations. Errors in interactions across the orders consistently stay low, which makes the model highly scalable to systems with many genes. The interactions defined by the averaging model are highly intuitive regardless of the orders. Yet, it is still a general linear model, so model fitting is easy and accurate using common statistical tools.

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The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication The serine/threonine protein kinase AKT is a critical mediator of growth factor signaling that broadly impacts mRNA translation, metabolism, programmed cell death and stress responses. Human cytomegalovirus (HCMV), a large dsDNA virus in the herpesvirus family, dramatically remodels host cell signaling pathways during lytic infection. Here, we show that AKT accumulates in an inactive form during HCMV infection, as indicated by hypo-phosphorylation at Thr308 and Ser473, as well as nuclear localization of FOXO3a. Moreover, we find that expression of constitutively active myristoylated AKT (myr-AKT) causes a 1-log defect in viral replication, accompanied by defects in viral DNA synthesis and viral late gene expression. These findings suggest that HCMV shutoff of AKT is not merely incidental to viral factors such as pUL38 that maintain high levels of mTORC1 activity independently of extracellular growth factor signals. Short interfering RNA knockdown of FOXO3a, but not FOXO1, phenocopied the replication defects seen during expression of myr-AKT, corroborating a role for FOXO3a during HCMV infection. Accordingly, a chimeric FOXO3a-estrogen receptor fusion protein, whose nuclear localization is regulated by the small molecule ligand 4-hydroxytamoxifen instead of AKT activity, rescues virtually all the replication defects induced by myr-AKT expression. Taken together, our results argue that HCMV dampens AKT signaling to promote nuclear localization of FOXO transcription factors, which are required for efficient viral replication.

IMPORTANCEEvidence from a diverse herpesvirus infection models suggests that the PI3K / AKT signaling pathway suppresses reactivation from latency, and that inactivation of the pathway stimulates viral lytic replication. Here, we show that AKT accumulates in an inactive state during HCMV infection of lytically permissive cells while the presence of constitutive AKT activity causes substantial viral replication defects. Although AKT phosphorylates a diverse array of cellular substrates, we identify an important role for the Forkhead box class O transcription factors. Our findings show that when FoxO3a nuclear localization is decoupled from its negative regulation by AKT, the viral replication defects observed in the presence of constitutively active AKT are reversed. Collectively, our results reveal that HCMV inactivates AKT to promote the nuclear localization of FOXO transcription factors, which strongly implies that FOXOs play critical roles in transactivating cellular and/or viral genes during infection.

sentences: Structure-based prediction of KDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. In particular, HDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as -tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of HDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of HDAC8 and other enzymes. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. In particular, we propose novel regulatory roles of HDAC6 in tumorigenesis and cancer cell survival via the regulation of the EGFR/Akt pathway activation process. The calibration process and comparison of the results between HDAC6 and HDAC8 highlight structural differences that explain the established promiscuity of HDAC6.

sentences: Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.

sentences: Recombination-independent recognition of DNA homology for meiotic silencing in Neurospora crassa Pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. While in some organisms meiotic pairing requires programmed DNA breakage and recombination, in many others it engages homologous chromosomes that remain apparently intact. The mechanistic nature of such recombination-independent pairing represents a fundamental question in molecular genetics. Using  meiotic silencing by unpaired DNA (MSUD) in Neurospora crassa as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesin complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be co-linear and must contain short tracts of sequence identity spaced apart with a periodicity of 21 or 22 base-pairs. By using these values as a guiding parameter in all-atom molecular modeling, we discover that homologous double-stranded DNA molecules can associate by forming quadruplex-based contacts with an interval of 2.5 helical turns, which requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. These results (i) reconcile genetic and biophysical lines of evidence for the existence of direct homologous dsDNA-dsDNA pairing, (ii) identify a role for this process in initiating post-transcriptional silencing, and (iii) suggest that chromosomes are cross-matched in meiosis by a precise mechanism that operates on intact double-stranded DNA molecules.

sentences: Non cell-autonomous effect of astrocytes on cerebral cavernous malformations Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by loss-of-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we report that proliferative astrocytes play a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2 and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilizes HIF-1 in astrocytes, resulting in increased VEGF production and expression of a "hypoxic" program under normoxic conditions. We show that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1 target gene and a known component of the hypoxic program, contributes to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevents progression of CCM lesions. Thus, non-cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.

sentences: Feature-based gating of cortical information transmission In highly developed visual systems, spatial- and feature-based attentional modulation interact to prioritize relevant information and suppress irrelevant details. We investigated the specific role and integration of these two attentional mechanisms in visual cortical area MST of rhesus monkeys. We show that spatial attention acts as a gate for information processing by providing unimpeded high-gain pass-through processing for all sensory information from attended visual locations. Feature-based attentional enhancement does not only show the known dependency on a match between the attended feature and a given cells selectivity, but surprisingly is restricted to those features for which a given cell contributes to perception. This necessitates a refinement of the feature-similarity gain model of attention and documents highly optimized attentional gating of sensory information for cortical processing. This gating is shaped by neuronal sensory preferences, behavioral relevance, and the causal link to perception of neurons that process this visual input.

sentences: Metal-Organic Framework Encapsulated Whole-Cell Vaccines Enhance Humoral Immunity against Bacterial Infection The increasing rate of resistance of bacterial infection against antibiotics requires next generation approaches to fight potential pandemic spread. The development of vaccines against pathogenic bacteria has been difficult owing, in part, to the genetic diversity of bacteria. Hence, there are many potential target antigens and little a priori knowledge of which antigen/s will elicit protective immunity. The painstaking process of selecting appropriate antigens could be avoided with whole-cell bacteria; however, whole-cell formulations typically fail to produce long-term and durable immune responses. These complications are one reason why no vaccine against any type of pathogenic E. coli has been successfully clinically translated. As a proof of principle, we demonstrate a method to enhance the immunogenicity of a model pathogenic E. coli strain by forming a slow releasing depot. The E. coli strain CFT073 was biomimetically mineralized within a metal-organic framework (MOF). This process encapsulates the bacteria within 30 minutes in water and at ambient temperatures. Vaccination with this new formulation substantially enhances antibody production and results in significantly enhanced survival in a mouse model of bacteremia compared to standard inactivated formulations.

sentences: Depriving mice of sleep also deprives of food Both sleep-wake behavior and circadian rhythms are tightly coupled to energy metabolism and food intake. Altered feeding times in mice are known to entrain clock-gene rhythms in brain and liver and sleep-deprived humans tend to eat more and gain weight. Previous observations in mice showing that sleep deprivation (SD) changes clock-gene expression might thus relate to altered food intake and not to the loss of sleep per se. Whether SD affects food intake in the mouse and how this might affect clock-gene expression is, however, unknown. We therefore quantified i) the cortical expression of the clock genes Per1, Per2, Dbp, and Cry1 in mice that had access to food or not during a 6h SD, and ii) food intake during baseline, SD, and recovery sleep. We found that food deprivation did not modify the SD-incurred clock-gene changes in the cortex. Moreover, we discovered that although food intake during SD did not differ from baseline, mice lost weight and increased food intake during subsequent recovery. We conclude that SD is associated with food deprivation and that the resulting energy deficit might contribute to the effects of SD that are commonly interpreted as a response to sleep loss.

sentences: A dynamic and combinatorial histone code drives malaria parasite asexual and sexual development A  histone code defines system-level crosstalk between histone post-translational modifications (PTMs) to induce specific biological outcomes. Proteome-scale information of co-existing PTM across the entire chromatin landscape of the malaria parasite, Plasmodium falciparum, was lacking. Here, we used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 72 PTMs on H3 and H3.3, of which 30 are novel to the parasite. Co-existing PTMs with unique stage distinction was identified, indicating a dynamic and complex histone code with increased connectivity of novel PTMs seen in gametocytes. Chromatin proteomics of a gametocyte-specific combination, H3R17me2K18acK23ac, identified a SAGA-like effector complex (including the transcription factor AP2-G2) tied to this combination to regulate gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with co-existing partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher order gene regulation for distinct proliferation and differentiation processes.

sentences: Nef enhances HIV-1 replication and infectivity independently of SERINC3 and SERINC5 in CEM T cells The authors have withdrawn their manuscript whilst they perform additional experiments to test some of their conclusions further. They found upon resequencing DNA from the cell lines used in their study that non-CRISPR edited genomes dominated the cultures. They believed they had confirmed complete editing in single-cell clones at the outset. Therefore, they are not confident based on their current experiments that the virologic effect of HIV-1 Nef is independent of SERINC3 and SERINC5 in T cells of the leukemic CEM line as they originally claimed. They are reengineering the cells to resolve this and apologize for any inconvenience that this has caused. Therefore, the authors do not wish this work to be cited as a reference for the project. If you have any questions, please contact the corresponding author.

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Viral manipulation of mechanoresponsive signaling disassembles processing bodies Processing bodies (PBs) are ribonucleoprotein granules that suppress cytokine mRNA translation that are targeted for disassembly by many viruses. Kaposis sarcoma-associated herpesvirus is the etiological agent of the inflammatory endothelial cancer, Kaposis sarcoma, and a PB-regulating virus. The virus encodes Kaposin B (KapB), which induces actin stress fibres (SFs) and cell spindling as well as PB disassembly. We now show that KapB-mediated PB disassembly requires actin rearrangements, RhoA effectors and the mechanoresponsive transcription activator, YAP. Moreover, ectopic expression of active YAP or exposure of ECs to mechanical forces caused PB disassembly in the absence of KapB and mechanoresponsive PB disassembly also required YAP. Using the viral protein KapB, we identified a new consequence of the exposure of cells to mechanical forces that alter actin dynamics and activate YAP, namely the disassembly of PBs.

ImportanceFor the first time, we demonstrate that processing bodies (PBs), cytoplasmic sites of RNA decay, are regulated by mechanical signaling events that alter actin dynamics and that this requires the mechanoresponsive transcription factor, YAP. Using the overexpression of a viral protein called KapB, known previously to mediate PB disassembly, we show that actin stress fibers (SFs) and the mechanoresponsive transcription factor, YAP, are required for PB loss. We also show that other established mechanical signals (shear stress or stiff extracellular matrix) that lead to the formation of SFs and activate YAP also cause PB disassembly. This is important because it means that KapB activates, from the inside out, a pathway that links cell shape to post-transcriptional gene regulation via cytoplasmic PBs.

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Conserved Structural Motifs in the Hammerhead Ribozyme of a Chloroplast Viroid Mimic tRNA Anticodon Structure to Hijack tRNA Ligase for Viroid Circularization Viroids belonging to the family Avsunviroidae contain hammerhead ribozymes that process to unit length the oligomeric RNAs of both polarities generated during the rolling-circle replication that occurs in chloroplasts of host plants. Linear products, with 5-hydroxyl and 2,3-phosphodiester termini, are then recognized and circularized by the host chloroplastic isoform of the tRNA ligase. Here we analyze the circularization process of eggplant latent viroid (ELVd), an asymptomatic viroid that infects eggplants (Solanum melongena L.), using an Escherichia coli co-expression system in which longer-than-unit linear ELVd (+) precursors are expressed along with the eggplant chloroplastic tRNA ligase. The RNA precursor contains two copies of the hammerhead ribozyme and yields the appropriate termini for the tRNA ligase-mediated ligation in bacteria. We have determined that the ligation efficiency is highly dependent on the presence of ribozyme sequences in the ligatable termini, since the circularization of a series of viroid variants in which the ligation position was rearranged increased substantially in the presence of these sequences. Further in silico analysis showed sequence and structure similarity between the hammerhead ribozyme catalytic pocket and the anticodon loop of tRNAs, both of which harbor a characteristic U-turn of the phosphodiester backbone. Directed mutagenesis in the ribozyme domain supports the role of this U-turn loop in the ligation process. We propose that, in addition to its self-cleavage function, the viroid ribozymes have evolved to mimic the structure of the tRNA anticodon loop to recruit host tRNA ligase for the circularization of the monomeric linear replication intermediates.

IMPORTANCEViroids are a very particular class of infectious agents because they only consist of a small RNA that, to our current knowledge, does not encode for proteins. Consequently, viroids parasite host factors and structures to mediate all processes in the infectious cycle. How these small infectious RNAs are able to hijack host resources is currently a mystery. In this work, we shed some light on the functionality of hammerhead ribozymes during replication of viroids that belong to the family Avsunviroidae, which replicate in the chloroplasts. Our findings suggest that, in addition to mediate self-cleavage of replication intermediates, hammerhead ribozymes also recruit tRNA ligase for monomer circularization, likely mimicking a common host tRNA structural motif.

sentences: Interface Refinement of Low-to-Medium Resolution Cryo-EM Complexes using HADDOCK2.4 A wide range of cellular processes require the formation of multimeric protein complexes. The rise of cryo-electron microscopy (cryo-EM) has enabled the structural characterization of these protein assemblies. The produced density maps can, however, still suffer from limited resolution, impeding the process of resolving structures at atomic resolution. In order to solve this issue, monomers can be fitted into low-to-medium resolution maps. Unfortunately, the produced models frequently contain atomic clashes at the protein-protein interfaces (PPIs) as intermolecular interactions are typically not considered during monomer fitting. Here, we present a refinement approach based on HADDOCK2.4 to remove intermolecular clashes and optimize PPIs. A dataset of 14 cryo-EM complexes was used to test eight protocols. The best performing protocol, consisting of a semi-flexible simulated annealing refinement with restraints on the centroids of the monomers, was able to decrease intermolecular atomic clashes by 98% without significantly deteriorating the quality of the cryo-EM density fit.

sentences: Mapping Brain-Behavior Space Relationships Along the Psychosis Spectrum Difficulties in advancing effective patient-specific therapies for psychiatric disorders highlight a need to develop a stable neurobiologically-grounded mapping between neural and symptom variation. This gap is particularly acute for psychosis-spectrum disorders (PSD). Here, in a sample of 436 cross-diagnostic PSD patients, we derived and replicated a dimensionality-reduced symptom space across hallmark psychopathology symptoms and cognitive deficits. In turn, these symptom axes mapped onto distinct, reproducible brain maps. Critically, we found that multivariate brain-behavior mapping techniques (e.g. canonical correlation analysis) do not produce stable results. Instead, we show that a univariate brain-behavioral space (BBS) can resolve stable individualized prediction. Finally, we show a proof-of-principle framework for relating personalized BBS metrics with molecular targets via serotonin and glutamate receptor manipulations and gene expression maps. Collectively, these results highlight a stable and data-driven BBS mapping across PSD, which offers an actionable path that can be iteratively optimized for personalized clinical biomarker endpoints.

sentences: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire B cells have been engineered ex vivo to express an HIV-1 broadly neutralizing antibody (bNAb). B-cell reprograming may be scientifically and therapeutically useful, but current approaches limit B-cell repertoire diversity and disrupt the organization of the heavy-chain locus. A more diverse and physiologic B-cell repertoire targeting a key HIV-1 epitope could facilitate evaluation of vaccines designed to elicit bNAbs, help identity more potent and bioavailable bNAb variants, or directly enhance viral control in vivo. Here we address the challenges of generating such a repertoire by replacing the heavy-chain CDR3 (HCDR3) regions of primary human B cells. To do so, we identified and utilized an uncharacterized Cas12a ortholog that recognizes PAM motifs present in human JH genes. We also optimized the design of 200 nucleotide homology-directed repair templates (HDRT) by minimizing the required 3-5 deletion of the HDRT-complementary strand. Using these techniques, we edited primary human B cells to express a hemagglutinin epitope tag and the HCDR3 regions of the bNAbs PG9 and CH01. Those edited with bNAb HCDR3 efficiently bound trimeric HIV-1 antigens, implying they could affinity mature in vivo in response to the same antigens. This approach generates diverse B-cell repertoires recognizing a key HIV-1 neutralizing epitope.

sentences: Protein N-glycosylation is essential for SARS-CoV-2 infection SARS-CoV-2 extensively N-glycosylates its spike proteins, which are necessary for host cell invasion and the target of both vaccines and immunotherapies. These sugars are predicted to help mediate spike binding to the host receptor by stabilizing its  open conformation and evading host immunity. Here, we investigated both the essentiality of the host N-glycosylation pathway and SARS-CoV-2 N-glycans for infection. Inhibition of host N-glycosylation using RNAi or FDA-approved drugs reduced virus infectivity, including that of several variants. Under these conditions, cells produced less virions and some completely lost their infectivity. Furthermore, partial deglycosylation of intact virions showed that surface-exposed N-glycans are critical for cell invasion. Altogether, spike N-glycosylation is a targetable pathway with clinical potential for treatment or prevention of COVID-19.

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The LysR-type transcriptional regulator BsrA (PA2121) controls vital metabolic pathways in Pseudomonas aeruginosa Pseudomonas aeruginosa, a facultative human pathogen causing nosocomial infections, has complex regulatory systems involving many transcriptional regulators. LTTR (LysR-Type Transcriptional Regulator) family proteins are involved in the regulation of various processes including stress responses, motility, virulence and amino acid metabolism. The aim of this study was to characterize the LysR-type protein BsrA (PA2121), previously described as a negative regulator of biofilm formation in P. aeruginosa. Genome wide identification of BsrA binding sites using ChIP-seq revealed 765 BsrA-bound regions in the P. aeruginosa PAO1161 genome, including 367 sites in intergenic regions. The motif T-N11-A was identified within sequences bound by BsrA. Transcriptomic analysis showed altered expression of 157 genes in response to BsrA excess, of which 35 had a BsrA binding site within their promoter regions, suggesting a direct influence of BsrA on the transcription of these genes. BsrA-repressed loci included genes encoding proteins engaged in key metabolic pathways such as the tricarboxylic acid cycle. The panel of loci possibly directly activated by BsrA, included genes involved in pili/fimbriae assembly as well as secretion and transport systems. In addition, DNA pull-down and regulatory analyses showed the involvement of PA2551, PA3398 and PA5189 in regulation of bsrA expression, indicating that this gene is part of an intricate regulatory network. Taken together, these findings reveal the existence of a BsrA regulon, which performs important functions in P. aeruginosa.

IMPORTANCEThis study shows that BsrA, a LysR-type transcriptional regulator from P. aeruginosa, previously identified as a repressor of biofilm synthesis, is part of an intricate global regulatory network. BsrA acts directly and/or indirectly as the repressor and/or activator of genes from vital metabolic pathways (e.g. pyruvate, acetate, tricarboxylic acid cycle), and is involved in control of transport functions and the formation of surface appendages. Expression of the bsrA gene is increased in the presence of antibiotics, which suggests its induction in response to stress, possibly reflecting the need to redirect metabolism under stressful conditions. This is particularly relevant for the treatment of infections caused by P. aeruginosa. In summary, the findings of this study demonstrate that the BsrA regulator performs important roles in carbon metabolism, biofilm formation and antibiotic resistance in P. aeruginosa.

sentences: Accurate estimation of cell-type resolution transcriptome in bulk tissue through matrix completion Single cell RNA-seq (scRNA-seq) has been widely used to uncover cellular heterogeneity, however, the constraints of cost make it impractical as a routine on large patient cohorts. Here we present ENIGMA, a method that accurately deconvolute bulk tissue RNA-seq into single cell-type resolution given the knowledge gained from scRNA-seq. ENIGMA applies a matrix completion strategy to minimize the distance between mixture transcriptome and weighted combination of cell type-specific expression, allowing quantification of cell type proportions and reconstruction of cell type-specific transcriptome. The superior performance of ENIGMA was validated in simulated and realistic datasets, including disease-related tissues, demonstrating its ability in novel biological findings.

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Regeneration of Plants from DNA-free Edited Grapevine Protoplasts CRISPR-Cas technology has widely extended the application fields of genome editing in plant breeding, making possible specific and minimal mutations within a genetic pool. With respect to standard genome editing technologies, CRISPR-Cas machinery can be introduced in the form of ribonucleoproteins (RNPs), thus avoiding the introduction of exogenous DNA into cells. The interest on the application of DNA-free delivery into plant cells is constantly increasing, especially in the case of valuable woody plants elite varieties where CRISPR-Cas9 technology would preserve their genotype, while still resulting into targeted genetic modifications. The use of single cells fits well the requirements of New Breeding Technologies, by ensuring both the CRISPR-Cas DNA-free delivery as RNPs and, since every plant will be regenerated from a single edited cell, the absence of chimerism. However, the use of protoplasts cell culture from woody plants is generally hampered by low editing efficiencies and an unsuccessful regenerative process.

We here describe a successful DNA-free methodology to obtain fully edited grapevine plants, regenerated from protoplasts obtained from V. vinifera cv. Crimson seedless L. embryogenic callus. The transfected protoplasts were edited on the Downy Mildew susceptibility gene VvDMR6-2. The regenerated edited plants exhibited homozygous deletions of 1bp or 2bp, and homozygous insertion of 1bp.

sentences: Whole-Genome Sequencing on 220 Alfalfa (Medicago sativa L.) Accessions Identified Association of DREB1C Gene with Fall Dormancy Height Fall dormancy (FD) is one of the most important traits of alfalfa (Medicago sativa) for cultivar selection to overcome winter stress. Although transcriptomics, proteomics analysis, and QTL mapping have revealed some important genes correlated with FD, the genetic architecture of this trait is still unclear. There are no applicable genes or markers for selection, which hinders progress in the genetic research and molecular breeding for the trait. We conducted whole-genome sequencing (WGS) on 220 alfalfa accessions at 10x depth. Among the 875,023 SNPs, four of them were associated with FD height using GWAS. One SNP located on chromosome 6 is in linkage disequilibrium with dehydration-responsive element-binding protein 1C (DREB1C). Furthermore, seven DREB genes are clustered in this region, one of which has previously been shown to enhance freezing tolerance in the model plant Medicago truncatula. The candidate genes uncovered by our research will benefit the transgenic and CRISPR-Cas9 research of FD in alfalfa. This gene will also be useful for molecular marker development and marker-associated breeding of FD for alfalfa.

sentences: TIR signaling promotes the interactions between EDS1/PAD4 and ADR1-L1 and oligomerization of ADR1-L1 Both plants and animals use nucleotide-binding leucine-rich repeat (NLR) immune receptors to perceive pathogens and trigger immunity. Toll/interleukin-1 receptor (TIR)-type plant NLRs (TNLs) require the lipase-like protein family members Enhanced Disease Susceptibility 1 (EDS1)/ Phytoalexin Deficient 4 (PAD4)/ Senescence-Associated Gene 101 (SAG101) and helper NLRs (hNLRs) for downstream signaling, the biochemical mechanisms of which remain unclear. Here, we report that TIR signaling promotes the association of EDS1 and PAD4 with hNLR ACTIVATED DISEASE RESISTANCE 1-Like 1 (ADR1-L1), and the oligomerization of ADR1-L1s for downstream immune activation and cell death.

sentences: Improving mathematical models of cancer by including resistance to therapy: a study in non-small cell lung cancer In this paper, a large dataset of 590 Non-Small Cell Lung Patients treated with either chemotherapy or immunotherapy was used to determine whether a game-theoretic model including both evolution of therapy resistance and cost of resistance provides a better fit than classical mathematical models of population growth (exponential, logistic, classic Bertalanffy, general Bertalanffy, Gompertz, general Gompertz). This is the first time a large clinical patient cohort (as opposed to only in-vitro data) has been used to apply a game-theoretic cancer model. The game-theoretic model provides a better fit to the tumor dynamics of the 590 Non-Small Cell Lung Cancer patients than any of the non-evolutionary population growth models. This is not simply due to having more parameters in the game-theoretic model. The game-theoretic model is able to fit accurately patients whose tumor burden exhibit a U-shaped trajectory over time. We then demonstrate how this game-theoretic model provides predictions of tumor growth based on just a few initial measurements. Assuming that treatment-specific parameters define the treatment impact completely, we then explore alternative treatment protocols and their impact on the tumor growth. As such, the model can be used to suggest patient-specific optimal treatment regimens with the goal of minimizing final tumor burden. Therapeutic protocols based on game-theoretic modeling can predict tumor growth, and improve patient outcome. The model invites evolutionary therapies that anticipate and steer the evolution of therapy resistance.

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Nuclear Lamina binds the EBV genome during latency and regulates viral gene expression The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.

AUTHOR SUMMARYEpstein-Barr virus (EBV) is a common herpesvirus that establishes a lifelong latent infection in a small fraction of B cells of the infected individuals. In most cases, EBV infection is asymptomatic; however, especially in the context of immune suppression, EBV latent infection is associated with several malignancies. In EBV+ cancer cells, latent viral gene expression plays an essential role in sustaining the cancer phenotype. We and others have established that epigenetic modifications of the viral genome are critical to regulating EBV gene expression during latency. Understanding how the EBV genome is epigenetically regulated during latent infection may help identify new specific therapeutic targets for treating EBV+ malignancies. The nuclear lamina is involved in regulating the composition and structure of the cellular chromatin. In the present study, we determined that the nuclear lamina binds the EBV genome during latency, influencing viral gene expression. Depleting one component of the nuclear lamina, lamin A/C, increased the expression of latent EBV genes associated with cellular proliferation, indicating that the binding of the nuclear lamina with the viral genome is essential to control viral gene expression in infected cells. Our data show for the first time that the nuclear lamina may be involved in the cellular response against EBV infection by restricting viral gene expression.

sentences: Identifying key federal, state, and private lands strategies for achieving 30x30 in the US Achieving ambitious conservation goals to conserve at least 30% of US lands and waters by 2030 ("30x30") will require a multi-scale baseline understanding of current protections, key decision makers, and policy tools for moving forward. To help conservationists and decision makers support the science-based call to address the biodiversity and climate crises, we analyze the current spatial patterns of imperiled species biodiversity and carbon stores in the U.S. relative to protected areas. Analyses demonstrate that 30x30 is numerically achievable nationally, but high spatial heterogeneity highlights the need for tailored approaches from a mix of authorities at federal, regional, and state scales. Critically, current land protections rarely overlap with areas essential for conserving imperiled species biodiversity and mitigating climate change. Nearly one-fifth of unprotected biodiversity hotspots and carbon-rich areas are also at risk of either land conversion or climate exposure by 2050. We discuss this baseline relative to key policy considerations for making practical, substantive progress toward the goal.

sentences: Aggregation is a context-dependent constraint on protein evolution Solubility is a requirement for many cellular processes. Loss of solubility and aggregation can lead to the partial or complete abrogation of protein function. Thus, understanding the relationship between protein evolution and aggregation is an important goal. Here, we analysed two deep mutational scanning experiments to investigate the role of protein aggregation in molecular evolution. In one data set, mutants of a protein involved in RNA biogenesis and processing, human TAR DNA binding protein 43 (TDP-43), were expressed in S. cerevisiae. In the other data set, mutants of a bacterial enzyme that controls resistance to penicillins and cephalosporins, TEM-1 beta-lactamase, were expressed in E. coli under the selective pressure of an antibiotic treatment. We found that aggregation differentiates the effects of mutations in the two different cellular contexts. Specifically, aggregation was found to be associated with increased cell fitness in the case of TDP-43 mutations, as it protects the host from aberrant interactions. By contrast, in the case of TEM-1 beta-lactamase mutations, aggregation is linked to a decreased cell fitness due to inactivation of protein function. Our study shows that aggregation is an important context-dependent constraint of molecular evolution and opens up new avenues to investigate the role of aggregation in different cellular contexts.

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4'-Fluorouridine is a broad-spectrum orally efficacious antiviral blocking respiratory syncytial virus and SARS-CoV-2 replication The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and the elderly. We describe 4-fluorouridine (4-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and SARS-CoV-2 with high selectivity index in cells and well-differentiated human airway epithelia. Polymerase inhibition in in vitro RdRP assays established for RSV and SARS-CoV-2 revealed transcriptional pauses at positions i or i+3/4 post-incorporation. Once-daily oral treatment was highly efficacious at 5 mg/kg in RSV-infected mice or 20 mg/kg in ferrets infected with SARS-CoV-2 WA1/2020 or variant-of-concern (VoC) isolate CA/2020, initiated 24 or 12 hours after infection, respectively. These properties define 4-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2 and related RNA virus infections.

One-Sentence Summary4-Fluorouridine is an orally available ribonucleoside analog that efficiently treats RSV and SARS-CoV-2 infections in vivo.

sentences: Alpha oscillations and stimulus-evoked activity dissociate metacognitive reports of attention, visibility and confidence in a rapid visual detection task Variability in the detection and discrimination of weak visual stimuli has been linked to oscillatory neural activity. In particular, the amplitude of activity in the alpha-band (8-12 Hz) has been shown to impact upon the objective likelihood of stimulus detection, as well as measures of subjective visibility, attention, and decision confidence. We aimed to clarify how preparatory alpha influences performance and phenomenology, by recording simultaneous subjective measures of attention and confidence (Experiment 1), or attention and visibility (Experiment 2) on a trial-by-trial basis in a visual detection task. Across both experiments, alpha amplitude was negatively and linearly correlated with the intensity of subjective attention. In contrast to this linear relationship, we observed a quadratic relationship between the strength of alpha oscillations and subjective ratings of confidence and visibility. We find that this same quadratic relationship links alpha amplitude to the strength of stimulus evoked responses. Visibility and confidence judgements corresponded to the strength of evoked responses, but confidence, uniquely, incorporated information about attentional state. As such, our findings reveal distinct psychological and neural correlates of metacognitive judgements of attentional state, stimulus visibility, and decision confidence.

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SPT6 loss Permits the Transdifferentiation of Keratinocytes into anIntestinal Fate that Recapitulates Barretts Metaplasia Transient depletion of the transcription elongation factor SPT6 in the keratinocyte has been recently shown to inhibit epidermal differentiation and stratification; instead, they transdifferentiate into a gut-like lineage. We show here that this phenomenon of transdifferentiation recapitulates Barretts metaplasia, the only human pathophysiologic condition in which a stratified squamous epithelium that is injured due to chronic acid reflux is trans-committed into an intestinal fate. The evidence we present here not only lend support to the notion that the keratinocytes are the cell of origin of Barretts metaplasia, but also provide mechanistic insights linking transient acid exposure, downregulation of SPT6, stalled transcription of the master regulator of epidermal fate TP63, loss of epidermal fate and metaplastic progression. Because Barretts metaplasia in the esophagus (BE) is a pre-neoplastic condition with no preclinical human models, these findings have a profound impact on the modeling Barretts metaplasia-in-a-dish.

GRAPHIC ABSTRACT

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C_LIO_LISuch transdifferentiation recapitulates Barretts metaplasia, not the healthy gut
C_LIO_LIAcid downregulates SPT6, which derails the expression and functions of TP63
C_LIO_LISuch downregulation precedes the metaplasia-dysplasia-neoplasia cascade
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sentences: Activity of CaMKIIa+ dorsal cochlear nucleus neurons are crucial for tinnitus perception but not for tinnitus induction The dorsal cochlear nucleus (DCN) is a region known to integrate somatosensory and auditory inputs and is identified as a potential key structure in the generation of phantom sound perception, especially noise-induced tinnitus. Yet, how altered homeostatic plasticity of the DCN induces and maintains the sensation of tinnitus is not clear. Here, we chemogenetically decrease activity of a subgroup of DCN neurons, Ca2+/Calmodulin kinase 2 (CaMKII) positive DCN neurons, using Gi-coupled human M4 Designer Receptors Exclusively Activated by Designer Drugs (hM4Di DREADDs), to investigate their role in noise-induced tinnitus. Mice were exposed to loud noise (9-11kHz, 90dBSPL, 1h, followed by 2h of silence) and auditory brainstem responses (ABRs) and gap prepulse inhibition of acoustic startle (GPIAS) were recorded two days before and two weeks after noise exposure to identify animals with a significantly decreased inhibition of startle, indicating tinnitus but without permanent hearing loss. Neuronal activity of CaMKII+ neurons expressing hM4Di in the DCN was lowered by administration of clozapine-N-oxide (CNO). We found that acutely decreasing firing rate of CaMKII+ DCN units decrease tinnitus-like responses (p = 0.038, n = 11 mice), compared to the control group that showed no improvement in GPIAS (control virus; CaMKII-YFP + CNO, p = 0.696, n = 7 mice). Extracellular recordings confirmed CNO to decrease unit firing frequency of CaMKII-hM4Di+ mice and alter best frequency and tuning width of response to sound. However, these effects were not seen if CNO had been previously administered during the noise exposure (n = 6 experimental and 6 control mice). Our results suggest that CaMKII-hM4Di positive cells in the DCN are not crucial for tinnitus induction but play a significant role in maintaining tinnitus perception in mice.

sentences: A Drosophila larvae-inspired vacuum-actuated soft robot Peristalsis is one of the most common locomotion patterns in limbless animals. This motion is generated by propagating muscular contraction and relaxation along the body axis. While the kinematics of peristalsis has been examined intensively, the kinetics and mechanical control of peristalsis remain unclear, partially due to the lack of suitable physical models to analyse the force and temporal control in soft-bodied animals' locomotion. Here, based on a soft-bodied animal, Drosophila larvae, we proposed a vacuum-actuated soft robot replicating their crawling behaviour. The soft structure, made with hyperelastic silicon rubber, was designed to mimic the larval hydrostatic structure. To estimate the adequate range of pressures and time scales for control of the soft robots, a numerical simulation by the finite element method was conducted. Pulse-Width-Modulation (PWM) was used to generate time-series signals to control the vacuum pressure in each segment. Based on this control system, the soft robots could exhibit the peristaltic pattern resembling fly larval crawling. The soft robots reproduced two previous experimental results on fly larvae: slower crawling speed in backward crawling than in forward crawling, and the involvement of segmental contraction duration and intersegmental delay in crawling speed. Furthermore, the soft robot provided a novel prediction that the larger the contraction force, the faster the crawling speed. These observations indicate that the use of soft robots could serve to examine the kinetics and mechanical regulation of crawling behaviour in soft-bodied animals.

sentences: Implication of synaptotagmins 4 and 7 in activity-dependent somatodendritic dopamine release Dopamine (DA) neurons can release DA not just from axon terminals, but also from their somatodendritic (STD) compartment thought a mechanism that is still incompletely understood. Using voltammetry in mouse mesencephalic brain slices, we find that STD DA release has low capacity, is stable in response to electrical but not optogenetic train pulses and shows a calcium sensitivity that is comparable to that of axonal release. It is also strikingly more resilient compared to axonal release in a 6- hydroxydopamine model of Parkinsons disease plasticity. We find that the molecular mechanism of STD DA release differs from axonal release with regards to the implication of synaptotagmin (Syt) calcium sensors. While individual constitutive knock-out Syt4 and Syt7 is not sufficient to reduce STD DA release, removal of both isoforms reduces this release by ~50%, leaving axonal release unimpaired. Our works unveils clear differences in the mechanisms of STD and axonal DA release.

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The population genomics of increased virulence and antibiotic resistance in human commensal Escherichia coli over 30 years in France Escherichia coli is a commensal species of the lower intestine, but also a major pathogen causing intestinal and extra-intestinal infections, increasingly prevalent and resistant to antibiotics. Most studies on genomic evolution of E. coli used isolates from infections. Here instead, we whole-genome sequenced a collection of 403 commensal E. coli isolated from fecal samples of healthy adult volunteers in France (1980-2010). These isolates were distributed mainly in phylogroups A and B2 (30% each) and belonged among 152 sequence types (STs), the five most frequent being ST10 (16.3%), ST73 (6.3%), ST95 (5.0%), ST69 (4.2%) and ST59 (3.9%), and 224 O:H serotypes. ST and serotype diversity increased over time. The O1, O2, O6 and O25-groups used in bioconjugate O-antigen vaccine against extraintestinal infections were found in 23% of the strains of our collection. Extra-intestinal virulence-associated genes and antibiotic resistances increased in frequency, suggesting positive selection on these genes. Evolution of virulence genes was driven by both clonal expansion of STs with more virulence genes ("ST-driven") and increases in gene frequency within STs independently of changes in ST frequencies ("gene-driven"), whereas the evolution of resistance was dominated by increases in frequency within STs ("gene-driven"). This study provides a unique picture of the phylogenomic evolution of E. coli in its human commensal habitat over 30 years and will have implications for the development of preventive strategies.

IMPORTANCEEscherichia coli is an opportunistic pathogen with the greatest burden of antibiotic resistance, one of the main causes of nosocomial infections and an increasing concern in an ageing population. Deciphering the evolutionary dynamics of virulence and antibiotic resistance in commensal E. coli is important to understand adaptation and anticipate future changes. The gut of vertebrates is the primary habitat of E. coli and probably where selection for virulence and resistance take place. Unfortunately, most whole-genome sequenced strains are isolated from pathogenic conditions. Here instead, we whole genome sequenced 403 E. coli commensals isolated from healthy French subjects on a 30-year period. Virulence and resistance genes increased in frequency both by clonal expansion of clones carrying them and by increases in frequency within clones. Such studies should be performed worldwide to have a broader picture of E. coli evolution and adaptation in commensalism.

sentences: DNA methylation is not a driver of gene expression reprogramming in young honey bee workers Intragenic DNA methylation, also called gene body methylation, is an evolutionarily-conserved epigenetic mechanism in animals and plants. In social insects, gene body methylation is thought to contribute to behavioral plasticity, for example between foragers and nurse workers, by modulating gene expression. However, recent studies have suggested that the majority of DNA methylation is sequence-specific, and therefore cannot act as a flexible mediator between environmental cues and gene expression. To address this paradox, we examined whole-genome methylation patterns in the brains and ovaries of young honey bee workers that had been subjected to divergent social contexts: the presence or absence of the queen. Although these social contexts are known to bring about extreme changes in behavioral and reproductive traits through differential gene expression, we found no significant differences between the methylomes of workers from queenright and queenless colonies. In contrast, thousands of regions were differentially methylated between colonies, and these differences were not associated with differential gene expression in a subset of genes examined. Methylation patterns were highly similar between brain and ovary tissues and only differed in nine regions. These results strongly indicate that DNA methylation is not a driver of differential gene expression between tissues or behavioral morphs. Finally, despite the lack of difference in methylation patterns, queen presence affected the expression of all four DNA methyltransferase genes, suggesting that these enzymes have roles beyond DNA methylation. Therefore, the functional role of DNA methylation in social insect genomes remains an open question.

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Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites The ring-like structural maintenance of chromosomes (SMC) complex MukBEF folds the genome of Escherichia coli and related bacteria into large loops, presumably by active DNA loop extrusion. MukBEF activity within the replication terminus macrodomain is suppressed by the sequence specific unloader MatP. Here we present the complete atomic structure of MukBEF in complex with MatP and DNA as determined by electron cryomicroscopy (cryo-EM). The complex binds two distinct DNA double helices corresponding to the arms of a plectonemic loop. MatP-bound DNA threads through the MukBEF ring, while the second DNA is clamped by the kleisin MukF, MukE and the MukB ATPase heads. Combinatorial cysteine cross-linking confirms this topology of DNA loop entrapment in vivo. Our findings illuminate how a class of near-ubiquitous DNA organizers with important roles in genome maintenance interacts with the bacterial chromosome.

HighlightsO_LIComplete atomic structures of the bacterial SMC complex MukBEF on and off DNA.
C_LIO_LIMukBEF entraps two DNA double helices when bound to the unloader MatP.
C_LIO_LIIn vivo topology of DNA loop entrapment determined by cysteine cross-linking.
C_LIO_LIArms of the DNA loop thread through separate compartments of MukBEF.
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Graphical abstract

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Hepatic MIR20B promotes nonalcoholic fatty liver disease by suppressing PPARA BackgroundNon-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation and imbalances in lipid metabolism in the liver. Although nuclear receptors (NRs) play a crucial role in hepatic lipid metabolism, the underlying mechanisms of NR regulation in NAFLD remain largely unclear.

MethodsUsing network analysis and RNA-seq to determine the correlation between NRs and microRNA in human NAFLD patients, we revealed that MIR20B specifically targets PPARA. MIR20B mimic and anti-MIR20B were administered to human HepG2 and Huh-7 cells and mouse primary hepatocytes as well as high fat diet (HFD)- or methionine-deficient diet (MCD)-fed mice to verify the specific function of MIR20B in NAFLD. We tested the inhibition of the therapeutic effect of a PPAR agonist, fenofibrate, by MIR20B and the synergic effect of combination of fenofibrate with anti-MIR20B.

ResultsWe revealed that MIR20B specifically targets PPARA through miRNA regulatory network analysis of nuclear receptor genes in NAFLD. The expression of MIR20B was upregulated in free fatty acid (FA)- treated hepatocytes and the livers of both obesity-induced mice and NAFLD patients. Overexpression of MIR20B significantly increased hepatic lipid accumulation and triglyceride levels. Furthermore, MIR20B significantly reduced FA oxidation and mitochondrial biogenesis by targeting PPARA. In MIR20B-introduced mice, the effect of fenofibrate to ameliorate hepatic steatosis was significantly suppressed. Finally, inhibition of MIR20B significantly increased FA oxidation and uptake, resulting in improved insulin sensitivity and a decrease in NAFLD progression. Moreover, combination of fenofibrate and anti-MIR20B exhibited the synergic effect on improvement of NAFLD in MCD-fed mice.

ConclusionsTaken together, our results demonstrate that the novel MIR20B targets PPARA, plays a significant role in hepatic lipid metabolism, and present an opportunity for the development of novel therapeutics for NAFLD.

FundingThis research was funded by Korea Mouse Phenotyping Project (2016M3A9D5A01952411), the National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1F1A1061267, 2018R1A5A1024340, NRF-2021R1I1A2041463, 2020R1I1A1A01074940), and the Future-leading Project Research Fund (1.210034.01) of UNIST.

sentences: Effects of age and surface instability on the control of the center of mass During standing, posture can be controlled by accelerating the Center of Mass (CoM) through shifting the center of pressure (CoP) within the base of support by applying ankle moments ("CoP mechanism"), or through the "counter-rotation mechanism", i.e., changing the angular momentum of segments around the CoM to change the direction of the ground reaction force. Postural control develops over the lifespan; at both the beginning and the end of the lifespan adequate postural control appears more challenging. In this study, we aimed to assess mediolateral balance performance and the related use of the postural control mechanisms in children, older adults and young adults when standing on different (unstable) surfaces. Sixteen pre-pubertal children (6-9y), 17 young adults (18-24y) and eight older adults (65-80y) performed bipedal upright standing trials of 16 seconds on a rigid surface and on three balance boards that could freely move in the frontal plane, varying in height (15-19 cm) of the surface of the board above the point of contact with the floor. Full body kinematics (16 segments, 48 markers, using SIMI 3D-motion analysis system (GmbH) and DeepLabCut and Anipose) were retrieved. Performance related outcome measures, i.e., the number of trials with balance loss and the Root Mean Square (RMS) of the time series of the CoM acceleration, the contributions of the CoP mechanism and the counter-rotation mechanism to the CoM acceleration in the frontal plane and selected kinematic measures, i.e. the orientation of the board and the head and the Mean Power Frequency (MPF) of the balance board orientation and the CoM acceleration were determined. Balance loss only occurred when standing on the highest balance board, twice in one older adult once in one young adult. In children and older adults, the RMS of the CoM accelerations were larger, corresponding to poorer balance performance. Across age groups and conditions, the contribution of the CoP mechanism to the total CoM acceleration was much larger than that of the counter-rotation mechanisms, ranging from 94%-113% vs 23%-38% (with totals higher than 100% indicating opposite effects of both mechanisms). Deviations in head orientation were small compared to deviations in balance board orientation. We hypothesize that the CoP mechanism is dominant, since the counter-rotation mechanism would conflict with stabilizing the orientation of the head in space.

sentences: Prolonging coagulant activity of factor Xa under hemophilic conditions by site-specific N-glycosylation of the surface-exposed autolysis loop The regulation of Factor X (FX) is critical to maintain hemostasis. To gain insights to the regulation of the active and zymogen form of coagulation FX, we probed specific molecular interactions by introducing novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. Introduction of N-glycans in the autolysis loop of these FX variants decreased Factor VIIa (FVIIa)-mediated activation ~3-fold and prothrombin activation 2- to 10-fold presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor Factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). Ki for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, while ATIII inhibition in the presence of the heparin-analogue Fondaparinux was modestly increased by 2- to 15-fold compared to wild type. To probe the in vitro hemostatic effect of the FX variants, the thrombin generation potential in FX-depleted plasma was evaluated. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FX variants with N-glycosylation at positions 148-150. These results indicate that residues of the surface-exposed autolysis loop and residues close by participate in FX activation, proteolytic activity and inhibition of FXa by TFPI and ATIII. In plasma-based assays, a modest decrease in FX-activation rate appeared to compensate for the collective reduction in inhibitor interactions.

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Repair of Mismatched Templates during Rad51-dependent Break-Induced Replication Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3 end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. When every 10th base is mismatched between donor and recipient, BIR is 44% efficient compared to fully homologous sequences; but BIR still occurs about 10% when every 6th base is mismatched. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, but where they are clustered with spacings of every 6th bp. These different arrangements of uneven mismatch distribution were in general less efficient in recombination as templates with evenly distributed mismatches. A donor with all 10 mismatches clustered every 6th base at the 3 invading end of the DSB was not impaired compared to the case where mismatches were clustered at the 5 end. These data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but sequence-specific factors are also important. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3 end. Mismatch incorporation depends on the 3 [->] 5 proofreading exonuclease activity of DNA polymerase {delta}, with little contribution from Msh2/Mlh1 mismatch repair proteins. Surprisingly, the probability of a mismatch 27 nt from the 3 end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase {delta} "chews back" the 3 end of the invading strand without any mismatch-dependent cues from the strand invasion structure.

Author SummaryDNA double-strand breaks (DSBs) are the most lethal forms of DNA damage and inaccurate repair of these breaks presents a serious threat to genomic integrity and cell viability. Break-induced replication (BIR) is a homologous recombination pathway that results in a nonreciprocal translocation of chromosome ends. We used budding yeast Saccharomyces cerevisiae to investigate Rad51-mediated BIR, where the invading 3 end of the DSB and a donor template share 108 bp of homology. We examined the tolerance of differently distributed mismatches on a homologous donor template and found that BIR efficiency was the same whether the mismatches were clustered at the 3 invading end or at the 5 end. We confirmed that mismatches are incorporated into the BIR product in a strongly polar fashion as far as about 40 nucleotides from the 3 end. We conclude that the proofreading activity of DNA polymerase {delta} "chews back" the 3 end of the invading strand even when the sequences removed have no mismatches for the first 26 nucleotides. These observations enrich our understanding of the details of Rad51-mediated strand invasion and provide insight into the mechanism of the 3 to 5 proofreading activity of DNA polymerase during homologous recombination.

sentences: Roseoflavin, a natural riboflavin analogue, possesses in vitro and in vivo antiplasmodial activity The ability of the human malaria parasite Plasmodium falciparum to access and utilise vital nutrients is critical to its growth and proliferation. Molecules that interfere with these process could potentially serve as antimalarials. We found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin metabolism and/or the utilisation of the riboflavin metabolites flavin mononucleotide and flavin adenine dinucleotide. An additional eight riboflavin analogues were evaluated, but none were found to be more potent than roseoflavin, nor was their activity on target. Focussing on roseoflavin, we tested its antimalarial activity in vivo against Plasmodium vinckei vinckei in mice. We found that roseoflavin decreased the parasitemia by 46-fold following a 4 day suppression test and, on average, increased the survival of mice by 4-5 days. Our data are consistent with riboflavin metabolism and/or the utilisation of riboflavin-derived cofactors being viable drug targets for the development of new antimalarials and that roseoflavin could serve as a potential starting point.

sentences: Transformation from local image contrast to location-independent numerosity tuning in human extrastriate cortex Human early visual cortex response amplitudes monotonically increase with numerosity (object number), regardless of object size and spacing. However, numerosity is typically considered a high-level visual or cognitive feature, while early visual responses follow image contrast in the spatial frequency domain. We found that, at fixed contrast, aggregate Fourier power (at all orientations and spatial frequencies) followed numerosity closely but nonlinearly with little effect of object size, spacing or shape. This would allow straightforward numerosity estimation from spatial frequency domain image representations. Using 7T fMRI, we showed monotonic responses originate in primary visual cortex (V1) at the stimuluss retinotopic location. Responses here and in neural network models followed aggregate Fourier power more closely than numerosity. Truly numerosity tuned responses emerged after lateral occipital cortex and were independent of retinotopic location. We propose numerositys straightforward perception and neural responses may have built on behaviorally beneficial spatial frequency analyses in simpler animals.

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satmut_utils: a simulation and variant calling package for multiplexed assays of variant effect BackgroundThe impact of thousands of individual genetic variants on molecular phenotypes for disease-relevant genes remains unknown. Multiplexed assays for variant effect (MAVEs) are highly scalable methods to annotate the relevant variants. However, current software methods for analyzing MAVEs lack standardized annotation, can require cumbersome configuration, and do not easily scale to large target regions.

ResultsHere, we present satmut_utils as a flexible solution for 1) simulation of saturation mutagenesis data; and 2) quantification of variants across four orders of magnitude from multiplexed assay data. Improvements of satmut_utils over existing solutions include support for multiple experimental strategies, unique molecular identifier-based consensus deduplication, and machine learning-based error correction. We developed a rigorous simulation workflow to validate the performance of satmut_utils and carried out the first benchmarking of existing software for variant calling. Finally, we used satmut_utils to determine the mRNA abundance of thousands of coding variants in cystathionine beta-synthase (CBS) by two library preparation methods. We identified an association between variants near chemical cofactor binding sites and decreased mRNA abundance. We also found a correlation between codon optimality and the magnitude of variant effects, emphasizing the potential of single-nucleotide variants to alter mRNA abundance.

Conclusionssatmut_utils enables high-performance analysis of saturation mutagenesis data, achieves unprecedented specificity through novel error correction approaches, and reveals the capability of single-codon variants to alter mRNA abundance in native coding sequences.

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Integrating genomics and multi-platform metabolomics enables metabolite QTL detection in breeding-relevant apple germplasm Research ConductedApple (Malus x domestica) has commercial and nutritional value, but breeding constraints of tree crops limit varietal improvement. Marker-assisted selection minimizes these drawbacks, but breeders lack applications for targeting fruit phytochemicals. To understand genotype-phytochemical associations in apples, we have developed a high-throughput integration strategy for genomic and multi-platform metabolomics data.

Methods124 apple genotypes, including members of three pedigree-connected breeding families alongside diverse cultivars and wild selections, were genotyped and phenotyped. Metabolite genome-wide association studies (mGWAS) were conducted with 10,000 single nucleotide polymorphisms and phenotypic data acquired via LC-MS and 1H NMR untargeted metabolomics. Putative metabolite quantitative trait loci (mQTL) were then validated via pedigree-based analyses (PBA).

Key ResultsUsing our developed method, 519, 726, and 177 putative mQTL were detected in LC-MS positive and negative ionization modes and NMR, respectively. mQTL were indicated on each chromosome, with hotspots on linkage groups 16 and 17. A chlorogenic acid mQTL was discovered on chromosome 17 via mGWAS and validated with a two-step PBA, enabling discovery of novel candidate gene-metabolite relationships.

Main ConclusionComplementary data from three metabolomics approaches and dual genomics analyses increased confidence in validity of compound annotation and mQTL detection. Our platform demonstrates the utility of multi-omics integration to advance data-driven, phytochemicalbased plant breeding.

sentences: A coarse-grained NADH redox model enables inference of subcellular metabolic fluxes from fluorescence lifetime imaging Mitochondrial metabolism is of central importance to diverse aspects of cell and developmental biology. Defects in mitochondria are associated with many diseases, including cancer, neuropathology, and infertility. Our understanding of mitochondrial metabolism in situ and dysfunction in diseases are limited by the lack of techniques to measure mitochondrial metabolic fluxes with sufficient spatiotemporal resolution. Herein, we developed a new method to infer mitochondrial metabolic fluxes in living cells with subcellular resolution from fluorescence lifetime imaging of NADH. This result is based on the use of a generic coarse-grained NADH redox model. We tested the model in mouse oocytes and human tissue culture cells subject to a wide variety of perturbations by comparing predicted fluxes through the electron transport chain (ETC) to direct measurements of oxygen consumption rate. Interpreting the FLIM measurements of NADH using this model, we discovered a homeostasis of ETC flux in mouse oocytes: perturbations of nutrient supply and energy demand do not change ETC flux despite significantly impacting NADH metabolic state. Furthermore, we observed a subcellular spatial gradient of ETC flux in mouse oocytes and found that this gradient is primarily a result of heterogeneous mitochondrial proton leak. We concluded from these observations that ETC flux in mouse oocytes is not controlled by energy demand or supply, but by the intrinsic rates of mitochondrial respiration.

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Prophages in the infant gut are largely induced, and may be functionally relevant to their hosts. BackgroundBacteriophages are the most abundant biological entity on the planet, and are key components of any ecosystem they are present in. The gut virome is increasingly being implicated in disease states although these studies largely focus on lytic phages in adults. Here we identify prophages from a large infant cohort and investigate their potential functions.

ResultsWe identified 10645 vOTUs from 662 metagenomes. No core virome was found: the most prevalent vOTU was identified in 70% of the samples. The most abundant and prevalent group of phages are a novel group closely related to Bacteroides phage Hanky p00. Functional annotation of this group revealed the presence of genes in the dDTP-L-rhamnose pathway, possibly involved in the production of capsular polysaccharides. We also found an abundance of diversity generating retroelements in the phages. Additionally, paired virome data allowed us to show that the majority of prophages are induced in at least one sample and that this is not affected by the use of antibiotics in the 4 weeks prior to sampling.

ConclusionsProphages in the infant gut are largely unique to the individual and not shared. Most of them appear to be induced and so may be key drivers in shaping the bacterial microbiome. The most abundant group of phages are novel, and possess elements that may allow them to maintain differentially susceptible subpopulations of their host bacterium; whilst also containing diversity generating retroelements that could expand their host range. Prophages are important components of the infant gut that may have far reaching influences on the composition and function of the microbiome.

sentences: IDENTIFYING PLANT GENES SHAPING MICROBIOTA COMPOSITION IN THE BARLEY RHIZOSPHERE A prerequisite to exploiting soil microbes for sustainable crop production is the identification of the plant genes shaping microbiota composition in the rhizosphere, the interface between roots and soil. Here we used metagenomics information as an external quantitative phenotype to map the host genetic determinants of the rhizosphere microbiota in wild and domesticated genotypes of barley, the fourth most cultivated cereal globally. We identified a small number of loci with a major effect on the composition of rhizosphere communities. One of those, designated the QRMC-3HS locus, emerged as a major determinant of microbiota composition. We then subjected soil-grown sibling lines harbouring contrasting alleles at QRMC-3HS and hosting contrasting microbiotas to comparative root RNA-seq profiling. This allowed us to identify three primary candidate genes, including a Nucleotide-Binding-Leucine-Rich-Repeat (NLR) gene in a region of structural variation of the barley genome. Our results provide novel insights into the footprint of crop improvement on the plants capacity of shaping rhizosphere microbes.

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Decoding COVID-19 mRNA Vaccine Immunometabolism in Central Nervous System: human brain normal glial and glioma cells by Raman imaging The paper presents the effect of COVID-19 mRNA (Pfizer/BioNT) vaccine on in vitro glial cells of the brain studied by means of Raman spectroscopy and imaging.. The results obtained for human brain normal and tumor glial cells of astrocytes, astrocytoma, glioblastoma incubated with the Covid-19 mRNA vaccine Pfizer/BioNT vaccine show alterations in the reduction-oxidation pathways associated with Cytochrome c.

We found that the Pfizer/BioNT vaccine down regulate the concentration of cytochrome c in mitochondria upon incubation with normal and tumorous glial cells. Concentration of oxidized form of cytochrome c in brain cells has been shown to decrease upon incubation the mRNA vaccine. Lower concentration of oxidized cytochrome c results in lower effectiveness of oxidative phosphorylation (respiration), reduced apoptosis and lessened ATP production. Alteration of Amide I concentration, which may reflect the decrease of mRNA adenine nucleotide translocator. Moreover, mRNA vaccine leads to alterations in biochemical composition of lipids that suggest the increasing role of signaling. mRNA vaccine produce statistically significant changes in cell nucleus due to histone alterations. The results obtained for mitochondria, lipid droplets, cytoplasm may suggest that COVID-19 mRNA (Pfizer/BioNT) vaccine reprograms immune responses. The observed alterations in biochemical profiles upon incubation with COVID-19 mRNA in the specific organelles of the glial cells are similar to those we observe for brain cancer vs grade of aggressiveness.

sentences: Extensive recombination-driven coronavirus diversification expands the pool of potential pandemic pathogens The ongoing SARS-CoV-2 pandemic is the third zoonotic coronavirus identified in the last twenty years. Enzootic and epizootic coronaviruses of diverse lineages also pose a significant threat to livestock, as most recently observed for virulent strains of porcine epidemic diarrhea virus (PEDV) and swine acute diarrhea-associated coronavirus (SADS-CoV). Unique to RNA viruses, coronaviruses encode a proofreading exonuclease (ExoN) that lowers point mutation rates to increase the viability of large RNA virus genomes, which comes with the cost of limiting virus adaptation via point mutation. This limitation can be overcome by high rates of recombination that facilitate rapid increases in genetic diversification. To compare dynamics of recombination between related sequences, we developed an open-source computational workflow (IDPlot) to measure nucleotide identity, locate recombination breakpoints, and infer phylogenetic relationships. We analyzed recombination dynamics among three groups of coronaviruses with noteworthy impacts on human health and agriculture: SARSr-CoV, Betacoronavirus-1, and SADSr-CoV. We found that all three groups undergo recombination with highly diverged viruses from sparsely sampled or undescribed lineages, which can disrupt the inference of phylogenetic relationships. In most cases, no parental origin of recombinant regions could be found in genetic databases, suggesting that much coronavirus diversity remains unknown. These patterns of recombination expand the genetic pool that may contribute to future zoonotic events. Our results also illustrate the limitations of current sampling approaches for anticipating zoonotic threats to human and animal health.

sentences: Automated strain separation in low-complexity metagenomes using long reads High-throughput short-read metagenomics has enabled large-scale species-level analysis and functional characterization of microbial communities. Microbiomes often contain multiple strains of the same species, and different strains have been shown to have important differences in their functional roles. Despite this, strain-level resolution from metagenomic sequencing remains challenging. Recent advances on long-read based methods enabled accurate assembly of bacterial genomes from complex microbiomes and an as-yet-unrealized opportunity to resolve strains. Here we present Strainberry, a metagenome assembly method that performs strain separation in single-sample low-complexity metagenomes and that relies uniquely on long-read data. We benchmarked Strainberry on mock communities and showed it consistently produces strain-resolved assemblies with near-complete reference coverage and 99.9% base accuracy. We also applied Strainberry on real datasets for which it improved assemblies generating 20-118% additional genomic material than conventional metagenome assemblies on individual strain genomes. Our results hence demonstrate that strain separation is possible in low-complexity microbiomes using a single regular long read dataset. We show that Strainberry is also able to refine microbial diversity in a complex microbiome, with complete separation of strain genomes. We anticipate this work to be a starting point for further methodological improvements aiming to provide better strain-resolved metagenome assemblies in environments of higher complexities.

sentences: A novel juxtamembrane basolateral targeting motif regulates TGF-β receptor signaling in Drosophila In polarized epithelial cells, receptor-ligand interactions can be restricted by different spatial distributions of the two interacting components, giving rise to an underappreciated layer of regulatory complexity. We explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue that requires the TGF-{beta} family member Dpp for growth and patterning. Dpp protein has been observed in a gradient within the columnar cells of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of two distinctly localized pools. We find the Dpp type II receptor Punt, but not the type I receptor Tkv, is enriched at the basolateral membrane, and depleted at the junctions and apical surface. Wit, a second type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified the BLT, a short juxtamembrane sequence required for basolateral targeting of Punt in both wing discs and mammalian MDCK cells, and that dominantly confers basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility and significant lethality. We also show that apicalized Punt does not produce an ectopic signal, indicating that the apical pool of Dpp is not a significant signaling source even when presented with Punt. Finally, we present evidence that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.

sentences: Dynamic neural reconstructions of attended object location and features using EEG We live in a dynamic world and our environment contains far more stimuli than our brain can process at one time. Visual attention allows us to select relevant information and ignore irrelevant information. What happens when attention is shifted from one item to another? To answer this question, it is critical to have tools that can accurately recover neural representations of both feature and location information, and do so across shifts of attention with high temporal resolution. In the current study, we used human electroencephalography (EEG) and machine learning methods to explore how neural representations of object features and locations update across dynamic shifts of attention. We demonstrate that EEG can be used to create simultaneous timecourses of the neural representations of attended features (timepoint-by-timepoint inverted encoding model reconstructions) and attended location (timepoint-by-timepoint decoding) during both stable periods and across dynamic spatial shifts of attention. On each trial two oriented gratings were presented that flickered at the same frequency but had different orientations; participants were cued to attend to one of them, and on half of the trials received a shift cue in the middle of the trial. We trained the models on a stable period from Hold attention trials, and then reconstructed/decoded the attended orientation/location information at each timepoint on Shift attention trials. Our results showed that both feature reconstructions and location decoding dynamically track the shift of attention, and that there may be timepoints during the shifting of attention when (1) feature and location representations become uncoupled, and (2) both the previously-attended and currently-attended orientations are represented with roughly equal strength. The results offer insight into our understanding of attentional shifts, and the noninvasive techniques developed in the current study lend themselves well to a wide variety of future applications.

sentences: Virucidal and antiviral activity of astodrimer sodium against SARS-CoV-2 in vitro An effective response to the ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will involve a range of complementary preventive modalities. The current studies were conducted to evaluate the in vitro SARS-CoV-2 antiviral and virucidal activity of astodrimer sodium, a dendrimer with broad spectrum antimicrobial activity, including against enveloped viruses in in vitro and in vivo models, that is marketed for antiviral and antibacterial applications. We report that astodrimer sodium inhibits replication of SARS-CoV-2 in Vero E6 and Calu-3 cells, with 50% effective concentrations (EC50) for i) reducing virus-induced cytopathic effect of 0.002 to 0.012 mg/mL in Vero E6 cells, and ii) infectious virus release by plaque assay of 0.019 to 0.031 mg/mL in Vero E6 cells and 0.031 to 0.045 mg/mL in Calu-3 cells. The selectivity index (SI) in these assays was as high as 2197. Astodrimer sodium was also virucidal, reducing SARS-CoV-2 infectivity by >99.9% (>3 log10) within 1 minute of exposure, and up to >99.999% (>5 log10) shown at astodrimer sodium concentrations of 10 to 30 mg/mL in Vero E6 and Calu-3 cell lines. Astodrimer sodium also inhibited infection in a primary human airway epithelial cell line. The data were similar for all investigations and were consistent with the potent antiviral and virucidal activity of astodrimer sodium being due to inhibition of virus-host cell interactions, as previously demonstrated for other viruses. Further studies will confirm if astodrimer sodium binds to SARS-CoV-2 spike protein and physically blocks initial attachment of the virus to the host cell. Given the in vitro effectiveness and significantly high SI, astodrimer sodium warrants further investigation for potential as a nasally administered or inhaled antiviral agent for SARS-CoV-2 prevention and treatment applications.

sentences: An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we developed a novel liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detected 679 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes.

sentences: Efferent control of hearing sensitivity and protection via inner ear supporting cells It is well-known that medial olivocochlear (MOC) efferent nerves innervate outer hair cells (OHCs) and inhibit OHC electromotility to control hearing sensitivity. Here, we report that the MOC nerve fibers associated with MOC neurotransmitter acetylcholine (ACh) receptors and presynaptic vesicular acetylcholine transporters (VAChT) also innervated the cochlear supporting cells (SCs). Application of ACh could evoke inward currents in SCs and reduced gap junctions (GJs) between them with long-lasting effect. We further found that uncoupling of GJs between SCs shifted OHC electromotility to the left hyperpolarization direction and enhanced the direct effect of ACh on OHC electromotility. Diminution of this control by deficiency of GJs between SCs compromised the regulation of active cochlear amplification and increased susceptibility to noise. These data suggest that the MOC efferent system has functional innervations with SCs. Such efferent innervation may have an important role in the protection of hearing from noise and slow efferent effect.

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In vitro reconstituted quorum sensing pathways enable rapid evaluation of quorum sensing inhibitors Quorum sensing, as inner- or inter-species microbial communication process orchestrated by diffusible autoinducers, typically results in collective pathogenic behaviours, being recognized as a promising druggable target for anti-virulence. Here, we reconstituted las and rhl quorum sensing pathways of Pseudomonas aeruginosa, mediated by acyl-homoserine lactones (AHLs) and LuxI/LuxR-family proteins, with fluorescence output in Escherichia coli cell-free expression system, offering a platform to rapidly evaluate quorum sensing inhibitors (QSIs) in vitro. Previously reported small-molecule quorum sensing inhibitors for interfering with P. aeruginosa quorum sensing systems were tested and showed mild to high on-target inhibition as well as off-target toxicity. Of note, quercetin displayed potent on-target inhibition to quorum sensing pathways as well as acceptable off-target toxicity to cell-free expression machinery. Upon our work, cell-free platform is anticipated to further facilitate automated and high-throughput drug screening, bridge in silico and in vivo drug-screening methods, and accelerate the upgrading of antimicrobial arsenal.

GRAPHICAL ABSTRACT

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The effects of an 8-week mindful eating intervention on anticipatory reward responses in striatum and midbrain Obesity is a highly prevalent disease, usually resulting from chronic overeating. Accumulating evidence suggests that increased neural responses during the anticipation of high-calorie food play an important role in overeating. A promising method for counteracting enhanced food anticipation in overeating might be mindfulness-based interventions (MBIs). However, the neural mechanisms by which MBIs can affect food reward anticipation are unclear. In this randomized, actively controlled study, the primary objective was to investigate the effect of an 8-week mindful eating intervention on reward anticipation. On the neural level, we hypothesized that mindful eating would decrease striatal reward anticipation responses. Additionally, responses in the midbrain - from which the reward pathways originate - were explored. Using functional magnetic resonance imaging (fMRI), we tested 58 healthy participants with a wide body mass index range (BMI: 19-35 kg/m2), motivated to change their eating behavior. During scanning they performed an incentive delay task, measuring neural reward anticipation responses to caloric and monetary cues before and after 8 weeks of mindful eating or educational cooking (active control). Compared with the educational cooking intervention, mindful eating affected neural reward anticipation responses, with relatively reduced caloric versus monetary reward responses. This effect was, however, not seen in the striatum, but only in the midbrain. The secondary objective was to assess temporary and long-lasting (one year follow-up) intervention effects on self-reported eating behavior and anthropometric measures (BMI, waist circumference, waist-to-hip-ratio (WHR)). We did not observe effects of the mindful eating intervention on eating behavior. Instead, the control intervention showed temporary beneficial effects on BMI, waist circumference, and diet quality, but not on WHR or self-reported eating behavior, as well as long-lasting increases in knowledge about healthy eating. These results suggest that an 8-week mindful eating intervention may have decreased the relative salience of food cues by affecting midbrain but not striatal reward responses. However, these exploratory results should be verified in confirmatory research.

The primary and secondary objectives of the study were registered in the Dutch Trial Register (NTR): NL4923 (NTR5025).

sentences: Azithromycin Plus Zinc Sulfate Rapidly and Synergistically Suppresses IκBα-Mediated In Vitro Human Airway Cell ACE2 Expression for SARS-CoV-2 Entry Large-scale efforts have been persistently undertaken for medical prophylaxis and treatment of COVID-19 disasters worldwide. A variety of novel viral spike protein-targeted vaccine preparations have recently been clinically distributed based on accelerated approval. We revisited the early but inconclusive clinical interest in the combination of azithromycin and zinc sulfate repurposing with safety advantages. In vitro proof of concept was provided for rapid and synergistic suppression of ACE2 expression following treatments in human airway cells, Calu-3 and H322M. The two representative ACE2-expressing human airway cells indicate the upper and lower respiratory tracts. Prophylactic and early therapeutic roles of azithromycin combined with zinc are proposed for virus cellular entry prevention potential bridging to effective antibody production.

sentences: Microbial helpers allow cyanobacteria to thrive in ferruginous waters The Great Oxidation Event (GOE) was a rapid accumulation of oxygen in the atmosphere as a result of the photosynthetic activity of cyanobacteria. This accumulation reflected the pervasiveness of O2 on the planets surface, indicating that cyanobacteria had become ecologically successful in Archean oceans. Micromolar concentrations of Fe2+ in Archean oceans would have reacted with hydrogen peroxide, a byproduct of oxygenic photosynthesis, to produce hydroxyl radicals, which cause cellular damage. Yet cyanobacteria colonized Archean oceans extensively enough to oxygenate the atmosphere, which likely required protection mechanisms against the negative impacts of hydroxyl radical production in Fe2+-rich seas. We identify several factors that could have acted to protect early cyanobacteria from the impacts of hydroxyl radical production and hypothesize that microbial cooperation may have played an important role in protecting cyanobacteria from Fe2+ toxicity before the GOE. We found that several strains of facultative anaerobic heterotrophic bacteria (Shewanella) with ROS defense mechanisms increase the fitness of cyanobacteria (Synechococcus) in ferruginous waters. Shewanella species with manganese transporters provided the most protection. Our results suggest that a tightly regulated response to prevent Fe2+ toxicity could have been important for the colonization of ancient ferruginous oceans, particularly in the presence of high manganese concentrations, and may expand the upper bound for tolerable Fe2+ concentrations for cyanobacteria.

sentences: Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing1-3. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary4,5. Chemical labeling-based single-cell approaches introduce hundreds of cells into the MS, but direct analysis of single cells has not yet reached the necessary sensitivity, robustness and quantitative accuracy to answer biological questions6,7. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography and a novel trapped ion mobility mass spectrometer, resulting in a more than ten-fold improved sensitivity. We accurately and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators such as CDK2NA, the E2 ubiquitin ligase UBE2S, DNA topoisomerases TOP2A/B and the chromatin regulator HMGA1. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable core proteome despite perturbation, while the transcriptome appears volatile. This emphasizes substantial regulation of translation and sets the stage for its elucidation at the single cell level. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material8, posttranslational modifications and small molecule studies to gain unprecedented insights into cellular heterogeneity in health and disease.

sentences: Targeting of Protein Kinase CK2 in Acute Myeloid Leukemia Cells Using the Clinical-Grade Synthetic-Peptide CIGB-300 Protein kinase CK2 has emerged as an attractive therapeutic target in acute myeloid leukemia (AML), advent that becomes particularly relevant since the treatment of this hematological neoplasia remains challenging. Here we explored for the first time the effect of the clinical-grade peptide-based CK2 inhibitor CIGB-300 on AML cells proliferation and viability. CIGB-300 internalization and subcellular distribution were also studied, and the role of B23/nucleophosmin 1 (NPM1), a major target for the peptide in solid tumors, was addressed by knock-down in model cell lines. Finally, pull-down experiments and phosphoproteomic analysis were performed to study CIGB-interacting proteins and identify the array of CK2 substrates differentially modulated after treatment with the peptide. Importantly, CIGB-300 elicited a potent anti-proliferative and proapoptotic effect in AML cells, with more than 80% of peptide transduced cells within three minutes. Unlike solid tumor cells, NPM1 did not appear to be a major target for CIGB-300 in AML cells. However, in vivo pull-down experiments and phosphoproteomic analysis evidenced that CIGB-300 targeted the CK2 catalytic subunit, different ribosomal proteins, and inhibited the phosphorylation of a common CK2 substrates array among both AML backgrounds. Remarkably, our results not only provide cellular and molecular insights unveiling the complexity of the CIGB-300 anti-leukemic effect in AML cells, but also reinforce the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy.

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A uniform benchmark for testing ssrA-derived degrons in the Escherichia coli ClpXP pathway The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder developments of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches.

SUMMARYThis work lays standards for assays used to compare engagement of SspB and ClpXP by a set of ssrA-derived degrons that can be used to fine-tune tools for protein stability control.

sentences: Plasma Membrane Calcium ATPase (PMCA) Regulates Stoichiometry of CD4+ T-cell Compartments Immune responses involve mobilization of T cells within naive and memory compartments. Tightly regulated Ca2+ levels are essential for balanced immune outcomes. How Ca2+ contributes to regulating compartment stoichiometry is unknown. Here, we show that plasma membrane Ca2+ ATPase 4 (PMCA4) is differentially expressed in human CD4+ T compartments yielding distinct store operated Ca2+ entry (SOCE) profiles. Modulation of PMCA4 yielded a more prominent increase of SOCE in memory than in naive CD4+ T cell. Interestingly, downregulation of PMCA4 reduced the effector compartment fraction and led to accumulation of cells in the naive compartment. In silico analysis and chromatin immunoprecipitation unraveled Ying Yang 1 (YY1) as a transcription factor regulating PMCA4 expression. Analyses of PMCA and YY1 expression patterns following activation and of PMCA promoter activity following downregulation of YY1 highlight repressive role of YY1 on PMCA expression. Our findings show that under the transcriptional control by YY1, PMCA4 adapts Ca2+ levels to cellular requirements during effector and quiescent phases and thereby represent a potential target to intervene with the outcome of the immune response.

sentences: Cell lineage as a predictor of immune response in neuroblastoma Immunotherapy for patients with neuroblastoma has met with limited success, partly due to an incomplete understanding of the mechanisms underlying immune responsiveness in this clinically and genetically heterogenic tumor. Here, we undertook an unbiased analysis using dimension reduction and UMAP visualization of transcriptional signatures derived from 498 primary neuroblastoma tumors. Four distinct clusters based on differentially expressed genes emerged, of which one, representing about 30% and comprising mainly of MYCN-nonamplified tumors, was notable for the high expression of genes associated with both immune response activation and suppression. This capacity to elicit a productive immune response resided exclusively in tumors with dominant populations of undifferentiated, neural crest-like or mesenchymal cells; by contrast, tumors comprising primarily of committed, adrenergic neuron-like cells were less immunogenic. Mesenchymal neuroblastoma cells were enriched for innate and adaptive immune gene signatures, demonstrated engagement with cytotoxic T and natural killer cells, and induced immune cell infiltration in an immunocompetent mouse model. Transcriptional or targeted therapy-induced reprogramming of adrenergic cells to the mesenchymal state led to reactivation of tumor cell-intrinsic immune genes. Key immune response genes in adrenergic tumor cells were found to be epigenetically silenced by the PRC2 complex, and such repression could be relieved by either mesenchymal cell state reprogramming or EZH2 inhibition, leading to increased activation of natural killer cells by the tumor cells. These data identify cell lineage as a major determinant of the immunogenic potential in neuroblastoma that could be used to stratify patients who are most likely to benefit from immunotherapy.

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Label-free Cell Tracking Enables Collective Motion Phenotyping in Epithelial Monolayers Collective cell migration is an umbrella term for a rich variety of cell behaviors, whose distinct character is essential for biological function, notably for cancer metastasis. One essential feature of collective behavior is the motion of cells relative to their immediate neighbors. We introduce an AI-based pipeline to segment and track cell nuclei from phase contrast images. Nuclei segmentation is based on a U-Net convolutional neural network trained on images with nucleus staining. Tracking, based on the Crocker-Grier algorithm, quantifies nuclei movement and allows for robust downstream analysis of collective motion. Since the AI algorithm required no new training data, our approach promises to be applicable to and yield new insights for vast libraries of existing collective motion images. In a systematic analysis of a cell line panel with oncogenic mutations, we find that the collective rearrangement metric, D2min, which reflects non-affine motion, shows promise as an indicator of metastatic potential.

Graphical Abstract

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C_LIO_LIAnalysis of motion of cells relative to nearby neighbors distinguishes weakly tumorigenic (KRas) and metastatic (KRas/PTEN-/-) cell lines.
C_LI

sentences: Ecological diversity exceeds evolutionary diversity in model ecosystems Understanding community saturation is fundamental to ecological theory. While investigations of the diversity of evolutionary stable states (ESSs) are widespread, the diversity of communities that have yet to reach an evolutionary endpoint is poorly understood. We use Lotka-Volterra dynamics and trait-based competition to compare the diversity of randomly assembled communities to the diversity of the ESS. We show that, with a large enough founding diversity (whether assembled at once or through sequential invasions), the number of long-time surviving species exceeds that of the ESS. However, the excessive founding diversity required to assemble a saturated community increases rapidly with the dimension of phenotype space. Additionally, traits present in communities resulting from random assembly are more clustered in phenotype space compared to random, though still markedly less ordered than the ESS. By combining theories of random assembly and ESSs we bring a new viewpoint to both the saturation and random assembly literature.

sentences: Evaluation of entomopathogenic nematodes against red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) The entomopathogenic nematodes play a pivotal role as bio-control agents of different species of insect pests including red palm weevil. In current investigations, infective capabilities of four species of entomopathogenic nematodes including Hetrerorhabditis bacteriophora, Steinernema feltiae, Steinernema glesri and Steinernema carpocapsae were evaluated against larvae, pupae and adult red palm weevil, under laboratory and field conditions. The pathogenic potential of selected nematode species was assessed based on dissection and adult emergence of weevils. Results indicated that S. carpocapsae and H. bacteriophora with 94.68 and 92.68% infection were found most effective EPN species against red palm weevil larvae. Focusing the adult emergence, aforementioned EPNs were found most pathogenic when pupae of red palm weevil were treated and resulted into 83.60 and 80.20% infested pupae. It was noted that the adult emergence is the better option for the evaluation of pathogenic potential of EPN compared to dissection of insect. The S. carpocapsae was found most effective against 6th instar larvae of red palm weevil and caused 100% mortality at 340 hours after treatment; whereas; S. glesri and S. feltiae were found least pathogenic and caused 70 and 76% mortality. All the evaluated nematode species were found highly infective under field conditions. The S. carpocapsae was found most pathogenic causing 83.60% mortality of red palm weevil. Overall; the tested nematodes were found most effective against larvae followed by adult weevils. The tested nematode species were found least effective against pupae of red palm weevil. Based on current findings, it is concluded that the tested species of nematodes can be used as sustainable option for the management of red palm weevil.

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Oiling the gears of memory: quercetin exposure during memory formation, consolidation, and recall enhances memory in Lymnaea stagnalis Memory formation (short-term, intermediate-term, and long-term) is an integral process of cognition which allows individuals to retain important information and is influenced by various intrinsic and extrinsic factors. A major extrinsic factor influencing cognition across taxa is diet, which may contain rich sources of molecular agents with antioxidant, anti-inflammatory, and memory enhancing properties that potentially enhance cognitive ability. A common and abundant flavonoid present in numerous food substances is quercetin (Q) which is also known to upregulate cyclic AMP response element binding protein (CREB) in several animals including our model system Lymnaea stagnalis. Since CREB is known to be involved in long term memory (LTM) formation, we investigated the role of Q-exposure on memory formation, consolidation, and recall during operant conditioning of aerial respiratory behaviour in Lymnaea. Snails were exposed to Q 3h before or after training to ascertain its effects on LTM. Additionally, we investigated the effect of the combined presentation of a single reinforcing stimulus (at 24h post-training or 24h before training) and Q-exposure on both LTM formation and reconsolidation. Our data indicate that Q-exposure acts on the different phases of memory formation, consolidation, and recall leading to enhanced LTM formation.

Summary StatementQuercetin enhances long-term memory formation acting on the different phases of memory formation, consolidation, and recall.

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Cryptic survival and an unexpected recovery of the long-tailed mayfly Palingenia longicauda (Olivier, 1791) (Ephemeroptera: Palingeniidae) in Southeastern Europe. O_LIOnce widespread in the large European rivers, Palingenia longicauda underwent a drastic range contraction as a result of the intense pollution and hydromorphological interventions of the 19th and 20th centuries. For the last decades it was considered to be restricted only to the Tisa River and its tributaries, and to the Raba River, but new reports indicated its presence in the Danube River in Hungary, in the Danube Delta in Romania and Ukraine, and in the Prut River in the Republic of Moldova.
C_LIO_LIThe objective of this study is to analyze the phylogeographic pattern between the two main eco-regions (Pannon and Pontic) of the species distribution, based on the combined mitochondrial COI (472 bp) and 16S (464 bp) sequences generated for individuals collected in Romania and Ukraine, and from publicly available ones, representing the Tisa catchment populations.
C_LIO_LIThe presence of viable populations in the Danube Delta and on the Prut River in Romania is confirmed, and additional presence on the Mure{square} and Bega rivers from Romania, and on the Styr and Horyn rivers in Northern Ukraine is shown.
C_LIO_LIThe phylogeographic results indicate that the presence of the analyzed populations are not the result of recent founding events from the Pannon region, confirming the survival and expansion of cryptic local lineages.
C_LIO_LIThe recent recovery of the species may be related to the improvement of water quality as a result of the implementation of the EU Water Framework Directive and the EU Floods Directive after 2000.
C_LI

sentences: Functional analysis of Ost3p and Ost6p containing yeast oligosaccharyltransferases The oligosaccharyltransferase (OST) is the central enzyme in the N-glycosylation pathway. It transfers a defined oligosaccharide from a lipid-linker onto the asparagine side chain of proteins. The yeast OST consists of eight subunits and exists in two catalytically distinct isoforms that differ in one subunit, Ost3p or Ost6p. The cryo-electron microscopy structure of the Ost6p containing complex was found to be highly similar to the Ost3p containing OST. OST enzymes with altered Ost3p/Ost6p subunits were generated and functionally analysed. The three C-terminal transmembrane helices were responsible for the higher turnover-rate of the Ost3p vs. the Ost6p containing enzyme in vitro and the more severe hypoglycosylation in Ost3p lacking strains in vivo. Glycosylation of specific OST target sites required the N-terminal thioredoxin domain of Ost3p or Ost6p. This Ost3p/Ost6p dependence was glycosylation site but not protein specific. We concluded that the Ost3p/Ost6p subunits modulate the catalytic activity of OST and provide additional specificity for OST substrate recognition.

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Calprotectin-mediated zinc chelation inhibits Pseudomonas aeruginosa protease activity in cystic fibrosis sputum Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to control cultures, but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc-starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to post-translationally inhibit zinc metalloprotease activity and thereby impact the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment.

ImportanceThe factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc-starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.

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ProteInfer: deep networks for protein functional inference Predicting the function of a protein from its amino acid sequence is a long-standing challenge in bioinformatics. Traditional approaches use sequence alignment to compare a query sequence either to thousands of models of protein families or to large databases of individual protein sequences. Here we instead employ deep convolutional neural networks to directly predict a variety of protein functions - EC numbers and GO terms - directly from an unaligned amino acid sequence. This approach provides precise predictions which complement alignment-based methods, and the computational efficiency of a single neural network permits novel and lightweight software interfaces, which we demonstrate with an in-browser graphical interface for protein function prediction in which all computation is performed on the users personal computer with no data uploaded to remote servers. Moreover, these models place full-length amino acid sequences into a generalised functional space, facilitating downstream analysis and interpretation. To read the interactive version of this paper, please visit https://google-research.github.io/proteinfer/

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/461077v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@74f8f1org.highwire.dtl.DTLVardef@183ae0borg.highwire.dtl.DTLVardef@1754f3org.highwire.dtl.DTLVardef@1ca54f5_HPS_FORMAT_FIGEXP  M_FIG C_FIG QR code for the interactive version of this preprint at https://google-research.github.io/proteinfer/

sentences: Wide-Field Multicontrast Nonlinear Microscopy for Histopathology A multicontrast polarimetric wide-field second harmonic generation (SHG) and multiphoton excitation fluorescence (MPF) microscope is optimized for large area imaging of hematoxylin and eosin (H&E) stained and unstained histology slides. The bleaching kinetics of MPF and SHG are examined with various laser intensities at different pulse repetition rates to determine the optimal wide-filed imaging conditions for H&E stained histology slides. Several polarimetric parameters are used to investigate the organization of extracellular matrix collagen in the histology samples.

sentences: Defining kinetic roles of transcriptional activators in the early Drosophila embryo Most animal transcription factors are categorized as activators or repressors without specifying their mechanisms of action. Defining their specific roles is critical for deciphering the logic of transcriptional regulation and predicting the function of regulatory sequences. Here, we define the kinetic roles of three activating transcription factors in the Drosophila embryo--Zelda, Bicoid and Stat92E--by introducing their binding sites into the even skipped stripe 2 enhancer and measuring transcriptional output with live imaging. We find that these transcription factors act on different subsets of kinetic parameters, and these subsets can change over the course of nuclear cycle (NC) 14. These transcription factors all increase the fraction of active nuclei. Zelda dramatically shortens the time interval between the start of NC 14 and initial activation, and Stat92E increases the duration of active transcription intervals throughout NC 14. Zelda also decreases the time intervals between instances of active transcription early in NC 14, while Stat92E does so later. Different transcription factors therefore play distinct kinetic roles in activating transcription; this has consequences for understanding both regulatory DNA sequences as well as the biochemical function of transcription factors.

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Phosphorylation of myosin A regulates Plasmodium sporozoite motility and is essential for efficient malaria transmission Malaria-causing parasites rely on an actin-myosin based motor for the invasion of different host cells as well as tissue traversal in mosquitoes and vertebrates. The unusual myosin A of Plasmodium spp. has a unique N-terminal extension which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19. Here, using the rodent-infecting P. berghei we show that serine 19 is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry and migration of salivary gland sporozoites in both 2D and 3D environments. Our data suggests that entry into salivary glands represents the strongest barrier in parasite transmission and hence is the key determinant for evolution of the motility and invasion machinery of these parasites.

HighlightsThe unusual N-terminal extension of Plasmodium myosin A is important for efficient gliding motility

Altering the kinetics of the myosin A power stroke impacts Plasmodium life cycle progression and sporozoite motility

Myosin A phosphorylation at serine 19 is important for malaria transmission by mosquitoes

Salivary gland invasion emerges as key selection step for evolution of the parasite motor

sentences: Predicting microbiome compositions through deep learning. Microbes can form complex communities that perform critical functions in maintaining the integrity of their environment or their hosts well-being. Rationally managing these microbial communities requires improving our ability to predict how different species assemblages affect the final species composition of the community. However, making such a prediction remains challenging because of our limited knowledge of the diverse physical, biochemical, and ecological processes governing microbial dynamics. To overcome this challenge, here we present a deep learning framework that automatically learns the map between species assemblages and community compositions from training data only, without knowledge of any of the above processes. First, we systematically validate our framework using synthetic data generated by classical population dynamics models. Then, we apply it to experimental data of both in vitro and in vivo communities, including ocean and soil microbial communities, Drosophila melanogaster gut microbiota, and human gut and oral microbiota. In particular, we show how our framework learns to perform accurate out-of-sample predictions of complex community compositions from a small number of training samples. Our results demonstrate how deep learning can enable us to understand better and potentially manage complex microbial communities.

sentences: Comprehensive dissection of immune microenvironment in the progression of early gastric cancer at spatial and single-cell resolution While the changes of tumor immune microenvironment (TME) have critical implications for most tumor progression, works that could reveal the compositions and immunity features of TME are needed. Profiling gastric malignant cells at single-cells resolution has shown the transcriptional heterogeneity is represented at different states of gastric cancer, implying that diverse cell states may exist, including immune cells, and all components in TME make some balances in early gastric cancer (EGC) progression. However, it remains unclear how immune cells contributing malignancy of gastritis, constituting general characteristics of gastric TME. Furthermore, the role of specific interactions among cells in gastric TME remains to be illustrated. Here, we performed spatial transcriptomes and single-cell RNA-seq analysis across 18 gastric samples, identifying 17 celltypes and reconstructing their location information. We found that immune cells represented different degree of dysregulations during the progression from non-atrophic gastritis (NAG), atrophic gastritis (AG) to EGC, including imbalance of cytotoxic and inhibitory effects in T cells, maturation inhibition in B cells and malignant genes up-regulated obviously in myeloid cells. Besides, pathway activities showed that hypoxia, reactive oxygen species and fatty metabolism signaling were activated from AG stage, which may accelerate progression of EGC. Moreover, cellular interactions further identified the roles of hypoxia in gastric TME. Overall, the multi-omics data presented in this study offer a comprehensive view of immune cell types, states changes and locations within the gastric tissues during the progression from NAG, AG to EGC, advancing our understanding of the composition and immunity of different gastric states, offering diagnostic and preventive thoughts for EGC.

sentences: A dentate gyrus-CA3 inhibitory circuit promotes evolution of hippocampal-cortical ensembles during memory consolidation Memories encoded in the dentate gyrus (DG) - CA3 circuit of the hippocampus are routed from CA1 to anterior cingulate cortex (ACC) for consolidation. Although CA1 parvalbumin inhibitory neurons (PV INs) orchestrate hippocampal-cortical communication, we know less about CA3 PV INs or DG - CA3 principal neuron - IN circuit mechanisms that contribute to evolution of hippocampal-cortical ensembles during memory consolidation. Using viral genetics to selectively mimic and boost an endogenous learning-dependent circuit mechanism, DG cell recruitment of CA3 PV INs and feed-forward inhibition (FFI) in CA3, in combination with longitudinal in vivo calcium imaging, we demonstrate that FFI facilitates formation and maintenance of context-associated neuronal ensembles in CA1. Increasing FFI in DG - CA3 promoted context specificity of neuronal ensembles in ACC over time and enhanced long-term contextual fear memory. In vivo LFP recordings in mice with increased FFI in DG - CA3 identified enhanced CA1 sharp-wave ripple - ACC spindle coupling as a potential network mechanism facilitating memory consolidation. Our findings illuminate how FFI in DG - CA3 dictates evolution of ensemble properties in CA1 and ACC during memory consolidation and suggest a teacher-like function for hippocampal CA1 in stabilization and re-organization of cortical representations.

sentences: The Phtheirospermum japonicum isopentenyltransferase PjIPT1a regulates host cytokinin responses in Arabidopsis The hemiparasitic plant Phtheirospermum japonicum is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyse the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analysed the subcellular localization, the enzymatic function, and contribution to inducing hypertrophy by studying CRISPR-Cas9 induced Phtheirospermum mutants. PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed isopentenyltransferases activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a with shared characteristics was also identified in the related weed Striga hermonthica. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.

sentences: Depletion of Sun1/2 Induces Heterochromatin Accrual in Mesenchymal Stem Cells during Adipogenesis Critical to the mechano-regulation of mesenchymal stem cells (MSC), Linker of the Nucleoskeleton and Cytoskeleton (LINC) complex transduces cytoskeletal forces to the nuclei. The LINC complex contains outer nuclear membrane Nesprin proteins that associate with the cytoskeleton and their inner nuclear membrane couplers, Sun proteins. In addition to coupling Nesprin-associated cytoskeletal elements to inner nuclear membrane, Sun proteins also function in regulating nuclear mechanics and chromatin tethering to inner nuclear membrane. This suggests that release of LINC-mediated cytoskeletal connections from cell nuclei may have different effects on chromatin organization and MSC differentiation than those due to ablation of intra-nuclear Sun proteins. To test this hypothesis we compared Sun1/2 depletion with expression of the dominant-negative KASH domain (dnKASH) that inhibits Nesprin-Sun association. In cells cultured under adipogenic conditions, disconnecting LINC from the cytoskeleton through dnKASH expression, increased adipogenic gene expression and fat droplet formation; heterochromatin H3K27 and H3K9 methylation was unaltered. In contrast, Sun1/2 depletion inhibited adipogenic gene expression and fat droplet formation; as well the anti-adipogenic effect of Sun1/2 depletion was accompanied by increased H3K9me3, which was enriched on Adipoq, silencing this fat locus. We conclude that releasing the nucleus from cytoskeletal constraints via dnKASH accelerates adipogenesis while depletion of Sun1/2 increases heterochromatin accrual on adipogenic genes in a fashion independent of LINC complex function. Therefore, while these two approaches both disable LINC functions, their divergent effects on the epigenetic landscape indicate they cannot be used interchangeably to study mechanical regulation of cell differentiation.

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The asgardarchaeal-unique contribution to protein families of the eukaryotic common ancestor was 0.3% Significance StatementEver since the first report of a new archaeal lineage, the asgardarchaea, their metagenome analyses have encouraged continued speculations on a type of cell biology ranging between that of prokaryotes and eukaryotes. While it appears a tempting notion, recent microscopic images of an asgardarchaeon suggest otherwise. We inspected the origin of eukaryotic protein families with respect to their distribution across bacteria and archaea. This reveals that the protein families shared exclusively between asgardarchaea and eukaryotes amounts to only 0.3% of the protein families conserved across all eukaryotes. Asgardarchaeal diversity is likely unrivaled across archaea, but their cell biology remains prokaryotic in nature and lends support for the importance of endosymbiosis in evolving eukaryotic traits.

SummaryThe difference between pro- and eukaryotic biology is evident in their genomes, cell biology, and evolution of complex and macroscopic body plans. The lack of intermediates between the two types of cells places the endosymbiotic acquisition of the mitochondrion through an archaeal host at the event horizon of eukaryote origin. The identification of eukaryote specific proteins in a new archaeal phylum, the asgardarchaea, has fueled speculations about their cellular complexity, suggesting they could be eukaryote-like. Here we analyzed the coding capacity of 150 eukaryotes, 1000 bacteria, and 226 archaea, including the only cultured member of the asgardarchaea, Candidatus Prometheoarchaeon syntrophicum MK-D1. Established clustering methods that recover endosymbiotic contributions to eukaryotic genomes, recover an asgardarchaeal-unique contribution of a mere 0.3% to protein families present in the last eukaryotic common ancestor, while simultaneously suggesting that asgardarchaeal diversity rivals that of all other archaea combined. Furthermore, we show that the number of homologs shared exclusively between asgardarchaea and eukaryotes is only 27 on average. Genomic and in particular cellular complexity remains a eukaryote-specific feature and, we conclude, is best understood as the archaeal hosts solution to housing an endosymbiont and not as a preparation for obtaining one.

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Accurate assignment of disease liability to genetic variants using only population data PurposeThe growing size of public variant repositories prompted us to test the accuracy of predicting pathogenicity of DNA variants using population data alone.

MethodsUnder the a priori assumption that the ratio of the prevalence of variants in healthy and affected populations form two distinct distributions (pathogenic and benign), we used a Bayesian method to assign probability of a variant belonging to either distribution.

ResultsThe approach, termed BayPR, accurately parsed 300 of 313 expertly curated cystic fibrosis transmembrane conductance regulator (CFTR) variants: 284 of 296 pathogenic/likely pathogenic (P/LP) variants in one distribution and 16 of 17 benign/likely benign (B/LB) variants in another. BayPR produced an area under the receiver operating curve (AUC) of 0.99 for 103 functionally-confirmed missense CFTR variants, equal to or exceeding ten commonly used algorithms (AUC range: 0.54 to 0.99). Application of BayPR to expertly curated variants in eight genes associated with seven Mendelian conditions assigned [≥]80% disease-causing probability to 1,350 of 1,374 (98.3%) P/LP variants and [≤]20% to 22 of 23 (95.7%) B/LB variants.

ConclusionAgnostic to variant type or functional effect, BayPR provides probabilities of pathogenicity for DNA variants responsible for Mendelian disorders using only variant counts in affected and unaffected population samples.

sentences: The molecular organization of flat and curved caveolae indicates bendable structural units at the plasma membrane. Caveolae are small coated inner plasma membrane invaginations found in many cell types. Their diverse functions span from endocytosis to signaling, regulating key cellular processes including lipid uptake, pathogen entry, and membrane tension. Caveolae undergo shape changes from flat to curved. It is unclear which proteins regulate this process. To address this gap, we studied the shapes of caveolae with platinum replica electron microscopy in six common cell types. Next, we developed a correlative multi-color stimulated emission depletion (STED) fluorescence and platinum replica EM imaging (CLEM) method to image caveolae-associated proteins at caveolae of different shapes at the nanoscale. Caveolins and cavins were found at all caveolae, independent of their curvature. EHD2, a classic caveolar neck protein, was strongly detected at both curved and flat caveolae. Both pacsin2 and the regulator EHBP1 were found only at a subset of caveolae. Pacsin2 was localized primarily to areas surrounding flat caveolae, whereas EHBP1 was mostly detected at spheres. Contrary to classic models, dynamin was absent from caveolae and localized only to clathrin-coated structures. Cells lacking dynamin showed no substantial changes to caveolae, suggesting that dynamin is not directly involved in caveolae curvature. Together, we provide a mechanistic map for the molecular control of caveolae shape by eight of the major caveolae-associated coat and regulatory proteins. We propose a model where caveolins, cavins, and EHD2 assemble as a cohesive structural unit regulated by more intermittent associations with pacsin2 and EHBP1. These complexes can flatten and curve, capturing membrane to enable lipid traffic and changes to the surface area of the cell.

sentences: Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the media, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3){Delta} pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylations state of proteins when heterologously expressed, since their biochemical and physiological properties are dependent on post-translational modification.

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Impacts of dams on freshwater turtles: a global review to identify conservation solutions Background and Research AimsDams impact freshwater ecosystems and biodiversity. Freshwater turtles are at direct and indirect risk due to changes caused by damming including the loss of terrestrial and aquatic nesting habitats, changes to food availability and blocking movement. Effective management of these impacts requires robust evidence in order to gain an understanding of conservation solutions that work.

MethodsWe reviewed the global scientific literature that evaluated the impact of dams on freshwater turtles, and carried out additional searches of literature published in seventeen languages for studies evaluating actions to mitigate dam impacts.

ResultsThe search produced 47 published articles documenting dam impacts on 30 freshwater turtle species from seven families (Chelidae, Chelydridae, Emydidae, Geoemydidae, Kinosternidae, Podocnemididae and Trionychidae) in 13 countries. Few studies were found from Europe and Asia and none from Africa. Most studies were from temperate latitudes, where studies focused more on adults and less threatened species compared with tropical latitudes. More than half of the studies (57%, n = 27) suggested actions to help mitigate dam impacts. Yet, only five studies (three temperate and two tropical) documented the effect of interventions (dam removal, flow management, artificial pond maintenance and community-based action).

ConclusionThese findings demonstrate a serious lack of documented evidence evaluating mitigation actions for dam impacts on freshwater turtles. Implications for Conservation: This lack of evidence reinforces the importance of strengthening and maintaining robust long-term studies needed to develop effective and adaptive conservation actions for this group of threatened vertebrates particularly in tropical regions.

sentences: Mechanism of negative membrane curvature generationby I-BAR domains The membrane sculpting ability of BAR domains has been attributed to the intrinsic curvature of their banana-shaped dimeric structure. However, there is often a mismatch between this intrinsic curvature and the diameter of the membrane tubules generated. I-BAR domains have been especially mysterious: they are almost flat but generate high negative membrane curvature. Here, we use atomistic implicit-solvent computer modeling to show that the membrane bending of the IRSP53 I-BAR domain is dictated by its higher oligomeric structure, whose curvature is completely unrelated to the intrinsic curvature of the dimer. Two other I-BARs gave similar results, whereas a flat F-BAR sheet developed a concave membrane binding interface, consistent with its observed positive membrane curvature generation. Laterally interacting helical spirals of I-BAR dimers on tube interiors are stable and have an enhanced binding energy that is sufficient for membrane bending to experimentally observed tubule diameters at a reasonable surface density.

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Publicly available transcriptomes provide the opportunity for dual RNA-Seq meta analysis in Plasmodium infection Dual RNA-Seq is the simultaneous transcriptomic analysis of interacting symbionts, for example, in malaria. Potential cross-species interactions identified by correlated gene expression might highlight interlinked signaling, metabolic or gene regulatory pathways in addition to physically interacting proteins. Often, malaria studies address one of the interacting organisms - host or parasite - rendering the other "contamination". Here we perform a meta-analysis using such studies for cross-species expression analysis.

We screened experiments for gene expression from host and Plasmodium. Out of 171 studies in Homo sapiens, Macaca mulatta and Mus musculus, we identified 63 potential studies containing host and parasite data. While 16 studies (1950 samples) explicitly performed dual RNA-Seq, 47 (1398 samples) originally focused on one organism. We found 915 experimental replicates from 20 blood studies to be suitable for co-expression analysis and used orthologs for meta-analysis across different host-parasite systems. Centrality metrics from the derived gene expression networks correlated with gene essentiality in the parasites. We found indications of host immune response to elements of the Plasmodium protein degradation system, an antimalarial drug target. We identified well-studied immune responses in the host with our co-expression networks as our approach recovers known broad processes interlinked between hosts and parasites in addition to individual host and parasite protein associations.

The set of core interactions represents commonalities between human malaria and its model systems for prioritization in laboratory experiments. Our approach might also allow insights into the transferability of model systems for different pathways in malaria studies.

ImportanceMalaria still causes about 400,000 deaths a year and is one the most studied infectious diseases. The disease is studied in mice and monkeys as lab models to derive potential therapeutic intervention in human malaria. Interactions between Plasmodium spp. and its hosts are either conserved across different host-parasite systems or idiosyncratic to those systems. Here we use correlation of gene expression from different RNA-Seq studies to infer common host-parasite interactions across human, mouse and monkey studies. We, firstly, find a set of very conserved interactors, worth further scrutiny in focussed laboratory experiments. Secondly, this work might help assess to which extent experiments and knowledge on different pathways can be transferred from models to humans for potential therapy.

sentences: Schizosaccharomyces pombe KAT5 contributes to resection and repair of a DNA double strand break Chromatin remodeling is essential for effective repair of a DNA double strand break. KAT5 (S. pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that coordinates various DNA damage response activities at a DNA double strand break (DSB), including histone remodeling and activation of the DNA damage checkpoint. In S. pombe, mutations in mst1+ causes sensitivity to DNA damaging drugs. Here we show that Mst1 is recruited to DSBs. Mutation of mst1+ disrupts recruitment of repair proteins and delays resection. These defects are partially rescued by deletion of pku70, which has been previously shown to antagonize repair by homologous recombination. These phenotypes of mst1 are similar to pht1-4KR, a non-acetylatable form of histone variant H2A.Z, which has been proposed to affect resection. Our data suggest that Mst1 functions to direct repair of DSBs towards homologous recombination pathways by modulating resection at the double strand break.

sentences: Genetic admixture history and forensic characteristics of Tibeto-Burman-speaking Qiang people explored via the newly developed Y-STR panel and genome-wide SNP data Fine-scale patterns of population genetic structure and diversity of ethnolinguistically diverse populations are important for biogeographical ancestry inference, kinship testing and also for the development and validation of new kits focused on forensic personal identification. Analyses focused on forensic markers and genome-wide SNP data can provide new insights into the origin, admixture processes and forensic characteristics of targeted populations. Qiang people with a large sample size among Tibeto-Burman-speaking populations widely reside in the middle latitude of the Tibetan Plateau. However, their genetic structure and forensic features have remained uncharacterized due to the paucity of comprehensive genetic analyses. Here, we first developed and validated the AGCU-Y30 Y-STR panel, which contains slowly and moderately mutating Y-STRs, and then we conducted comprehensive population genetic analyses based on Y-STRs and genome-wide SNPs to explore the admixture history of Qiang people and their neighbours. The validated results of this panel showed that the new Y-STR kit was sensitive and robust enough for forensic applications. Haplotype diversity (HD) ranging from 0.9932 to 0.9996 and allelic frequencies ranging from 0.001946 to 0.8326 in 514 Qiang people demonstrated that all included markers were highly polymorphic in Tibeto-Burman people. Population genetic analyses based on Y-STRs (RST, FST, MDS, NJ, PCA and MJNs) revealed that the Qiang people harboured a paternally close relationship with lowland Tibetan-Yi corridor populations. Furthermore, we made a comprehensive population admixture analysis among Eurasian modern and ancient populations based on the shared alleles. We determined that the Qiang people were a genetically admixed population and showed the closest relationship with Tibetan and Neolithic Yellow River farmers. Admixture modelling showed that Qiang people shared the primary ancestry with Tibetan and was derived from North China, supporting the hypothesis of common origin between Tibetan and Qiang people.

sentences: Synthetic Nanobody-Functionalized Nanoparticles for Accelerated Development of Rapid, Accessible Detection of Viral Antigens Successful control of emerging infectious diseases requires accelerated development of fast, affordable, and accessible assays to be widely implemented at a high frequency. Here we present a generalizable assay platform, nanobody-functionalized nanoparticles for rapid, electronic detection (Nano2RED), demonstrated in the detection of Ebola and COVID-19 antigens. To efficiently generate high-quality affinity reagents, synthetic nanobody co-binders and mono-binders with high affinity, specificity, and stability were selected by phage display screening of a vastly diverse, rationally randomized combinatorial library, bacterially expressed and site-specifically conjugated to gold nanoparticles (AuNPs) as multivalent in-solution sensors. Without requiring fluorescent labelling, washing, or enzymatic amplification, these AuNPs reliably transduce antigen binding signals upon mixing into physical AuNP aggregation and sedimentation processes, displaying antigen-dependent optical extinction readily detectable by spectrometry or simple electronic circuitry. With nanobodies against an Ebola virus secreted glycoprotein (sGP) and a SARS-CoV-2 spike protein receptor binding domain (RBD) as targets, Nano2RED showed a high sensitivity (limit of detection of [~]10 pg/mL for sGP and [~]40 pg/mL for RBD in diluted human serum), a high specificity, and a large dynamic range ([~]7 logs). Unlike conventional assays where slow mass transport for surface binding limits the assay time, Nano2RED features fast antigen diffusion at micrometer scale, and can be accelerated to deliver results within a few minutes. The rapid detection, low material cost (estimated < $0.01 per test), inexpensive and portable readout system (< $5 and < 100 cm3), and digital data output, make Nano2RED particularly suitable for screening of patient samples with simplified operation and accelerated data transmission. Our method is widely applicable for prototyping diagnostic assays for other antigens from new emerging viruses.

sentences: Nm-Nano: Predicting 2'-O-methylation (Nm) Sites in Nanopore RNA Sequencing Data Nm (2-O-methylation) is one of the most abundant modifications of mRNAs and non-coding RNAs occurring when a methyl group (-CH3) is added to the 2 hydroxyl (-OH) of the ribose moiety. This modification can appear on any nucleotide (base) regardless of the type of nitrogenous base, because each ribose sugar has a hydroxyl group and so 2-O-methyl ribose can occur on any base. Nm modification has a great contribution in many biological processes such as the normal functioning of tRNA, the protection of mRNA against degradation by DXO, and the biogenesis and specificity of rRNA. Recently, the single-molecule sequencing techniques for long reads of RNA sequences data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications on the molecule that is being sequenced, but to our knowledge there was only one research attempt that applied this technology to predict the stoichiometry of Nm-modified sites in RNA sequence of yeast cells. To this end, in this paper, we extend this research direction by proposing a bio-computational framework, Nm-Nano for predicting Nm sites in Nanopore direct RNA sequencing reads of human cell lines. Nm-Nano framework integrates two supervised machine learning (ML) models for predicting Nm sites in Nanopore sequencing data, namely Xgboost and Random Forest (RF). Each model is trained with set of features that are extracted from the raw signal generated by the Oxford Nanopore MinION device, as well as the corresponding basecalled k-mer resulting from inferring the RNA sequence reads from the generated Nanopore signals. The results on two benchmark data sets generated from RNA Nanopore sequencing data of Hela and Hek293 cell lines show a great performance of Nm-Nano. In independent validation testing, Nm-Nano has been able to identify Nm sites with a high accuracy of 93% and 88% using Xgboost and RF models respectively by training each model with Hela benchmark dataset and testing it for identifying Nm sites on Hek293 benchmark dataset. Thus, Nm-Nano outperforms the Nm sites predictors existing in the literature (not relying on Nanopore technology) that were only limited to predict Nm sites on short reads of RNA sequences and unable to predict Nm sites on long RNA sequence reads. By deploying Nm-Nano to predict Nm sites in Hela cell line, it was revealed that a total of 196 genes was identified to have the most abundance of Nm modification among all other genes that have been modified by Nm in this cell line. Similarly, deploying Nm-Nano to predict Nm sites in Hek393 cell line revealed that a total of 196 genes line was identified to have the most abundance of Nm modification among all other genes that have been modified by Nm in this cell line. According to this, a significant enrichment of a wide range of functional processes like high confidences (adjusted p-val < 0.05) enriched ontologies that were more representative of Nm modification role in immune response and cellular homeostasis were revealed in Hela cell line, and "MHC class 1 protein complex", "mitotic spindle assembly", "response to glucocorticoid", and "nucleocytoplasmic transport" were revealed in Hek293 cell line. The source code of Nm-Nano can be freely accessed at https://github.com/Janga-Lab/Nm-Nano.

sentences: Locally-adaptive inversions modulate genetic variation at different geographic scales in a seaweed fly Across a species range, multiple sources of environmental heterogeneity, at both small and large scales, create complex landscapes of selection, which may challenge adaptation, particularly when gene flow is high. One key to multidimensional adaptation may reside in the heterogeneity of recombination along the genome. Structural variants, like chromosomal inversions, reduce recombination, increasing linkage disequilibrium among loci at a potentially massive scale. In this study, we examined how chromosomal inversions shape genetic variation across a species range, and ask how their contribution to adaptation in the face of gene flow varies across geographic scales. We sampled the seaweed fly Coelopa frigida along a bioclimatic gradient stretching across 10{degrees} of latitude, a salinity gradient and a range of heterogeneous, patchy habitats. We generated a chromosome-level genome assembly to analyse 1,446 low-coverage whole genomes collected along those gradients. We found several large non-recombining genomic regions, including putative inversions. In contrast to the collinear regions, inversions and low recombining regions differentiated populations more strongly, either along an ecogeographic cline or at a fine-grained scale. These genomic regions were associated with environmental factors and adaptive phenotypes, albeit with contrasting patterns. Altogether, our results highlight the importance of recombination in shaping adaptation to environmental heterogeneity at local and large scales.

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Characteristics and data reporting of rare disease clinical trials: Getting better but still room for improvement BackgroundIt is estimated that there are more than 7,000 rare diseases (RDs) worldwide, impacting the lives of approximately 400 million people and only 5% have an approved therapy. Facing special challenges, including patient scarceness, incomplete knowledge of the natural history and only few specialized clinical sites, clinical trials (CT) are limited, making the data from trials critical for research and clinical care. Despite the introduction of the U.S. Food and Drug Administration Amendment Act (FDAAA) in 2007 requiring certain CTs to post results on the registry ClinicalTrials.gov within 12 months following completion, compliance has been reportedly poor. Here, we describe general characteristics of RD CTs, identify trends, and evaluate result reporting practices under the FDAAA aiming to draw awareness to the problem of non-compliance.

MethodsCTs conducted between 2008 and 2015 were extracted from the public U.S. trial registry ClinicalTrials.gov using the text mining software I2E (Linguamatics). Disease names were matched with rare disease names from the Orphanet Rare Disease Ontology (ORDO, v2.5, Orphanet). Statistical analyses and data visualization were performed using GraphPad Prism 7 and R (v3.5). The Students t-test was employed to calculate significance using p-value cut-offs of <0.05 or <0.001.

ResultsWe analyzed 1,056 RD CTs of which 55.7% were phase 2, 7.7% phase 2/3 and 36.7% phase 3 trials. The studies were mostly one- and two-armed experimental CTs with the majority (60.2%) being funded by industry. Cystic fibrosis and sickle cell disease represented the most frequently investigated diseases (25.0% and 16.5%). Industry-led phase 2 RD CTs were significantly (p<0.0001) shorter than their equivalent led by academia/non-profit (22 vs. 33 months). Screening CTs completed before the end of 2015, we found that of the 725 analyzed studies, 55.2% predominantly phase 2 CTs, did not report results. Taking their potential applicability to the FDAAA into account, 25.2% industry-funded and 28.0% academia/non-profit-funded trials failed to disclose results on ClinicalTrial.gov.

ConclusionRD CTs tend to be comparatively small, industry-funded studies focusing on genetic and neurologic conditions. Sponsor-related differences in study design, duration, and enrollment were observed. There are still substantial shortcomings when it comes to result publication.

sentences: High school science fair: Experiences of two groups of undergraduate bioscience students Science fairs offer potential opportunities for students to learn first-hand about the practices of science. Over the past six years we have been carrying out voluntary and anonymous surveys with regional and national groups of high school and post high school students to learn about their high school science fair experiences regarding help received, obstacles encountered, and opinions about the value and impact of science fair. Understanding what students think about science fairs will help educators make science fairs more effective learning opportunities. In this paper, we focus on the findings with two national groups of post high school students - undergraduate research fellows (SURF students) who did research at UT Southwestern Medical Center during 2014-2019 and undergraduates biology students attending the 2019 Howard Hughes Medical Institute Science Education Alliance (SEA) summer symposium. About 25% of the students who completed surveys indicated that they had participated in high school science fair, but more than half attended high schools where science fairs were unavailable. Effectively, 6 out of every 10 students participated in science fair if available. Students who could have participated in high school science fair but chose not to do so identified not enough time and coming up with their project idea as major reasons why not. About half the SURF students favored requiring non-competitive science fair regardless whether they themselves had participated in science fair. On the other hand, less than 1 in 5 thought that competitive science fair should be required. Introduction to the scientific process and general learning were mentioned most frequently as the reasons to require non-competitive science fair; these reasons were mentioned rarely in connection with competitive science fair. Unlike the national cohort of high school students we surveyed previously, who mostly did science fair in 9th and 10th grades, SURF students participated in science fair throughout high school and were twice as likely as high school students to have carried out science fair more than once. In conclusion, our findings suggest that participation of the undergraduate bioscience majors in high school science fairs occurs far more frequently than recognized previously. The findings emphasize further the importance of incentivizing rather than requiring science fair participation, especially in 9th and 10th grades, and the potential value of developing non-competitive science fairs.

sentences: Insights into Long-term Acclimation Strategies of Grapevines in Response to Multi-decadal Cyclical Drought The Australian wine industry is currently under pressure to sustain its profitability due to climate change. Therefore, there is a pressing need to explore grapevine genetic diversity and identify superior clones with improved drought resistance. We previously characterised more than 15,000 dry-farmed (for over 65 years) Cabernet Sauvignon clones in a vineyard and identified three drought-tolerant (DT) clones, which can maintain significantly higher intrinsic water use efficiency (WUEi) under limited soil moisture than drought-sensitive (DS) clones. To understand whether DT clones grown under multi-decadal cyclical drought can prime their vegetatively-propagated clonal progenies for future drought events, in this study, all DT and DS vegetative progenies were propagated with commercial clones in the glasshouse. Their physiological and molecular responses were investigated under well-watered and two recurrent drought (D1 and D2) conditions. We observed that concentration of a natural priming agent, {gamma}-amino butyric acid (GABA), were significantly higher in all DT progenies relative to other progenies under drought. Both commercial and DT progenies exhibited improved gas exchange, photosynthetic performance and WUEi under recurrent drought events relative to DS progenies. Our results suggest that DT progenies have adapted to be in a "primed state" to withstand future drought events.

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Combined effects of 12-week yoga warm-up on athletic performance in male high school track and field athletes AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSPracticing yoga could improve balance and flexibility, but its positive significance as a long-term warm-up for formal training was uncertain. We hypothesized that practicing yoga during warm-up might positively affect balance, flexibility, and speed performance in male high school track and field athletes.

MethodsOver a 12-week period, athletes in a yoga group (YG) (n=10) practiced yoga for 15 min 4 times a week during warm-up, while athletes in a dynamic stretching group (DSG) (n=10) practiced 15 min of dynamic stretching. Except for the warm-up activities, the training content of the two groups of students was the same. we tested performance indicators immediately before and after the intervention, including lower extremity flexibility test (right hip active flexion range), lower extremity balance test [using surface electromyography (sEMG) to measure right leg tibialis anterior (TA) activation during one-leg stance (OLS) with eyes closed], and speed performance test (100-meter and 800-meter tests).

ResultsWe performed between-group and within-group comparisons for indicators of two groups by using SPSS (version 26.0). Within-group comparisons showed a significant improvement in flexibility (P=0.002) and balance (P=0.003) in YG, but no significant change in DSG, after the 12-week intervention. In addition, speed performance of both YG (100m, P=0.026; 800m, P=0.045) and DSG (100m, P=0.029; 800m, P=0.006) was significantly improved. Between-group comparison showed that YG had a significant advantage in 800m (P=0.045) and flexibility (P=0.031).

ConclusionsThese data suggested that practicing yoga as a long-term warm-up could help male high school track and field athletes improve lower body flexibility and 800m speed. In addition, yoga had a certain positive effect on balance, but it was not significant overall.

sentences: Elucidation of SARS-CoV-2 budding mechanisms through molecular dynamics simulations of M and E protein complexes SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratorys refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics which inhibit coronavirus budding.

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Best templates outperform homology models in predicting the impact of mutations on protein stability MotivationPrediction of protein stability change upon mutation ({Delta}{Delta}G) is crucial for facilitating protein engineering and understanding of protein folding principles. Robust prediction of protein folding free energy change requires the knowledge of protein three-dimensional (3D) structure. Unfortunately, protein 3D structure is not always available. In this case, one can still predict the protein stability change by constructing a homology model of the protein; however, the accuracy of homology model-based {Delta}{Delta}G predictions is unknown. The perspectives of using 3D structures of the best templates are also unclear.

ResultsTo investigate these questions, we used the most popular and accurate publicly available tools: FoldX for stability change prediction and I-Tasser for homology modeling. We found that both homology models and best templates worsen the {Delta}{Delta}G prediction, with best templates performing 1.5 times better than homology models. For AlphaFold models, we also found that the best templates seem to outperform protein models. Our findings imply using the 3D structures of the best templates for {Delta}{Delta}G prediction if the 3D protein structure is unavailable.

Contactd.ivankov@skoltech.ru

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uniForest: an unsupervised machine learning technique to detect outliers and restrict variance in microbiome studies Isolation Forests is an unsupervised machine learning technique for detecting outliers in continuous datasets that does not require an underlying equivariant or Gaussian distribution and is suitable for use on small datasets. While this procedure is widely used across quantitative fields, to our knowledge, this is the first attempt to solely assess its use for microbiome datasets. Here we present uniForest, an interactive Python notebook (which can be run from any desktop computer using the Google Colaboratory web service) for the processing of microbiome outliers. We used uniForest to apply Isolation Forests to the Healthy Human Microbiome project dataset and imputed outliers with the mean of the remaining inliers to maintain sample size and assessed its prowess in variance reduction in both community structure and derived ecological statistics (-diversity). We also assessed its functionality in anatomical site differentiation (pre- and postprocessing) using principal component analysis, dissimilarity matrices, and ANOSIM. We observed a minimum variance reduction of 81.17% across the entire dataset and in alpha diversity at the Phylum level. Application of Isolation Forests also separated the dataset to an extremely high specificity, reducing variance within taxa samples by a minimum of 81.33%.

It is evident that Isolation Forests are a potent tool in restricting the effect of variance in microbiome analysis and has potential for broad application in studies where high levels of microbiome variance is expected. This software allows for clean analyses of otherwise noisy datasets.

sentences: Single-cell RNA sequencing unveils intestinal eosinophil development and specialization Eosinophils are an integral part of the gastrointestinal (GI) immune system that contribute to homeostatic and inflammatory processes. Here, we investigated the existence of functional subsets that carry out specialized tasks in health and disease. We used single-cell transcriptomics and high-dimensional flow cytometry to delineate murine eosinophil subpopulations and their ontogenetic relationship in the steady state and during infection and inflammation. Profiling of eosinophils from bone marrow, blood, spleen and several GI tissues revealed five distinct subsets representing consecutive developmental and maturation stages across organs, each controlled by a specific set of transcription factors. Furthermore, we discovered a highly adapted PD-L1+CD80+ eosinophil subset in the GI tract, characterized by its immune regulatory properties, bactericidal activity upon challenge infection and tissue-protective function during inflammation. Our data provide a framework for the characterization of eosinophil subsets in GI diseases and highlight their crucial contribution to homeostasis, immune regulation and host defense.

sentences: Fast and parallel nanoscale 3D tracking of heterogeneous mammalian chromatin dynamics Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel 3D tracking of multiple chromosomal loci of choice over many thousands of frames on various time scales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking of both fast dynamics (50 Hz) and over time scales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent , and a transition to more isotropic dynamics in 3D after such treatment. We think that our methodology in the future will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.

sentences: An interplay between cellular growth and atypical fusion defines morphogenesis of a modular glial niche Neural stem cells (NSCs) are embedded in a multi-layered, intricate cellular microenvironment supporting their activity, the niche. Whilst shape and function are inseparable, the morphogenetic aspects of niche development are poorly understood. Here, we use the formation of the glial network of a NSC niche to investigate acquisition of architectural complexity. Cortex glia (CG) in Drosophila regulate neurogenesis and build a reticular structure around NSCs. We first show that individual CG cells grow tremendously to ensheath several NSC lineages, eventually spanning the entire tissue while partitioning the NSC population. Elaborate proliferative mechanisms convert these cells into syncytia rich in cytoplasmic bridges. Unexpectedly, CG syncytia further undergo homotypic cell-cell fusion, relying on defined molecular players of cell fusion such as cell surface receptors and actin regulators. Exchange of cellular components is however dynamic in space and time, a previously unreported unique mechanism. This atypical cell fusion remodels cellular borders, restructuring the CG syncytia. Ultimately, the coordination of growth and fusion builds the multi-level architecture of the niche, and creates a modular, spatial partition of the NSC population. Our findings provide novel insights into how a niche forms and organises while developing intimate contacts with a stem cell population.

sentences: A Genome-Phenome Association study in native microbiomes identifies a mechanism for cytosine modification in DNA and RNA Shotgun metagenomic sequencing is a powerful approach to study microbiomes in an unbiased manner and of increasing relevance for identifying novel enzymatic functions. However, the potential of metagenomics to relate from microbiome composition to function has thus far been underutilized. Here, we introduce the Metagenomics Genome-Phenome Association (MetaGPA) study framework, which allows to link genetic information in metagenomes with a dedicated functional phenotype. We applied MetaGPA to identify enzymes associated with cytosine modifications in environmental samples. From the 2365 genes that met our significance criteria, we confirm known pathways for cytosine modifications and proposed novel cytosine-modifying mechanisms. Specifically, we characterized and identified a novel nucleic acid modifying enzyme, 5-hydroxymethylcytosine carbamoyltransferase, that catalyzes the formation of a previously unknown cytosine modification, 5-carbamoyloxymethylcytosine, in DNA and RNA. Our work introduces MetaGPA as a novel and versatile tool for advancing functional metagenomics.

sentences: MDR1 Drug Efflux Pump Promotes Intrinsic and Acquired Resistance to PROTACs in Cancer Cells PROTACs (Proteolysis-Targeting Chimeras) represent a promising new class of drugs that selectively degrade proteins of interest from cells. PROTACs targeting oncogenes are avidly being explored for cancer therapies, with several currently in clinical trials. Drug resistance represents a significant challenge in cancer therapies, and the mechanism by which cancer cells acquire resistance to PROTACs remains poorly understood. Using proteomics, we discovered acquired and intrinsic resistance to PROTACs in cancer cells can be mediated by upregulation of the drug efflux pump MDR1. PROTAC-resistant cells could be re-sensitized to PROTACs through co-administering MDR1 inhibitors. Notably, co-treatment of MDR1-overexpressing colorectal cancer cells with MEK1/2 or KRASG12C degraders and the dual ErbB receptor/MDR1 inhibitor lapatinib exhibited potent drug synergy due to simultaneous blockade of MDR1 and ErbB receptor activity. Together, our findings suggest that concurrent blockade of MDR1 will likely be required in combination with PROTACs to achieve durable protein degradation and therapeutic response in cancer.

sentences: EZH2 Abundance Regulated by UHRF1/UBE2L6/UBR4 Ubiquitin System is the Potential Therapeutic Target to Trigger Pigmented Phenotype in Melanoma Cellular heterogeneity in cancer is linked to disease progression and therapy response, although the mechanisms regulating distinct cellular states within tumours are not well understood. To address this, we identified melanin pigment content as a major source of phenotypic and functional heterogeneity in melanoma and compared RNAseq data from high (HPC) and low pigmented melanoma cells (LPC), revealing the polycomb repressor complex protein, EZH2, as a master regulator of these states. EZH2 protein, but not RNA expression, was found to be upregulated in LPCs and inversely correlated with melanin in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA strategy or DZNep, MS1943 that reduces EZH2 protein levels, significantly inhibited cell growth in LPCs by hampering ribosome biogenesis. In addition, decline in EZH2 protein level induces pigmented cell phenotype by inducing melanin biosynthesis. Proteasomal inhibitor, MG132 treatment induced EZH2 protein levels in HPCs prompted us to look for differentially regulated ubiquitin system proteins in HPC vs LPCs. UBE2L6, E2 conjugating enzyme has been shown to be downregulated significantly in LPCs by UHRF1-mediated CpG methylation. Both biochemical assays and animal studies demonstrated that UBE2L6 expression decline, in turn, promotes EZH2 protein stability due to lack of ubiquitination on K381 residue in LPCs. UBR4 cooperates with UBE2L6 to facilitate this ubiquitination process. Targeting UHRF1/UBE2L6/UBR4 axis can be a better treatment option to trigger HPC state in melanoma in which conventional EZH2 inhibitors are ineffective.

sentences: The influence of saccades on insect flight control In a way analogous to human vision, the fruit fly Drosophila melanogaster and many other flying insects generate smooth and saccadic movements to stabilize and shift their gaze in flight, respectively. It has been hypothesized that this combination of continuous and discrete movements benefits both flight stability and performance, particularly at high frequencies or speeds. Here we develop a hybrid control system model to explore the effects of saccades on the yaw stabilization reflex of D. melanogaster. Inspired from experimental data, the model includes a first order plant, a Proportional-Integral (PI) continuous controller, and a saccadic reset system that fires based on the integrated error of the continuous controller. We explore the gain, delay and switching threshold parameter space to quantify the optimum regions for stability and performance. We show that the addition of saccades to a continuous controller provides benefits to both stability and performance across a range of frequencies. Our model suggests that Drosophila operate near its optimal switching threshold for its experimental gain set. We also show that based on experimental data, D. melanogaster operates in a region that trades off performance and stability. This trade-off increases flight robustness to compensate for environmental uncertainties such as wing damage.

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A Marmoset Model for Mycobacterium avium Complex Pulmonary Disease RationaleMycobacterium avium complex, is the most common nontuberculous mycobacterial respiratory pathogen in humans. Disease mechanisms are poorly understood due to the absence of a reliable animal model for M. avium complex pulmonary disease.

ObjectiveAssess the susceptibility, immunologic and histopathologic responses of the common marmoset (Callithrix jacchus) to M. avium complex pulmonary infection.

Methods7 adult female marmosets underwent endobronchial inoculation with 108 colony-forming units of M. intracellulare and were monitored for 30 or 60 days. Prior to infection, chest radiograph and serum cytokines were assessed; serum cytokines were also monitored weekly for 30 days. At sacrifice 30 days (3 animals) or 60 days (4 animals) after infection, chest radiograph, serum and bronchoalveolar lavage cytokines, histopathology, and cultures of the bronchoalveolar lavage, lungs, liver, and kidney were analyzed.

Measurements and Main ResultsFive of seven animals (two at 30 days and three at 60 days of infection) had positive lung cultures for M. intracellulare. Extra-pulmonary cultures were positive in three animals. All animals appeared healthy throughout the study. All five animals with positive lung cultures had radiographic changes consistent with pneumonitis. At 30 days, those with M. intracellulare lung infection showed granulomatous inflammation while at 60 days there was less inflammatory change, but bronchiectasis was noted. The cytokine response in the bronchoalveolar lavage fluid was uniformly greater in the animals with positive M. intracellulare cultures than those without a productive infection with greater levels at 30-days compared to 60-days. Similarly, serum cytokines were more elevated in the animals that had positive M. intracellulare cultures compared to those without a productive infection, peaking 14-21 days after inoculation.

ConclusionEndobronchial instillation of M. intracellulare resulted in pulmonary mycobacterial infection in marmosets with a differential immune response, radiographic and histopathologic abnormalities, and an indolent course consistent with M. avium complex lung infection in humans.

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Genome sequencing of 196 Treponema pallidum strains from six continents reveals additional variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains - including 191 T. pallidum subsp. pallidum - sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

Author SummaryEach year, millions of new cases of venereal and congenital syphilis, caused by the bacterium Treponema pallidum (T. pallidum) subsp. pallidum, are diagnosed worldwide, resulting in significant morbidity and mortality. Alongside endemic circulation of syphilis in low-income countries, disease resurgence in high-income nations has underscored the need for a vaccine. Due to prior technological limitations in culturing and sequencing the organism, the extent of the genetic diversity within modern strains of T. pallidum subsp. pallidum remains poorly understood, hampering development of a broadly protective vaccine. In this study, we obtained 196 near-complete T. pallidum genomes directly from clinical swabs from eight countries across six continents. Of these, 191 were identified as T. pallidum subsp. pallidum, including 90 Nichols clade genomes. Bayesian analysis revealed a high degree of variance in mutation rate among subclades. Interestingly, a Nichols subclade with a particularly high mutation rate harbors a non-synonymous mutation in a putative DNA repair helicase. Coupling sequencing data with protein structure prediction, we identified multiple novel amino acid variants in several proteins previously identified as potential vaccine candidates. Our data help inform current efforts to develop a broadly protective syphilis vaccine.

sentences: Bone marrow adipocytes drive the development of tissue invasive Ly6Chigh monocytes during obesity During obesity and high fat-diet (HFD) feeding in mice, sustained low-grade inflammation includes not only increased pro-inflammatory macrophages in the expanding adipose tissue, but also bone marrow (BM) production of invasive Ly6Chigh monocytes. As BM adiposity also accrues with HFD, we explored the relationship between the gains in BM white adipocytes and invasive Ly6Chigh monocytes in vivo and through ex vivo paradigms. We find a temporal and causal link between BM adipocyte whitening and the Ly6Chigh monocyte surge, preceding the adipose tissue macrophage rise during HFD. Phenocopying this, ex vivo treatment of BM cells with conditioned media from BM adipocytes or from bona fide white adipocytes favoured Ly6Chigh monocyte preponderance. Notably, the switch towards the Ly6Chigh subset was preceded by monocyte metabolic reprogramming towards glycolysis, reduced oxidative potential and increased mitochondrial fission. In sum, short-term HFD changes BM cellularity, resulting in local adipocyte whitening driving a gradual increase and activation of invasive Ly6Chigh monocytes.

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Paradoxes and synergies: optimizing management of a deadly virus in an endangered carnivore O_LIPathogen management strategies in wildlife are typically accompanied by an array of uncertainties such as the efficacy of vaccines or potential unintended consequences of interventions. In the context of such uncertainties, models of disease transmission can provide critical insight for optimizing pathogen management, especially for species of conservation concern. The endangered Florida panther experienced an outbreak of feline leukemia virus (FeLV) in 2002-04, and continues to be affected by this deadly virus. Ongoing management efforts aim to mitigate the effects of FeLV on panthers, but with limited information about which strategies may be most effective and efficient.
C_LIO_LIWe used a simulation-based approach to determine optimal FeLV management strategies in panthers. We simulated use of proactive FeLV management strategies (i.e., proactive vaccination) and several reactive strategies, including reactive vaccination and test-and-removal. Vaccination strategies accounted for imperfect vaccine-induced immunity, specifically partial immunity in which all vaccinates achieve partial pathogen protection. We compared the effectiveness of these different strategies in mitigating the number of FeLV mortalities and the duration of outbreaks.
C_LIO_LIResults showed that inadequate proactive vaccination can paradoxically increase the number of disease-induced mortalities in FeLV outbreaks. These effects were most likely due to imperfect vaccine immunity causing vaccinates to serve as a semi-susceptible population, thereby allowing outbreaks to persist in circumstances otherwise conducive to fadeout. Combinations of proactive vaccination with reactive test-and-removal or vaccination, however, had a synergistic effect in reducing impacts of FeLV outbreaks, highlighting the importance of using mixed strategies in pathogen management.
C_LIO_LISynthesis and applications: Management-informed disease simulations are an important tool for identifying unexpected negative consequences and synergies among pathogen management strategies. In particular, we find that imperfect vaccine-induced immunity necessitates further consideration to avoid unintentionally worsening epidemics in some conditions. However, mixing proactive and reactive interventions can improve pathogen control while mitigating uncertainties associated with imperfect interventions.
C_LI

sentences: Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids In mammals, circadian clocks are strictly suppressed during early embryonic stages as well as pluripotent stem cells, by the lack of CLOCK/BMAL1 mediated circadian feedback loops. During ontogenesis, the innate circadian clocks emerge gradually at a late developmental stage, then, with which the circadian temporal order is invested in each cell level throughout a body. Meanwhile, in the early developmental stage, a segmented body plan is essential for an intact developmental process and somitogenesis is controlled by another cell-autonomous oscillator, the segmentation clock, in the posterior presomitic mesoderm (PSM). In the present study, focusing upon the interaction between circadian key components and the segmentation clock, we investigated the effect of the CLOCK/BMAL1 on the segmentation clock Hes7 oscillation, revealing that the expression of functional CLOCK/BMAL1 severely interferes with the ultradian rhythm of segmentation clock in induced PSM and gastruloids. RNA sequencing analysis showed that the premature expression of CLOCK/BMAL1 affects the Hes7 transcription and its regulatory pathways. These results suggest that the suppression of CLOCK/BMAL1-mediated transcriptional regulation during the somitogenesis may be inevitable for intact mammalian development.

sentences: In silico analyses of maleidride biosynthetic gene clusters Maleidrides are a family of structurally related fungal natural products, many of which possess diverse, potent bioactivities. Previous identification of several maleidride biosynthetic gene clusters, and subsequent experimental work, has determined the  core set of genes required to construct the characteristic medium-sized alicyclic ring with maleic anhydride moieties. Through genome mining, this work has used these core genes to discover ten entirely novel maleidride biosynthetic gene clusters, amongst both publicly available genomes, and encoded within the genome of the previously un-sequenced epiheveadride producer Wicklowia aquatica CBS125634. We have undertaken phylogenetic analyses and comparative bioinformatics on all known and putative maleidride biosynthetic gene clusters to gain further insights regarding these unique biosynthetic pathways.

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High-frequency oscillations and replay in a two-population model of hippocampal region CA1 Hippocampal sharp wave/ripple oscillations are a prominent pattern of collective activity, which consists of a strong overall increase of activity with onmodulated (140 - 200 Hz) ripple oscillations. Despite its prominence and its experimentally demonstrated importance for memory consolidation, the mechanisms underlying its generation are to date not understood. Several models assume that recurrent networks of inhibitory cells alone can explain the generation and main characteristics of the ripple oscillations. Recent experiments, however, indicate that in addition to inhibitory basket cells, the pattern requires in vivo the activity of the local population of excitatory pyramidal cells. Here we study a model for networks in the hippocampal region CA1 incorporating such a local excitatory population of pyramidal neurons and investigate its ability to generate ripple oscillations using extensive simulations. We find that with biologically plausible values for single neuron, synapse and connectivity parameters, random connectivity and absent strong feedforward drive to the inhibitory population, oscillation patterns similar to in vivo sharp wave/ripples can only be generated if excitatory cell spiking is triggered by short pulses of external excitation. Specifically, whereas temporally broad excitation can lead to high-frequency oscillations in the ripple range, sparse pyramidal cell activity is only obtained with pulse-like external CA3 excitation. Further simulations indicate that such short pulses could originate from dendritic spikes in the apical or basal dendrites of CA1 pyramidal cells, which are triggered by coincident spike arrivals from hippocampal region CA3. Finally we show that replay of sequences by pyramidal neurons and ripple oscillations can arise intrinsically in CA1 due to structured connectivity that gives rise to alternating excitatory pulse and inhibitory gap coding; the latter implies phases of silence in specific basket cell groups and selective disinhibition of groups of pyramidal neurons. This general mechanism for sequence generation leads to sparse pyramidal cell and dense basket cell spiking, does not rely on synfire chain-like feedforward excitation and may be relevant for other brain regions as well.

Author summaryDuring certain phases of sleep, rest and consummatory behavior the hippocampus brain area of many species, including humans, is known to intermittently generate strong high frequency oscillations. These oscillations are important for memory formation and consolidation. To date, the mechanisms underlying their generation remain incompletely understood. We find that in unstructured networks carefully designing how excitation is transmitted in the hippocampus is required for the generation of robust fast oscillations in its main output region. Broad, temporally extended excitation of cells results in unrealistic single cell activity, whereas temporally narrow input that differs from cell to cell gives rise to oscillations with realistic single cell and network activity. We show that the biophysical mechanism to generate the required temporally narrow excitation may be related to spiking events in the dendrites, which are triggered by coincident input. Our results in structured networks suggest that the interplay of hippocampal excitation and inhibition can serve as a means to generate robust sequential activity, which is thought to be crucial for memory formation and recall. The sequence generation mechanism also leads to strong high frequency oscillations with sparse excitatory cell and frequent inhibitory cell spiking, as observed in the hippocampus.

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

sentences: Defining the Immune Responses for SARS-CoV-2-Human Macrophage Interactions Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end organ malfunctions. All follow an abortive viral infection. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding consequent end-organ tissue damage.

sentences: Condensation of LINE-1 is required for retrotransposition LINE-1 (L1) is the only autonomously active retrotransposon in the human genome, and accounts for 17% of the human genome. The L1 mRNA encodes two proteins, ORF1p and ORF2p. ORF1p is a homotrimeric RNA-binding protein that plays a critical role in assembling functional L1 ribonucleoprotein (RNP) complexes. Here we show that condensation of ORF1p is required for L1 retrotransposition. Using a combination of biochemical reconstitution and live-cell imaging, we demonstrate that RNA binding, electrostatic interactions, and trimer conformational dynamics together tune the properties of ORF1p assemblies to allow for efficient L1 condensate formation in cells. Furthermore, we directly relate the dynamics of ORF1p assembly to the ability to complete the entire retrotransposon life-cycle. Mutations that prevented ORF1 condensation led to loss of retrotransposition activity, while orthogonal restoration of coiled-coil conformational flexibility rescued both condensation and retrotransposition. Based on these observations, we propose that ORF1p oligomerization on L1 RNA drives the formation of a dynamic L1 condensate that is essential for retrotransposition.

sentences: Non-clinical safety profile and pharmacodynamics of two formulations of the anti-sepsis drug candidate Rejuveinix Here, we demonstrate that the two distinct formulations of our anti-sepsis drug candidate Rejuveinix (RJX) have a very favorable safety profile in Wistar Albino rats at dose levels comparable to the projected clinical dose levels. 14-day treatment with RJX-P or RJX-B similarly elevated the day 15 tissue levels of the antioxidant enzyme superoxide dismutase (SOD) as well as ascorbic acid in both the lungs and liver in a dose-dependent fashion. The activity of SOD and ascorbic acid levels were significantly higher in tissues of RJX-P or RJX-B treated rats than vehicle-treated control rats (p <0.0001). There was no statistically significant difference between tissues SOD activity or ascorbic acid levels of rats treated with RJX-P vs. rats treated with RJX-B (p >0.05). The observed elevations of the SOD and ascorbic acid levels were transient and were no longer detectable on day 28 following a 14-day recovery period. These results demonstrate that RJX-P and RJX-B are bioequivalent relative to their pharmacodynamic effects on tissue SOD and ascorbic acid levels.

sentences: Multiple classes of bactericidal antibiotics cause DNA double strand breaks in Staphylococcus aureus Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA and other macromolecules. However, the type of DNA damage that arises and the mechanisms used by bacteria to repair it are largely unclear. We found that several different classes of antibiotic triggered dose-dependent DNA damage in Staphylococcus aureus, including some bacteriostatic drugs. Damage was heterogenous and varied in magnitude between strains. However, antibiotic-triggered DNA damage led to double strand breaks, the processing of which by the RexAB helicase/nuclease complex triggered the SOS response and reduced staphylococcal susceptibility to most of the antibacterials tested. In most cases, DNA DSBs occurred under aerobic but not anaerobic conditions, suggesting a role for ROS. We conclude that DNA double strand breaks are a common occurrence during bacterial exposure to several different antibiotic classes and that repair of this damage by the RexAB complex promotes bacterial survival.

sentences: Smc3 acetylation, Pds5 and Scc2 control the translocase activity that establishes cohesin dependent chromatin loops Chromosome spatial organization and dynamics influence DNA-related metabolic processes. SMC complexes like cohesin are essential instruments of chromosome folding. Cohesin-dependent chromatin loops bring together distal loci to regulate gene transcription, DNA repair and V(D)J recombination processes. Here we characterize further the roles of members of the cohesin holocomplex in regulating chromatin loop expansion, showing that Scc2, which stimulates cohesin ATPase activity, is essential for the translocation process required to extend DNA loop length. Eco1-dependent acetylation of Smc3 during S phase counteracts this activity through the stabilization of Pds5, to finely tune loop sizes and stability during G2. Inhibiting Pds5 in G2 leads to a strong enlargement of pre-established, stable DNA loops, in a Scc2-dependent manner. Altogether, the study strongly supports a Scc2-mediated translocation process driving expansion of DNA loops in living cells.

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Figbird: A probabilistic method for filling gaps in genome assemblies MotivationAdvances in sequencing technologies have led to sequencing of genomes of a multitude of organisms. However, draft genomes of many of these organisms contain a large number of gaps due to repeats in genomes, low sequencing coverage and limitations in sequencing technologies. Although there exist several tools for filling gaps, many of these do not utilize all information relevant to gap filling.

ResultsHere, we present a probabilistic method for filling gaps in draft genome assemblies using second generation reads based on a generative model for sequencing that takes into account information on insert sizes and sequencing errors. Our method is based on the expectation-maximization (EM) algorithm unlike the graph based methods adopted in the literature. Experiments on real biological datasets show that this novel approach can fill up large portions of gaps with small number of errors and misassemblies compared to other state of the art gap filling tools.

Availability and ImplementationThe method is implemented using C++ in a software named "Filling Gaps by Iterative Read Distribution (Figbird)", which is available at: https://github.com/SumitTarafder/Figbird.

Contactatif@cse.buet.ac.bd

Supplementary informationSupplementary data are available at Bioinformatics online.

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Associations between ADHD symptom remission and white matter microstructure: a longitudinal analysis BackgroundAttention-deficit hyperactivity disorder (ADHD) is associated with white matter (WM) microstructure. Our objective was to investigate how WM microstructure is longitudinally related to symptom remission in adolescents and young adults with ADHD.

MethodsWe obtained diffusion-weighted imaging (DWI) data from 99 participants at two time points (mean age baseline: 16.91 years, mean age follow-up: 20.57 years). We used voxel-wise Tract-Based Spatial Statistics (TBSS) with permutation-based inference to investigate associations of inattention (IA) and hyperactivity-impulsivity (HI) symptom change with fractional anisotropy (FA) at baseline, follow-up, and change between time points.

ResultsRemission of combined HI and IA symptoms was significantly associated with reduced FA at follow-up in the left superior longitudinal fasciculus and the left corticospinal tract (CST) (PFWE=0.038 and PFWE=0.044, respectively), mainly driven by an association between HI remission and follow-up CST FA (PFWE=0.049). There was no significant association of combined symptom decrease with FA at baseline or with changes in FA between the two assessments.

ConclusionsIn this longitudinal DWI study of ADHD using dimensional symptom scores, we show that greater symptom decrease is associated with lower follow-up FA in specific WM tracts. Altered FA thus may appear to follow, rather than precede, changes in symptom remission. Our findings indicate divergent WM developmental trajectories between individuals with persistent and remittent ADHD, and support the role of prefrontal and sensorimotor tracts in the remission of ADHD.

sentences: Complex Activity and Short-term Memories in Reciprocally Connected Cerebral Organoids Macroscopic axonal connections in the human brain distribute information and neuronal activity across the brain. Although brain organoid technologies have recently provided novel avenues to investigate human brain function by replicating small neuronal networks of the brain in vitro, functionality of the macroscopic connections between distant regions has not been investigated with organoids. Here, we describe the neural activity of human cerebral organoids reciprocally connected by a bundle of axons. The connected organoids produced significantly more intense and complex oscillatory activity than conventional cerebral organoids. Optogenetic manipulations revealed that the connected organoids could respond to external stimuli and maintain the elevated frequency of neuronal activity for a short term, indicating that the connected organoids exhibit plasticity as a neuronal network. Our findings highlight functional importance of inter-regional connections and suggest that connected organoids can be used as a powerful tool for investigating the development and functions of macroscopic circuits in the human brain. can allow researchers to develop and test compounds to modulate activity of in vitro neuronal network models in unprecedented ways.

sentences: Roosting ecology and the evolution of bat landing maneuvers Biomechanics is poised at the intersection of organismal form, function, and ecology, and forms a practical lens through which to investigate evolutionary linkages among these factors. We conducted the first evolutionary analysis of bat flight dynamics by examining the phylogenetic patterning of landing mechanics. We discovered that bats perform stereotyped maneuvers that are correlated with landing performance quantified as impact force, and that these are linked with roosting ecology, a critical aspect of bat biology. Our findings suggest that bat ancestors performed simple, four-limbed landings, similar to those performed by gliding mammals, and that more complex landings evolved in association with novel roost types. This explicit connection between ecology and biomechanics presents the opportunity to identify traits that are associated with a locomotor behavior of known ecological relevance, thus laying the foundation for a broader understanding of the evolution of flight and wing architecture in this extraordinarily successful mammalian lineage.

sentences: Influence of High Concentrations of Copper Sulfate on In Vitro Adventitious Organogenesis of Cucumis sativus L. The effects of different concentrations of copper sulfate (0.2 to 5 mg L-1) on in vitro callus and shoot formation of cucumber was investigated. Four-day-old cotyledon explants from the inbred line  Wisconsin 2843 and the commercial cultivars  Marketer and  Negrito were used. The results on callus-derived shoots showed that the optimal concentration of CuSO4 added to Murashige & Skoog (MS)-derived shoot induction medium containing 0.5 mg L-1 IAA and 2.5 mg L-1 BAP was 8-200 fold greater than in standard MS medium, and was genotype dependent. The highest genotypes response on shoot frequency and shoot number was achieved in this order by  Marketer,  Negrito and  Wisconsin 2843 with 1, 0.2 y 5 mg L-1 CuSO4, respectively. The genotype with the lowest control performance demanded the highest concentration of CuSO4 for its optimal morphogenic response - 6- and 10-fold more in shoot frequency and shoot number, respectively. The other cultivars registered a 2-fold increase in both variables. All explants formed callus and the response on callus extension varied among cultivars. The regression analysis showed a statistically significant relationship between shoot number and concentrations of CuSO4 and absence of association with callus extension. The present results indicate that application of specific concentrations of CuSO4 higher than in standard MS medium, increases adventitious cucumber shoot organogenesis.

sentences: Another mystery snail in the Adirondacks: DNA barcoding reveals the first record of Sinotaia cf. quadrata (Caenogastropoda: Viviparidae) from North America Alien molluscs pose a serious threat to global freshwater diversity and have been implicated in many ecosystem-altering invasion events over the past few decades. Biomonitoring surveys are therefore a key tool for ensuring biosecurity in diversity hotspots and vulnerable habitats. In this study, we use DNA barcoding to provide the first record of the viviparid, Sinotaia cf. quadrata from North America. Reciprocal monophyly and low genetic divergence (uncorrected p-distance: 0.004) with a Bellamya quadrata individual from the type region (China) provides strong support for this identification. The species was recovered as part of a routine biomonitoring survey of the Adirondack region of northern New York. Only three adults were recovered (no populations or juveniles) indicating that the discovery represents a very recent arrival. Considering the proximity of the sampling site from the massive St. Lawrence River, it is likely that S. cf. quadrata was introduced into the St. Lawrence, probably via the aquarium plant trade, and was able to spread into smaller river system in northern New York and possibly other border states. This record represents the fourth alien viviparid, the third of which is of Asian origin, that have made its way to New York waters. Future biomonitoring efforts for the upcoming summer period will involve targeted searches for S. cf. quadrata to determine the extent of its spread in the region.

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Enhanced fitness of SARS-CoV-2 variant of concern B.1.1.7, but not B.1.351, in animal models Emerging variants of concern (VOCs) drive the SARS-CoV-2 pandemic. We assessed VOC B.1.1.7, now prevalent in several countries, and VOC B.1.351, representing the greatest threat to populations with immunity to the early SARS-CoV-2 progenitors. B.1.1.7 showed a clear fitness advantage over the progenitor variant (wt-S614G) in ferrets and two mouse models, where the substitutions in the spike glycoprotein were major drivers for fitness advantage. In the "superspreader" hamster model, B.1.1.7 and wt-S614G had comparable fitness, whereas B.1.351 was outcompeted. The VOCs had similar replication kinetics as compared to wt-S614G in human airway epithelial cultures. Our study highlights the importance of using multiple models for complete fitness characterization of VOCs and demonstrates adaptation of B.1.1.7 towards increased upper respiratory tract replication and enhanced transmission in vivo.

Summary sentenceB.1.1.7 VOC outcompetes progenitor SARS-CoV-2 in upper respiratory tract replication competition in vivo.

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Reproducible Coactivation Patterns of Functional Brain Networks Reveal the Aberrant Dynamic State Transition in Schizophrenia It is well documented that massive dynamic information is contained in the resting-state fMRI. Recent studies have identified recurring states dominated by similar coactivation patterns (CAP) and revealed their temporal dynamics. However, the reproducibility and generalizability of the CAP analysis is unclear. To address this question, the effects of methodological pipelines on CAP are comprehensively evaluated in this study, including preprocessing, network construction, cluster number and three independent cohorts. The CAP state dynamics are characterized by fraction of time, persistence, counts, and transition probability. Results demonstrate six reliable CAP states and their dynamic characteristics are also reproducible. The state transition probability is found to be positively associated with the spatial similarity. Furthermore, the aberrant CAP in schizophrenia has been investigated by using the reproducible method on three cohorts. Schizophrenia patients spend less time in CAP states that involve the fronto-parietal network, but more time in CAP states that involve the default mode and salience network. The aberrant dynamic characteristics of CAP are correlated with the symptom severity. These results reveal the reproducibility and generalizability of the CAP analysis, which can provide novel insights into the neuropathological mechanism associated with aberrant brain network dynamics of schizophrenia.

HighlightsO_LIThree coactivation patterns (CAPs) pairs with opposite coactivation profiles were identified, and the between-state transition probability was positively correlated with their spatial similarity.
C_LIO_LIGood spatial and temporal reproducibility and generalizability of CAPs were achieved under varied analytic methods and independent cohorts.
C_LIO_LISchizophrenia patients showed altered temporal dynamics not only within the triple-network but also other primary and higher-order networks.
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sentences: Genomic phylogeography of the White-crowned Manakin  Pseudopipra pipra (Aves: Pipridae) illuminates a continental-scale radiation out of the Andes The complex landscape history of the Neotropics has generated opportunities for population isolation and diversification that place this region among the most species-rich in the world. Detailed phylogeographic studies are required to uncover the biogeographic histories of Neotropical taxa, to identify evolutionary correlates of diversity, and to reveal patterns of genetic connectivity, disjunction, and potential differentiation among lineages from different areas of endemism. The White-crowned Manakin (Pseudopipra pipra) is a small suboscine passerine bird that is broadly distributed through the subtropical rainforests of Central America, the lower montane cloud forests of the Andes from Colombia to central Peru, the lowlands of Amazonia and the Guianas, and the Atlantic forest of southeast Brazil. Pseudopipra is currently recognized as a single, polytypic biological species. We studied the effect of the Neotropical landscape on genetic and phenotypic differentiation within this species using genomic data derived from double digest restriction site associated DNA sequencing (ddRAD), and mitochondrial DNA. Most of the genetic breakpoints we identify among populations coincide with physical barriers to gene flow previously associated with avian areas of endemism. The phylogenetic relationships among these populations imply a novel pattern of Andean origination for this group, with subsequent diversification into the Amazonian lowlands. Our analysis of genomic admixture and gene flow reveals a complex history of introgression between some western Amazonian populations. These reticulate processes confound our application of standard concatenated and coalescent phylogenetic methods and raise the question of whether a lineage in the western Napo area of endemism should be considered a hybrid species. Lastly, analysis of variation in vocal and plumage phenotypes in the context of our phylogeny supports the hypothesis that Pseudopipra is a species-complex composed of at least 8, and perhaps up to 17 distinct species which have arisen in the last [~]2.5 Ma.

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A machine learning model for screening of body fluid cytology smears IntroductionBody fluid cytology is one of the commonest investigations performed in indoor patients, both for diagnosis of suspected carcinoma as well as staging of known carcinoma. Carcinoma is diagnosed in body fluids by the pathologist through microscopic examination and searching for malignant epithelial cell clusters. The process of screening body fluid smears is a time consuming and error prone process.

AimWe have attempted to construct a machine learning model which can screen body fluid cytology smears for malignant cells.

Materials and methodsMGG stained Ascitic / pleural fluid cytology smears were included from 21 cases (14 malignant, 07 benign) in this study. A total of 693 microphotographs were taken at 40x magnification at the same illumination and after correction of white balance. A Magnus Microphotography system was used for photography. The images were split into the training set (195 images), test set (120 images) and validation set (378 images).

A machine learning model, a convolutional neural network, was developed in the Python programming language using the Keras deep learning library. The model was trained with the images of the training set. After completion of training, the model was evaluated on the test set of images.

ResultsEvaluation of the model on the test set produced a sensitivity of 97.87%, specificity 85.26%, PPV 95.18%, NPV 93.10% In 06 images, the model has failed to detect singly scattered malignant cells/ clusters. 14 (3.7%) false positives was reported by the model.

The machine learning model shows potential utility as a screening tool. However, it needs improvement in detecting singly scattered malignant cells and filtering inflammatory infiltrate.

sentences: Structural Basis for Allosteric Control of the SERCA-Phospholamban Membrane Complex by Ca2+ and cAMP-dependent Phosphorylation Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to {beta}-adrenergic stimulation, thus modulating cardiac output during the fight- or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzymes function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLNs cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzymes active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally-modified bitopic membrane proteins.

sentences: Cropping systems impact changes in soil fungal, but not prokaryote, alpha-diversity and community composition stability over a growing season in a long-term field trial. Crop harvest followed by a fallow period can act as a disturbance on soil microbial communities. Cropping systems intended to improve alpha-diversity of communities may also confer increased compositional stability during succeeding growing seasons. Over a single growing season in a long-term (18 year) agricultural field experiment incorporating conventional (CON), conservation (CA), organic (ORG) and integrated (INT) cropping systems, temporal changes in prokaryote, fungal and arbuscular mycorrhizal fungi (AMF) communities were investigated overwinter, during crop growth and at harvest. While certain prokaryote phyla were influenced by cropping system (e.g. Acidobacteria), the community as a whole was primarily driven by temporal changes over the growing season as distinct overwinter and crop-associated communities, with the same trend observed regardless of cropping system. Species- rich prokaryote communities were most stable over the growing season. Cropping system exerted a greater effect on fungal communities, with alpha-diversity highest and temporal changes most stable under CA. CON was particularly detrimental for alpha-diversity in AMF communities, with AMF alpha-diversity and stability improved under all other cropping systems. Practices that promoted alpha-diversity tended to also increase the similarity and temporal stability of soil fungal (and AMF) communities during a growing season, while prokaryote communities were largely insensitive to management.

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GBSmode: a pipeline for haplotype-aware analysis of genotyping-by-sequencing data 2AO_SCPLOWBSTRACTC_SCPLOW2.1 BO_SCPLOWACKGROUNDC_SCPLOWGenotyping-by-sequencing (GBS) has revolutionised molecular genetic analysis. It enables simultaneous genotyping of thousands of DNA markers in the genome of any species. In contrast to whole-genome shotgun sequencing, GBS exploits a restriction enzyme to reduce genome complexity and directs the sequencing to begin at fixed digestion sites. However, currently used tools for the analysis of GBS data, such as SAMtools, often neglect the fundamental technical differences between GBS and shotgun sequencing.

2.2 RO_SCPLOWESULTSC_SCPLOWHere we present GBSmode, a dedicated pipeline to call DNA sequence variants using whole-read information from GBS data. It removes false positives by incorporating biological features such as the ploidy level and the number of possible alleles in the population under investigation. Comparison of GBSmode with SAMtools in an F2 population of rice (Oryza sativa L.) showed both identified a similar number of polymorphisms (13,449 and 14,445, respectively) with a high overlap (8,143). However, differences were found in the number of read misalignments (8.0% and 14.3% for GBSmode and SAMtools, respectively) and genotyping errors (5.0% and 8.3% for GBSmode and SAMtools, respectively). Further tests in a bi-parental F1 population of cassava (Manihot esculenta Crantz) showed GBSmode found 31,489 polymorphic loci, whereas the number was higher with SAMtools (43,860). However, this difference was mainly attributable to GBSmode rejecting 11,695 loci that were biologically not possible.

2.3 CO_SCPLOWONCLUSIONSC_SCPLOWThis study shows that GBSmode is a versatile tool for the analysis of GBS data. Moreover, GBSmode was able to reduce genotyping errors arising from read misalignments by combining haplotype data with biological information. Whilst other tools may find more markers, GBSmode is designed for accuracy.

sentences: Probing DNA - transcription factor interactions using single-molecule fluorescence detection in nanofluidic devices Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, we combine nanofluidic devices and camera-based, single-molecule Forster resonance energy transfer (smFRET) detection to study the interactions between plant transcription factors of the auxin response family (ARF) and DNA oligonucleotides that contain target DNA response elements. In particular, we show that the binding of the unlabelled ARF DNA binding domain (ARF-DBD) to donor and acceptor labelled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, our data on fluorescently labelled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element.

sentences: RNA polymerase II clusters form in line with liquid phase wetting of chromatin It is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state and subsequent pause release to begin transcript elongation. Pol II associates with macromolecular clusters during recruitment, but it remains unclear how Pol II recruitment and pause release might affect these clusters. Here, we show that clusters exhibit morphologies that are in line with wetting of chromatin by a liquid phase enriched in recruited Pol II. Applying instantaneous structured illumination microscopy and stimulated emission double depletion microscopy to pluripotent zebrafish embryos, we find recruited Pol II associated with large clusters, and elongating Pol II with dispersed clusters. A lattice kinetic Monte Carlo model representing recruited Pol II as a liquid phase reproduced the observed cluster morphologies. In this model, chromatin is a copolymer chain containing regions that attract or repel recruited Pol II, supporting droplet formation by wetting or droplet dispersal, respectively.

sentences: Kinetochore- and chromosome-driven transition of microtubules into bundles promotes spindle assembly Mitotic spindle assembly is crucial for chromosome segregation and relies on bundles of microtubules that extend from the poles and overlap in the middle. However, how these structures form remains poorly understood. Here we show that overlap bundles arise through a network-to-bundles transition driven by kinetochores and chromosomes. STED super-resolution microscopy revealed that PRC1-crosslinked microtubules initially form loose arrays, which become rearranged into bundles. Kinetochores promote microtubule bundling by lateral binding via CENP-E/kinesin-7 in an Aurora B-regulated manner. Bundle separation is driven by steric interactions of the bundle-associated chromosomes at the spindle midplane since spindles with uncondensed or uncongressed chromosomes are narrower. In agreement with experiments, theoretical modeling suggests that bundles arise through competing attractive and repulsive mechanisms. Finally, perturbation of overlap bundles led to inefficient correction of erroneous kinetochore-microtubule attachments. Thus, kinetochores and chromosomes drive coarsening of a uniform microtubule array into overlap bundles, which promote not only spindle formation but also chromosome segregation fidelity.

sentences: Identification of a new, Rab14-dependent, endo-lysosomal pathway Cells can endocytose material from the surrounding environment. Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Several endocytosis pathways have been described (e.g. clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway). Besides possible recycling routes to the plasma membrane and various organelles, these pathways all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs) used in research and clinic, we have discovered a second endocytic pathway that moves material to late endosomes/lysosomes and that is fully independent of Rab5 and Rab7 but requires the Rab14 protein. This newly identified pathway differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already and is not affected by a series of compounds that inhibit the Rab5-dependent pathway. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins. Rab14 is expressed by the last eukaryotic common ancestor. The Rab14-dependent pathway may therefore correspond to a primordial endosomal pathway taken by cationic cargos.

sentences: ClusterMap: multi-scale clustering analysis of spatial gene expression Quantifying RNAs in their spatial context is crucial to understanding gene expression and regulation in complex tissues. In situ transcriptomic methods generate spatially resolved RNA profiles in intact tissues. However, there is a lack of a unified computational framework for integrative analysis of in situ transcriptomic data. Here, we present an unsupervised and annotation-free framework, termed ClusterMap, which incorporates physical proximity and gene identity of RNAs, formulates the task as a point pattern analysis problem, and thus defines biologically meaningful structures and groups. Specifically, ClusterMap precisely clusters RNAs into subcellular structures, cell bodies, and tissue regions in both two- and three-dimensional space, and consistently performs on diverse tissue types, including mouse brain, placenta, gut, and human cardiac organoids. We demonstrate ClusterMap to be broadly applicable to various in situ transcriptomic measurements to uncover gene expression patterns, cell-cell interactions, and tissue organization principles from high-dimensional transcriptomic images.

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Uncovering the modified immunopeptidome reveals insights into principles of PTM-driven antigenicity Antigen processing and presentation are critical for modulating tumor-host interactions. While post-translational modifications (PTMs) can alter the binding and recognition of antigens, their identification remains challenging. Here we uncover the role PTMs may play in antigen presentation and recognition in human cancers by profiling 29 different PTM combinations in immunopeptidomics data from multiple clinical samples and cell lines. We established and validated an antigen discovery pipeline and showed that newly identified modified antigens from a murine cancer model are cancer-specific and can elicit T cell killing. Systematic analysis of PTMs across multiple cohorts defined new haplotype preferences and binding motifs in association with specific PTM types. By expanding the antigenic landscape with modifications, we uncover disease-specific targets, including thousands of novel cancer-specific antigens and reveal insight into PTM-driven antigenicity. Collectively, our findings highlight an immunomodulatory role for modified peptides presented on HLA I, which may have broad implications for T-cell mediated therapies in cancer and beyond.

SignificanceMajor efforts are underway to identify cancer-specific antigens for personalized immunotherapy. Here, we enrich the immunopeptidome landscape by uncovering thousands of novel putative antigens that are post-translationally modified. We define unique preferences for PTM-driven alterations affecting HLA binding and TCR recognition, which in turn alter tumor-immune interactions.

Conflict of interest statementAuthors declare no conflicts of interest.

sentences: Long-range promoter-enhancer contacts are conserved during evolution and contribute to gene expression robustness Gene expression is regulated through complex molecular interactions, involving cis-acting elements that can be situated far away from their target genes. Data on long-range contacts between promoters and regulatory elements is rapidly accumulating. However, it remains unclear how these regulatory relationships evolve and how they contribute to the establishment of robust gene expression profiles. Here, we address these questions by comparing genome-wide maps of promoter-centered chromatin contacts in mouse and human. We show that there is significant evolutionary conservation of cis-regulatory landscapes, indicating that selective pressures act to preserve not only regulatory element sequences but also their chromatin contacts with target genes. The extent of evolutionary conservation is remarkable for long-range promoter-enhancer contacts, illustrating how the structure of regulatory landscapes constrains large-scale genome evolution. We show that the evolution of cis-regulatory landscapes, measured in terms of distal element sequences, synteny or contacts with target genes, is significantly associated with gene expression evolution.

sentences: Biomolecular solid-state NMR spectroscopy at highest field: the gain in resolution at 1200 MHz Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins and viral capsids and others. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

sentences: Reversible blood-brain barrier opening utilizing the membrane active peptide melittin in vitro and in vivo The blood-brain barrier (BBB) tightly controls entry of molecules and cells into the brain, restricting the delivery of therapeutics. Blood-brain barrier opening (BBBO) utilizes reversible disruption of cell-cell junctions between brain microvascular endothelial cells to enable transient entry into the brain. Development of BBBO techniques has been hindered by a lack of physiological models for in vitro study. Here, we utilize an in vitro tissue-engineered microvessel model to demonstrate that melittin, a membrane active peptide present in bee venom, supports BBBO. From endothelial and neuronal viability studies, we identify the accessible concentration range for BBBO. We then use a tissue-engineered model of the human BBB to optimize dosing and elucidate the mechanism of opening. Melittin and other membrane active variants transiently increase paracellular permeability via disruption of cell-cell junctions. In mice, we demonstrate a minimum clinically effective intra-arterial dose of 3 M{middle dot}min melittin, which is reversible within one day and neurologically safe. Melittin-induced BBBO represents a novel platform for delivery of therapeutics into the brain.

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

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

sentences: Sex-specific regulation of binge drinking and affective behaviors by subcortical serotonin 5HT2c receptors Serotonin 5HT2c receptors have been implicated in the pathophysiology of both mood disorders and substance use disorders, but the precise cells and circuits mediating the effects on behavior have yet to be identified. In this study, we employed anatomical tracing, in-vivo fiber photometry with both calcium and serotonin sensors, electrophysiology, chemogenetics, and genetic manipulations of the 5HT2c receptor to identify two populations of neurons expressing serotonin 5HT2c receptors, one in the lateral habenula and one in the bed nucleus of the stria terminalis, that sex-specifically co-regulate affective behaviors and are modulated by binge alcohol consumption. These findings may have implications for the development of sex-specific treatments for mood disorders and substance use disorders targeting the brains serotonin system.

sentences: An immunogenic model of KRAS-mutant lung cancer for study of targeted therapy and immunotherapy combinations Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumour regression but ultimately fail to cure these cancers, leading to intense interest in how best to combine them with other treatments, such as immunotherapies. However, preclinical systems for studying the interaction of lung tumours with the host immune system are inadequate, in part due to the low tumour mutational burden in genetically engineered mouse models. Here we set out to develop mouse models of mutant KRAS-driven lung cancer with an elevated tumour mutational burden by expressing the human DNA cytosine deaminase, APOBEC3B, to mimic the mutational signature seen in human lung cancer. This failed to substantially increase clonal tumour mutational burden and autochthonous tumours remained refractory to immunotherapy. However, by establishing clonal cell lines from these tumours we generated an immunogenic syngeneic transplantation model of KRAS mutant lung adenocarcinoma that was sensitive to immunotherapy. Unexpectedly, we found that anti-tumour immune responses were not directed against neoantigens but instead targeted derepressed endogenous retroviral antigens. The ability of KRASG12C inhibitors to cause regression of KRASG12C-expressing versions of these tumours was markedly potentiated by the adaptive immune system, providing a unique opportunity for the study of combinations of targeted and immunotherapies in immune-hot lung cancer.

sentences: Tuned inhibitory firing rate and connection weights as emergent network properties Excitatory cortical neurons show clear tuning to stimulus features, but the tuning properties of inhibitory neurons are ambiguous and have been the subject of a long debate. While inhibitory neurons have been considered to be largely untuned [1-4], recent studies show that some parvalbumin expressing (PV) neurons do show feature selectivity and participate in co-tuned subnetworks with pyramidal cells in which PV cells show high response similarity to the excitatory (E) neurons [5, 6]. Given shared input from layer 4 that drives feature tuning in excitatory subnetworks, we demonstrate that homeostatic regulation of postsynaptic firing rate governing the synaptic dynamics of the connections from PV to E cells, in combination with heterogeneity in the excitatory postsynaptic potentials (EPSP) that impinge on PV cells, results in the self-organization of PV subnetworks. We reconcile different experimental findings by showing that feature tuning of PV cells is an emerging network property that may be driven by synaptic heterogeneity, and can be inferred using population-level measures, while pairwise individual-level measures may fail to reveal inhibitory tuning. We show that such co-tuning can enhance network stability at the cost of response salience.

sentences: Every cog and wheel: Unraveling biocomplexity at the genomic and phenotypic level in a population complex of Chinook salmon. Genomic diversity is the fundamental building block of biodiversity and the necessary ingredient for adaptation. Our rapidly increasing ability to quantify functional, compositional, and structural genomic diversity of populations forces the question of how to balance conservation goals - should the focus be on important functional diversity and key life history traits or on maximizing genomic diversity as a whole? Specifically, the intra-specific diversity (biocomplexity) comprised of phenotypic and genetic variation can determine the ability of a population to respond to changing environmental conditions. Here, we explore the biocomplexity of Californias Central Valley Chinook salmon (Oncorhynchus tshawytscha) population complex at the genomic level. Notably, despite apparent gene flow among individuals with the same migration (life history) phenotypes inhabiting different tributaries, each group is characterized by a surprising component of unique genomic diversity. Our results emphasize the importance of formulating conservation goals focused on maintaining biocomplexity at both the phenotypic and genotypic level. Doing so will maintain the species adaptive potential and increase the probability of persistence of the population complex despite changing environmental pressures.

sentences: Mutational Changes and Enhanced Cell Migration during Transformation of Breast Non-tumorigenic Cells to Tumorigenic Cells Although cancer stem cells (CSCs) play a major role in tumor initiation and progression, their mechanisms in invasiveness and metastasis are unclear. Tumor microenvironment signals, such as extracellular matrix (ECM) composition, significantly influence cell and tissue behaviors. Unfortunately, these signals are often lost in in vitro cell culture. The current study determines putative CSC populations and contrasts genomic changes in human breast epithelial tumorigenic versus non-tumorigenic cells (both derived from the same normal parental stem cells) and investigates the single-cell migration properties on ECM-mimetic platforms. Whole exome sequencing data indicate that tumorigenic cells have a higher mutation burden than non-tumorigenic cells, and mutations exclusive to tumorigenic cells exhibit higher predictive deleterious scores. ECM-mimetic topography selectively enhances migration speed of tumorigenic cells but not of non-tumorigenic cells. The enhanced migratory ability of tumorigenic cells is lost in conventional two-dimension culture conditions. A wide distribution of tumorigenic single-cell migration speeds suggests heterogeneity in cellular sensing of contact guidance cues. This study identifies mutations acquired during breast tumorigenesis, which can be associated with enhanced invasiveness of breast tumorigenic cells. Results demonstrate that the nanotopographically defined platform can be applied to recapitulate an ECM structure for investigating cellular migration in the simulated tumor microenvironment.

sentences: Impact of a native hemiparasitic plant on invasive and native hosts in the field. There is increasing evidence that native parasitic plants are showing potential as biocontrols for plant invasions which are a major global threat to biodiversity. However, for this potential to be realised, the range of invasive hosts that can be controlled needs to be identified while also evaluating any potential off-target effects the parasite may pose. To address this, we investigated the impact of the Australian native shoot hemiparasitic plant, Cassytha pubescens on the major invasive species Rubus anglocandicans and two native shrubs, Acacia pycnantha and Bursaria spinosa in naturally occurring populations in southern Australia. We measured foliar predawn and midday quantum yield, electron transport rate (ETR), stomatal conductance (gs), carbon isotope composition and foliar nitrogen concentration [N] of uninfected and infected plants and, apart from gs, also for C. pubescens. Infection significantly decreased predawn and midday quantum yield, ETR and gs of invasive R. anglocandicans. In contrast, infection had no effect on predawn and midday quantum yield, ETR or carbon isotope composition of the native hosts. However, C. pubescens had a significant negative effect on native host gs, and a positive effect on host [N]. Parasite stem [N] was significantly higher when infecting A. pycnantha than B. spinosa. These results strengthen evidence for native parasitic plants having greater impact on invasive hosts while having mild off-target effects on native hosts and thus, show potential to mitigate plant invasions and help protect biodiversity.

sentences: Mind the gap: Nodes of Ranvier are remodeled by chronic psychosocial stress and neuronal activity Differential expression of myelin-related genes and changes in myelin thickness have been demonstrated in mice after chronic psychosocial stress, a risk factor for anxiety disorders. To determine whether and how stress affects structural remodeling of nodes of Ranvier, another form of myelin plasticity, we developed a 3D reconstruction analysis of node morphology in C57BL/6NCrl and DBA/2NCrl mice. We identified strain-dependent effects of chronic stress on node morphology, including elongation of paranodes in the medial prefrontal cortex (mPFC) in DBA/2NCrl mice. Furthermore, chronic chemogenetic activation of the ventral hippocampus-to-mPFC pathway resulted in increased risk assessment behavior and shortened paranodes specifically in stimulated axons, providing a direct link between anxiety-like behavior and remodeling of the nodes. Altogether, our data demonstrate genetic regulation of nodal remodeling in stress and suggest an activity-dependent regulation of paranodes in anxiety-related circuits. Nodal remodeling may thus contribute to the aberrant circuit function associated with anxiety disorders.

sentences: Identification of key interactions of benzimidazole resistance-associated amino acid mutations in Ascaris β-tubulins by molecular docking simulations Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to {beta}-tubulin proteins and destabilising microtubules. Three mutations in the {beta}-tubulin protein family are associated with BZ resistance. Seven shared {beta}-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all {beta}-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two {beta}-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris {beta}-tubulin isotype A clusters with helminth {beta}-tubulins previously shown to interact with BZ. Molecular dynamics simulations using {beta}-tubulin isotype A highlighted the key role of amino acid E198 in BZ-{beta}-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with {beta}-tubulins have been identified and show how mutations can lead to resistance in nematodes.

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CINmetrics: An R package for chromosomal instability analysis Genomic instability has been an important hallmark in cancer and more recently in neurodegenrative diseases. Chromosomal instability, as a measure of genomic instability, has been used to characterize clinical and biological phenotypes associated with these diseases by measuring structural and numerical chromosomal alterations. There have been multiple Chromosomal Instability Scores developed across many studies in the literature; however, these scores have not been compared because of a lack of single tool available to calculate these various metrics. Here, we provide an R package CINmetrics, that calculates six different chromosomal instability scores and allows direct comparison between them. We also show how these scores differ by applying CINmetrics to breast cancer data from The Cancer Genome Atlas (TCGA).

AvailabilityThe package is available on CRAN at https://cran.r-project.org/package=CINmetrics and on github at https://github.com/lasseignelab/CINmetrics

sentences: Cue overlap supports pre-retrieval selection in episodic memory: ERP evidence People often want to recall only currently relevant events, but this selective remembering is not always possible. We contrasted two candidate mechanisms: the overlap between retrieval cues and stored memory traces, and the ease of recollection. In two preregistered experiments (Ns = 28) we used event-related potentials (ERPs) to quantify pre-retrieval selection and the goal states - retrieval orientations - thought to achieve this selection. Participants viewed object pictures or heard object names, and one of these sources was designated as targets in each memory test. We manipulated cue overlap by probing memory with visual names (Experiment 1) or line drawings (Experiment 2). Results revealed that regardless of which source was targeted, the left parietal ERP effect indexing recollection was selective when test cues overlapped more with the targeted than non-targeted information, despite consistently better memory for pictures. ERPs for unstudied items were also more positive-going when cue overlap was high, suggesting that engagement of retrieval orientations reflected availability of external cues matching the targeted source. The data support the view that selection can act prior to recollection if there is sufficient overlap between retrieval cues and targeted versus competing memory traces.

sentences: Your head is there to move you around: Goal-driven models of the primate dorsal pathway Neurons in the dorsal visual pathway of the mammalian brain are selective for motion stimuli, with the complexity of stimulus representations increasing along the hierarchy. This progression is similar to that of the ventral visual pathway, which is well characterized by artificial neural networks (ANNs) optimized for object recognition. In contrast, there are no image-computable models of the dorsal stream with comparable explanatory power. We hypothesized that the properties of dorsal stream neurons could be explained by a simple learning objective: the need for an organism to orient itself during self-motion. To test this hypothesis, we trained a 3D ResNet to predict an agents self-motion parameters from visual stimuli in a simulated environment. We found that the responses in this network accounted well for the selectivity of neurons in a large database of single-neuron recordings from the dorsal visual stream of non-human primates. In contrast, ANNs trained on an action recognition dataset through supervised or self-supervised learning could not explain responses in the dorsal stream, despite also being trained on naturalistic videos with moving objects. These results demonstrate that an ecologically relevant cost function can account for dorsal stream properties in the primate brain.

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Microbial cysteine degradation is a source of hydrogen sulfide in oxic freshwater lakes The sulfur-containing amino acid cysteine is abundant in the environment including in freshwater lakes. Biological degradation of cysteine can result in hydrogen sulfide (H2S), a toxic and ecologically relevant compound that is a central player in biogeochemical cycling in aquatic environments, including freshwater lakes. Here, we investigated the ecological significance of cysteine in oxic freshwater lake environments, using model systems of isolated cultures, controlled growth experiments, and multi-omics. We screened bacterial isolates enriched from natural lake water for their ability to produce H2S when provided cysteine. In total, we identified 29 isolates that produced H2S and belonged to the phylum proteobacteria Bacteroidetes, Proteobacteria and Actinobacteria. To understand the genomic and genetic basis for cysteine degradation and H2S production, we further characterized 3 freshwater isolates using whole-genome sequencing, and quantitatively tracked cysteine and H2S levels over their growth ranges: Stenotrophomonas maltophila, Stenotrophomonas bentonitica (Gammaproteobacteria) and Chryseobacterium piscium (Bacteroidetes). We observed a decrease in cysteine and increase in H2S, and identified genes involved in cysteine degradation in all 3 genomes. Finally, to assess the presence of these organisms and genes in the environment, we surveyed a five-year time series of metagenomic data from the same isolation source at Lake Mendota and identified their presence throughout the time series. Overall, our study shows that sulfur-containing amino acids can drive microbial H2S production in oxic environments. Future considerations of sulfur cycling and biogeochemistry in oxic environments should account for H2S production from degradation of organosulfur compounds.

ImportanceHydrogen sulfide (H2S), a naturally occurring gas with biological origins, can be toxic to living organisms. In aquatic environments, H2S production typically originates from anoxic (lacking oxygen) environments such as sediments, or the bottom layers of lakes. However, the degradation of sulfur-containing amino acids such as cysteine, which all cells and life forms rely on, can be a source of ammonia and H2S in the environment. Unlike other approaches for the biological production of H2S such as dissimilatory sulfate reduction, cysteine degradation can occur in the presence of oxygen. Yet, little is known about how cysteine degradation influences sulfur availability and cycling in freshwater lakes. In our study, we found that multiple bacteria originating from a freshwater lake can not only produce H2S, but do so in oxic conditions. Overall, our study highlights the ecological importance of oxic H2S production in natural ecosystems, and necessitates a change in our outlook of sulfur biogeochemistry.

sentences: Latent circuit inference from heterogeneous neural responses during cognitive tasks Higher cortical areas carry a wide range of sensory, cognitive, and motor signals supporting complex goal-directed behavior. These signals are mixed in heterogeneous responses of single neurons tuned to multiple task variables. Dimensionality reduction methods used to analyze neural responses rely merely on correlations, leaving unknown how heterogeneous neural activity arises from connectivity to drive behavior. Here we present a framework for inferring a low-dimensional connectivity structure--the latent circuit--from high-dimensional neural response data. The latent circuit captures mechanistic interactions between task variables and their mixed representations in single neurons. We apply the latent circuit inference to recurrent neural networks trained to perform a context-dependent decision-making task and find a suppression mechanism in which contextual representations inhibit irrelevant sensory responses. We validate this mechanism by confirming the behavioral effects of patterned connectivity perturbations predicted by the latent circuit structure. Our approach can reveal interpretable and causally testable circuit mechanisms from heterogeneous neural responses during cognitive tasks.

sentences: Adeno-associated virus (AAV) reduces cortical dendritic complexity in a TLR9-dependent manner Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery in the nervous system. AAVs are widely used in research and are the basis of multiple FDA-approved gene therapies. Here, we find that the immune response to AAVs genome reduces dendritic complexity in mammalian cortex. Dendritic loss associated with AAV-mediated gene delivery occurs at experimentally-relevant titers, cannot be explained by responses to transgene expression or surgery, and is not restricted to a particular capsid serotype, encoded transgene, promoter, or production facility. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) and upregulation of immune molecules that can limit dendritic complexity and synaptic transmission. Blocking detection of unmethylated CpG-rich DNA via Toll-like receptor 9 (TLR9) protects dendritic complexity, suggesting that immunodetection of a core feature of the AAV genome triggers dendritic loss. These results reveal previously unsuspected impacts of AAV on neuronal structure and function and identify TLR9 inhibitors as important tools to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.

sentences: In for a pound, in for a penny: How the opportunity to gain reward influences the competition for memory resources When people encounter items that they believe will help them gain reward, they later remember them better than others. A recent model of emotional memory, the emotional Context Maintenance and Retrieval model (eCMR), predicts that these effects would be stronger when stimuli that predict high and low reward can compete with each other both during encoding and retrieval. We tested this prediction in two experiments. Participants were promised {pound}1 for remembering some pictures, but only a few pence for remembering others. Their recall of the content of the pictures they saw was tested after one minute and in Experiment 2, also after 24 hours. Memory at immediate test showed effects of list composition. Recall of stimuli that predicted high reward was greater than of stimuli that predicted lower reward, but only when high- and low-reward items were studied and recalled together, not when they were studied and recalled separately. More high-reward items in mixed lists were forgotten over a 24-hour retention interval compared to items studied in other conditions, but reward did not modulate the forgetting rate, a null effect which should be replicated in a larger sample. These results confirm eCMRs predictions, although further research is required to compare that model against alternatives.

sentences: Characterization of metabolic compartmentalization in the liver using spatially resolved metabolomics Cells adapt their metabolism to physiological stimuli, and metabolic heterogeneity exists between cell types, within tissues, and subcellular compartments. The liver plays an essential role in maintaining whole-body metabolic homeostasis and is structurally defined by metabolic zones. These zones are well-understood on the transcriptomic level, but have not been comprehensively characterized on the metabolomic level. Mass spectrometry imaging (MSI) can be used to map hundreds of metabolites directly from a tissue section, offering an important advance to investigate metabolic heterogeneity in tissues compared to extraction-based metabolomics methods that analyze tissue metabolite profiles in bulk. We established a workflow for the preparation of tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI and achieved broad coverage of central carbon, nucleotide, and lipid metabolism pathways. We used this approach to visualize the effect of nutrient stress and excess on liver metabolism. Our data revealed a highly organized metabolic compartmentalization in livers, which becomes disrupted under nutrient stress conditions. Fasting caused changes in glucose metabolism and increased the levels of fatty acids in the circulation. In contrast, a prolonged high-fat diet (HFD) caused lipid accumulation within liver tissues with clear zonal patterns. Fatty livers had higher levels of purine and pentose phosphate related metabolites, which generates reducing equivalents to counteract oxidative stress. This MALDI MSI approach allowed the visualization of liver metabolic compartmentalization at high resolution and can be applied more broadly to yield new insights into metabolic heterogeneity in vivo.

sentences: Voltage-Gated Potassium Channel Kv1.3 as a Therapeutic Target for Pancreatic Ductal Adenocarcinoma The mitochondrial voltage-gated potassium channel, Kv1.3, has been emerged as an attractive oncologic target but its function in pancreas cancer (PDAC) is unknown. In this study we evaluated tissue expression of Kv1.3 in resected PDAC from 55 patients and tumor inhibition in orthotopic mouse models using the recently developed Kv1.3 inhibitors PCARBTP and PAPTP. Immunohistochemistry of 55 human PDAC specimens showed that all tumors expressed Kv1.3 with 60% of tumor specimens having high Kv1.3 expression. In pancreas tumor models (Pan02 cells injected into C57BL/6 mice), PCARBTP and PAPTP treatment resulted in tumor reductions of 87% and 70%, respectively. When combined with gemcitabine/abraxane, this increased to 95% and 80% without resultant organ toxicity. In vivo models indicated PCARBTP-mediated cell death occurred through the p38-MAPK pathway. In vitro-generated resistant clones to PCARBTP escaped cell death through upregulation of the anti-oxidant system as determined using SWATH-MS analysis. These data show Kv1.3 is highly expressed in resected human PDAC and the use of novel mitochondrial Kv1.3 inhibitors combined with cytotoxic chemotherapies might be novel, effective treatment for PDAC.

sentences: Estimating the Dynamic Range of Quantitative Single-Molecule Localization Microscopy In recent years, there have been significant advances in quantifying molecule copy number and protein stoichiometry with single-molecule localization microscopy (SMLM). However, as the density of fluorophores per diffraction-limited spot increases, distinguishing between detection events from different fluorophores becomes progressively more difficult, affecting the accuracy of such measurements. Although essential to the design of quantitative experiments, the dynamic range of SMLM counting techniques has not yet been studied in detail. Here we provide a working definition of the dynamic range for quantitative SMLM in terms of the relative number of missed localizations or blinks, and explore the photophysical and experimental parameters that affect it. We begin with a simple two-state model of blinking fluorophores, then extend the model to incorporate photobleaching and temporal binning by the detection camera. From these models, we first show that our estimates of the dynamic range agree with realistic simulations of the photoswitching. We find that the dynamic range scales inversely with the duty cycle when counting both blinks and localizations. Finally, we validate our theoretical approach on dSTORM datasets of photo-switching Alexa647 dyes. Our results should help guide researchers in designing and implementing SMLM-based molecular counting experiments.

sentences: Genetic instability from a single S-phase after whole genome duplication Diploid and stable karyotypes are associated with health and fitness in animals. In contrast, whole genome duplications (WGDs) - doubling full chromosome content - are linked to genetic instability (GIN) and frequently found in human cancers 1-3. It has been established that WGDs fuel chromosome instability through abnormal mitosis 4-8, however, the immediate consequences of tetraploidy in the first interphase are not known. This is an essential question because single WGD events such as cytokinesis failure can promote tumorigenesis 9. Here, we found that newly born tetraploid human cells undergo high rates of DNA damage during DNA replication in the first S-phase. Using DNA combing and single cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we found that these defects result from the lack of protein mass scaling up at the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the GIN landscape that favors tumorigenesis after tetraploidization.

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Competing interactions give rise to two-state behavior and switch-like transitions in charge-rich intrinsically disordered proteins The most commonly occurring intrinsically disordered proteins (IDPs) are polyampholytes, which are defined by the duality of low net charge per residue and high fractions of charged residues. Recent experiments have uncovered nuances regarding sequence-ensemble relationships of model polyampholytic IDPs. These include differences in conformational preferences for sequences with lysine vs. arginine, and the suggestion that well-mixed sequences form a range of conformations, including globules, conformations with ensemble averages that are reminiscent of ideal chains, or self-avoiding walks. Here, we explain these observations by analyzing results from atomistic simulations. We find that polyampholytic IDPs generally sample two distinct stable states, namely globules and self-avoiding walks. Globules are favored by electrostatic attractions between oppositely charged residues, whereas self-avoiding walks are favored by favorable free energies of hydration of charged residues. We find sequence-specific temperatures of bistability at which globules and self-avoiding walks can coexist. At these temperatures, ensemble averages over coexisting states give rise to statistics that resemble ideal chains without there being an actual counterbalancing of intra-chain and chain-solvent interactions. At equivalent temperatures, arginine-rich sequences tilt the preference toward globular conformations whereas lysine-rich sequences tilt the preference toward self-avoiding walks. We also identify differences between aspartate and glutamate containing sequences, whereby the shorter aspartate sidechain engenders preferences for metastable, necklace-like conformations. Finally, although segregation of oppositely charged residues within the linear sequence maintains the overall two-state behavior, compact states are highly favored by such systems.

Significance StatementIntrinsically disordered regions (IDRs) of proteins, when tethered to folded domains, function either as flexible tails or as linkers between domains. Most IDRs are polyampholytes that comprise a mixture of oppositely charged residues. Recent measurements of tethered polyampholytes showed that tendency of arginine- and lysine-rich sequences to behave very differently from one another. Using computer simulations, we show that these differences are determined by differences in free energies of hydration, steric volumes, and other considerations. Further, the interplay between electrostatic attractions and favorable free energies of hydration creates distinct stable states for polyampholytic IDRs. These findings have implications for switch-like transitions and the regulation of effective concentrations of interaction motifs by IDRs.

sentences: FEASIBILITY OF VASCULAR REMODELING PARAMETER ESTIMATION FOR ASSESSING HYPERTENSIVE PREGNANCY DISORDERS Hypertensive pregnancy disorders, such as preeclampsia, are leading sources of both maternal and fetal morbidity in pregnancy. Non-invasive imaging, such as ultrasound and magnetic resonance imaging (MRI), is an important tool in predicting and monitoring these high risk pregnancies. While imaging can measure hemodynamic parameters, such as uterine artery pulsatility and resistivity indices, the interpretation of such metrics for disease assessment rely on ad-hoc standards, which provide limited insight to the physical mechanisms underlying the emergence of hypertensive pregnancy disorders. To provide meaningful interpretation of measured hemodynamic data in patients, advances in computational fluid dynamics can be brought to bear. In this work, we develop a patient-specific computational framework that combines Bayesian inference with a reduced-order fluid dynamics model to infer remodeling parameters, such as vascular resistance, compliance and vessel cross-sectional area, known to be related to the development of hypertension. The proposed framework enables the prediction of hemodynamic quantities of interest, such as pressure and velocity, directly from sparse and noisy MRI measurements. We illustrate the effectiveness of this approach in two systemic arterial network geometries: an aorta with carotid and a maternal pelvic arterial network. For both cases, the model can reconstruct the provided measurements and infer parameters of interest. In the case of the maternal pelvic arteries, the model can make a distinction between the pregnancies destined to develop hypertension and those that remain normotensive, expressed through the value range of the predicted absolute pressure.

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Single-cell atlas of mouse limb development reveals a complex spatiotemporal dynamics of skeleton formation Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex nonconsecutive three-dimensional pattern.

Single-cell RNA sequencing of the developing mouse forelimb revealed three progenitor states: naive, proximal and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments.

Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest a new model for limb skeleton development.

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Haisu: Hierarchical Supervised Nonlinear Dimensionality Reduction We propose a novel strategy for incorporating hierarchical supervised label information into nonlinear dimensionality reduction techniques. Specifically, we extend t-SNE, UMAP, and PHATE to include known or predicted class labels and demonstrate the efficacy of our approach on multiple single-cell RNA sequencing datasets. Our approach, "Haisu," is applicable across domains and methods of nonlinear dimensionality reduction. In general, the mathematical effect of Haisu can be summarized as a variable perturbation of the high dimensional space in which the original data is observed. We thereby preserve the core characteristics of the visualization method and only change the manifold to respect known or assumed class labels when provided. Our strategy is designed to aid in the discovery and understanding of underlying patterns in a dataset that is heavily influenced by parent-child relationships. We show that using our approach can also help in semi-supervised settings where labels are known for only some datapoints (for instance when only a fraction of the cells is labeled). In summary, Haisu extends existing popular visualization methods to enable a user to incorporate known, relevant relationships via a user-defined hierarchical distancing factor.

Availabilitygithub.com/Cobanoglu-Lab/Haisu

sentences: Cardiotoxic effects of methamphetamine associated with electrophysiological and epigenetic aberrations in Zebrafish Long-term methamphetamine (Meth) abuse damages functional and molecular changes in the brain that causes chronic and relapsing disease. In this study, we sought to investigate a relationship between cardiotoxicity and arrhythmia with associated Meth abuse in zebrafish to identify and to understand the adverse cardiac symptoms associated with Meth as well as to assess the applicability of zebrafish as an appropriate model for cardiac-related drug screening studies. Over a two-week duration, zebrafish were first treated with various concentrations of Meth, ranging from 0 to 50 M. Immediately after treatment, zebrafish underwent electrocardiogram (ECG) measurement for electrophysiological analysis. Results show that a higher incidence of increased heart rate over the duration of the experiment, corroborating with results from previous human case studies involving Meth users. However, abnormalities commonly cited in those same case studies, such as prolongation of QTc, were not significantly presented in obtained ECG recordings. We have also conducted genetic, epigenetic, and histochemical analysis in an attempt to understand the cardiotoxic effects of Meth on zebrafish cardiac function. These results suggested myocardial damage and decrease in gene expression associated with normal physiological function. Finally, this paper provides insights into potential reasons for the apparent discrepancies in our data with prior research as well as an outlook of zebrafish cardiotoxic drug screening studies.

sentences: Microtubule plus-end regulation by centriolar cap proteins Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, grow very slowly and are very stable. The complex of centriolar proteins CP110 and CEP97 forms a cap that stabilizes the distal centriole end and prevents its over-elongation. Here, we used in vitro reconstitution assays to show that whereas CEP97 does not interact with microtubules directly, CP110 specifically binds microtubule plus ends, potently blocks their growth and induces microtubule pausing. Cryo-electron tomography indicated that CP110 binds to the luminal side of microtubule plus ends and reduces protofilament peeling. Furthermore, CP110 directly interacts with another centriole biogenesis factor, CPAP/SAS- 4, which tracks growing microtubule plus ends, slows down their growth and prevents catastrophes. CP110 and CPAP synergize in inhibiting plus-end growth, and this synergy depends on their direct binding. Together, our data reveal a molecular mechanism controlling centriolar microtubule plus- end dynamics and centriole biogenesis.

sentences: TraB is a novel component of the ER-Mitochondria contact site (EMCS) with dual roles in ER-Mitochondrial tethering and mitophagy ER-mitochondria contact sites (EMCSs) are important for mitochondrial function. Here, we have identified a novel eukaryotic EMCS complex, comprising a family of uncharacterised mitochondrial outer-membrane proteins, TraB1 and the ER protein, VAP27-1. In Arabidopsis, there are two TraB1 isoforms and the trab1a/trab1b double mutant exhibits abnormal mitochondrial morphology, strong starch accumulation and impaired energy metabolism, indicating that these proteins are essential for normal mitochondrial function. Moreover, TraB1 proteins also interact with ATG8 in order to regulate mitochondrial degradation (mitophagy). The turnover of depolarised mitochondria is significantly reduced in both trab1a/b and VAP27 mutants (vap27-1/3/4/6) under mitochondrial stress conditions, with an increased population of dysfunctional mitochondria present in the cytoplasm. Consequently, plant recovery after stress is significantly perturbed. A similar phenotype is found in both autophagy mutants (atg5 and atg7), suggesting that TraB1 regulated mitophagy and ER-mitochondrial tethering are two closely related processes, necessary for normal mitochondrial function. Taken together, we ascribe a dual role to TraB1 which is a novel component of the EMCS complex in eukaryotes, regulating both tethering of the mitochondria to the ER and mitophagy.

sentences: IRONMAN interacts with OsHRZ1 and OsHRZ2 to maintain Fe homeostasis IRONMAN is a family of small peptides which positively regulate the Fe deficiency response. However, the molecular mechanism by which OsIMA1 and OsIMA2 regulate Fe homeostasis was unclear. Here, we reveal that OsIMA1 and OsIMA2 interact with the potential Fe sensors, OsHRZ1 and OsHRZ2. OsIMA1 and OsIMA2 contain a conserved 17-amino acid C-terminal region which is responsible for the interactions with OsHRZ1 and OsHRZ2. The OsIMA1 overexpressing plants have the increased seed Fe concentration and the reduced fertility, as observed in the hrz1-2 loss-of-function mutant plants. Moreover, the expression trends of Fe deficiency inducible genes in the OsIMA1 overexpressing plants are the same to those in the hrz1-2. Co-expression assays suggest that OsHRZ1 and OsHRZ2 promote the degradation of OsIMA1 proteins. As the interaction partners of OsHRZ1, the OsPRI proteins also interact with OsHRZ2. The conserved C-terminal region of four OsPRIs contributes to the interactions with OsHRZ1 and OsHRZ2. An artificial IMA (aIMA) derived from the C-terminal of OsPRI1 can be also degraded by OsHRZ1. Moreover, the aIMA overexpressing rice plants accumulate more Fe without reduction of fertility. This work establishes the link between OsIMAs and OsHRZs, and develops a new strategy for Fe fortification in rice.

sentences: Sources of variation in the spectral slope of the sleep EEG Building on previous work linking changes in the electroencephalogram (EEG) spectral slope to arousal level, Lendner et al. (2021) reported that wake, non rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep exhibit progressively steeper 30-45 Hz slopes, interpreted in terms of increasing cortical inhibition. Here we sought to replicate Lendner et al.s scalp EEG findings (based on 20 individuals) in a larger sample of 11,630 individuals from multiple cohorts in the National Sleep Research Resource (NSRR). In a final analytic sample of N = 10,255 distinct recordings, there was unambiguous statistical support for the hypothesis that, within individuals, the mean spectral slope grows steeper going from wake to NREM to REM sleep. We found that the choice of mastoid referencing scheme modulated the extent to which electromyogenic or electrocardiographic artifacts were likely to bias 30-45 Hz slope estimates, as well as other sources of technical, device-specific bias. Nonetheless, within individuals, slope estimates were relatively stable over time. Both cross-sectionally and longitudinal, slopes tended to become shallower with increasing age, particularly for REM sleep; males tended to show flatter slopes than females across all states. Although conceptually distinct, spectral slope did not predict sleep state substantially better than other summaries of the high frequency EEG power spectrum (>20 Hz, in this context) including beta band power, however. Finally, to more fully describe sources of variation in the spectral slope and its relationship to other sleep parameters, we quantified state-dependent differences in the variances (both within and between individuals) of spectral slope, power and interhemispheric coherence, as well as their covariances. In contrast to the common conception of the REM EEG as relatively wake-like (i.e.  paradoxical sleep), REM and wake were the most divergent states for multiple metrics, with NREM exhibiting intermediate profiles. Under a simplified modelling framework, changes in spectral slope could not, by themselves, fully account for the observed differences between states, if assuming a strict power law model. Although the spectral slope is an appealing, theoretically inspired parameterization of the sleep EEG, here we underscore some practical considerations that should be borne in mind when applying it in diverse datasets. Future work will be needed to fully characterize state-dependent changes in the aperiodic portions of the EEG power spectra, which appear to be consistent with, albeit not fully explained by, changes in the spectral slope.

sentences: Cauliflower Mosaic Virus Utilizes Processing Bodies to Escape Translational Repression in Arabidopsis Viral infections impose extraordinary RNA stress on a cell, triggering cellular RNA surveillance pathways like RNA decapping, nonsense-mediated decay and RNA silencing. Viruses need to maneuver between these pathways to establish infection and succeed in producing high amounts of viral proteins. Processing bodies (PBs) are integral to RNA triage in eukaryotic cells with several distinct RNA quality control pathways converging for selective RNA regulation. In this study, we investigate the role of Arabidopsis thaliana PBs during Cauliflower Mosaic Virus (CaMV) infection. We find that several PB components are co-opted into viral replication factories and support virus multiplication. This pro-viral role was not associated with RNA decay pathways but instead, we could establish PB components as essential helpers in viral RNA translation. While CaMV is normally resilient to RNA silencing, PB dysfunctions expose the virus to this pathway, similar to previous observations on transgenes. Transgenes, however, undergo RNA Quality Control dependent RNA degradation, whereas CaMV RNA remains stable but becomes translationally repressed through decreased ribosome association, revealing a unique dependence between PBs, RNA silencing and translational repression. Together, our study shows that PB components are co-opted by the virus to maintain efficient translation, a mechanism not associated with canonical PB functions.

sentences: Rapid auxin-mediated phosphorylation of Myosin regulates trafficking and polarity in Arabidopsis The signaling molecule auxin controls plant development through a well-known transcriptional mechanism that regulates many genes. However, auxin also triggers cellular responses within seconds or minutes, and mechanisms mediating such fast responses have remained elusive. Here, we identified an ultrafast auxin-mediated protein phosphorylation response in Arabidopsis roots that is largely independent of the canonical TIR1/AFB receptors. Among targets of this novel response are Myosin XI and its adaptor protein MadB2. We show that their auxin-mediated phosphorylation regulates trafficking and polar, subcellular distribution of PIN auxin transporters. This phosphorylation-based auxin signaling module is indispensable during developmental processes that rely on auxin-mediated PIN repolarization, such as termination of shoot gravitropic bending or vasculature formation and regeneration. Hence, we identified a fast, non-canonical auxin response targeting multiple cellular processes and revealed auxin-triggered phosphorylation of a myosin complex as the mechanism for feedback regulation of directional auxin transport, a central component of auxin canalization, which underlies self-organizing plant development.

sentences: DYNAMICS OF NEUROMUSCULAR TRANSMISSION REPRODUCED BY CALCIUM-DEPENDENT SERIAL TRANSITIONS IN THE VESICLE FUSION COMPLEX Neuromuscular transmission, from spontaneous release to facilitation and depression was accurately reproduced by a mechanistic kinetic model of sequential maturation transitions in the molecular fusion complex. The model incorporates three predictions. First, sequential calcium-dependent forward transitions take vesicles from docked to pre-primed to primed states, followed by fusion. Second, pre-priming and priming are reversible. Third, fusion and recycling are unidirectional. The model was fed with experimental data from previous studies while the backward ({beta}) and recycling ({rho}) rate constant values were fitted. Classical experiments were successfully reproduced when every forward () rate constant had the same value, and both backward rate constants were 50-100 times larger. Such disproportion originated an abruptly decreasing gradient of resting vesicles from docked to primed states. Simulations also predict that: i. Spontaneous release reflects primed to fusion spontaneous transitions. ii. Calcium elevations synchronize the series of forward transitions that lead to fusion. iii Facilitation reflects a transient increase of priming following calcium-dependent transitions. iv. Backward transitions and recycling restore the resting state. v. Depression reflects backward transitions and slow recycling after intense release. Such finely-tuned kinetics offers a mechanism for collective non-linear transitional adaptations of a homogeneous vesicle pool to an ever-changing pattern of electrical activity.

sentences: Unsupervised clustering of track-weighted dynamic functional connectivity reveals white matter substrates of functional connectivity dynamics The contribution of structural connectivity to functional connectivity dynamics is still far from being fully elucidated. Herein, we applied track-weighted dynamic functional connectivity (tw-dFC), a model integrating structural, functional, and dynamic connectivity, on high quality diffusion weighted imaging and resting-state fMRI data from two independent repositories. The tw-dFC maps were analyzed using independent component analysis, aiming at identifying spatially independent white matter components which support dynamic changes in functional connectivity. Each component consisted of a spatial map of white matter bundles that show consistent fluctuations in functional connectivity at their endpoints, and a time course representative of such functional activity. These components show high intra-subject, inter-subject, and inter-cohort reproducibility. We provided also converging evidence that functional information about white matter activity derived by this method can capture biologically meaningful features of brain connectivity organization, as well as predict higher-order cognitive performance.

sentences: Targeting glucose metabolism sensitizes pancreatic cancer to MEK inhibition Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and identify druggable targets for tumor maintenance. This is especially challenging in the context of PDAC, in which activating mutations of KRAS oncogene (KRAS*) dominate the genetic landscape. By using an inducible KrasG12D-driven p53 deficient PDAC mouse model (iKras model), we demonstrate that RAF-MEK-MAPK signaling is the major effector for oncogenic Kras-mediated tumor maintenance. However, MEK inhibition has minimal therapeutic effect as single agent for PDAC both in vitro and in vivo. Although MEK inhibition partially downregulates the transcription of glycolysis genes, it surprisingly fails to suppress the glycolysis flux in PDAC cell, which is a major metabolism effector of oncogenic KRAS. Accordingly, In vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MAPK inhibition. Furthermore, inhibition of glucose metabolism with low dose 2-deoxyglucose (2DG) in combination with MEK inhibitor dramatically induces apoptosis in KrasG12D-driven PDAC cell in vitro, inhibits xenograft tumor growth and prolongs the overall survival of genetically engineered mouse PDAC model. Molecular and metabolism analyses indicate that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal ER stress. Together, our work suggests that combinatory inhibition of glycolysis and MAPK pathway may serve as an alternative approach to target KRAS-driven PDAC.

sentences: Long-term stability of neural activity in the motor system How an established behavior is retained and stably produced by a nervous system in constant flux remains a mystery. One possible solution is to fix the activity patterns of single neurons in the relevant circuits. Alternatively, activity in single cells could drift over time provided that the population dynamics are constrained to produce stable behavior. To arbitrate between these possibilities, we recorded single unit activity in motor cortex and striatum continuously for several weeks as rats performed stereotyped motor behaviors - both learned and innate. We found long-term stability in behaviorally-locked single neuron activity patterns across both brain regions. A small amount of neural drift observed over weeks of recording could be explained by concomitant changes in task-irrelevant behavioral output. These results suggest that stereotyped behaviors are generated by stable neural activity patterns.

sentences: Electrophysiological correlates of proactive and reactive inhibition in a modified visual Go/NoGo task Impulse control is crucial for everyday functioning in modern society. People with borderline personality disorder (BPD) suffer from impulse control impairment. According to the theory of dual mechanisms of control, motor impulse control can be divided into proactive and reactive modes. Proactive inhibition is involved before an event that might require inhibitory control. Reactive inhibition is initiated after the occurrence of an event that requires inhibitory control. Few studies have focused on proactive inhibition in relation to impaired impulse control, moreover electrophysiological evidence is scarce. Therefore, in the search for electrophysiological correlates of proactive and reactive inhibitions, we assessed event-related potentials elicited during a modified emotionally neutral visual Go/NoGo task in 28 clinically impulsive BPD patients and 35 healthy control (HC) subjects. In both groups, proactive inhibition was associated with enhanced late prestimulus activity and a suppressed poststimulus N2 component. In both groups, reactive inhibition was associated with enhanced poststimulus N2 and P3 components. We found no electrophysiological differences between HC subjects and BPD patients and both groups performed similarly in the task. Hence, the clinically observed impulse control impairment in BPD might act through different mechanisms other than altered inhibitory control in an emotionally neutral task.

sentences: Local CpG density affects the trajectory of age-associated epigenetic changes DNA methylation is an epigenetic mark associated with gene repression and genome stability. Its pattern in the genome is disrupted with age and these changes can be used to statistically predict age with epigenetic clocks. Rates of aging inferred from these clocks correlate with human health. However, the molecular mechanisms underpinning age-associated DNA methylation changes are unknown. Local DNA sequence plays a strong role in programming DNA methylation levels at individual loci independently of age, but its influence on age-associated DNA methylation changes is unknown. We analysed longitudinal human DNA methylation trajectories at 345,895 CpGs from 600 individuals aged between 67 and 80 to understand the factors responsible for age-associated epigenetic changes at individual CpGs in the genome. We show that changes in methylation with age are especially apparent at 8,322 low CpG density loci. Using SNP data from the same individuals we demonstrate that DNA methylation trajectories are affected by local sequence polymorphisms at 1,487 loci with low CpG density. More generally, we find that local CpG density is a strong determinant of a CpGs methylation trajectory and that CpGs located in low CpG density regions are particularly prone to change. Overall, our results demonstrate that local DNA sequence influences age-associated DNA methylation changes in humans in vivo. We suggest that this occurs because interactions between CpGs reinforce maintenance of methylation patterns in CpG dense regions.

sentences: Epigenetic MRI: Noninvasive Imaging of DNA Methylation in the Brain Both neuronal and genetic mechanisms regulate brain function. While there are excellent methods to study neuronal activity in vivo, there are no nondestructive methods to measure global gene expression in living brains. Here we present a method, epigenetic magnetic resonance imaging (eMRI), that overcomes this limitation via direct imaging of DNA methylation, a major gene expression regulator. eMRI exploits the methionine metabolic pathways for DNA methylation to label genomic DNA through 13C-enriched diets. A novel 13C magnetic resonance spectroscopic imaging method then maps the spatial distribution of labeled DNA. We validated eMRI using pigs, whose brains have stronger similarity to humans in volume and anatomy than rodents, and confirmed efficient 13C labeling of brain DNA. We also discovered strong regional differences in global DNA methylation. Just as MRI measurements of regional neuronal activity have had a transformational effect on neuroscience, we expect that the eMRI signal as a surrogate for regional gene expression will enable many new investigations into the roles of gene expression in human brain function, behavior, and disease.

sentences: CALR frameshift mutations in MPN patient-derived iPS cells accelerate maturation of megakaryocytes Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor, and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem (iPS) cells to establish a MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in CD15+ granulocytic cells derived from homozygous CALR-mutant iPS cells, rescued by repairing the mutation using CRISPR/Cas9. iPS cell-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelets protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated vs. unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis, dependent on its zygosity and found known and novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.

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Decorating chromatin for enhanced genome editing using CRISPR-Cas9 CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile way to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K4me3 and H3K36me3 shown to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in DNA to double the observed HDR efficiency and increase the HDR:indel ratio by 3-fold compared to that induced by Cas9 alone. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin structure for the choice of DNA repair pathway during CRISPR-Cas genome editing and provide a new strategy to increase the efficiency of HDR.

Significance StatementCRISPR-Cas-mediated homology-directed repair (HDR) enables precision genome editing for diverse research and clinical applications, but HDR efficiency is often low due to competing end-joining pathways. Here, we describe a simple strategy to influence DNA repair pathway choice and improve HDR efficiency by engineering CRISPR-Cas9-methyltransferase fusion proteins. This strategy highlights the impact of histone modifications on DNA repair following CRISPR-Cas-induced double-stranded breaks and adds to the CRISPR genome editing toolbox.

sentences: Host phenology can select for multiple stable parasite virulence strategies Host phenology is an important driver of parasite transmission dynamics and evolution. Prior research has demonstrated that host phenology can drive monocyclic, obligate-killer parasites to evolve an intermediate virulence strategy where all parasites kill their host just before the season ends to limit the death of parasite progeny in the environment. The impact of host seasonality on parasites that are not constrained to a monocyclic life-cycle, however, cannot be inferred from these results. Here we present a mathematical model that demonstrates that many, but not all, seasonal host activity patterns support multiple evolutionarily stable parasite strategies (ESS), although these strategies cannot coexist in the same system. The specific monocyclic and polycyclic parasite evolutionarily stable strategies in each phenological pattern are interspersed with less-fit mono- and polycyclic strategies (evolutionary repellors). The ESS that dominates each system at equilibrium is a function of the strategy of the parasite introduced into the system. The results demonstrate that host phenology can, in theory, maintain diverse parasite strategies among isolated geographic locations.

sentences: Seasonal migration as a life history trait facilitating adaptation to climate change Predicting the geographic range of species and their response to variation in climate are entwined goals in conservation and evolutionary ecology. Species distribution models (SDMs) are foundational in this effort and used to visualize the geographic range of species as the spatial representation of its realized niche, or when based only on climate, its climate niche. SDMs can also forecast shifts in species range given climate change, but often lack of empirical support for causal links between climate and demography, yielding uncertain predictions. We addressed such uncertainties whilst also exploring the role of migration and resident life-histories in climate adaptation in mobile animals using 48 years of detailed demographic and climate data for song sparrows (Melospiza melodia), a polytypic species that varies in migratory life history. We developed SDMs representing demographic and climate niches of migratory and resident populations in western North America from California (CA) to Alaska (AK) using data from a focal population in British Columbia (BC) and 1.2 million citizen science observations. Distributions of resident and migrant populations predicted by each model agreed strongly (72.8%) in the region of our focal population, but less well in regions with dissimilar climates. Mismatches were largest in CA, smaller in AK, but in all cases supported the hypothesis that climate influences the evolution of migration and limits year-round residency. Our results imply that migrants predominated in our focal population a century ago, but that climate change has favored range expansions by non-migratory phenotypes and facilitated an upward shift in the elevational range of residents. We suggest long-term studies are crucial to evaluating the predictions of SDMs positing causal links between climatic conditions and species demography. We found such links to be robust regionally and particularly useful to elucidating the potential for migration or residence to facilitate adaptation to climate change.

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DNMT family induced down-regulation of NDRG1 via DNA methylation and clinicopathological significance in gastric cancer BackgroundAberrant DNA methylation of tumor suppressor genes is a common event in the development and progression of gastric cancer(GC). Our previous study showed NDRG1, which could suppress cell invasion and migration, was frequently down-regulated by DNA methylation of its promoter in GC.

Purpose and MethodsTo analyze the relationship between the expression and DNA methylation of NDRG1 and DNA methyltransferase (DNMT) family. We performed a comprehensive comparison analysis using 407 patients including sequencing analysis data of GC from TCGA.

ResultsNDRG1 was negatively correlative to DNMT1 (p =0.03), DNMT3A(p =0.01), DNMT3B(p =0.88), respectively. Whereas, the DNA methylation of NDRG1 was positively correlative to DNMT family(DNMT1 p<0.01, DNMT3A p<0.001, DNMT3B p=0.57, respectively). NDRG1 expression was significantly inverse correlated with invasion depth (p =0.023), and DNMT1 was significantly positive correlated with the degree of tumor cell differentiation (p =0.049). DNMT3B was significantly correlated with tumor cell differentiation (p =0.030). However, there was no association between the expression of DNMT3A and clinicopathological features. The univariate analysis showed that NDRG1and DNMTs had no association with prognosis of GC patients. But, multivariate analysis showed DNMT1 was significantly correlated with prognosis of GC patients.

ConclusionThese data suggest that down-regulation of NDRG1 in gastric cancer is due to DNA methylation of NDRG1 gene promoter via DNMT family. The demethylating agent maybe a potential target drug for GC patients.

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Frequent development of broadly neutralizing antibodies in early life in a large cohort of HIV-infected children Recent studies conducted in small cohorts of children have indicated that broadly neutralizing antibodies (bnAbs) may develop earlier after HIV infection compared to adults. To define the frequency and kinetics of bnAb responses in a larger pediatric cohort, we evaluated plasma from 212 ART-naive, children living with HIV aged 1 to 3 years. Neutralization breadth and potency was assessed using a panel of 10 tier-2 viruses and compared to those of adults with chronic HIV. Further, the magnitude, epitope specificity and IgG subclass distribution of Env-specific antibodies were also assessed using a binding antibody multiplex assay. We found that 1-year-old children demonstrated neutralization breadth comparable to that of chronically-infected adults, and breadth continued to increase with age such that the pediatric cohort overall exhibited significantly greater neutralization breadth than adults (p= 0.014). Similarly, binding antibody responses increased with age, and the levels in 2 to 3 year-old children were comparable to those of adults. Overall, there was no significant difference in antibody specificities or IgG subclass distribution between the pediatric and adult cohorts. Interestingly, the neutralization activity was mapped to a single epitope (CD4 binding site, V2 or V3 glycans) in only 5 of 38 pediatric broadly neutralizing samples, suggesting a polyclonal neutralization response may develop in most children. These results contribute to a growing body of evidence suggesting that the early life immune system may present advantages for the development of an effective HIV vaccine.

Author summaryUnderstanding the development of broadly neutralizing antibodies during natural HIV infection is critical to provide insights for the development of an effective vaccine. In this study, we used a large cohort (n=212) of HIV infected children aged 1-3 years old to define the frequency and kinetics of broad neutralization in comparison to adults with chronic HIV. We also evaluated the magnitude and epitope specificity of HIV Env-specific antibodies in children and adults. We observed that both the binding and neutralizing antibody responses increased with age. However, by one year of age, children had neutralization breadth comparable to that of chronically infected adults; and overall, 1-3 year-old children demonstrated higher neutralization breadth than adults. Only slight differences in epitope-specificity and IgG subclass distribution were observed between children and adults, suggesting that these factors are not major contributors to the observed age-related difference in neutralization breadth. The epitope specificity of the neutralizing antibodies could not be mapped in the majority of children, suggesting that their response may be polyclonal. Our study contributes to the body of work indicating a potential advantage of the early life immune system for the elicitation of broad neutralization.

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Dynamic behavioral and molecular changes induced by chronic stress exposure in mice Depression is a leading cause of disabilities around the world, and the underlying mechanisms involved in its pathophysiology are broad and complex. Exposure to chronic stress is a risk factor for developing depressive-symptoms and contributes to cellular and molecular changes precipitating the emergence of symptoms. In the brain, excitatory neurons, inhibitory interneurons and supporting astroglial cells are all sensitive to chronic stress exposure and are known to be impaired in depression.

Using an animal model of chronic stress, we assessed the impact of variable durations of chronic stress on the emergence of behavioral deficits and associated molecular changes in the prefrontal cortex (PFC), brain region highly sensitive to stress and impaired in depression. Mice were exposed to up to 35 days of chronic restraint stress and were assessed weekly on behavioral tests measuring anxiety and anhedonia. PFC Protein and RNA levels of specific markers of excitatory, inhibitory synapses and astroglia were quantified using western blot and qPCR, respectively. Correlation and integrative network analyses were used to investigated the impact of chronic stress on the different compartments.

Results showed that chronic stress induces anxiety-like behaviors within 7 days, while anhedonia-like behaviors were observed only after 35 days. At the molecular level, alterations of many markers were observed, in particular with longer exposure to chronic stress. Finally, correlation analyses and integrative network analyses revealed that male and female mice react differently to chronic stress exposure and that some markers seem to be more correlated to behaviors deficits in males than in females.

Our study demonstrate that chronic induces a dynamic changes that can be observed at the behavioral and molecular levels, and that male and female mice, while exhibiting similar symptoms, have different underlying pathologies.

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Complex developmental and transcriptional dynamics underlie pollinator-driven evolutionary transitions in nectar spur morphology in Aquilegia (columbine) PremiseDetermining the developmental programs underlying morphological variation is key to elucidating the evolutionary processes that generated the stunning biodiversity of the angiosperms. Here, we characterize the developmental and transcriptional dynamics of the elaborate petal nectar spur of Aquilegia (columbine) in species with contrasting pollination syndromes and spur morphologies.

MethodsWe collected petal epidermal cell number and length data across four Aquilegia species, two with the short, curved nectar spurs of the bee-pollination syndrome, and two with the long, straight spurs of the hummingbird syndrome. We also performed RNA-seq on A. brevistyla (bee) and A. canadensis (hummingbird) distal and proximal spur compartments at multiple developmental stages. Finally, we intersected these datasets with a previous QTL mapping study on spur length and shape to identify new candidate loci.

ResultsThe differential growth between the proximal and distal surfaces of curved spurs is primarily driven by differential cell division. However, independent transitions to straight spurs in the hummingbird syndrome have evolved by increasing differential cell elongation between spur surfaces. The RNA-seq data reveal these tissues to be transcriptionally distinct, and point to auxin signaling as being involved with the differential cell elongation responsible for the evolution of straight spurs. We identify several promising candidate genes for future study.

ConclusionsOur study, taken together with previous work in Aquilegia, reveals the complexity of the developmental mechanisms underlying trait variation in this system. The framework we have establish here will lead to exciting future work examining candidate genes and processes involved in the rapid radiation of the genus.

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Time-resolved parameterization of aperiodic and periodic brain activity Macroscopic neural dynamics comprise both aperiodic and periodic components. Recent advances in parameterizing neural power spectra offer practical tools for evaluating these features separately. Although neural signals vary dynamically and express non-stationarity in relation to ongoing behaviour and perception, current methods yield static spectral decompositions. Here, we introduce Spectral Parameterization Resolved in Time (SPRiNT) as a novel method for decomposing complex neural dynamics into periodic and aperiodic spectral elements in a time-resolved manner. First, we demonstrate, with naturalistic synthetic data, SPRiNTs capacity to reliably recover time-varying spectral features and emphasize SPRiNTs specific strengths over other time-frequency parameterization approaches based on wavelets. Second, we use SPRiNT to further illustrate how aperiodic spectral features fluctuate across time in resting-state electroencephalography (n = 178) and relate changes in aperiodic parameters to participants demographics and behaviour. Lastly, we use SPRiNT to demonstrate aperiodic dynamics related to movement behaviour in intracranial recordings in rodents. We foresee SPRiNT responding to growing neuroscientific interests in the parameterization of time-varying neural power spectra and advancing the quantitation of complex neural dynamics at the natural time scales of behaviour.

Significance StatementThe new method and reported findings address a growing interest in neuroscience for research tools that can reliably decompose brain activity at the mesoscopic scale into interpretable components. We show that the new approach proposed herein is capable of tracking transient, dynamic spectral (aperiodic and periodic) components across time, both in synthetic simulated and experimental data. We anticipate that this novel technique, SPRiNT, will enable new neuroscience inquiries that reconcile multifaceted neural dynamics with complex behaviour.

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Differential microRNA expression analyses across two brain regions in Alzheimer's disease BackgroundDysregulation of microRNAs (miRNAs) is involved in the pathogenesis of neurodegenerative diseases, including Alzheimers disease (AD). Hitherto, sample sizes from differential miRNA expression studies in AD are exceedingly small aggravating any biological inference. To overcome this limitation, we investigated six candidate miRNAs in a large collection of brain samples.

MethodsBrain tissue was derived from superior temporal gyrus (STG) and entorhinal cortex (EC) from 99 AD patients and 91 controls. Expression of six miRNAs was examined by qPCR (STG) or small RNA sequencing (EC). Brain region-dependent differential miRNA expression was investigated in a transgenic AD mouse model using qPCR and FISH. Total RNA sequencing was used to assess differential expression of miRNA target genes.

ResultsMiR-129-5p, miR-132-5p, and miR-138-5p were significantly downregulated in AD vs. controls both in STG and EC, while miR-125b-5p and miR-501-3p showed no evidence for differential expression in this dataset. In addition, miR-195-5p was significantly upregulated in EC but not STG in AD patients. The brain region-specific pattern of miR-195-5p expression was corroborated in vivo in transgenic AD mice. Total RNA sequencing identified several novel and functionally interesting target genes of these miRNAs involved in synaptic transmission (GABRB1), the immune-system response (HCFC2) or AD-associated differential methylation (SLC16A3).

ConclusionsUsing two different methods (qPCR and small RNA-seq) in two separate brain regions in 190 individuals we more than doubled the available sample size for most miRNAs tested. Differential gene expression analyses confirm the likely involvement of miR-129-5p, miR-132-5p, miR-138-5p, and miR-195-5p in AD pathogenesis and highlight several novel potentially relevant target mRNAs.

FundingThis work was supported by the Deutsche Forschungsgemeinschaft (DFG) and the National Science Foundation China (NSFC) as a Joint Sino-German research project ("MiRNet-AD", #391523883). Additional support was provided by the DFG Research Infrastructure NGS_CC (project 407495230) as part of the Next Generation Sequencing Competence Network (#423957469) and the Cure Alzheimer s Fund (CAF) as part of the CIRCUITS consortium project.

sentences: The human visual system preserves the hierarchy of 2-dimensional pattern regularity Symmetries are present at many scales in images of natural scenes. A large body of literature has demonstrated contributions of symmetry to numerous domains of visual perception. The four fundamental symmetries, reflection, rotation, translation and glide reflection, can be combined in exactly 17 distinct ways. These wallpaper groups represent the complete set of symmetries in 2D images and have recently found use in the vision science community as an ideal stimulus set for studying the perception of symmetries in textures. The goal of the current study is to provide a more comprehensive description of responses to symmetry in the human visual system, by collecting both brain imaging (Steady-State Visual Evoked Potentials measured using high-density EEG) and behavioral (symmetry detection thresholds) data using the entire set of wallpaper groups. This allows us to probe the hierarchy of complexity among wallpaper groups, in which simpler groups are subgroups of more complex ones. We find that this hierarchy is preserved almost perfectly in both behavior and brain activity: A multi-level Bayesian GLM indicates that for most of the 63 subgroup relationships, subgroups produce lower amplitude responses in visual cortex (posterior probability: > 0.95 for 56 of 63) and require longer presentation durations to be reliably detected (posterior probability: > 0.95 for 49 of 63). This systematic pattern is seen only in visual cortex and only in components of the brain response known to be symmetric-specific. Our results show that representations of symmetries in the human brain are precise and rich in detail, and that this precision is reflected in behavior. These findings expand our understanding of symmetry perception, and open up new avenues for research on how fine-grained representations of regular textures contribute to natural vision.

sentences: Human gut bacteria produce TH17-modulating bile acid metabolites The microbiota plays a pivotal role in gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17a (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. While it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown. Furthermore, it was not clear whether 3-oxoLCA and other immunomodulatory bile acids are associated with gut inflammatory pathologies in humans. Using a high-throughput screen, we identified human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Like 3-oxoLCA, isoLCA suppressed TH17 differentiation by inhibiting ROR{gamma}t (retinoic acid receptor-related orphan nuclear receptor {gamma}t), a key TH17 cell-promoting transcription factor. Levels of both 3-oxoLCA and isoLCA and the 3-hydroxysteroid dehydrogenase (3-HSDH) genes required for their biosynthesis were significantly reduced in patients with inflammatory bowel diseases (IBD). Moreover, levels of these bile acids were inversely correlated with expression of TH17 cell-associated genes. Overall, our data suggest that bacterially produced TH17 cell-inhibitory bile acids may reduce the risk of autoimmune and inflammatory disorders such as IBD.

sentences: Identification of Cell-Free DNA Methylation Patterns Unique to the Human Left Ventricle as a Potential Indicator of Acute Cellular Rejection Increased levels of donor-derived cell-free DNA (dd-cfDNA) in recipient plasma have been associated with rejection after transplantation. DNA sequence differences have been used to distinguish between donor and recipient but epigenetic differences could also potentially identify dd-cfDNA. This pilot study aimed to identify ventricle-specific differentially methylated regions of DNA (DMRs) that could be detected in cfDNA. We identified 24 ventricle-specific DMRs and chose two for further study, one on chromosome 9 and one on chromosome 12. The specificity of both DMRs for the left ventricle was confirmed using genomic DNA from multiple human tissues. Serial matched samples of myocardium (n=33) and plasma (n=24) were collected from stable adult heart transplant recipients undergoing routine endomyocardial biopsy for rejection surveillance. Plasma DMR levels increased with biopsy-proven rejection grade for individual patients. Mean cellular apoptosis in biopsy samples increased significantly with rejection severity (2.4%, 4.4% and 10.0% for ACR 0R, 1R and 2R, respectively) but did not show a consistent relationship with DMR levels. We identified multiple DNA methylation patterns unique to the human ventricle and conclude that epigenetic differences in cfDNA populations represent a promising alternative strategy for the non-invasive detection of rejection.

sentences: Antibacterial effects of propolis and brood comb extracts on the causative agent of European Foulbrood (Melissococcus plutonius) in honey bees (Apis mellifera) Among a long list of parasites and pathogens that threaten the European honey bee (Apis mellifera), European Foulbrood (EFB) has become an urgent apiary disease, as epidemic outbreaks are becoming increasingly common. EFB is a bacterial disease of larval honey bees, caused by a gram-positive, anaerobic bacterium, Melissococcus plutonius. The most effective current treatment for EFB, oxytetracycline hydrochloride, can disrupt the bee microbiome, cause bee mortality and residues may persist in honey harvested for human consumption. In this study, we explore the efficacy of more sustainable bee-derived solutions, including propolis, honey comb and brood comb ethanol extracts. Using a series of dilutions of these extracts, we determined the minimum inhibitory concertation (MIC) of each bee-derived product on M. plutonius, as well as two model bacterial species, Staphylococcus saprophyticus (gram-positive) and Escherichia coli (gram-negative). Overall, we found that propolis extract was most effective at inhibiting growth of gram-positive bacteria, and that M. plutonious was also susceptible to honey comb (MIC = 16.00 mg/mL) and brood comb (MIC = 45.33 mg/mL) extracts, but at much higher concentrations than that of propolis (MIC = 1.14 mg/mL).

sentences: Nonlinear Stability Analysis for Artificial Kidney Multi-compartmental Models This paper addresses a new global stability analysis for a specific class of nonlinear multi-compartment models with non-positive flows and whose balances are described by equilibrium sets. We apply the stability analysis to our physiological-based model of extracellular fluid used during dialysis therapy in end-stage kidney disease patients. To gain an in-depth understanding of the risk associated with fluid removal by the artificial kidney during the short time (3-5hrs) of the dialysis therapy, we use the stability results to analyze the solutions behavior of our model under standard ultrafiltration and patient-specific ultrafiltration profiles. Therefore, the standard ultrafiltration profiles do not guarantee optimal outcomes, and we highly recommend incorporating physiological insights into the ultrafiltration profiles to improve outcomes.

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Simulated poaching affects global connectivity and efficiency in social networks of African savanna elephants - an exemplar of how human disturbance impacts group-living species Selective harvest, such as poaching, impacts group-living animals directly through mortality of individuals with desirable traits, and indirectly by altering the structure of their social networks. Understanding the relationship between the structural network changes and group performance in wild animals remains an outstanding problem. To address this knowledge gap, we evaluate the immediate effect of disturbance on group sociality in African savanna elephants -- an example, group-living species threatened by poaching. Drawing on static association data from one free ranging population, we constructed 100 virtual networks; performed a series of experiments  poaching the oldest, socially central or random individuals; and quantified the immediate change in the theoretical indices of network connectivity and efficiency of social diffusion. Although the virtual networks never broke down, targeted elimination of the socially central conspecifics, regardless of age, decreased network connectivity and efficiency. These findings hint at the need to further study resilience by modeling network reorganization and interaction-mediated socioecological learning, empirical data permitting. Our work is unique in quantifying connectivity together with global efficiency in multiple virtual networks that represent the sociodemographic diversity of elephant populations likely found in the wild. The basic design of our simulation platform makes it adaptable for hypothesis testing about the consequences of anthropogenic disturbance or lethal management on social interactions in a variety of group-living species with limited, real-world data.

Author SummaryWe consider the immediate response of animal groups to human disturbance by using the African savanna elephant as an example of a group-living species threatened by poaching. Previous research in one elephant population showed that poaching-induced mortality reduced social interaction among distantly related elephants, but not among close kin. Whether this type of resilience indicates that affected populations function similarity before and after poaching is an open problem. Understanding it is important because poaching often targets the largest and most socially and ecologically experienced group members. Drawing on empirical association data, we simulated poaching in 100 virtual elephant populations and eliminated the most senior or sociable members. Targeted poaching of sociable conspecifics was more impactful. Although it did not lead to population breakdown, it hampered theoretical features of interspecific associations that in other systems have been associated with group cohesion and the efficiency of transferring social information. Our findings suggest that further inquiry into the relationship between resilience to poaching and group performance is warranted. In addition, our simulation platform offers a generalizable basis for hypothesis testing in other social species, wild or captive, subject to exploitation by humans.

sentences: Uncovering Differences in Hydration Free Energies for Model Compound Mimics of Charged Sidechains of Amino Acids Free energies of hydration are of fundamental interest for modeling and understanding conformational and phase equilibria of macromolecular solutes in aqueous phases. Of particular relevance to systems such as intrinsically disordered proteins are the free energies of hydration and hydration structures of model compounds that mimic charged sidechains of Arg, Lys, Asp, and Glu. Here, we deploy a Thermodynamic Cycle based Proton Dissociation (TCPD) approach in conjunction with data from direct measurements to obtain estimates for the free energies of hydration for model compounds that mimic the sidechains of Arg+, Lys+, Asp-, and Glu-. Irrespective of the choice made for the hydration free energy of the proton, the TCPD approach reveals clear trends regarding the free energies of hydration for Arg+, Lys+, Asp-, and Glu-. These trends include asymmetries between the hydration free energies of acidic (Asp- and Glu-) and basic (Arg+ and Lys+) residues. Further, the TCPD analysis, which relies on a combination of experimental data, shows that the free energy of hydration of Arg+ is less favorable than that of Lys+. We sought a physical explanation for the TCPD derived trends free energies of hydration. To this end, we performed temperature dependent calculations of free energies of hydration and analyzed hydration structures from simulations that use the polarizable AMOEBA (Atomic Multipole Optimized Energetics for Biomolecular Applications) forcefield and water model. At 298 K, the AMOEBA model generates estimates of free energies of hydration that are consistent with TCPD values with a free energy of hydration for the proton of {approx} -259 kcal / mol. Analysis of temperature dependent simulations leads to a structural explanation for the observed differences in free energies of hydration of ionizable residues and reveals that the heat capacity of hydration is positive for Arg+ and Lys+ and negative for Asp- and Glu-.

sentences: Coiled coil control of growth factor and inhibitor-dependent EGFR trafficking anddegradation EGFR exhibits biased signaling, whereby growth factor or mutation-dependent changes in receptor conformation and/or dynamics elicit distinct intracellular outcomes. We report that many outcomes associated with activated EGFR are controlled by a two-state coiled coil switch located within the juxtamembrane segment (JM), an essential component of the cytosolic dimer interface. The position of this switch defines the path of endocytic trafficking and whether or not EGFR is degraded within lysosomes. JM coiled coil identity also predicts kinase-independent effects of oncogenic EGFR mutations and clinically relevant tyrosine kinase inhibitors (TKIs) that promote efficient, lysosome-based EGFR degradation. These findings provide a model for biased EGFR signaling, insights into kinase-independent activities of EGFR and clinically relevant TKIs, and identify new strategies for modulating protein lifetime.

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Effective mechanical potential of cell-cell interaction explains basic structural units of three-dimensional morphogenesis Mechanical forces of cell-cell interactions have been suggested to be critical for the emergence of diverse three-dimensional morphologies of multicellular organisms. The direct evaluation of the forces in living systems has been difficult due to technical limitations. Here, we developed a framework for inferring and modeling mechanical forces of cell-cell interactions. First, by analogy to coarse-grained models in molecular and colloidal sciences, cells were assumed to be spherical particles, where the mean forces (i.e. effective forces) of pairwise cell-cell interactions were considered. Then, the forces were statistically inferred from live imaging data, and subsequently, we successfully detected effective mechanical potentials of cell-cell interactions as a function of the cell-cell distances in Madin-Darby canine kidney (MDCK) cells, C.elegans early embryos, and mouse pre-implantation embryos. The qualitative and quantitative differences in the inferred potentials can be a control parameter for morphological transition during the mouse compaction process, and can also reproduce various three-dimensional morphologies including aggregates, cavities, tubes, cups, and two-dimensional sheets, which constitute basic structures observed during morphogenesis. We propose that effective potentials of cell-cell interactions are measurable, and their qualitative and quantitative features are critical for the emergence of diverse three-dimensional morphogenesis.

Significance statementEmergence of diverse morphologies of multicellular organisms is one of the most intriguing phenomena in nature. Mechanical forces generated by cells play central roles in morphogenesis, however, their measurement is technically limited. Furthermore, due to the complex situations in living systems, a model for describing the emergent properties of multicellular systems has not been established. Here, we developed a method for inferring mechanical potential energy of cell-cell interactions, and showed that the quantitative differences in the potential is alone sufficient to describe basic three-dimensional morphologies observed during embryogenesis and organogenesis. This framework sheds light on the emergent properties of multicellular systems.

sentences: Genome-wide association study on 13,167 individuals identifies regulators of hematopoietic stem and progenitor cell levels in human blood Understanding how hematopoietic stem and progenitor cells (HSPCs) are regulated is of central importance for the development of new therapies for blood disorders and stem cell transplantation. To date, HSPC regulation has been extensively studied in vitro and in animal models, but less is known about the mechanisms in vivo in humans. Here, in a genome-wide association study on 13,167 individuals, we identify 9 significant and 2 suggestive DNA sequence variants that influence HSPC (CD34+) levels in human blood. The identified loci associate with blood disorders, harbor known and novel HSPC genes, and affect gene expression in HSPCs. Interestingly, our strongest association maps to the PPM1H gene, encoding an evolutionarily conserved serine/threonine phosphatase never previously implicated in stem cell biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD34+ cell levels downregulates PPM1H. By functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates a MYB transcription factor binding site in PPM1H intron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, rs772557-A selectively increases HSPC subpopulations in which the MYB site is active, and PPM1H shRNA- knockdown increased CD34+ and CD34+90+ cell proportions in umbilical cord blood cultures. Our findings represent the first large-scale association study on a stem cell trait, illuminating HSPC regulation in vivo in humans, and identifying PPM1H as a novel inhibition target that can potentially be utilized clinically to facilitate stem cell harvesting for transplantation.

sentences: Identification of Escherichia coli Genes Essential for High-Level, Clinically Relevant, Resistance to Antibiotics Antibiotic resistance is one of the biggest public health challenges of our time. Here we present a novel approach to recognizing molecular mechanisms essential for maintaining high-level clinically relevant antibiotic resistance. To identify essential genes in this context, we subjected Escherichia coli EV18, a strain highly resistant to quinolones, to random transposon mutagenesis. This strains unique advantage is that the screen is performed at very high concentrations of the antibiotic, non-permissive for most strains. The transposons insertion affected the transcription of five genes required for the maintenance of resistance in EV18. Three of these genes (YihO, YhdP, and WaaY) have not been previously identified as essential for high-level antimicrobial resistance (AMR). Our work provides a new perspective to identify and characterize novel players crucial for maintaining AMR in E. coli.

sentences: Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of twelve known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represent structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites.

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Increasing Animal Cognition Research in Zoos Animal cognition covers various mental processes including perception, learning, decision-making and memory, and animal behavior is often used as a proxy for measuring cognition. Animal cognition/behavior research has multiple benefits; it provides fundamental knowledge on animal biology and evolution but can also have applied conservation and welfare applications. Zoos provide an excellent yet relatively untapped resource for animal cognition research, because they house a wide variety of species - many of which are under threat - and allow close observation and relatively high experimental control compared to the wild. Multi-zoo collaboration leads to increased sample size and species representation, which in turn leads to more robust science. However, there are salient challenges associated with zoo-based cognitive research, which are subject-based (e.g., small sample sizes at single zoos, untrained/unhabituated subjects, site effects) and human-based (e.g., time restrictions, safety concerns, and perceptions of animals interacting with unnatural technology or apparatus). We aim to increase the understanding and subsequent uptake of animal cognition research in zoos, by transparently outlining the main benefits and challenges. Importantly, we use our own research (1) a study on novelty responses in hornbills, and (2) a multi-site collaboration called the  ManyBirds project to demonstrate how challenges may be overcome. These potential options include using  drop and go apparatuses that require no training, close human contact or animal separation. This article is aimed at zoo animal care and research staff, as well as external researchers interested in zoo-based studies.

RESEARCH HIGHLIGHTSO_LIZoos are an excellent yet relatively untapped resource for animal cognition research.
C_LIO_LIZoo cognition research has historically been challenging, and traditional laboratory paradigms often do not translate well to the majority of zoos.
C_LIO_LISalient challenges of zoo-based cognitive research can be overcome by using less restrictive test apparatuses, limiting animal training and isolation, and subscribing to multi-zoo collaborative programs.
C_LI

sentences: DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as Alk target in the Drosophila visceral mesoderm. Development of the midgut visceral muscle of Drosophila crucially depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signalling, which is needed to specify founder cells (FCs) in the circular visceral mesoderm (VM). While activation of the Alk receptor by its ligand Jelly Belly (Jeb) is well characterized, only a small number of target molecules have been identified. Here, we assayed RNA polymerase II (Pol II) occupancy in VM cells by using the targeted DamID (TaDa) approach. To identify Alk targets we employed comparative analysis of embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differential expression of a number of genes, including the Snail/Scratch family transcription factor Kahuli (Kah). Upon further in vivo validation, we confirmed that Alk signalling regulates Kah mRNA expression in the VM. We show that Kah mutants display defects in the formation of midgut constrictions, similar to that of pointed (pnt) mutants. Analysis of publicly available ChIP data defined a Kah target-binding site similar to that of Snail. In addition, we compared genes that were differentially expressed in Kah mutants with publicly available Kah- and Pnt-ChIP datasets identifying a set of common target genes putatively regulated by Kah and Pnt in midgut constriction. Taken together, we (i) report a rich dataset of Alk responsive loci in the embryonic VM, (ii) provide the first functional characterization of the Kah transcription factor, identifying a role in embryonic midgut constriction, and (iii) suggest a model in which Kah and Pnt cooperate in embryonic midgut morphogenesis.

sentences: Parametric tests for Leave-One-Out Inter-Subject Correlations in fMRI provide adequate Type I error control while providing high sensitivity The inter-subject correlation (ISC) of fMRI data of different subjects performing the same task is a powerful way to localize and differentiate neural processes caused by a stimulus from those that spontaneously or idiosyncratically take place in each subject. The wider adoption of this method has however been impeded by the lack of widely available tools to assess the significance of the observed correlations. Several non-parametric approaches have been proposed, but these approaches are computationally intensive, challenging to implement, and sensitive methods to correct for multiple comparison across voxels are not yet well established. More widely available, and computationally simple, parametric methods have been criticized theoretically on the basis that dependencies in the data could inflate false positives. Here, we therefore endeavored to assess the actual performance of parametric tests on leave-one-out ISC values in two ways. First, we assess whether parametric tests protect against Type I error by assessing how often they find significant clusters of synchronized activity in publicly available datasets, in which such synchronization should not occur. This includes three resting state datasets, and one dataset in which participants did view movies, but where we randomly select segments that were not taken at the same time in the same movie. Contrary to what has been suspected, we find that parametric tests with corrections for multiple comparisons do protect appropriately against Type I error in that data. This was true for FDR correction at the voxel level at q < 0.05, with a minimum cluster-size of k = 20 voxels, FWE correction at the voxel level at  < 0.05, with a minimum cluster-size of k = 5, and for correction at the cluster-level with punc < 0.001 with k = max(20, FWEc). Second, we assessed how these parametric tests compare with non-parametric methods when it comes to detecting ISC when participants actually did watch the same movies. We used a dataset including 150 participants viewing two movies, and used a bootstrapping thresholding of the ISC in the entire dataset to outline our best guess of the network of brain regions that truly synchronize while viewing the movies. We then drew subsamples of between 10 and 50 participants from the entire dataset, calculated the ISC, and thresholded it using our candidate methods. We find that FDR thresholding with k = 20 in particular, was substantially more sensitive than bootstrapping methods in detecting this network even in smallish samples of N = 20 participants typical of cognitive neuroscience studies, while at the same time retaining appropriate specificity. Because the parametric tests we show to perform well are more readily available to the neuroscience community than the non-parametric tests previously championed, we trust that this finding paves the way to a wider adoption of ISC, and empowers a wider range of neuroimagers to use ISC to tackle the challenges of naturalistic neuroscience. In particular in the context of often limited sample sizes and modest effect sizes in cognitive neuroscience, we trust that using FDR correction in particular will help neuroimagers identify the contribution of higher brain regions that process stimuli in more loosely timed fashions, more effectively than non-parametric alternatives.

sentences: The Epigenetic Pacemaker is a more sensitive tool than penalized regression for identifying moderators of epigenetic aging Epigenetic clocks, DNA methylation based chronological age prediction models, are commonly employed to study age related biology. The error between the predicted and observed age is often interpreted as a form of biological age acceleration and many studies have measured the impact of environmental and other factors on epigenetic age. Epigenetic clocks are fit using approaches that minimize the error between the predicted and observed chronological age and as a result they reduce the impact of factors that may moderate the relationship between actual and epigenetic age. Here we compare the standard methods used to construct epigenetic clocks to an evolutionary framework of epigenetic aging, the epigenetic pacemaker (EPM) that directly models DNA methylation as a function of a time dependent epigenetic state. We show that the EPM is more sensitive than epigenetic clocks for the detection of factors that moderate the relationship between actual age and epigenetic state (ie epigenetic age). Specifically, we show that the EPM is more sensitive at detecting sex and cell type effects in a large aggregate data set and in an example case study is more sensitive sensitive at detecting age related methylation changes associated with polybrominated biphenyl exposure. Thus we find that the pacemaker provides a more robust framework for the study of factors that impact epigenetic age acceleration than traditional clocks based on linear regression models.

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Relative model selection can be sensitive to multiple sequence alignment uncertainty BackgroundMultiple sequence alignments (MSAs) represent the fundamental unit of data inputted to most comparative sequence analyses. In phylogenetic analyses in particular, errors in MSA construction have the potential to induce further errors in downstream analyses such as phylogenetic reconstruction itself, ancestral state reconstruction, and divergence time estimation. In addition to providing phylogenetic methods with an MSA to analyze, researchers must also specify a suitable evolutionary model for the given analysis. Most commonly, researchers apply relative model selection to select a model from candidate set and then provide both the MSA and the selected model as input to subsequent analyses. While the influence of MSA errors has been explored for most stages of phylogenetics pipelines, the potential effects of MSA uncertainty on the relative model selection procedure itself have not been explored.

ResultsWe assessed the consistency of relative model selection when presented with multiple perturbed versions of a given MSA. We find that while relative model selection is mostly robust to MSA uncertainty, in a substantial proportion of circumstances, relative model selection identifies distinct best-fitting models from different MSAs created from the same set of sequences. We find that this issue is more pervasive for nucleotide data compared to amino-acid data. However, we also find that it is challenging to predict whether relative model selection will be robust or sensitive to uncertainty in a given MSA.

ConclusionsWe find that that MSA uncertainty can affect virtually all steps of phylogenetic analysis pipelines to a greater extent than has previously been recognized, including relative model selection.

sentences: Targeting Stem-loop 1 of the SARS-CoV-2 5'UTR to suppress viral translation and Nsp1 evasion SARS-CoV-2 is a highly pathogenic virus that evades anti-viral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the mRNA entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the SL1 region of the SARS-CoV-2 5 UTR is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides (ASOs) inhibits viral translation and makes SARS-CoV-2 5 UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus own virulence mechanism against itself could force a critical trade off between drug resistance and pathogenicity.

sentences: Bioluminescent zebrafish transplantation model for drug discovery In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing.

sentences: A Unified Model for Entrainment by Circadian Clocks: Dynamic Circadian Integrated Response Characteristic (dCiRC) Circadian rhythm is a ubiquitous phenomenon, and it is observed in all biological kingdoms. In nature, their primary characteristic or phenotype is the phase of entrainment. There are two main hypotheses related to how circadian clocks entrain, parametric and non-parametric models. The parametric model focuses on the gradual changes of the clock parameters in response to the changing ambient condition, whereas the non-parametric model focuses on the instantaneous change of the phase of the clock in response to the zeitgeber. There are ample empirical data supporting both models. However, only recently has a unifying model been proposed, the circadian integrated response characteristic (CiRC). In the current study, we developed a system of ordinary differential equations, dynamic CiRC (dCiRC), that describes parameters of circadian rhythms and predicts the phase of entrainment in zeitgeber cycles. dCiRC mathematically extracts the underlying information of velocity changes of the internal clock that reflects the parametric model and the phase shift trajectory that reflects the non-parametric model from phase data under entraining conditions. As a proof of concept, we measured clock parameters of 26 Neurospora crassa ecotypes in both cycling and constant conditions using dCiRC. Our data showed that the morning light shortens the period of the clock while the afternoon light lengthens it. We also found that individual ecotypes have different strategies of integrating light effects to accomplish the optimal phase of entrainment, a model feature that is consistent with our knowledge of how circadian clocks are organized and encoded. The unified model dCiRC will provide new insights into how circadian clocks function under different zeitgeber conditions. We suggest that this type of model may be useful in the advent of chronotherapies.

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Phosphatidylserine within the Viral Membrane Enhances Chikungunya Virus Infectivity in a Cell-type Dependent Manner Chikungunya virus (CHIKV), an alphavirus of the Togaviridae family, is the causative agent of the human disease chikungunya fever (CHIKF), which is characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals exist for CHIKV. Preventing the attachment of viral particles to host cells is an attractive intervention strategy. Viral entry of enveloped viruses from diverse families including Filoviridae and Flaviviridae is mediated or enhanced by phosphatidylserine receptors (PSRs). PSRs facilitate the attachment of enveloped viruses to cells by binding to exposed phosphatidylserine (PS) in the viral lipid membrane - a process termed viral apoptotic mimicry. To investigate the role of viral apoptotic mimicry during CHIKV infection, we produced viral particles with discrete amounts of exposed PS on the virion envelope by exploiting the cellular distribution of phospholipids at the plasma membrane. We found that CHIKV particles containing high outer leaflet PS (produced in cells lacking flippase activity) were more infectious in Vero cells than particles containing low levels of outer leaflet PS (produced in cells lacking scramblase activity). However, the same viral particles were similarly infectious in NIH3T3 and HAP1 cells, suggesting PS levels can influence infectivity only in cells with high levels of PSRs. Interestingly, PS-dependent CHIKV entry was observed in mosquito Aag2 cells, but not C6/36 cells. These data demonstrate that CHIKV entry via viral apoptotic mimicry is cell-type dependent. Furthermore, viral apoptotic mimicry has a mechanistic basis to influence viral dynamics in vivo in both the human and mosquito host.

ImportanceOutbreaks of Chikungunya virus (CHIKV) have occurred throughout Africa, Asia, and Europe. Climate change permits the expansion of Aedes mosquito vectors into more temperate regions, broadening the geographic range and increasing the frequency of future human outbreaks. The molecular basis underlying the broad host and cellular tropism of CHIKV remains unresolved. While several host molecules have been implicated in CHIKV viral attachment and entry, the role of lipid-mediated attachment (viral apoptotic mimicry) is unclear. We observed that higher levels of externalized phosphatidylserine (PS) in the viral lipid bilayer correlated with enhanced CHIKV infectivity in mammalian cells abundant with PS receptors and lacking alternative attachment factors. Interestingly, CHIKV infection in mosquito Aag2 cells was also affected by viral PS accessibility. This study further delineates the role of virus-cell attachment molecules in CHIKV infection. Viral apoptotic mimicry has potential to influence CHIKV dynamics in vivo in both the human and mosquito host.

sentences: Local and substrate-specific S-palmitoylation determines subcellular localization of Gαo Peripheral membrane proteins (PMPs) associate with cellular membranes through post-translational modifications like S-palmitoylation. The Golgi apparatus is generally viewed as the transitory station where palmitoyl acyltransferases (PATs) modify PMPs, which are then transported to their ultimate destinations such as the plasma membrane (PM). However, little substrate specificity among the many PATs has been determined. Here we describe the inherent partitioning of Go - -subunit of heterotrimeric Go proteins - to PM and Golgi, independent from Golgi-to-PM transport. A minimal code within Go N-terminus governs its compartmentalization and re-coding produces G protein versions with shifted localization. We establish the S-palmitoylation at the outer nuclear membrane assay ("SwissKASH") to probe substrate specificity of PATs in intact cells. With this assay, we show that PATs localizing to different membrane compartments display remarkable substrate selectivity, which is the basis for PMP compartmentalization. Our findings uncover a mechanism governing protein localization and establish the basis for innovative drug discovery.

sentences: LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans SARS-CoV-2 infection is mediated by the entry receptor ACE2. Although attachment factors and co-receptors facilitating entry are extensively studied, cellular entry factors inhibiting viral entry are largely unknown. Using a surfaceome CRISPR activation screen, we identified human LRRC15 as an inhibitory receptor for SARS-CoV-2 entry. LRRC15 directly binds to the receptor-binding domain (RBD) of spike protein with a moderate affinity and inhibits spike-mediated entry. Analysis of human lung single cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not co-expressed in the same cell types in the lung. Strikingly, expression of LRRC15 in ACE2-negative cells blocks spike-mediated viral entry in ACE2+ cell in trans, suggesting a protective role of LRRC15 in a physiological context. Therefore, LRRC15 represents an inhibitory receptor for SARS-CoV-2 regulating viral entry in trans.

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Novel RT-ddPCR assays for determining the transcriptional profile of SARS-CoV-2 The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription.

Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.

HighlightsO_LIWe developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2
C_LIO_LIOur panel targets nongenic and genic regions in genomic and subgenomic RNAs
C_LIO_LIAll assays detect 1-10 copies and are linear over 3-4 orders of magnitude
C_LIO_LIAll assays correlated with the clinical Abbott SARS-CoV-2 Viral Load Assay
C_LIO_LIClinical samples showed higher copy numbers for targets at the 3 end of the genome
C_LI

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Colonial choanoflagellate isolated from Mono Lake harbors a microbiome Choanoflagellates offer key insights into bacterial influences on the origin and early evolution of animals. Here we report the isolation and characterization of a new colonial choanoflagellate species, Barroeca monosierra, that, unlike previously characterized species, harbors a microbiome. B. monosierra was isolated from Mono Lake, California and forms large spherical colonies that are more than an order of magnitude larger than those formed by the closely related Salpingoeca rosetta. By designing fluorescence in situ hybridization probes from metagenomic sequences, we found that B. monosierra colonies are colonized by members of the halotolerant and closely related Saccharospirillaceae and Oceanospirillaceae, as well as purple sulfur bacteria (Ectothiorhodospiraceae) and non-sulfur Rhodobacteraceae. This relatively simple microbiome in a close relative of animals presents a new experimental model for investigating the evolution of stable interactions among eukaryotes and bacteria.

IMPORTANCEThe animals and bacteria of Mono Lake (California) have evolved diverse strategies for surviving the hypersaline, alkaline, arsenic-rich environment. We sought to investigate whether the closest living relatives of animals, the choanoflagellates, exist among the relatively limited diversity of organisms in Mono Lake. We repeatedly isolated members of a single species of choanoflagellate, which we have named Barroeca monosierra, suggesting that it is a stable and abundant part of the ecosystem. Characterization of B. monosierra revealed that it forms large spherical colonies that each contain a microbiome, providing an opportunity to investigate the evolution of stable physical associations between eukaryotes and bacteria.

sentences: A model of egocentric to allocentric understanding in mammalian brains In the mammalian brain, allocentric representations support efficient self-location and flexible navigation. A number of distinct populations of these spatial responses have been identified but no unified function has been shown to account for their emergence. Here we developed a network, trained with a simple predictive objective, that was capable of mapping egocentric information into an allocentric spatial reference frame. The prediction of visual inputs was sufficient to drive the appearance of spatial representations resembling those observed in rodents: head direction, boundary vector, and place cells, along with the recently discovered egocentric boundary cells, suggesting predictive coding as a principle for their emergence in animals. Strikingly, the network learned a solution for head direction tracking and stabilisation convergent with known biological connectivity. Moreover, like mammalian representations, responses were robust to environmental manipulations, including exposure to novel settings. In contrast to existing reinforcement learning approaches, agents equipped with this network were able to flexibly reuse learnt behaviours --adapting rapidly to unfamiliar environments. Thus, our results indicate that these representations, derived from a simple egocentric predictive framework, form an efficient basis-set for cognitive mapping.

sentences: Infection and vaccine-induced neutralizing antibody responses to the SARS-CoV-2 B.1.617.1 variant SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.

sentences: Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key-residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression.

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The molecular chaperone Cysteine String Protein is required to stabilize trans-SNARE complexes during human sperm acrosomal exocytosis Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to assure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine String Protein (CSP) is a member of the Hsp40 co-chaperones, and for more than 20 years, most research published focused on CSPs role in synapsis. However, the participation of molecular chaperones in acrosomal exocytosis is poorly understood. Using western blot and indirect immunofluorescence, we showed that CSP is present in human sperm, is predominantly bound to membranes, and is palmitoylated. Moreover, using electron microscopy and functional assays, we reported that sequestration of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, our data demonstrated that CSP is necessary and mediates the trans-SNARE complex assembly between the outer acrosomal and plasma membranes, thereby regulating human sperm acrosomal exocytosis. Understanding CSPs role is critical in identifying new biomarkers and generating new rational-based approaches to treating male infertility.

Summary statementCysteine String Protein is necessary and mediates the trans-SNARE complex assembly between the outer acrosomal and plasma membranes, thereby regulating human sperm acrosomal exocytosis.

sentences: Coordinated activation of both TGFβ and BMP canonical pathways regulates autophagy and tissue regeneration in acetaminophen induced liver injury Transforming Growth Factor-{beta}s (TGF{beta}s)/Activins and Bone Morphogenetic Proteins (BMPs) have been implicated in numerous aspects of hepatic pathophysiology. However, the way by which hepatocytes integrate and decode the interplay between the TGF{beta}/Activin and BMP branches in health and disease is still not fully understood. To address this, TGF{beta}/BMP Smad- responsive double transgenic reporter mice were generated and utilized to map patterns of TGF{beta}- and/or BMP-pathway activation during acetaminophen- induced liver injury. TGF{beta} signaling was blocked either pharmacologically or by Smad7 over-expression and the transcriptomes of canonical TGF{beta}- and/or BMP4-treated hepatospheres and Smad7-treated livers were analyzed to highlight TGF{beta}-superfamily-regulated pathways and processes. Acetaminophen administration led to dynamically evolving, stage- and context-specific, patterns of hepatic TGF{beta}/Activin and BMP-reporter expression. TGF{beta}-superfamily signaling was activated in an autophagy prone zone at the borders between healthy and injured tissue. Inhibition of TGF{beta}-superfamily signaling attenuated autophagy, exacerbated liver histopathology, and finally led to accelerated tissue-recovery. Hallmarks of this process were the paraptosis-like cell death and the attenuation of immune and reparatory cell responses. Transcriptomic analysis highlighted autophagy as a prominent TGF{beta}1- and BMP4-regulated process and recognized Trp53inp2 as the top TGF{beta}-superfamily-regulated autophagy-related gene. Collectively, these findings implicate the coordinated activation of both canonical TGF{beta}-superfamily signalling branches in balancing autophagic response and tissue-reparatory and -regenerative processes upon acetaminophen-induced hepatotoxicity, highlighting opportunities and putative risks associated with their targeting for treatment of hepatic diseases.

sentences: The retrosplenial cortex combines internal and external cues to encode head velocity during navigation The extent to which we successfully navigate the environment depends on our ability to continuously track our heading direction and speed. Angular head velocity (AHV) cells, which encode the speed and direction of head turns during navigation, are fundamental to this process, yet the mechanisms that determine their function remain unknown. By performing chronic single-unit recordings in the retrosplenial cortex (RSP) of the mouse and tracking the activity of individual AHV neurons between freely moving and head-restrained conditions, we find that vestibular inputs dominate AHV signalling. In addition, we discover that self-generated optic flow input onto these neurons increases the gain and signal-to-noise ratio of angular velocity coding during free exploration. Psychophysical experiments and neural decoding further reveal that vestibular-visual integration increases the perceptual accuracy of egocentric angular velocity and the fidelity of its representation by RSP ensembles. We propose that while AHV coding is dependent on vestibular input, it also uses vision to maximise navigation accuracy in nocturnal and diurnal environments.

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Kasugamycin is a novel chitinase 1 inhibitor with strong antifibrotic effects on pulmonary fibrosis RationalePulmonary fibrosis is a devastating lung disease with few therapeutic options. Chitinase 1 (CHIT1), an 18 glycosyl hydrolase family member, contributes to the pathogenesis of pulmonary fibrosis through regulation of Transforming Growth Factor (TGF)-{beta} signaling and effector function. Therefore, CHIT1 is a potential therapeutic target of pulmonary fibrosis.

ObjectivesThis study aimed to identify and characterize a druggable CHIT1 inhibitor with strong antifibrotic activity and minimal toxicity for therapeutic application to pulmonary fibrosis.

MethodsExtensive screening of small molecule libraries identified the aminoglycoside antibiotic Kasugamycin as a potent CHIT1 inhibitor.

Measurements and Main ResultsElevated levels of CHIT1 were detected in the lungs of patients with pulmonary fibrosis. In vivo bleomycin- and TGF-{beta}-stimulated murine models of pulmonary fibrosis, Kasugamycin showed impressive anti-fibrotic effects in both preventive and therapeutic conditions. In vitro studies also demonstrated that Kasugamycin inhibits fibrotic macrophage activation, fibroblast proliferation and myofibroblast transformation. Null mutation of transforming growth factor beta associated protein 1 (TGFBRAP1), a recently identified CHIT1 interacting signaling molecule, phenocopied antifibrotic effects of Kasugamycin in in vivo lungs and in vitro fibroblasts responses. Kasugamycin inhibits physical association between CHIT1 and TGFBRAP1, suggesting that antifibrotic effect of Kasugamycin is mediated through regulation of TGFBRAP1, at least in part.

ConclusionsThese studies demonstrate that Kasugamycin is a novel CHIT1 inhibitor with strong antifibrotic effect that can be further developed as an effective and safe therapeutic drug for pulmonary fibrosis.

sentences: Capturing hidden regulation based on noise change of gene expression level from single cell RNA-seq in yeast Recent progress in high throughput single cell RNA-seq (scRNA-seq) has activated the development of data-driven inferring methods of gene regulatory networks. Most network estimations assume that perturbations produce downstream effects. However, the effects of gene perturbations are sometimes compensated by a gene with redundant functionality (functional compensation). In order to avoid functional compensation, previous studies constructed double gene deletions, but its vast nature of gene combinations was not suitable for comprehensive network estimation. We hypothesized that functional compensation may emerge as a noise change without mean change (noise-only change) due to varying physical properties and strong compensation effects. Here, we show compensated interactions, which are not detected by mean change, are captured by noise-only change quantified from scRNA-seq. We investigated whether noise-only change genes caused by a single deletion of STP1 and STP2, which have strong functional compensation, are enriched in redundantly regulated genes. As a result, noise-only change genes are enriched in their redundantly regulated genes. Furthermore, novel downstream genes detected from noise change are enriched in "transport", which is related to known downstream genes. Herein, we suggest the noise difference comparison has the potential to be applied as a new strategy for network estimation that capture even compensated interaction.

sentences: Characterization of spatial dynamics of fMRI data in white matter using diffusion-informed white matter harmonics In this work, we leverage the Laplacian eigenbasis of voxel- wise white matter (WM) graphs derived from diffusion- weighted MRI data, dubbed WM harmonics, to characterize the spatial structure of WM fMRI data. Our motivation for such a characterization is based on studies that show WM fMRI data exhibit a spatial correlational anisotropy that coincides with underlying fiber patterns. By quantifying the energy content of WM fMRI data associated with subsets of WM harmonics across multiple spectral bands, we show that the data exhibits notable subtle spatial modulations under functional load that are not manifested during rest. WM har- monics provide a novel means to study the spatial dynamics of WM fMRI data, in such way that the analysis is informed by the underlying anatomical structure.

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Experimental evolution in TP53 deficient gastric organoids recapitulates tumorigenesis The earliest events during human tumor initiation are poorly characterized but may hold clues as to how to detect and prevent malignancy. Here we model occult pre-neoplasia by engineering TP53 deficiency, a common and early event in gastric cancer, into human gastric organoids. By performing experimental evolution in multiple clonally derived cultures over two years we define causal relationships between this common initiating genetic lesion and resulting phenotypes. TP53 loss elicited progressive aneuploidy, including copy number alterations and complex structural variants that are common in gastric cancers and which follow preferred temporal orders. Longitudinal single cell sequencing of TP53 deficient gastric organoids similarly indicates progression towards malignant transcriptional programs. Moreover, lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programs repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a striking degree of phenotypic convergence in pre-malignant epithelial organoids, implying that the earliest stages of tumorigenesis may be predictable while illuminating evolutionary constraints and barriers to malignant transformation.

Graphical abstract

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sentences: Evolutionary determinants of non-seasonal breeding in wild chacma baboons Animal reproductive phenology varies from strongly seasonal to non-seasonal, sometimes among closely related or sympatric species. While the extent of reproductive seasonality is often attributed to environmental seasonality, this fails to explain many cases of non-seasonal breeding in seasonal environments. We investigated the evolutionary determinants of non-seasonal breeding in a wild primate, the chacma baboon (Papio ursinus), living in a seasonal environment with high climatic unpredictability. We tested three hypotheses proposing that non-seasonal breeding has evolved in response to (1) climatic unpredictability, (2) reproductive competition between females favouring birth asynchrony, and (3) individual, rank-dependent variations in optimal reproductive timing. We found strong support for an effect of reproductive asynchrony modulated by rank: (i) birth synchrony is costly to subordinate females, lengthening their interbirth intervals, and (ii) females delay their reproductive timings (fertility periods and conceptions) according to other females in the group to stagger conceptions. These results indicate that reproductive competition generates reproductive asynchrony, weakening the intensity of reproductive seasonality at the population level. This study emphasizes the importance of sociality in mediating the evolution of reproductive phenology in gregarious organisms, a result of broad significance for understanding key demographic parameters driving population responses to increasing climatic fluctuations.

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Title-Effects of Catha Edulis Forsk on Spatial Cognition and correlation with serum electrolytes in Wild- type Male White Albino Rats BackgroundThe burdens of psychostimulant use disorders are becoming a worldwide problem. One of the psychostimulants widely consumed in Ethiopia and East African countries is Catha edulis Forsk (khat). However, no studies have been conducted on the cognitive effects of khat and its correlation with serum electrolytes. The present study was aimed to evaluate the effects of khat on cognitive functions and its correlation with serum electrolytes.

Materials and MethodsA total of 36 adult (7-8 weeks) wild-type male Swiss albino rats weighing between 213 and 229g were used in this study. The rats were received crude khat extract subchronically (kesc) (100 mg/kg, 200 mg/kg and 300 mg/kg b.w), khat juice (khJ 2.5 mL/kg) and 2% tween 80 in distilled water (T80W- v/v, vehicle) and khat extract subacutely (kesa) (300 mg/kg). Spatial learning and memory were measured using Morris water maze model and serum electrolytes were measured using Cobas 6000. The data were analyzed using SPSS version 21.0 and Microsoft Excel.

ResultsSpatial learning was improved with trials across the groups, while average escape latency (s) and swim path-length (cm) of rats that received kesc 200 mg/kg (p<0.001 and p<0.001) and kesc 300 mg/kg (p<0.01 and p<0.001) was significantly greater than rats that received the vehicle. However, there was no significant difference in the latency between rats that received kesa 300mg/kg and vehicle (p>0.05). Thigmotaxis was significantly higher in rats that received all doses of khat extract (p<0.001). The time spent in the target quadrant in rats that received kesc 300 mg/kg was significantly reduced (p<0.05). Serum calcium level was inversely correlated with the escape latency (R=-0.417, p<0.05) in rats that received khat.

Conclusionskhat extract and juice administered subchronically, but not subacute administration, impaired learning and memory in rats and was associated with serum calcium reduction. The neuronal basis for such alteration should be investigated.

sentences: Cancer Hallmarks Define a Continuum of Plastic Cell States between Small Cell Lung Cancer Archetypes Small Cell Lung Cancer (SCLC) tumors are heterogeneous mixtures of transcriptional subtypes. Understanding subtype dynamics could be key to explaining the aggressive properties that make SCLC a recalcitrant cancer. Applying archetype analysis and evolutionary theory to bulk and single-cell transcriptomics, we show that SCLC cells reside within a cell-state continuum rather than in discrete subtype clusters. Gene expression signatures and ontologies indicate each vertex of the continuum corresponds to a functional phenotype optimized for a cancer hallmark task: three neuroendocrine archetypes specialize in proliferation/survival, inflammation and immune evasion, and two non-neuroendocrine archetypes in angiogenesis and metabolic dysregulation. Single cells can trade-off between these defined tasks to increase fitness and survival. SCLC cells can easily transition from specialists that optimize a single task to generalists that fall within the continuum, suggesting that phenotypic plasticity may be a mechanism by which SCLC cells become recalcitrant to treatment and adaptable to diverse microenvironments. We show that plasticity is uncoupled from the phenotype of single cells using a novel RNA-velocity-based metric, suggesting both specialist and generalist cells have the capability of becoming destabilized and transitioning to other phenotypes. We use network simulations to identify transcription factors such as MYC that promote plasticity and resistance to treatment. Our analysis pipeline is suitable to elucidate the role of phenotypic plasticity in any cancer type, and positions SCLC as a prime candidate for treatments that target plasticity.

sentences: Sulfation of glycosaminoglycans depends on catalytic activity of a lithium-inhibited phosphatase Golgi-resident bisphosphate nucleotidase 2 (BPNT2) is a member of a family of magnesium-dependent/lithium-inhibited phosphatases that share a three-dimensional structural motif that directly coordinates metal binding to effect phosphate hydrolysis. BPNT2 is responsible for the breakdown of 3-phosphoadenosine-5-phosphate (PAP), a by-product of glycosaminoglycan (GAG) sulfation. Disruption of BPNT2 in mice leads to skeletal abnormalities due to impaired GAG sulfation, especially chondroitin-4-sulfation. Mutations in BPNT2 have also been found to underlie a chondrodysplastic disorder in humans. The precise mechanism by which loss of BPNT2 impairs sulfation remains unclear. Here, we utilize an in vitro approach using mouse embryonic fibroblasts (MEFs) to test the hypothesis that catalytic activity of BPNT2 is required for GAG sulfation. We show that a catalytic-dead Bpnt2 construct (D108A) does not rescue impairments in intracellular or secreted sulfated GAG, including decreased chondroitin-4-sulfate, present in Bpnt2-knockout MEFs. We also demonstrate that missense mutations in Bpnt2 which are adjacent to the catalytic site (and known to cause chondrodysplasia in humans) recapitulate defects in overall GAG sulfation and chondroitin-4-sulfation in MEF cultures. We further show that treatment of MEFs with lithium inhibits GAG sulfation, and that this effect depends on the presence of BPNT2. This work demonstrates that the catalytic activity of an enzyme potently inhibited by lithium can modulate GAG sulfation and therefore extracellular matrix composition, revealing new insights into lithium pharmacology and the pathophysiology of psychiatric disorders responsive to lithium.

sentences: A modified porous silicon microparticle promotes mucosal delivery of SARS-CoV-2 antigen and induction of potent and durable systemic and mucosal T helper 1 skewed protective immunity Development of optimal SARS-CoV-2 vaccines to induce potent, long-lasting immunity and provide cross-reactive protection against emerging variants remains a high priority. Here, we report that a modified porous silicon microparticle (mPSM)-adjuvanted SARS-CoV-2 receptor-binding domain (RBD) vaccine activated dendritic cells and generated more potent and durable SARS-CoV-2-specific systemic humoral and type 1 helper T (Th) cell-mediated immune responses than alum-formulated RBD following parenteral vaccination, and protected mice from SARS-CoV-2 and Beta variant infection. mPSM facilitated the uptake of SARS-CoV-2 RBD antigens by nasal and airway epithelial cells. Parenteral and intranasal prime and boost vaccinations with mPSM-RBD elicited potent systemic and lung resident memory T and B cells and SARS-CoV-2 specific IgA responses, and markedly diminished viral loads and inflammation in the lung following SARS-CoV-2 Delta variant infection. Our results suggest that mPSM can serve as potent adjuvant for SARS-CoV-2 subunit vaccine which is effective for systemic and mucosal vaccination.

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Re-expressing coefficients from regression models for inclusion in a meta-analysis BackgroundQuantitative summaries of study results, or meta-analyses, have proven useful in several fields, but pose special challenges when applied to non-randomized studies of exposure in relation to outcome. One such issue arises when summarizing regression results when some studies have log-transformed exposure while others have not. Methods of re-expression have recently been developed, with the goal of generating comparable results across studies regardless of data transformation. We examined the validity of three re-expression methods using simulations and real data examples.

MethodsSimulations were developed to examine the validity of the re-expression methods when the exposure had a skewed distribution. We also identified 15 published studies for which we were able to either access the data and replicate the result, or which had provided regression coefficients ({beta}) from analyses with both log-transformed and untransformed exposure data. For the simulated and real data we re-expressed {beta} as though it had been based on untransformed data, and vice versa. We compared the re-expressed results to fitted regression coefficients. The proportional difference from the fitted {beta} was used to quantitate bias. We also examined the effect of influential observations on the success of the re-expression using the real data examples.

ResultsIn the simulated and real data examples, all three re-expression methods usually gave biased results. The method of Rodriguez-Barranco et al. (2017) tended to give less biased results but with a wider range of bias. For example, in the real data, when re-expressing results as if they had been fit to untransformed exposure, the median estimate was 22% too high, with a range of -40% to 1,904%. In some analyses, the presence of influential observations had a large effect on the validity of the re-expression.

ConclusionsWhen the distribution of exposure is skewed, the re-expression methods examined are likely to give biased results. The bias varied by method, the direction of the re-expression, skewness, influential observations, and in some cases, the median exposure. Meta-analysts using any of these re-expression methods may want to consider the uncertainty, the likely direction and degree of bias, and conduct sensitivity analyses on the re-expressed results.

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Intercellular crosstalk regulating ARRB2/RARRES1 is involved in transition from fibrosis to cancer Progressive fibrogenesis in chronic liver injury is often associated with cancer development. Beta-arrestin-2 (ARRB2) is a regulator of the profibrotic Angiotensin II type 1 receptor (AGTR1). The role of ARRB2 in liver fibrosis and in the transition from fibrosis to cancer is not fully understood and was investigated in this study.

This study demonstrates that upregulation of the retinoic acid receptor responder 1 (RARRES1) in HSC mediated by ARRB2 leads to fibrosis. This process is driven by exosomal ARRB2 transfer to HSC, major fibrosis contributors, from injured hepatocytes, which highly express ARRB2. By contrast, downregulation of RARRES1 in hepatocytes induces malignant transformation and hepatocellular carcinoma (HCC) development. Consequently, Arrb2-deficient mice show higher number and size of liver tumors than wild-type mice in a hepatocellular carcinoma model with fibrosis. The identified relationship between ARRB2 and RARRES1 was observed in at least two species, including human cells and tissues in fibrosis and HCC and has a predictive value for survival in cancer patients. This study describes the discovery of a novel molecular pathway mediating the transition from fibrosis to cancer offering potential diagnostics and therapeutics.

sentences: High throughput RNA sequencing discovers symptomatic and latent viruses: an example from ornamental Hibiscus Hibiscus rosa-sinensis L. (Hibiscus, Malvaceae) is an ornamental species grown widely in amenity plantings. We collected leaves on an urban roadside pavement (sidewalk) near a market in Guangzhou which showed multiple symptoms of leaf rolling, deformation and chlorosis. Initial evaluation by electron microscopy using negative staining of drip preparations revealed the presence of tobamovirus-like particles. Total RNA was extracted, and, unusually, without any RNA selection based on sequence, was used for cDNA library construction and high-throughput survey sequencing. From the 814 Mb of clean sequence data (from 2,712,161 paired reads of 150 bp) reads representing chloroplast, ribosomal, and mitochondrial genes were filtered out, eliminating 79.1% of reads. 1,135,848 x 150 bp of the sequence was retained and screened for viral sequences. Assembly of these sequences detected nine virus species from seven virus genera comprising three tobamoviruses, namely, Tobacco mosaic virus, Tobacco mild green mosaic virus and Hibiscus latent Singapore virus, Turnip mosaic virus (Potyvirus), Potato virus M (Carlavirus), Hibiscus chlorotic ringspot virus (Betacarmovirus), Fabavirus sp (Fabavirus), Cotton leaf curl Multan virus (Begomovirus) and a putative mitoviruses replicating in mitochondria, Chenopodium quinoa mitovirus 1. Mapping the reads to complete virus reference sequences showed high and uniform coverage of the genomes from 3,729 x coverage for Turnip mosaic virus to 22 x for Cotton leaf curl Multan virus. By comparison, nuclear reference genes actin showed 14 x coverage and polyubiquitin 27 x. Notable variants from reference sequences (SNPs) were identified. With the low cost of sequencing and potential for semi-automated bioinformatic pipelines, the whole-RNA approach has huge potential for identifying multiple undiagnosed viruses in ornamental plants, resulting in the ability to take preventive measures in production facilities against spread and to product quality for the mutual benefit of producers and consumers.

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Detecting Flavobacterial Fish Pathogens in the Environment Using High-Throughput Community Analysis Diseases caused by the fish pathogens Flavobacterium columnare and Flavobacterium psychrophilum are major contributors of preventable losses in the aquaculture industry. The persistent and difficult to control infections caused by these bacteria make timely intervention and prophylactic elimination of pathogen reservoirs important measures to combat these disease-causing agents. In the present study, we present two independent assays for detecting these pathogens in a range of environmental samples. Natural water samples were inoculated with F. columnare and F. psychrophilum cells, and pathogen levels were detected using Illumina MiSeq sequencing and droplet digital PCR. Both detection methods accurately identified pathogen-positive samples and showed good agreement in quantifying each pathogen. Additionally, the real-world application of these approaches was demonstrated using environmental samples collected at a rainbow trout aquaculture facility. These results show that both methods can serve as useful tools for surveillance efforts in aquaculture facilities, where the early detection of these flavobacterial pathogens may direct preventative measures to reduce disease occurrence.

ImportanceEarly detection of a deadly disease outbreak in a population can be the difference between mass fatality or mitigated effects. In the present study, we evaluated and compared two techniques for detecting economically impactful aquaculture pathogens. We demonstrate that one of these techniques, 16S rRNA gene sequencing using Illumina MiSeq technology, provides the ability to accurately detect two fish pathogens, F. columnare and F. psychrophilum, while simultaneously profiling the native microbial community. The second technique, droplet digital PCR, is commonly used for pathogen detection, and the results obtained using the assays we designed with this method served to validate those obtained using the MiSeq method. These two methods offer distinct advantages. The MiSeq method pairs pathogen detection and microbial community profiling to answer immediate and long-term fish health concerns, while droplet digital PCR method provides fast and highly sensitive detection that is useful for surveillance and rapid clinical responses.

sentences: Staring at the naked Goddess. Unraveling structure and reactivity of Artemis endonuclease interacting with a DNA double strand. Artemis is an endonuclease responsible for breaking hairpin DNA strand during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, or the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulation, both classically and at hybrid quantum mechanics/ molecular mechanics level, we evidence the stable interaction modes of Artemis with a model DNA strand. We also analyze the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.

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The temporal transcriptomic signature of cartilage formation Chondrogenesis is a multistep process whereby cartilage progenitor cells generate a tissue with distinct structural and functional properties. Although several approaches to cartilage regeneration rely on the differentiation of implanted progenitor cells, the temporal transcriptomic landscape of in vitro chondrogenesis in different models has not been reported. Using RNA sequencing, we examined the differences between gene expression patterns during early cartilage formation in micromass cultures of embryonic limb bud-derived progenitors. Principal component analysis indicated a progressively different and distinct transcriptome during chondrogenesis. Differentially expressed genes (DEGs) based on pairwise comparisons of samples from consecutive days were classified into clusters and analysed. We confirmed the involvement of the top DEGs in chondrogenic differentiation with pathway analysis. We discovered several chondrogenesis-associated transcription factors and new collagen subtypes not previously linked to cartilage formation. These results provide, for the first time, a detailed insight into the molecular mechanism of in vitro chondrogenesis and provide a new gold standard for the temporal transcriptomic landscape of chondrogenic differentiation that may serve as a platform for new approaches in cartilage tissue engineering.

Highlights O_LIChondroprogenitors derived from chicken embryonic limb buds were differentiated into chondrocytes.
C_LIO_LIRNA-sequencing revealed expression levels of 24,145 transcripts.
C_LIO_LIWe examined the global expression patterns during chondrogenesis and defined 6 clusters.
C_LIO_LIUsing WGCNA analysis, we created a module of genes that had similar patterns to the chondrogenic transcription factor SOX9.
C_LIO_LIWe created a list of transcription factors with a hitherto unexplored role in chondrogenesis.
C_LI

sentences: OCT4 interprets and enhances nucleosome flexibility Pioneer transcription factors are proteins that induce cellular identity transitions by binding to inaccessible regions of DNA in nuclear chromatin. They contribute to chromatin opening and recruit other factors to regulatory DNA elements. The structural features and dynamics modulating their interaction with nucleosomes are still unresolved. From a combination of experiments and molecular simulations, we reveal here how the pioneer factor and master regulator of pluripotency, Oct4, interprets and enhances nucleosome structural flexibility. The magnitude of Oct4s impact on nucleosome dynamics depends on the binding site position and the mobility of the unstructured tails of nucleosomal histone proteins. Oct4 propagates and stabilizes open nucleosome conformations by specific sequence recognition and nonspecific DNA exploration. Our findings provide a structural basis for the versatility of transcription factors in engaging with nucleosomes and have implications for understanding how pioneer factors induce chromatin dynamics.

sentences: Examining Blunted Initial Response to Reward and Recent Suicidal Ideation in Children and Adolescents Using Event-Related Potentials: Failure to Conceptually Replicate Across Two Independent Samples A recent study by Tsypes and colleagues (2019) found that children with recent suicidal ideation had blunted neural reward processing, as measured by the reward positivity (RewP), compared to matched controls, and that this difference was driven by reduced neural responses to monetary loss, rather than by blunted neural response to monetary reward. Here, we aimed to conceptually replicate and extend these findings in two samples (n = 275, 27 with suicidal ideation; and n = 235, 48 with suicidal ideation at T1) of children and adolescents (11 to 15 years and 8 to 15 years, respectively). Results from both samples showed no evidence that children and adolescents with suicidal ideation have abnormal reward or loss processing, nor that reward processing predicts suicidal ideation two years later. The results highlight the need for greater statistical power, as well as continued research examining the neural underpinnings of suicidal thoughts and behaviors.

sentences: A mycobacterial glycine-rich protein governs actin-based motility Mycobacterium marinum, a close relative of the significant human pathogen Mycobacterium tuberculosis, polymerizes host actin at the bacterial surface to drive intracellular movement and cell-to-cell spread during infection. Here, we report the identification and characterization of MirA, the M. marinum actin-based motility factor. MirA is a member of the glycine-rich PE_PGRS family of ESX-5-secreted proteins. MirA uses an amphipathic helix to anchor into the mycobacterial outer membrane and, surprisingly, also the surface of host lipid droplet organelles. The glycine-rich PGRS domain in MirA directly binds and activates host N-WASP to stimulate actin polymerization through the Arp2/3 complex, directing both bacterial and lipid droplet actin- based motility. MirA is dissimilar to known N-WASP activating ligands and may represent a new class of microbial and host actin regulator. Additionally, the MirA-N-WASP interaction represents a model to understand how the enigmatic PE_PGRS proteins contribute to mycobacterial pathogenesis.

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Global structural stability and the role of cooperation in mutualistic system Dynamical systems on graphs allow to describe multiple phenomena from different areas of Science. In particular, many complex systems in Ecology are studied by this approach. In this paper we analize the mathematical framework for the study of the structural stability of each stationary point, feasible or not, introducing a generalization for this concept, defined as Global Structural Stability. This approach would fit with the proper mathematical concept of structural stability, in which we find a full description of the complex dynamics on the phase space due to nonlinear dynamics. This fact can be analyzed as an informational field grounded in a global attractor whose structure can be completely characterized. These attractors are stable under perturbation and suppose the minimal structurally stable sets. We also study in detail, mathematically and computationally, the zones characterizing different levels of biodiversity in bipartite graphs describing mutualistic antagonistic systems of population dynamics. In particular, we investigate the dependence of the region of maximal biodiversity of a system on its connectivity matrix. On the other hand, as the network topology does not completely determine the robustness of the dynamics of a complex network, we study the correlation between structural stability and several graph measures. A systematic study on synthetic and biological graphs is presented, including 10 mutualistic networks of plants and seed-dispersal and 1000 random synthetic networks. We compare the role of centrality measures and modularity, concluding the importance of just cooperation strength among nodes when describing areas of maximal biodiversity. Indeed, we show that cooperation parameters are the central role for biodiversity while other measures act as secondary supporting functions.

Author summaryWe introduce the concept of Global Structural Stability as a proper mathematical concept to fully understand biodiversity in some ecological systems. Our concept retakes the definitions in the classical works of R. Thom [1] and Andronov-Pontryagin [2]. Moreover, there exists a close relation between the structure of a complex network, described as a graph, and its associated dynamics. Mutualistic networks introduce cooperation links between two groups of species, as plant and pollinators or seed-dispersal. The understanding of organizational aspects leading to maximizing biodiversity is one of the more important research areas in Theoretical and Applied Ecology. In this work we introduce a systematic study on different graph measures in order to identify optimal organization for maximal biodiversity (defined as structural stability). Our results conclude that, for mutualistic systems, the strength in cooperation parameters are the core fact, i.e., cooperation is the real fact optimizing biodiversity among other possible structural configurations.

sentences: The critical role of spiny stellate cells in seizure onset based on dynamic analysis of a neural mass model Growing evidence suggests that excitatory neurons in the brain play a significant role in seizure generation. Nonetheless, spiny stellate cells are cortical excitatory non-pyramidal neurons in the brain, which their basic role in seizure occurrence is not well understood. In the present research, we study the critical role of spiny stellate cells or the excitatory interneurons (EI), for the first time, in epileptic seizure generation using an extended neural mass model inspired by Liu and Wang model in 2017. Applying bifurcation analysis on this modified model, we investigated the rich dynamics corresponding to the epileptic seizure onset and transition between interictal and ictal states caused by EI connectivity to other cell types. Our results indicate that the transition between interictal and ictal states (preictal signal) corresponds to a supercritical Hopf bifurcation, and thus, the extended model suggests that before seizure onset, the amplitude and frequency of neural activities gradually increase. Moreover, we showed that 1) the altered function of GABAergic and glutamatergic receptors of EI can cause seizure, and 2) the pathway between the thalamic relay nucleus and EI facilitates the transition from interictal to the ictal activity by decreasing the preictal period. Thereafter, we considered both sensory and cortical periodic inputs to study model responses to various harmonic stimulations. Bifurcation analysis of the model, in this case, suggests that the initial state of the model might be the main cause for the transition between interictal and ictal states as the stimulus frequency changes. The extended thalamocortical model shows also that the amplitude jump phenomenon and nonlinear resonance behavior result from the preictal state of the modified model. These results can be considered as a step forward to a deeper understanding of the mechanisms underlying the transition from normal activities to epileptic activities.

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cytoNet: Spatiotemporal Network Analysis of Cell Communities We introduce cytoNet, a cloud-based tool to characterize cell populations from microscopy images. cytoNet quantifies spatial topology and functional relationships in cell communities using principles of network science. Capturing multicellular dynamics through graph features, cytoNet also evaluates the effect of cell-cell interactions on individual cell phenotypes. We demonstrate cytoNets capabilities in four case studies: 1) characterizing the temporal dynamics of neural progenitor cell communities during neural differentiation, 2) identifying communities of pain-sensing neurons in vivo, 3) capturing the effect of cell community on endothelial cell morphology, and 4) investigating the effect of laminin 4 on perivascular niches in adipose tissue. The analytical framework introduced here can be used to study the dynamics of complex cell communities in a quantitative manner, leading to a deeper understanding of environmental effects on cellular behavior. The versatile, cloud-based format of cytoNet makes the image analysis framework accessible to researchers across domains.

Availability and ImplementationQutubLab.org/how | cytoNet contact: cytoNetProject@gmail.com Brain Initiative Alliance Toolmaker cytoNet site: https://www.braininitiative.org/toolmakers/resources/cytonet/

Author / Lay SummarycytoNet provides an online tool to rapidly characterize relationships between objects within images and video frames. To study complex tissue, cell and subcellular topologies, cytoNet integrates vision science with the mathematical technique of graph theory. This allows the method to simultaneously identify environmental effects on single cells and on network topology. cytoNet has versatile use across neuroscience, stem cell biology and regenerative medicine. cytoNet applications described in this study include: (1) characterizing how sensing pain alters neural circuit activity, (2) quantifying how vascular cells respond to neurotrophic stimuli overexpressed in the brain after injury or exercise, (3) delineating features of fat tissue that may confer resistance to obesity and (4) uncovering structure-function relationships of human stem cells as they transform into neurons.

sentences: Myelin imaging of the basal forebrain in first-episode psychosis Cholinergic dysfunction has been implicated in the pathophysiology of psychiatric disorders such as schizophrenia, depression, and bipolar disorder. The basal forebrain (BF) cholinergic nuclei, defined as cholinergic cell groups Ch1-3 and Ch4 (Nucleus Basalis of Meynert; NBM), provide extensive cholinergic projections to the rest of the brain. Here, we examined microstructural neuroimaging measures of the cholinergic nuclei in patients with untreated psychosis ([~] 31 weeks of psychosis, <2 defined daily dose of antipsychotics) and used Magnetic Resonance Spectroscopy (1H-MRS) and transcriptomic data to support our findings. We used a cytoarchitectonic atlas of the BF to map the nuclei and obtained measures of myelin (quantitative T1, or qT1 as myelin surrogate) and microstructure (axial diffusion; AxD). In a clinical sample (n=85; 29 healthy controls, 56 first-episode psychosis), we found significant correlations between qT1 and 1H-MRS-based dorsal anterior cingulate choline in healthy controls, while this relationship was disrupted in FEP. Case-control differences in qT1 and AxD were observed in the Ch1-3, with increased qT1 (reflecting reduced myelin content) and AxD (reflecting reduced axonal integrity). We found clinical correlates between left NBM qT1 with manic symptom severity, and AxD with negative symptom burden in FEP. Intracortical and subcortical myelin maps were derived and correlated with BF myelin. BF-cortical and BF-subcortical myelin correlations demonstrate known projection patterns from the BF. Using data from the Allen Human Brain Atlas, cholinergic nuclei showed significant enrichment for schizophrenia and depression-related genes. Cell-type specific enrichment indicated enrichment for cholinergic neuron markers as expected. Further relating the neuroimaging correlations to transcriptomics demonstrated links with cholinergic receptor genes and cell type markers of oligodendrocytes and cholinergic neurons, providing biological validity to the measures. These results provide genetic, neuroimaging, and clinical evidence for cholinergic dysfunction in schizophrenia and other psychiatric disorders such as depression.

sentences: Antimicrobial activity and degradation of superhydrophobic magnesium substrates in bacterial media The interest in magnesium-based materials is promoted by their biocompatibility, bioresorbability, and by their recently found antibacterial potential. Until now the widespread use of magnesium alloys in different corrosive environments was inhibited by their weakly controllable degradation rate and poorly understood microbiologically induced corrosion behavior. To better understand the degradation and usability of magnesium-based alloys, in this study we have fabricated the superhydrophobic coatings on top of magnesium-based alloy and analyzed the behavior of this alloy in bacterial dispersions of Pseudomonas aeruginosa and Klebsiella pneumoniae cells in phosphate buffered saline. It was shown that immersion of such coatings into bacterial dispersions causes notable changes in the morphology of the samples, dependent on the bacterial dispersion composition and the type of bacterial strain. The interaction of superhydrophobic coatings with the bacterial dispersion caused the formation of biofilms and sodium polyphosphate films, which provided enhanced barrier properties for magnesium dissolution and hence for dispersion medium alkalization, eventually leading to inhibition of magnesium substrate degradation. Electrochemical data obtained for superhydrophobic samples continuously contacted with the corrosive bacterial dispersions during 48 h indicated a high level of anti-corrosion protection.

sentences: Mucoricin is a Ricin-Like Toxin that is Critical for the Pathogenesis of Mucormycosis Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We now demonstrate that Mucorales fungi produce a toxin that plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro, and prevent hypovolemic shock, organ necrosis, and death in mice with mucormycosis. RNAi inhibition of the toxin in Rhizopus delemar, compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin, ricin, including the ability to inhibit protein synthesis by its N-glycosylase activity, the existence of a motif that mediates vascular leak, and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross-react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name "mucoricin" for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin- like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

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MONI: A Pangenomics Index for Finding MEMs Recently, Gagie et al. proposed a version of the FM-index, called the r-index, that can store thousands of human genomes on a commodity computer. Then Kuhnle et al. showed how to build the r-index efficiently via a technique called prefix-free parsing (PFP) and demonstrated its effectiveness for exact pattern matching. Exact pattern matching can be leveraged to support approximate pattern matching but the r-index itself cannot support efficiently popular and important queries such as finding maximal exact matches (MEMs). To address this shortcoming, Bannai et al. introduced the concept of thresholds, and showed that storing them together with the r-index enables efficient MEM finding -- but they did not say how to find those thresholds. We present a novel algorithm that applies PFP to build the r-index and find the thresholds simultaneously and in linear time and space with respect to the size of the prefix-free parse. Our implementation called MONI can rapidly find MEMs between reads and large sequence collections of highly repetitive sequences. Compared to other read aligners - PuffAligner, Bowtie2, BWA-MEM, and CHIC - MONI used 2-11 times less memory and was 2-32 times faster for index construction. Moreover, MONI was less than one thousandth the size of competing indexes for large collections of human chromosomes. Thus, MONI represents a major advance in our ability to perform MEM finding against very large collections of related references.

AvailabilityMONI is publicly available at https://github.com/maxrossi91/moni.

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Successful IL-12 cancer immunotherapy requires NK cell-derived CCL5 for anti-tumor DC-T cell crosstalk T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the IFN{gamma}-IL-12 pathway relies on the ability of a population of tissue-resident NK (trNK) cells to orchestrate an anti-tumor microenvironment. Particularly, utilizing an engineered adenoviral platform, we show that paracrine IL-12 enhances functional DC-CD8 T cell interactions to generate adaptive anti-tumor immunity. This effect depends on the abundance of trNK cells and specifically their capacity to produce the cDC1-chemoattractant CCL5. Failure to respond to IL-12 and other IFN{gamma}-inducing therapies such as immune checkpoint blockade in tumors with low trNK cell infiltration could be overcome by intra-tumoral delivery of CCL5. Our findings reveal a novel barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC crosstalk can be enhanced to promote anti-tumor immunity and overcome resistance.

SignificanceWe identified the lack of CCL5-producing, tissue-resident NK (trNK) cells as a barrier to T cell-focused therapies. While IL-12 induces anti-tumoral DC-T cell crosstalk in trNK cellrich tumors, resistance to IL-12 or anti-PD-1 in trNK cellpoor tumors can be overcome by the additional delivery of CCL5.

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microRNA775 targets a β-(1,3)-galactosyltransferase to regulate growth and development in Arabidopsis thaliana MicroRNAs are critical regulators of gene expression in plants and other organisms, and are involved in regulating plethora of developmental processes. Evolutionarily, miRNAs can be ancient and conserved across species or recently evolved and young, which are not conserved across diverse plant groups. miR775 is a non-conserved miRNA identified only in Arabidopsis thaliana. Here, we investigated the functional significance of miR775 in A. thaliana and observed that miR775 targets a probable {beta}-(1,3)-galactosyltransferase gene at post transcriptional level. Phenotypic analysis of miR775 over-expression lines and the target mutant suggested miR775 regulates rosette size by elongating petiole length and increasing leaf area. Further, the expression of miR775 was found to be up-regulated in response to UV-B and hypoxia. Our results also suggest that miR775 regulated {beta}-(1,3)-galactosyltransferase may involve in regulating the {beta}-(1,3)-galactan content of arabinogalactans. Collectively, our findings establish a role of miR775 in regulating growth and development in A. thaliana.

HighlightsO_LIThe role of an uncharacterized microRNA, miR775 has been explored
C_LIO_LImiR775 targets a probable {beta}-(1,3)-galactosyltransferase involved in complex carbohydrate biosynthesis
C_LIO_LImiR775 regulates rosette size in A. thaliana and may play role under UV light and hypoxia
C_LI

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Evidence of MHC class I and II influencing viral and helminth infection via the microbiome in a non-human primate Until recently, the study of major histocompability complex (MHC) mediated immunity has focused on the direct link between MHC variability and susceptibility to parasite infection. However, MHC genes can also influence host health indirectly through the sculpting of the bacterial community that in turn shape immune responses. We investigated the links between MHC class I and II gene variability gut microbiome diversity and micro- (adenovirus, AdV) and macro- (helminth) parasite infection probabilities in a wild population of non-human primates, mouse lemurs of Madagascar. This setup encompasses a plethora of underlying interactions between parasites, microbes and adaptive immunity in natural populations. Both MHC classes explained shifts in microbiome composition and the effect was driven by a few select microbial taxa. Among them were three taxa (Odoribacter, Campylobacter and Prevotellaceae-UCG-001) which were in turn linked to AdV and helminth infection status, evidence of the indirect effect of the MHC via the microbiome. Our study provides support for the coupled role of MHC variability and microbial flora as contributing factors of parasite infection.

Author summaryThe selective pressure of the major histocompatibility complex (MHC) on microbial communities, and the potential role of this interaction in driving parasite resistance has been largely neglected. Using a natural population of the primate Microcebus griseorufus, we provide evidence of two outstanding findings: that MHCI and MHCII diversity shapes the composition of the gut microbiota; and that select taxa associated with MHC variability predicted adenovirus and helminth infection status. Our study highlights the importance of incorporating the microbiome when investigating parasite-mediated MHC selection.

sentences: Single-cell analyses of axolotl forebrain organization, neurogenesis, and regeneration Salamanders are important tetrapod models to study brain organization and regeneration, however the identity and evolutionary conservation of brain cell types is largely unknown. Here, we delineate cell populations in the axolotl telencephalon during homeostasis and regeneration, representing the first single-cell genomic and spatial profiling of an anamniote tetrapod brain. We identify glutamatergic neurons with similarities to amniote neurons of hippocampus, dorsal and lateral cortex, and conserved GABAergic neuron classes. We infer transcriptional dynamics and gene regulatory relationships of postembryonic, region-specific direct and indirect neurogenesis, and unravel conserved signatures. Following brain injury, ependymoglia activate an injury-specific state before reestablishing lost neuron populations and axonal connections. Together, our analyses yield key insights into the organization, evolution, and regeneration of a tetrapod nervous system.

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Towards Synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression BackgroundBiocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient concurrent extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.

ResultsP. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter mhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.

ConclusionOur results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

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Nutrient cycling is an important mechanism for homeostasis in plant cells Homeostasis in living cells refers to the steady state of internal, physical, and chemical conditions. It is maintained by self-regulation of the dynamic cellular system. In order to gain insight into homeostatic mechanisms that keep cytosolic nutrient concentrations in plant cells within a homeostatic range, I performed computational cell biology experiments. Systems of membrane transporters were modelled mathematically followed by the simulation of their dynamics. The detailed analyses of  what-if scenarios demonstrate that a single transporter type for a nutrient, irrespective whether it is a channel or a co-transporter, is not sufficient to set a desired cytosolic concentration. A cell cannot flexibly react on different external conditions. At least two different transporter types for the same nutrient are required, which are energized differently. The gain of flexibility in adjusting the nutrient concentration was accompanied by the establishment of energy-consuming nutrient cycles at the membrane suggesting that these sometimes called  futile cycles are not as futile as they appear. This understanding may help in future to design new strategies for increasing nutrient use efficiency of crop plants taking into account the complex interplay of transporter networks at the cellular level.

One sentence summaryFirst principles of membrane transport explain why the maintenance of a constant cytosolic nutrient concentration is often accompanied by the  futile cycling of the nutrient across the membrane.

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phenopype: a phenotyping pipeline for Python O_LIDigital images are an intuitive way to capture, store and analyze organismal phenotypes. Many biologists are taking images to collect high-dimensional phenotypic information from specimens, which are key to investigating complex ecological, evolutionary and developmental phenomena, such as relationships between trait diversity and ecosystem function, multivariate natural selection, or developmental plasticity. As a consequence, images are being collected at ever increasing rates, but extraction of the contained phenotypic information still poses a veritable analytical bottleneck.
C_LIO_LIphenopype is a high throughput phenotyping pipeline for the programming language Python that aims at alleviating this bottleneck. The package facilitates immediate extraction of high dimensional phenotypic data from laboratory grade digital images with low levels of background noise and complexity. At the core, phenopype provides functionality for rapid signal processing based image preprocessing and segmentation, followed by trait extraction, visualization and numerous options for data export. The functionality is provided by wrapping low-level Python computer vision libraries (e.g. OpenCV) into accessible functions, facilitating their incorporation into scientific image analysis workflows. In addition, phenopype provides a project management ecosystem to further simplify rapid data collection and to increase reproducibility.
C_LIO_LIphenopype offers two different workflows that support users during different stages of scientific image analysis. The low-throughput workflow uses regular Python syntax and has greater flexibility at the cost of reproducibility, which is suitable for prototyping during the initial stage of a research project. The high-throughput workflow allows users to specify and store image-specific settings for analysis in human-readable YAML format, and then execute all functions in one step by means of an interactive parser. This approach facilitates rapid program-user interactions during batch processing, and greatly increases scientific reproducibility.
C_LIO_LIOverall, phenopype intends to make the features of powerful but technically involved low-level CV libraries available to biologists with little or no Python coding experience. Therefore, phenopype is aiming to augment, rather than replace the utility of existing Python CV libraries, allowing biologists to focus on rapid and reproducible data collection. Furthermore, image annotations produced by phenopype can be used as training data, thus presenting a stepping stone towards the application of deep learning architectures.
C_LI

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Multi-Study Learning for Real-time Neurochemical Sensing in Humans using the "Study Strap Ensemble" We propose the "study strap ensemble," which combines advantages of two common approaches to fitting prediction models when multiple training datasets ("studies") are available: pooling studies and fitting one model versus averaging predictions from multiple models each fit to individual studies. The study strap ensemble fits models to bootstrapped datasets, or "pseudo-studies." These are generated by resampling from multiple studies with a hierarchical resampling scheme that generalizes the randomized cluster bootstrap. The study strap is controlled by a tuning parameter that determines the proportion of observations to draw from each study. When the parameter is set to its lowest value, each pseudo-study is resampled from only a single study. When it is high, the study strap ignores the multi-study structure and generates pseudo-studies by merging the datasets and drawing observations like a standard bootstrap. We empirically show the optimal tuning value often lies in between, and prove that special cases of the study strap draw the merged dataset and the set of original studies as pseudo-studies. We extend the study strap approach with an ensemble weighting scheme that utilizes information in the distribution of the covariates of the test dataset.

Our work is motivated by neuroscience experiments using real-time neurochemical sensing during awake behavior in humans. Current techniques to perform this kind of research require measurements from an electrode placed in the brain during awake neurosurgery and rely on prediction models to estimate neurotransmitter concentrations from the electrical measurements recorded by the electrode. These models are trained by combining multiple datasets that are collected in vitro under heterogeneous conditions in order to promote accuracy of the models when applied to data collected in the brain. A prevailing challenge is deciding how to combine studies or ensemble models trained on different studies to enhance model generalizability.

Our methods produce marked improvements in simulations and in this application. All methods are available in the studyStrap CRAN package.

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Complexity of genomic epidemiology of carbapenem-resistant Klebsiella pneumoniae isolates in Colombia urges the reinforcement of whole genome sequencing based surveillance programs BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is an emerging public health problem. This study explores the specifics of CRKP epidemiology in Colombia based on whole genome sequencing (WGS) of the National Reference Laboratory at Instituto Nacional de Salud (INS)s 2013-2017 sample collection.

MethodsA total of 425 CRKP isolates from 21 departments were analyzed by HiSeq-X10(R)Illumina high-throughput sequencing. Bioinformatic analysis was performed, primarily using the pipelines developed collaboratively by the National Institute for Health Research Global Health Research Unit (GHRU) on Genomic Surveillance of AMR, and AGROSAVIA.

ResultsOf the 425 CRKP isolates, 91.5% were carbapenemase-producing strains. The data support a recent expansion and the endemicity of CRKP in Colombia with the circulation of 7 high-risk clones, the most frequent being CG258 (48.39% of isolates). We identified genes encoding carbapenemases blaKPC-3, blaKPC-2, blaNDM-1, blaNDM-9, blaVIM-2, blaVIM-4, and blaVIM-24, and various mobile genetic elements (MGE). The virulence of CRKP isolates was low, but colibactin (clb3) was present in 25.2% of isolates, and a hypervirulent CRKP clone (CG380) was reported for the first time in Colombia. ST258, ST512, and ST4851 were characterized by low levels of diversity in the core genome (ANI> 99.9%).

ConclusionsThe study outlines complex CRKP epidemiology in Colombia. CG258 expanded clonally and carries specific carbapenemases in specific MGEs, while the other high-risk clones (CG147, CG307, and CG152) present a more diverse complement of carbapenemases. The specifics of the Colombian situation stress the importance of WGS-based surveillance to monitor evolutionary trends of STs, MGE, and resistance and virulence genes.

summaryIn Colombia, the dissemination of carbapenemases in carbapenem-resistant Klebsiella pneumoniae is attributed to horizontal gene transfer and successful circulation of CG258, and, to a lesser extent, other clones such as ST307, ST147, and ST152.

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Transposable elements contribute to genome dynamics and gene expression variation in the fungal plant pathogen Verticillium dahliae Transposable elements (TEs) are a major source of genetic and regulatory variation in their host genome and are consequently thought to play important roles in evolution. Many fungal and oomycete plant pathogens have evolved dynamic and TE-rich genomic regions containing genes that are implicated in host colonization. TEs embedded in these regions have typically been thought to accelerate the evolution of these genomic compartments, but little is known about their dynamics in strains that harbor them. Here, we used whole-genome sequencing data of 42 strains of the fungal plant pathogen Verticillium dahliae to systematically identify polymorphic TEs that may be implicated in genomic as well as in gene expression variation. We identified 2,523 TE polymorphisms and characterize a subset of 8% of the TEs as dynamic elements that are evolutionary younger, less methylated, and more highly expressed when compared with the remaining 92% of the TE complement. As expected, the dynamic TEs are enriched in the dynamic genomic regions. Besides, we observed an association of dynamic TEs with pathogenicity-related genes that localize nearby and that display high expression levels. Collectively, our analyses demonstrate that TE dynamics in V. dahliae contributes to genomic variation, correlates with expression of pathogenicity-related genes, and potentially impacts the evolution of dynamic genomic regions.

Significance statementTransposable elements (TEs) are ubiquitous components of genomes and are major sources of genetic and regulatory variation. Many plant pathogens have evolved TE-rich genomic regions containing genes with roles in host colonization, and TEs are thought to contribute to accelerated evolution of these dynamic regions. We analyzed the fungal plant pathogen Verticillium dahliae to identify TE variation between strains and to demonstrate that polymorphic TEs have specific characteristic that separates them from the majority of TEs. Polymorphic TEs are enriched in dynamic genomic regions and are associated with structural variants and highly expressed pathogenicity-related genes. Collectively, our results provide evidence for the hypothesis that dynamic TEs contribute to increased genomic diversity, functional variation, and the evolution of dynamic genomic regions.

sentences: A novel mobile RND-type efflux pump gene cluster, tmexC3D2-toprJ3, confers tigecycline resistance in Pseudomonas alcaligenes Tigecycline exhibits promising activity against multidrug-resistant Gram-negative bacteria (MDR-GNB), whose infections are difficult to treat with antimicrobials. However, mobile tigecycline resistance genes, such as tmexCD-toprJ, have emerged in Enterobacterales isolated in China, Vietnam, and possibly other countries in the world. In this study, we investigated tigecycline-nonsusceptible GNB in Japan. Eight tigecycline- and carbapenem-nonsusceptible isolates of Pseudomonas alcaligenes were obtained from sewage water from a medical institution in Japan in 2020. Whole genome analysis of all P. alcaligenes isolates was performed using short-read sequencing, and the isolate KAM426 was further analyzed using long-read sequencing. For important antimicrobial resistance genes, analysis of surrounding structures and comparison with similar sequences in the public genome database were performed. We identified a novel hybrid type of tmexCD-toprJ gene cluster, tmexC3D2-toprJ1b consisting of tmexC3, tmexC2, and toprJ1b, in phylogenetically clonal isolates of P. alcaligenes. The complete genome sequence of KAM426 revealed that this isolate co-harbors tmexC3D2-toprJ1b and two copies of the carbapenemase gene blaIMP-1 on the chromosome. tmexC3D2-toprJ1b in KAM426 was flanked by the IS5/IS1182 family transposase gene, suggesting that the gene cluster was acquired by horizontal gene transfer (HGT). tmexC3D2-toprJ1b seemed to have spread to other Pseudomonas species such as Pseudomonas aeruginosa via HGT mediated by mobile gene elements such as a plasmid. This study identified tmexCD-toprJ-like tigecycline resistance genes in Japan for the first time and suggests that diverse tmexCD-toprJ-like gene clusters, including tmexC3D2-toprJ1b, have spread among MDR-GNB worldwide. Further epidemiological genomic studies in clinical and environmental settings are needed.

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Coordinated DNA and Histone Dynamics Drive Accurate Histone H2A.Z Exchange Nucleosomal histone H2A is exchanged for its variant H2A.Z by the SWR1 chromatin remodeler, but the mechanism and timing of histone exchange remain unclear. Here, we quantify DNA and histone dynamics during histone exchange in real-time using a three-color single-molecule FRET assay. We show that SWR1 operates with timed precision to unwrap DNA with large displacement from one face of the nucleosome, remove H2A-H2B from the same face, and rewrap DNA, all within 2.3 seconds. Such productive DNA unwrapping requires full SWR1 activation and differs from unproductive, smaller-scale DNA unwrapping caused by SWR1 binding alone. On an asymmetrically positioned nucleosome, SWR1 intrinsically senses long-linker DNA to preferentially exchange H2A.Z on the distal face as observed in vivo. The displaced H2A-H2B dimer remains briefly associated with the SWR1-nucleosome complex and is dissociated by histone chaperones. These findings reveal how SWR1 coordinates DNA unwrapping with histone dynamics to rapidly and accurately place H2A.Z at physiological sites on chromatin.

One-Sentence SummaryMulticolor single-molecule FRET reveals how SWR1 unwraps DNA to exchange nucleosomal H2A-H2B for H2A.Z-H2B.

sentences: A comparison between time-constrained counts and line transects as methods to estimate butterfly diversity in tropical habitats Long-term species monitoring programmes have revealed catastrophic insect population declines and disruption of biological communities that are contributing to biodiversity loss. Such discoveries have been possible because of standardised methods, such as line transects, of counting butterflies and other insects. However, line transects are not feasible in many tropical and mountainous habitats, so alternative methods must be explored. To tackle this issue, we devised time-constrained (30-min) counts and compared butterfly diversity as estimated through this method with that estimated through line transects in three tropical habitats in India (evergreen forest, dry deciduous forest and an urban woodland). We tested the efficacy of the two methods to sample species richness and abundance, as well as numbers of rare, endemic and specialist butterflies. We observed greater species richness, and more species of habitat specialists and endemics per sample in time-constrained counts in evergreen forest, but not in the other two habitats. Thus, time-constrained counts were more efficient at detecting species in the species-rich evergreen habitat. Apart from this difference, the two sampling methods captured similar levels of species richness and other measures of diversity. Our study thus shows that time-constrained counts is a suitable if not a superior alternative to line transects to conduct butterfly diversity surveys and population monitoring in complex tropical landscapes. Due to methodological flexibility and simplicity, this method may be particularly useful to study the impacts of climate change, habitat fragmentation and land use practices on butterfly conservation in populous and tech-ready tropical countries using citizen science frameworks.

sentences: Epigenetic changes in autoimmune monocytes contribute to disease and can be targeted by JAKinhibition How the local inflammatory environment regulates epigenetic changes in the context of autoimmune diseases remains unclear. Here we assessed the transcriptional and active enhancer profile of monocytes derived from the inflamed joints of Juvenile Idiopathic Arthritis (JIA) patients, a model well-suited for studying autoimmune diseases. RNA-sequencing analysis demonstrated that synovial-derived monocytes display an activated phenotype, characterized by increased expression of surface activation markers and multiple chemokines and cytokines. This increased expression is regulated on the epigenetic level, as ChIP-sequencing indicated increased H3K27 acetylation of genes upregulated in synovial monocytes. IFN signaling-associated genes are increased and epigenetically altered in synovial monocytes, suggesting a role for IFN in establishing the inflammatory phenotype. Treatment of human synovial monocytes with the Janus-associated kinase (JAK) inhibitor Ruxolitinib, which inhibits IFN signaling, transformed the activated enhancer landscape and reduced disease-associated gene expression, thereby inhibiting the inflammatory phenotype. Taken together, these data provide novel insight into epigenetic regulation of autoimmune disease-patient derived monocytes and suggest that altering the epigenetic profile can be a therapeutic approach for the treatment of autoimmune diseases.

sentences: The organizational principles of de-differentiated topographic maps Topographic maps are a fundamental feature of cortex architecture in the mammalian brain. One common theory is that the de-differentiation of topographic maps links to impairments in everyday behavior due to less precise functional map readouts. Here, we tested this theory by characterizing de-differentiated topographic maps in primary somatosensory cortex (SI) of younger and older adults by means of ultra-high resolution functional magnetic resonance imaging together with perceptual finger individuation and hand motor performance. Older adults SI maps showed similar amplitude and size to younger adults maps, but presented with less representational similarity between distant fingers. Larger population receptive field sizes in older adults maps did not correlate with behavior, whereas reduced cortical distances between D2 and D3 related to worse finger individuation but better motor performance. Our data uncover the drawbacks of a simple de-differentiation model of topographic map function, and motivate the introduction of feature-based models of cortical reorganization.

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In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center at the Channel Entrance Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as  minimized or  expanded based on whether assembly requires only a core set of signature subunits or additional system-specific components. The prototypical  minimized systems mediating Agrobacterium tumefaciens T-DNA transfer and conjugative transfer of plasmids pKM101 and R388 are built from 12 subunits generically named VirB1-VirB11 and VirD4. In this study, we visualized the pKM101-encoded T4SS in the native context of the bacterial cell envelope by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388, a large substructure of which was previously purified and analyzed by negative-stain electron microscopy (nsEM). The IMC of the in situ T4SSpKM101 machine is highly symmetrical and exhibits 6-fold symmetry, dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three  expanded T4SSs previously visualized by in situ CryoET, but strikingly differs from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Together, our findings support a unified architectural model for all T4SSs assembled in vivo regardless of their classification as  minimized or  expanded: the signature VirB4-like ATPases invariably are arranged as central hexamers of dimers at the entrances to the T4SS channels.

SignificanceBacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and firmly support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate substrate entry into and passage through T4SS channels.

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mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the hosts intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, in order to unravel mGBP2 functions, we report Galectin-9 (Gal9) and cytoskeleton-associated protein 4 (Ckap4) as critical mGBP2 interaction partners engaged for immunity to T. gondii. Interestingly, Gal9 and Ckap4 also accumulate and colocalize with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 and Ckap4 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that mGBP2 engages Gal9 and Ckap4 and that Gal9 and Ckap4 are critical factors for the mGBP2 coordinated cell autonomous host defense mechanism against T. gondii.

Significance StatementWe have previously shown that mGBP2 is an essential IFN{gamma} induced protein for defense against Toxoplasma gondii infection. mGBP2 is rapidly recruited to the parasitophorous vacuole (PV) of intracellular T. gondii and is required for the rupture and/or permeabilization of the PV membrane. Subsequently mGBP2 attacks the parasite membrane. Up to now, no data about other mGBP2 interaction partners and their molecular requirement for T. gondii control has been made available.

Here, we have identified Gal9 and Ckap4 as interacting proteins of mGBP2. Gal9 and Ckap4 are essential in the mGBP2 mediated attack of the T. gondii PV. Both proteins localize at the T. gondii PV in close association with mGBP2, as shown by state-of-the-art super resolution microscopy. In addition, T. gondii growth cannot be controlled anymore in Gal9 and Ckap4 deficient cell lines, comparable to mGBP2 deficient cells.

Thus, we have identified novel effector proteins in the intricate cell autonomous immune machinery against intracellular pathogens.

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Evaluation and Optimization of High-Field Asymmetric Waveform Ion Mobility Spectrometry for Multiplexed Quantitative Site-specific N-glycoproteomics The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified IgM protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass-spectrometry-based qualitative and quantitative N-glycoproteomics.

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sentences: Canonical BMP2/4 signaling degrades SOX2 in glioblastoma propagating cells. The effect of Bone Morphogenetic Proteins (BMPs) on glioblastoma propagation cells (GPCs) is being debated [1-5]. We observed that exposure of GPCs to BMP2/4 initiates a rapid proteasomal degradation of the pluripotency factor SOX2 followed by the differentiation of GPCs into glial cells using 5 primary human glioblastoma lines tested. Enforced expression of SOX2 in GPCs antagonized BMP2/4-induced gliogenesis and reduction of proliferation. Both outcomes are consistent with the role of SOX2 in normal neurogenesis [6-8]. Using gene expression analysis, we observed that level of SOX2 in different glioblastoma lines tracks with the levels of oncogenes H/K/N-RAS and OLIG2. Our work provides support for BMP2/4-SOX2 axis in GPCs and points to the proteasome-regulated degradation of SOX2 that initiates glycogenic differentiation similar to that observed during normal development [6, 7]. Our work supports a potential for developing novel therapeutic strategies aimed at differentiation of GPC into non-tumorigenic glia.

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Clones and Clusters of Antimicrobial-Resistant Klebsiella from Southwestern Nigeria IntroductionKlebsiella pneumoniae is a World Health Organization high-priority antibiotic-resistant pathogen. However, little is known about the population structure and evolution of Klebsiella circulating in Nigeria.

MethodsWe performed whole genome sequencing (WGS) of 141 Klebsiella isolated between 2016 and 2018 from clinical specimens at 3 antimicrobial-resistance (AMR) sentinel surveillance tertiary hospitals in southwestern Nigeria. We conducted in silico multilocus sequence typing, AMR gene, virulence gene, plasmid, and K and O loci profiling, as well as phylogenetic analyses, using publicly available tools and Nextflow pipelines.

ResultsPhylogenetic analysis revealed that the majority of the 134 K. pneumoniae and 5 K. quasipneumoniae isolates from Nigeria characterized are closely related to globally disseminated multidrug-resistant clones. Of the 39 K. pneumoniae sequence types (STs) identified, the most common were ST307 (15%), ST5241 (12%), ST15 (~9%), and ST25 (~6%). ST5241, one of 10 novel STs detected, is a single locus variant of ST636 carrying dfrA14, tetD, qnrS, and oqxAB resistance genes. The extended-spectrum {beta} lactamase (ESBL) gene blaCTX_M-15 was seen in 72 % of K. pneumoniae genomes, while 8% encoded a carbapenemase. Four likely outbreak clusters from one facility, within STs 17, 25, 307, and 5241, were ESBL but not carbapenemase-bearing clones.

ConclusionThis study uncovered known and novel K. pneumoniae lineages circulating in Nigeria that include multidrug-resistant ESBL producers. Carbapenemase-producing isolates remain uncommon. WGS retrospectively identified outbreak clusters, pointing to the value of genomic approaches in AMR surveillance for improving infection prevention and control in Nigerian hospitals.

summaryWe performed whole genome sequencing (WGS) of 141 Klebsiella isolated in 2016-2018 at 3 antimicrobial-resistance (AMR) sentinel surveillance tertiary hospitals in southwestern Nigeria. This study uncovered known and novel K. pneumoniae lineages circulating in Nigeria that include multidrug-resistant ESBL producers.

FUNDINGThis work was supported by Official Development Assistance (ODA) funding from the National Institute of Health Research [16/136/111: NIHR Global Health Research Unit on Genomic Surveillance of Antimicrobial Resistance].

This research was commissioned by the National Institute of Health Research using Official Development Assistance (ODA) funding. INO is an African Research Leader supported by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement that is also part of the EDCTP2 program supported by the European Union. The funders had no role in the content, crafting or submission of this paper. The views expressed in this publication are those of the authors and not necessarily those of the funders or their affiliates.

CONFLICT OF INTERESTThe authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

sentences: Disentangling hand and tool processing: distal effects of neuromodulation The neural processing within a brain region that responds to more than one object category can be separated by looking at the horizontal modulations established by that region, which suggests that local representations can be affected by connections to distal areas, in a category-specific way. Here we first wanted to test whether by applying transcranial direct current stimulation (tDCS) to a region thatre sponds both to hands and tools (posterior middle temporal gyrus; pMTG), while participants performed either a hand- or tool-related training task, we would be able to specifically target the trained category, and thereby dissociate the overlapping neural processing. Second, we wanted to see if these effects were limited to the target area or extended to distal but functionally connected brain areas. After each combined tDCS and training session, participants therefore viewed images of tools, hands, and animals, in an fMRI scanner. Using multivoxel pattern analysis, we found that tDCS stimulation to pMTG indeed improved the classification accuracy between tools vs. animals, but only when combined with a tool training task (not a hand training task). However, surprisingly, tDCS stimulation to pMTG also improved the classification accuracy between hands vs. animals when combined with a tool training task (not a hand training task). Our findings suggest that overlapping but functionally-specific networks can be separated by using a category-specific training task together with tDCS - a strategy that can be applied more broadly to other cognitive domains using tDCS - and demonstrates the importance of horizontal modulations in objectcategory representations.

sentences: Structure of the stress-related LHCSR1 complex determined by an integrated computational strategy Light-harvesting complexes (LHCs) are pigment-protein complexes whose main function is to capture sunlight and transfer the energy to reaction centers of photosystems. In response to varying light conditions, LH complexes also play photoregulation and photoprotection roles. In algae and mosses, a sub-family of LHCs, Light-Harvesting complex stress related (LHCSR), is responsible for photoprotective quenching. Despite their functional and evolutionary importance, no direct structural information on LHCSRs is available that can explain their unique properties. In this work we propose a structural model of LHCSR1 from the moss P. Patens, obtained through an integrated computational strategy that combines homology modeling, molecular dynamics, and multiscale quantum chemical calculations. The model is validated by reproducing the spectral properties of LHCSR1. Our model reveals the structural specificity of LHCSR1, as compared with the CP29 LH complex, and poses the basis for understanding photoprotective quenching in mosses.

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Protein preference and elevated plasma FGF21 induced by dietary protein restriction is similar in both male and female mice Animals that are moderately protein restricted respond to this dietary stress by increasing consumption of protein-containing foods. This is true in many species, including rodents. Rodent models of protein restriction have typically relied on only male subjects, and there are plausible reasons why female rodents may respond differently to dietary protein restriction. To address this gap in knowledge, the current experiments examined protein preference after two weeks on a 5% protein diet or 20% protein control diet, in male and female mice. We found that female mice, like male mice, increase consumption of 4% casein (protein) relative to 4% maltodextrin (carbohydrate) when presented with both simultaneously. Interestingly, this increased consumption was due to more bursts in females and more licks per burst in males, indicating possible differences in mechanism by which increased intake is achieved. Stage of the estrous cycle did not affect female responses. Moreover, we measured plasma FGF21 - a hormone induced by protein restriction and necessary for protein preference - in male and female mice. Here, we found no statistical differences between protein-restricted males, females in diestrus, or females in proestrus. In non-restricted mice FGF21 levels were low, but significantly higher in females in proestrus than females in diestrus or males. Overall, these experiments highlight the importance of including female subjects in studies of food choice and macronutrient restriction.

Highlights- Protein-restricted male and female mice exhibit protein preference
- Estrous cycle stage does not influence protein preference
- Estrous cycle stage does not affect plasma FGF21 during protein restriction
- Non-restricted females in proestrus have higher FGF21 than those in diestrus or males

sentences: Convergent genomic signatures of adaptation to an extreme environment in cyprinoid fishes Cyprinoids are a worldwide distributed and diverse group of freshwater fish with more than 3,000 species. Although primarily freshwater, some cyprinoid species had convergently evolved to thrive in extreme environments and underlying genetic mechanisms remain unclear. Here, we leveraged 32 cyprinoid fish genomes to identify common genomic changes associated with convergent adaptation to highly saline and alkaline water in two East Asian cyprinoid fish species, Gymnocypris przwalskii and Leuciscus waleckii, representing two independent extremophile fish lineages. We found that genome-wide rate of nonsynonymous substitution and signal of intensified selection is higher in extremophile relative to non-extremophile fish taxa. We further tested gene-wide molecular convergence and found hundreds of genes tended to experience convergent shifts in selections in extremophile fish taxa, including convergent acceleration and positive selection. These genes were associated with several key functions, such as nervous system development, reproduction, ion transport and immune response, and included genes that previously have been implicated for saline or alkaline tolerance in fish. Additionally, comparative transcriptomic analyses defined the convergent roles of differentially expressed genes under selection in extremophile fish taxa during convergent adaptation. Taken together, our work provides insights into the genomic basis of convergent adaptation to extreme environments in fish.

sentences: Reversible adhesion by type IV pili leads to formation of irreversible localized clusters Despite the fact a fundamental first step in the physiopathology of many disease-causing bacteria is the formation of long-lived, localized, multicellular clusters, the spatio-temporal dynamics of the cluster formation process, particularly on host tissues, remains poorly understood. Experiments on abiotic surfaces suggest that the colonization of a surface by swimming bacteria requires i) irreversible adhesion to the surface, ii) cell proliferation, and iii) a phenotypic transition from an initial planktonic state. Here, we investigate how Pseudomonas aeruginosa (PA) infects a polarized MDCK epithelium and show that contrary to what has been reported on the colonization of abiotic surfaces, PA forms irreversible bacterial clusters on apoptotic epithelial cell without requiring irreversible adhesion, cell proliferation, or a phenotypic transition. By combining experiments and a mathematical model, we reveal that the cluster formation process is regulated by type IV pili (T4P). Furthermore, we unveil how T4P quantitatively operate during adhesion on the biotic surface, finding that it is a stochastic process that involves an activation time, requires the retraction of pili, and results in reversible adhesion with a characteristic attachment time. Using a simple kinetic model, we explain how such reversible adhesion process leads to the formation of irreversible bacterial clusters and quantify the cluster growth dynamics.

sentences: Diversity and determinants of recombination landscapes in flowering plants During meiosis, crossover rates are not randomly distributed along the chromosome and therefore they locally influence the creation of novel genotypes and the efficacy of selection. To date, the broad diversity of recombination landscapes among plants has rarely been investigated, undermining the overall understanding of the constraints driving the evolution of crossover frequency and distribution. The determinants that shape the local crossover rate and the diversity of the resulting landscapes among species and chromosomes still need to be assessed in a formal comparative genomic approach. We gathered genetic maps and genomes for 57 flowering plant species, corresponding to 665 chromosomes, for which we estimated large-scale recombination landscapes. Chromosome length drives the basal recombination rate for each species, but within species we were intrigued to notice that the chromosome-wide recombination rate is proportional to the relative size of the chromosome. Moreover, for larger chromosomes, crossovers tend to accumulate at the ends of the chromosome leaving the central regions as recombination-free regions. Based on identified crossover patterns and testable predictions, we proposed a conceptual model explaining the broad-scale distribution of crossovers where both telomeres and centromeres are important. Finally, we qualitatively identified two recurrent crossover patterns among species and highlighted that these patterns globally correspond to the underlying gene distribution. In addition to the positive correlation between recombination and gene density, we argue that crossover patterns are essential for the efficiency of chromosomal genetic shuffling, even though the ultimate evolutionary potential forged by the diversity of recombination landscapes remains an open question.

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High-affinity iron and calcium transport pathways are involved in U(VI) uptake in the budding yeast Saccharomyces cerevisiae Uranium (U) is a naturally-occurring radionuclide toxic for living organisms that can take it up. To date, the mechanisms of U uptake are far from being understood. Here, we used the yeast Saccharomyces cerevisiae as a unicellular eukaryote model to identify U assimilation pathways. Thus, we have identified, for the first time, transport machineries capable of transporting U in a living organism. First, we evidenced a metabolism-dependent U transport in yeast. Then, competition experiments with essential metals allowed us to identify calcium, iron and copper entry pathways as potential routes for U uptake. The analysis of various metal transport mutants revealed that mid1{Delta}, cch1{Delta} and ftr1{Delta} mutants, affected in calcium (Mid1/Cch1 channel) and Fe(III) (Ftr1/Fet3 complex) transport, respectively, exhibited highly reduced U uptake rates and accumulation, demonstrating the implication of these import systems in U uptake. Finally, expression of the Mid1 gene into the mid1{Delta} mutant restored U uptake levels of the wild type strain, underscoring the central role of the Mid1/Cch1 calcium channel in U absorption process in yeast. Our results also open up the opportunity for rapid screening of U-transporter candidates by functional expression in yeast, before their validation in more complex higher eukaryote model systems.

HighlightsO_LILiving yeast Saccharomyces cerevisiae is able to take up U
C_LIO_LIAvailability of a metabolizable substrate stimulates U uptake
C_LIO_LICalcium, iron and copper inhibit U uptake
C_LIO_LIStrains deleted in Mid1/Cch1 calcium channel and Ftr1 iron permease are affected in U uptake
C_LIO_LIExpression of MID1 gene in mid1{Delta} strain restore wild type levels of U uptake
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Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.

ImportanceUltrastructural changes to the morphology and organization of cellular compartments during herpes simplex virus-1 (HSV-1) infection have not previously been studied under near-physiological conditions. In this study, soft X-ray tomography was used to image the ultrastructure of vitrified cells during HSV-1 infection, identifying striking changes to the abundance and organization of cytoplasmic vesicles and mitochondria. The concentration of vesicles in the juxtanuclear region increased with time post infection, which could represent an increasing supply of vesicles to support capsid envelopment, and there is a transient increase in the size of lipid droplets in infected cells. Furthermore, we show that mitochondria elongate and form highly-branched networks as infection progresses. These findings offer insight into stages of virion morphogenesis and the cellular response to infection, highlighting the utility of cryo-soft-X-ray tomography for monitoring the near-native state ultrastructure of infected cells.

sentences: Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

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Vitamin C functions as double-edge sword on cancer progression depending on ERK activation or inhibition mediated by its receptor SVCT2 The effect of Vitamin C (Vc) in oncotherapy was controversial for decades. And hyperactivation of extracellular signal-regulated kinase (ERK) drove tumorigenesis. Herein, we demonstrated that Vc activated ERK through sodium-dependent Vc transporter 2 (SVCT2), while high-dose Vc resulted in persistent ERK feedback inhibition following activation. Extracellular Vc binding to SVCT2 initiated ERK activation, then transmembrane transport of Vc induced dimerization of SVCT2. Activated ERK phosphorylated protein tyrosine phosphatase non-receptor type 12 (PTPN12) at Ser434 and inhibited PTPN12 activity, thus enhancing phosphorylation of Janus kinase 2 (JAK2), which phosphorylated growth factor receptor bound protein 2 (GRB2) at Tyr160 to promote GRB2 dimers dissociation and recruitment of GRB2 to SVCT2, leading to further ERK activation. Different cancers have different sensitivities to Vc, the dose effects of Vc on cancer phenotypes depended on that ERK was activated or inhibited. These findings suggest SVCT2 is a Vc receptor mediating the ERK-PTPN12-JAK2-GRB2-ERK positive feedback loop and a potential target for oncotherapy.

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sentences: RPRM negatively regulates ATM levels involving its phosphorylation mediated by CDK4/CDK6 Sensitizing cancer cells to radio- and chemotherapy remains a hot topic in cancer treatment. Here it is identified that Protein Reprimo (RPRM) negatively regulates the levels of ataxia-telangiectasia mutated (ATM) protein kinase, a master regulator of DNA damage response (DDR) in the presence of DNA double-strand breaks (DSBs), resulting in impaired DNA repair efficiency and enhanced cellular sensitivity to genotoxic agents. Mechanistically, although RPRM is primarily located in cytoplasm, it rapidly translocates to nucleus shortly after induced by X-irradiation, interacts with ATM and promotes the nuclear export and proteasomal degradation of ATM. The nuclear translocation of RPRM is associated with its phosphorylation at serine 98, which is mediated by cyclin-dependent kinases 4/6 (CDK4/6). Inhibition of CDK4/6 stabilizes RPRM and promotes its nuclear import, in turn enhances the nuclear export of ATM and the reduction of ATM levels. As a result, RPRM overexpression and its phosphorylation inhibition sensitize cells to genotoxic agents. Moreover, RPRM deficiency significantly increases resistance to radiation-induced damage both in vitro and in vivo. These findings establish a crucial regulatory mechanism in which ATM is negatively modulated by RPRM, suggesting that RPRM may serve as a novel target for both cancer therapy and radiation protection.

sentences: Galvanic current activates the NLRP3 inflammasome to promote type I collagen production in tendon The NLRP3 inflammasome coordinates inflammation in response to different pathogen- and damage-associated molecular patterns, being implicated in different infectious, chronic inflammatory, metabolic and degenerative diseases. In chronic tendinopathies lesions, different non-resolving mechanisms produce a degenerative condition that impairs tissue healing, complicating their clinical management. Percutaneous needle electrolysis consist in the application of a galvanic current and is emerging as a novel treatment for tendinopathies. Here we found that galvanic current activates the NLRP3 inflammasome and the induction of an inflammatory response promoting a collagen-mediated regeneration of the tendon. This study establish the molecular mechanism of percutaneous electrolysis for the treatment of chronic lesions and the beneficial effects of an induced inflammasome-related response.

sentences: A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19 Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective siRNA therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle delivery system. Multiple small-interfering RNAs (siRNAs) targeting highly conserved regions of the SARS-CoV-2 virus were screened and three candidate siRNAs emerged that effectively inhibit virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel lipid nanoparticle formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.

sentences: Human Lung-resident Mucosal-Associated Invariant T cells are Abundant, Express Antimicrobial Proteins, and are Cytokine Responsive Mucosal-associated Invariant T (MAIT) cells are an innate-like T cell subset that recognize a broad array of microbial pathogens, including respiratory pathogens. Here we investigate the transcriptional profile of MAIT cells localized to the human lung, and postulate that MAIT cells may play a role in maintaining homeostasis at this mucosal barrier. Using the MR1/5-OP-RU tetramer, we identified MAIT cells and non-MAIT CD8+ T cells in lung tissue not suitable for transplant from human donors. We used RNA-sequencing of MAIT cells compared to non-MAIT CD8+ T cells to define the transcriptome of MAIT cells in the human lung. We show that, as a population, lung MAIT cells are polycytotoxic, secrete the directly antimicrobial molecule IL-26, express genes associated with persistence, and selectively express cytokine and chemokine-related molecules distinct from other lung-resident CD8+ T cells, such as interferon-{gamma}- and IL-12-receptors. These data highlight MAIT cells predisposition to rapid pro-inflammatory cytokine responsiveness and antimicrobial mechanisms in human lung tissue, concordant with findings of blood-derived counterparts, and support a function for MAIT cells as early sensors in the defense of respiratory barrier function.

sentences: Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin Neisseria gonorrhoeae (GC) establishes symptomatic infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli ablation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shed from the cervix, but neither in the ectocervix nor the endocervix, by immunofluorescence microscopy. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Thus, polarized expression of ezrin at the apical surface of epithelial cells inhibits GC invasion, while non-polarized expression of ezrin promotes GC invasion by driving actin accumulation and microvilli elongation.

sentences: A flow-based latent state generative model of neural population responses to natural images We present a joint deep neural system identification model for two major sources of neural variability: stimulus-driven and stimulus-conditioned fluctuations. To this end, we combine (1) state-of-the-art deep networks for stimulus-driven activity and (2) a flexible, normalizing flow-based generative model to capture the stimulus-conditioned variability including noise correlations. This allows us to train the model end-to-end without the need for sophisticated probabilistic approximations associated with many latent state models for stimulus-conditioned fluctuations. We train the model on the responses of thousands of neurons from multiple areas of the mouse visual cortex to natural images. We show that our model outperforms previous state-of-the-art models in predicting the distribution of neural population responses to novel stimuli, including shared stimulus-conditioned variability. Furthermore, it successfully learns known latent factors of the population responses that are related to behavioral variables such as pupil dilation, and other factors that vary systematically with brain area or retinotopic location. Overall, our model accurately accounts for two critical sources of neural variability while avoiding several complexities associated with many existing latent state models. It thus provides a useful tool for uncovering the interplay between different factors that contribute to variability in neural activity.

sentences: Nuclear Factor I in neurons, glia and during the formation of Muller glia-derived progenitor cells in avian, porcine and primate retinas The regenerative potential of Muller glia (MG) is extraordinary in fish, poor in chick and terrible in mammals. In the chick model, MG readily reprogram into proliferating Muller glia-derived progenitor cells (MGPCs), but neuronal differentiation is very limited. The factors that suppress the neurogenic potential of MGPCs in the chick are slowly being revealed. Isoforms of Nuclear Factor I (NFI) are cell-intrinsic factors that limit neurogenic potential; these factors are required for the formation of MG in the developing mouse retina (Clark et al., 2019) and deletion of these factors reprograms MG into neuron-like cells in mature mouse retina (Hoang et al., 2020). Accordingly, we sought to characterize the patterns of expression NFIs in the developing, mature and damaged chick retina. In addition, we characterized patterns of expression of NFIs in the retinas of large mammals, pigs and monkeys. Using a combination of single cell RNA-sequencing (scRNA-seq) and immunolabeling we probed for patterns of expression. In embryonic chick, levels of NFIs are very low in early E5 (embryonic day 5) retinal progenitor cells (RPCs), up-regulated in E8 RPCs, further up-regulated in differentiating MG at E12 and E15. NFIs are maintained in mature resting MG, microglia and neurons. Levels of NFIs are reduced in activated MG in retinas treated with NMDA and/or insulin+FGF2, and further down-regulated in proliferating MGPCs. However, levels of NFIs in MGPCs were significantly higher than those seen in RPCs. Immunolabeling for NFIA and NFIB closely matched patterns of expression revealed in different types of retinal neurons and glia, consistent with findings from scRNA-seq. In addition, we find expression of NFIA and NFIB through progenitors in the circumferential marginal zone at the far periphery of the retina. We find similar patterns of expression for NFIs in scRNA-seq databases for pig and monkey retinas. Patterns of expression of NFIA and NFIB were validated with immunofluorescence in pig and monkey retinas wherein these factors were predominantly detected in MG and a few types of inner retinal neurons. In summary, NFIA and NFIB are prominently expressed in developing chick retina and by mature neurons and glia in the retinas of chicks, pigs and monkeys. Although levels of NFIs are decreased in chick, in MGPCs these levels remain higher than those seen in neurogenic RPCs. We propose that the neurogenic potential of MGPCs in the chick retina is suppressed by NFIs.

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Adolescent Development of Biological Rhythms: Estradiol Dependence and Effects of Combined Contraceptives PurposeAdolescence is a period of continuous development, including the maturation of endogenous rhythms across systems and timescales. Although these dynamic changes are well recognized, their continuous structure and hormonal dependence have not been systematically characterized. Given the well-established link between core body temperature (CBT) and reproductive hormones in adults, we hypothesized that high-resolution CBT can be applied to passively monitor pubertal development and disruption with high fidelity.

MethodsTo examine this possibility, we used signal processing to investigate the trajectory of CBT rhythms at the within-day (ultradian), daily (circadian), and ovulatory timescales, their dependence on estradiol, and the effects of hormonal contraceptives.

ResultsPuberty onset was marked by a rise in fecal estradiol (fE2), followed by an elevation in CBT and circadian power. This time period marked the commencement of 4-day rhythmicity in fE2, CBT, and ultradian power marking the onset of the estrous cycle. The rise in circadian amplitude was accelerated by E2 treatment, indicating a role for this hormone in rhythmic development. Contraceptive administration in later adolescence reduced CBT and circadian power and resulted in disruption to 4-day cycles that persisted after discontinuation.

ConclusionsOur data reveal with precise temporal resolution how biological rhythms change across adolescence and demonstrate a role for E2 in the emergence and preservation of multiscale rhythmicity. These findings also demonstrate how hormones delivered exogenously in a non-rhythmic pattern can disrupt rhythmic development. These data lay the groundwork for a future in which temperature metrics provide an inexpensive, convenient method for monitoring pubertal maturation and support the development of hormone therapies that better mimic and support human chronobiology.

sentences: Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy Selective autophagy of damaged mitochondria, intracellular pathogens, protein aggregates, endoplasmic reticulum, and other large cargoes is essential for health. The presence of cargo initiates phagophore biogenesis, which entails the conjugation of ATG8/LC3 family proteins to membrane phosphatidylethanolamine. Current models suggest that the presence of clustered ubiquitin chains on a cargo triggers a cascade of interactions from autophagic cargo receptors through the autophagy core complexes ULK1 and class III PI 3-kinase complex I (PI3KC3-C1), WIPI2, and the ATG7, ATG3, and ATG12-ATG5-ATG16L1 machinery of LC3 lipidation. This model was tested using giant unilamellar vesicles (GUVs), GST-Ub4 as a model cargo, the cargo receptors NDP52, TAX1BP1, and OPTN, and the autophagy core complexes. All three cargo receptors potently stimulated LC3 lipidation on GUVs. NDP52- and TAX1BP1-induced LC3 lipidation required the ULK1 complex together with all other components, however, ULK1 kinase activity was dispensable. In contrast, OPTN bypassed the ULK1 requirement completely. These data show that the cargo-dependent stimulation of LC3 lipidation is a common property of multiple autophagic cargo receptors, yet the details of core complex engagement vary considerably and unexpectedly between the different receptors.

sentences: Delta breakthrough infections elicit potent, broad and durable neutralizing antibody responses The SARS-CoV-2 Delta variant is currently responsible for most infections worldwide, including among fully vaccinated individuals. Although these latter infections are associated with milder COVID-19 disease relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by Delta breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants of concern than those observed in subjects who were infected only or received only two doses of COVID-19 vaccine. However, wee show that Delta breakthrough cases, subjects who were vaccinated after SARS-CoV-2 infection and individuals vaccinated three times (without infection) have serum neutralizing activity of comparable magnitude and breadth indicate that multiple types of exposure or increased number of exposures to SARS-CoV-2 antigen(s) enhance spike-specific antibody responses. Neutralization of the genetically divergent SARS-CoV, however, was moderate with all four cohorts examined, except after four exposures to the SARS-CoV-2 spike, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

sentences: Heat efficiently inactivates coronaviruses inside vehicles Heat is an established method to inactivate coronaviruses, and there is utility in using heat to reduce viral load on common touch points in vehicles exposed to a person shedding SARS-CoV-2. As SARS-CoV-2 is a Biosafety level (BSL)-3 pathogen, real world testing of heat as a sanitation method for public and private vehicles becomes a challenge, requiring a surrogate coronavirus that can be handled safely outside of a BSL-3 facility. In this study, we used Bovine Coronavirus (BCoV) as a surrogate for SARS-CoV-2 to test the efficacy of heat-based betacoronavirus inactivation. In vitro, a 30-minute exposure to 56{degrees}C completely inactivated BCoV in solution, and a 15-minute exposure reduced recovery of BCoV >1000-fold. When heated to 56{degrees}C for 15 minutes, the infectivity of BCoV spotted and dried on typical porous and non-porous automobile interior materials was reduced by 99 - 99.99%. When BCoV was spotted and dried on hard plastic (seat) material placed inside an out of service transit bus, 56{degrees}C heat for 30 minutes reduced BCoV infectivity 85 - 99.5%. Thus, 56{degrees}C is an accessible, rapid, and effective method to inactivate coronaviruses inside motor vehicles.

sentences: PFTK1 kinase regulates axogenesis during development via RhoA activation. PFTK1/Eip63E is a member of the Cyclin-dependent kinases (CDKs) family and plays an important role in normal cell cycle progression. Eip63E expresses primarily in postnatal and adult nervous system in Drosophila melanogaster but its role in CNS development remains unknown. We sought to understand the function of Eip63E in the CNS by studying the fly ventral nerve cord during development. Our results demonstrate that Eip63E regulates axogenesis in neurons and its deficiency leads to neuronal defects. Functional interaction studies performed using the same system identify an interaction between Eip63E and the small GTPase Rho1. Furthermore, deficiency of Eip63E homolog in mice, PFTK1, in a newly generated PFTK1 knockout mice results in increased axonal outgrowth confirming that the developmental defects observed in the fly model are due to defects in axogenesis. Importantly, RhoA phosphorylation and activity is affected by PFTK1 in primary neuronal cultures. We here report that GDP bound inactive RhoA is a substrate of PFTK1 and PFTK1 phosphorylation maintains RhoA activity. In conclusion, our work establishes an unreported role of PFTK1 in axon development mediated by phosphorylation and activation of GDP-bound RhoA. The results presented add to our understanding of the role of Cdks in the maintenance of RhoA mediated axon growth and its impact on CNS development and axonal regeneration.

sentences: Serum CSF1 levels are elevated in patients following partial hepatectomy and acetaminophen induced acute liver failure: Reanalysis of previous data A previous study analysed serum CSF1 concentration in patients who had undergone partial hepatectomy and patients with acute liver failure (ALF) induced by acetaminophen. A correlation between low CSF1 and poor prognosis was reported in ALF. In May 2018, an allegation of research misconduct was made against the second author of the published study, D.J.Antoine, who carried out the CSF1 analysis. The current study describes the independent reanalysis of circulating CSF1 concentration in sera from the same patient cohorts. Patient serum CSF1 levels increased following partial hepatectomy. By contrast to the published report, serum CSF1 concentration was greatest in ALF patients who died compared to survivors. The results confirm the relationship between liver injury and circulating serum CSF1.

sentences: An ancient polymorphic regulatory region within the BDNF gene associated with obesity modulates anxiety-like behaviour in mice and humans. Obesity and anxiety are morbidities notable for their increased impact on society during the recent COVID-19 pandemic. Understanding the mechanisms governing susceptibility to these conditions will increase quality of life and our resilience to future pandemics. In the current study we explored the function of a highly conserved regulatory region (BE5.1) within the BDNF gene that harbours a polymorphism strongly associated with obesity (rs10767664; p=4.69x10-26). Analysis in primary cells suggested that the major T-allele of BE5.1 was an enhancer whereas the obesity associated A-allele was not. However, CRISPR/CAS9 deletion of BE5.1 from the mouse genome (BE5.1KO) produced no significant effect on the expression of BDNF transcripts in the hypothalamus, no change in weight gain after 28 days and only a marginally significant increase in food intake. Nevertheless, transcripts were significantly increased in the amygdala of female mice and elevated zero maze and marble burying tests demonstrated a significant increase in anxiety-like behaviour that could be reversed by diazepam. Consistent with these observations, human GWAS cohort analysis demonstrated a significant association between rs10767664 and anxiousness in human populations. Intriguingly, interrogation of the human GTEx eQTL database demonstrated no effect on BDNF mRNA levels associated with rs10767664 but a highly significant effect on BDNF-antisense (BDNF-AS) gene expression and splicing suggesting a possible mechanism. We discuss our findings within the context of the known function and regulation of BDNF in obesity and anxiety whilst exploring the validity of interrogating GWAS data using comparative genomics and functional analysis using CRISPR genome editing in mice.

sentences: Multivariate genome-wide association study identifies 780 unique genetic loci associated with cortical morphology. Brain morphology has been shown to be highly heritable, yet only a small portion of the heritability is explained by the genetic variants discovered so far. Here we exploit the distributed nature of genetic effects across the brain and apply the Multivariate Omnibus Statistical Test (MOSTest) to genome-wide association studies (GWAS) of vertex-wise structural magnetic resonance imaging (MRI) cortical measures from N=35,657 participants in the UK Biobank. We identified 695 loci for cortical surface area and 539 for cortical thickness, in total 780 unique genetic loci associated with cortical morphology. This reflects an approximate 10-fold increase compared to the commonly applied univariate GWAS methods. Power analysis indicates that applying MOSTest to vertex-wise structural MRI data triples the effective sample size compared to conventional univariate GWAS approaches. Functional follow up including gene-based analyses implicate 10% of all protein-coding genes and point towards pathways involved in neurogenesis and cell differentiation.

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MutationalPatterns: The one stop shop for the analysis of mutational processes BackgroundThe collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data.

ResultsHere, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights.

ConclusionsThis novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns.

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Nuclear receptor ligand screening in an iPSC-derived in vitro blood-brain barrier model identifies new contributors to leptin transport BackgroundThe peptide hormone leptin exerts its function in the brain to reduce food intake and increase energy expenditure to prevent obesity. However, most obese subjects reflect the resistance to leptin even with elevated serum leptin. Considering that leptin must cross the blood-brain barrier (BBB) in several regions to enter the brain parenchyma, altered leptin transport through the BBB might play an important role in leptin resistance and other biological conditions. Here, we report the use of a human induced pluripotent stem cell (iPSC)-derived BBB model to explore mechanisms that influence leptin transport.

MethodsiPSCs were differentiated into brain microvascular endothelial cell (BMEC)-like cells using standard methods. BMEC-like cells were cultured in Transwell filters, treated with ligands from a nuclear receptor agonist library, and assayed for leptin transport using an enzyme-linked immune sorbent assay. RNA sequencing was further used to identify differentially regulated genes and pathways. The role of a select hit in leptin transport was tested with the competitive substrate assay and after gene knockdown using CRISPR techniques.

ResultsFollowing a screen of 73 compounds, 17{beta}-estradiol was identified as a compound that could significantly increase leptin transport. RNA sequencing revealed many differentially expressed transmembrane transporters after 17{beta}-estradiol treatment. Of these, cationic amino acid transporter-1 (CAT-1, encoded by SLC7A1) was selected for follow-up analyses due to its high and selective expression in BMECs in vivo. Treatment of BMEC-like cells with CAT-1 substrates, as well as knockdown of CAT-1 expression via CRISPR-mediated epigenome editing, yielded significant increases in leptin transport.

ConclusionsA major female sex hormone, as well as an amino acid transporter, were revealed as regulators of leptin BBB transport in the iPSC-derived BBB model. Outcomes from this work provide insights into regulation of peptide hormone transport across the BBB.

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T-cell Repertoire Characteristics of Asymptomatic and Re-detectable Positive COVID-19 Patients BackgroundThe prevention of COVID-19 pandemic is highly complicated by the prevalence of asymptomatic and recurrent infection. Many previous immunological studies have focused on symptomatic and convalescent patients, while the immune responses in asymptomatic patients and re-detectable positive cases remain unclear.

MethodsHere we comprehensively analyzed the peripheral T-cell receptor (TCR) repertoire of 54 COVID-19 patients in different phases, including asymptomatic, symptomatic, convalescent and re-detectable positive cases.

ResultsWe found progressed immune responses from asymptomatic to symptomatic phase. Furthermore, the TCR profiles of re-detectable positive cases were highly similar to those of asymptomatic patients, which could predict the risk of recurrent infection.

ConclusionTherefore, TCR repertoire surveillance has the potential to strengthen the clinical management and the immunotherapy development for COVID-19.

FundingThe Science and Technology Innovation Project of Foshan Municipality (2020001000431) and the National Key Research and Development Project (2020YFA0708001).

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Protein interaction analysis of Plasmodium falciparum circumsporozoite protein variants with human immunoproteins explains RTS,S vaccine efficacy in Ghana BackgroundThe worlds first malaria vaccine RTS,S provides only partial protection against Plasmodium falciparum infections. The explanation for such low efficacy is unclear. This study examined the associations of parasite genetic variations with binding affinity to human immunological proteins including human leukocyte antigen (HLA) and T cell receptors (TCR) involved in RTS,S-induced immune responses.

MethodsMultiplicity of infections was determined by amplicon deep sequencing of merozoite surface protein 1 (PfMSP1). Genetic variations in the C-terminal of circumsporozoite protein (PfMSP1) gene were examined across 88 samples of P. falciparum collected from high and low transmission settings of Ghana. Binding interactions of PfMSP1 variants and HLA/TCR were analyzed using NetChop and HADDOCK predictions. Anti-CSP IgG levels were measured by ELISA in a subset of 10 samples.

FindingsHigh polyclonality was detected among P. falciparum infections. A total 27 CSP haplotypes were detected among samples. A significant correlation was detected between the CSP and MSP multiplicity of infection (MOI). No clear clustering of haplotypes was observed by geographic regions. The number of genetic differences in PfCSP between 3D7 and non-3D7 variants does not influence binding interactions to HLA/T cells nor anti-CSP IgG levels. Nevertheless, PfCSP peptide length significantly affects its molecular weight and binding affinity to the HLA.

InterpretationsThe presence of multiple non-3D7 strains among P. falciparum infections in Ghana impact the effectiveness of RTS,S. Longer PfCSP peptides may elicit a stronger immune response and should be considered in future version RTS,S. The molecular mechanisms of RTS,S cell-mediated immune responses related to longer CSP peptides warrants further investigations.

sentences: DNA sliding and loop formation by E. coli SMC complex: MukBEF SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

sentences: Metabolism of glucose activates TORC1 through multiple mechanisms in Saccharomyces cerevisiae Target of Rapamycin Complex 1 (TORC1) is a conserved eukaryotic protein complex that links the presence of nutrients with cell growth. In Saccharomyces cerevisiae, TORC1 activity is positively regulated by the presence of amino acids and glucose in the medium. However, mechanisms underlying nutrient-induced TORC1 activation remain poorly understood. By utilizing a TORC1 activation assay, we demonstrate that differential metabolism of glucose activates TORC1 through three distinct pathways in yeast. The first  canonical Rag GTPase-dependent pathway requires conversion of glucose to fructose 1,6-bisphosphate which activates TORC1 via the Rag GTPase heterodimer Gtr1GTP/Gtr2GDP. The second  non-canonical Rag GTPase-dependent pathway requires conversion of glucose to glucose 6-phosphate which activates TORC1 via Gtr1GTP/Gtr2GTP. The third  Rag GTPase-independent pathway requires complete glycolysis and vacuolar ATPase reassembly for TORC1 activation. Glucose-induced TORC1 activation can be uncoupled from glucose-induced AMPK inactivation. We have established a roadmap to deconstruct the link between glucose metabolism and TORC1 activation.

sentences: RNA sequencing on muscle biopsy from a 5-week bedrest study reveals the effect of exercise and potential interactions with dorsal root ganglion neurons Lack of physical activity is a predictor of poor health outcomes that can be prevented or reversed by exercise. Sedentary lifestyle, chronic disease or microgravity can cause muscle deconditioning that then has an impact on other physiological systems. An example is the nervous system, which is adversely affected by decreased physical activity resulting in increased incidence of neurological problems such as chronic pain. We sought to better understand how this might occur by conducting RNA sequencing experiments on muscle biopsies from human volunteers in a 5-week bed-rest study with an exercise intervention arm. We also used a computational method for examining ligand-receptor interactions between muscle and human dorsal root ganglion (DRG) neurons, the latter of which play a key role in nociception and are generators of signals responsible for chronic pain. We identified 1352 differentially expressed genes (DEGs) in bed rest subjects without an exercise intervention but only 132 DEGs in subjects with the intervention. Thirty-six genes were shared between the exercise and no intervention groups. Among 591 upregulated muscle genes in the no intervention arm, 26 of these were ligands that have receptors that are expressed by human DRG neurons. We detected a specific splice variant of one of these ligands, placental growth factor (PGF), in deconditioned muscle that binds to neuropilin 1, a receptor that is highly expressed in DRG neurons and known to promote neuropathic pain. We conclude that exercise intervention protects muscle from deconditioning transcriptomic changes, and prevents changes in expression of ligands that might sensitize DRG neurons that promote pain. Our work creates a set of actionable hypotheses to better understand how deconditioned muscle may influence the function of sensory neurons that innervate the entire body.

sentences: On the generality and cognitive basis of base-rate neglect Base rate neglect refers to peoples apparent tendency to underweight or even ignore base rate information when estimating posterior probabilities for events, such as the probability that a person with a positive cancer-test outcome actually does have cancer. While often replicated, almost all evidence for the phenomenon comes from studies that used problems with extremely low base rates, high hit rates, and low false alarm rates. It is currently unclear whether the effect generalizes to reasoning problems outside this "corner" of the entire problem space. Another limitation of previous studies is that they have focused on describing empirical patterns of the effect at the group level and not so much on the underlying strategies and individual differences. Here, we address these two limitations by testing participants on a broader problem space and modelling their responses at a single-participant level. We find that the empirical patterns that have served as evidence for base-rate neglect generalize to a larger problem space, albeit with large individual differences in the extent with which participants "neglect" base rates. In particular, we find a bi-modal distribution consisting of one group of participants who almost entirely ignore the base rate and another group who almost entirely account for it. This heterogeneity is reflected in the cognitive modeling results: participants in the former group were best captured by a linear-additive model, while participants in the latter group were best captured by a Bayesian model. We find little evidence for heuristic models. Altogether, these results suggest that the effect known as "base-rate neglect" generalizes to a large set of reasoning problems, but varies largely across participants and may need a reinterpretation in terms of the underlying cognitive mechanisms.

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Prediction Errors Disrupt Hippocampal Representations and Update Episodic Memories The brain supports adaptive behavior by generating predictions, learning from errors, and updating memories to incorporate new information. Prediction error, or surprise, triggers learning when reality contradicts expectations. Prior studies have shown that the hippocampus signals prediction errors, but the hypothesized link to memory updating has not been demonstrated. In a human fMRI study, we elicited mnemonic prediction errors by interrupting familiar narrative videos immediately before the expected endings. We found that prediction error reversed the relationship between univariate hippocampal activation and memory: greater hippocampal activation predicted memory preservation after expected endings, but memory updating after surprising endings. In contrast to previous studies, we showed that univariate activation was insufficient for understanding hippocampal prediction error signals. We explained this surprising finding by tracking both the evolution of hippocampal activation patterns and connectivity between the hippocampus and neuromodulatory regions. We found that hippocampal activation patterns stabilized as each narrative episode unfolded, suggesting sustained episodic representations. Prediction errors disrupted these sustained representations, and the degree of disruption predicted memory updating. The relationship between hippocampal activation and subsequent memory depended on concurrent basal forebrain activation, supporting the idea that cholinergic modulation regulates attention and memory. We conclude that prediction errors create conditions that favor memory updating, prompting the hippocampus to abandon ongoing predictions and make memories malleable.

SignificanceOur brains draw on memories to predict the future; when our predictions are incorrect, we must update our memories to improve future predictions. Past studies have demonstrated that the hippocampus signals prediction error, or surprise, but have not linked this neural signal to memory updating. Here, we uncover this missing connection: We show that mnemonic prediction errors change the role of the hippocampus, reversing the relationship between hippocampal activation and memory outcomes. We examine the mechanisms of this shift in neural processing, showing that prediction errors disrupt the temporal continuity of hippocampal patterns. We propose that prediction errors disrupt sustained representations and enable memory updating. Our findings bear implications for improving education, understanding eyewitness memory distortion, and treating pathological memories.

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Intranasal oxytocin reduces pre-courtship aggression and increases paternal response in California mice (Peromyscus californicus) Oxytocin (OXT) is a neuropeptide that can facilitate prosocial behavior and decrease social stress and anxiety. We investigated whether acute pulses of intranasal (IN) OXT influenced social behavior during social challenges that are likely to occur throughout the lifespan of a wild mouse. To test this, we examined the acute effects of IN OXT in the male California mouse (Peromyscus californicus), a monogamous, biparental, and territorial rodent, using a within-subjects longitudinal design. Social challenges included a pre-courtship male-female encounter conducted during the initial aggressive and not the following affiliative phase of courtship, same-sex resident intruder test, and parental care test, with each test and dose separated by at least two weeks. Males were treated with intranasal infusions of 0.8 IU/kg OXT or saline controls 5-min before each behavioral test, receiving a total of three treatments of either IN OXT or saline control. We predicted that IN OXT would 1) decrease aggression and increase affiliation during the pre-courtship aggression phase, 2) increase aggression during resident intruder paradigms and 3) increase paternal care and vocalizations during a paternal care test. As predicted, during pre-courtship aggression with a novel female, IN OXT males displayed less contact aggression than control males, although with no change in affiliative behavior. However, post-pairing, during the resident intruder test, IN OXT males did not differ from control males in contact aggression. During the paternal care test, IN OXT males were quicker to approach their pups than control males but did not differ in vocalizations produced, unlike our previous research demonstrating an effect on vocalizations in females. In summary, during pre-courtship aggression and the paternal care test, IN OXT promoted prosocial approach; however, during the resident intruder test IN OXT did not alter social approach. These data suggest that IN OXT promotes prosocial approach specifically in social contexts that can lead to affiliation.

HIGHLIGHTSO_LIIN OXT attenuates male aggression during pre-courtship encounters
C_LIO_LIIN OXT does not attenuate male aggression during resident intruder encounters
C_LIO_LIIN OXT increases paternal responsiveness during a paternal care challenge
C_LIO_LIIN OXT in fathers does not influence total paternal care or vocalizations
C_LI

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sentences: MR-APSS: a unified approach to Mendelian Randomization accounting for pleiotropy and sample structure using genome-wide summary statistics Mendelian Randomization (MR) is a valuable tool for inferring causal relationships among a wide range of traits using summary statistics from genome-wide association studies (GWASs). Existing summary-level MR methods often rely on strong assumptions, resulting in many false positive findings. To relax MR assumptions, ongoing research has been primarily focused on accounting for confounding due to pleiotropy. Here we show that sample structure is another major confounding factor, including population stratification, cryptic relatedness, and sample overlap. We propose a unified MR approach, MR-APSS, which (i) accounts for pleiotropy and sample structure simultaneously by leveraging genome-wide information; and (ii) allows to include more genetic variants with moderate effects as instrument variables (IVs) to improve statistical power without inflating type I errors. We first evaluated MR-APSS using comprehensive simulations and negative controls, and then applied MR-APSS to study the causal relationships among a collection of diverse complex traits. The results suggest that MR-APSS can better identify plausible causal relationships with high reliability. In particular, MR-APSS can perform well for highly polygenic traits, where the IV strengths tend to be relatively weak and existing summary-level MR methods for causal inference are vulnerable to confounding effects.

sentences: A novel method for robust estimation of ants' walking speed and curvature on convoluted trajectories derived from their gait pattern Accurate measurements of travel distance and speed are crucial for the analysis of animal movements. Measuring the movements of ants entails measuring the change in locations registered at time intervals. This process involves dilemma of setting the proper time window: a short time window is vulnerable to spatial errors in observation, while a long time window leads to underestimation of the travel distance. To overcome these difficulties, we propose a novel algorithm that successively interpolates two consecutive points of ants trajectory for a given time window by embracing the alternating tripod gait of ants. We demonstrate that this algorithm is more reliable compared to the conventional method of travel distance estimation based on the sum of the consecutive straight-line displacements (SLD). After obtaining speed estimates for a range of sampling time windows, we applied a fitting method that can estimate the actual speed without prior knowledge of spatial error distribution. We compared results from several methods of speed and curvature extracted from the empirical data of ant trajectories. We encourage empirical scientists to utilize the proposed methods rather than the conventional SLD method of speed estimation as this process is a more reliable and subjective selection of the sampling time window can be avoided.

sentences: Progressive polysomal association of Upf1, Upf2, and Upf3 as ribosomes traverse mRNA coding regions Upf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function.

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Sequence based prediction of protein phase separation into disordered condensates using machine learning Several proteins which are responsible for neuro-degenrerative disorders (Alzheimers, Parkinsons etc) are shown to undergo a mechanism known as liquid liquid phase separation (LLPS). We in this research build a predictor which would answer whether a protein molecule would undergo LLPS or not. For this we used some protein sequences for which we already knew the answer. The ones who undergo LLPS were considered as the positive set and the ones who do not, were taken as the negative set. Depending on the knowledge of amino-acid sequences we identified some relevant variables in the context of LLPS e.g. number of amino acids, length of the best pairings, average register shifts. Using these variables we built a number of scoring functions which were basically analytic functions involving these variables and we also combined some scores already existing in the literature. We considered a total of 43636 protein sequences, among them only 121 were positive. We applied logistic regression and performed cross validation, where 25% of the data were used as the training set and the performance of the obtained results were tested on the remaining 75% of the data. In the training process, we used Simplex algorithm to maximize area under the curve (AUC) in receiver operator characteristics (ROC) space for each of the scores we defined. The optimised parameters were then used to evaluate AUC on the test set to check the accuracy. The best performing score was identified as the predicting model to answer the question whether a protein chain would undergo phase separating behavior or not.

Author summaryLiquid-liquid phase separation (LLPS) is a classic subject in polymer physics. The essen- tial physics is aptly captured within the Flory-Huggins (FH) approach, a simple lattice mean-field theory. Evidence has been mounting in the last decade that protein LLPS underlies the formation of membrane-less organelles (MLOs) in living cells, explaining why proteins and other bio-molecules can remain in a dense liquid condensate without diffusing away. Intrinsically disordered regions (IDRs), with a sequence-intrinsic prefer- ence for conformational heterogeneity or disorder under native conditions, are enriched in proteins that have the ability of switching on LLPS. The detailed understanding of the biological function of disordered bio-molecular condensates, whose formation is driven by LLPS, is currently the focus of a major effort in cell biology. Several key proteins in neuro-degenerative disorders are components of MLOs, and a further liquid-to-solid transition to unsoluble amyloid aggregates may be triggered by pathogenic mutants. Non-equilibrium active processes are also known to drive non trivial spatio-temporal organization patterns in MLOs. In this research we aim to predict which proteins can undergo LLPS in physiological conditions in living cells, and the corresponding phase behavior, based on theoretical tools and on the knowledge of the amino-acid sequence alone. We derive specific knowledge-based potentials for the different kind of short-range interactions that are believed to drive protein LLPS and use them to build a simple yes/no predictor of phase separation in physiological conditions.

sentences: Agrobacterium mediated transformation and deciphering SNPs in TaLr67 gene homeologs for gene editing in wheat. Diseases adversely affect grain yield of crop plants. Leaf rust is a major disease of wheat. As race-specific resistance breaks down, introduction of newer sources of resistance often from older accessions is necessary. Linkage drag from the donor accessions adversely impacts grain yield. As a result, CIMMYT breeding effort has shifted to using durable resistance, also referred to as race non-specific host resistance, which allows slow rusting but maintains grain yield. One of the key genes for durable resistance is Lr67 and the resistant form of this gene is absent in CIMMYT elite lines. Hence, we have initiated efforts to convert the susceptible copy of Lr67 into its resistant form directly in these elite lines using gene editing. This would eliminate backcrossing and thus save time as well as eliminate linkage drag that would accompany the resistant copy of the gene if it were to be introgressed from an older accession. As first steps, we have isolated and sequenced the genomic copies from each of the A, B, and D genome of the Lr67 gene from three elite lines and an experimental line. Identification of more than 50 single nucleotide polymorphisms (SNPs) in the open reading frames (ORF) among these lines would be useful in designing the guide RNA molecules with precision. Further, we have streamlined genetic transformation of these elite lines, a prerequisite step for gene editing.

sentences: KAT5 activity regulates G0-like states in human gliomas In solid tumors, G0-like cell populations likely play important roles in maintaining cellular heterogeneity and promoting recurrence after standard of care. However, little is known about the mechanisms of tumor cell G0 ingress and egress. To discover regulators of G0-like states for glioblastoma (GBM), we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) for genes that trap cells in G0-like states when inhibited. We identify the protein acetyltransferase KAT5 as a key regulator of G0 and cell cycle dynamics in GSCs and GSC-derived tumors. In primary gliomas, KAT5low cells display G0-like properties, while overall KAT5 activity increases from low to high grade tumors. Further, we find that KAT5 activity suppresses the emergence of non-dividing subpopulations with oligodendrocyte progenitor and radial glial cell characteristics both in vitro and in a GSC tumor model. These results reveal that KAT5 activity regulates transitions between non-dividing/slow cycling, neurodevelopmental, and proliferative states in GBM tumors.

sentences: Discovery and functional assessment of a novel adipocyte population driven by intracellular Wnt/β-catenin signaling in mammals Wnt/{beta}-catenin signaling has been well established as a potent inhibitor of adipogenesis. Here, we identified a population of adipocytes that exhibit persistent activity of Wnt/{beta}-catenin signaling, as revealed by the Tcf/Lef-Gfp reporter allele, in embryonic and adult mouse fat depots, named as Wnt+ adipocytes. We showed that the {beta}-catenin-mediated signaling activation in these cells is Wnt ligand- and receptor-independent but relies on AKT/mTOR pathway and is essential for cell survival. Such adipocytes are distinct from classical ones in transcriptomic and genomic signatures and can be induced from various sources of mesenchymal stromal cells including human cells. Genetic lineage-tracing and targeted cell ablation studies revealed that these adipocytes convert into beige adipocytes directly and are also required for beige fat recruitment under thermal challenge, demonstrating both cell autonomous and non-cell autonomous roles in adaptive thermogenesis. Furthermore, mice bearing targeted ablation of these adipocytes exhibited glucose intolerance, while mice receiving exogenously supplied such cells manifested enhanced glucose utilization. Our studies uncover a unique adipocyte population in regulating beiging in adipose tissues and systemic glucose homeostasis.

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Neural tracking of the fundamental frequency of the voice: the effect of voice characteristics Traditional electrophysiological methods to study temporal auditory processing of the fundamental frequency of the voice (f0) often use unnaturally repetitive stimuli. In this study, we investigated f0 processing of meaningful continuous speech. EEG responses evoked by stories in quiet were analysed with a novel method based on linear modelling that characterizes the neural tracking of the f0. We studied both the strength and the spatio-temporal properties of the f0-tracking response. Moreover, different samples of continuous speech (six stories by four speakers: two male and two female) were used to investigate the effect of voice characteristics on the f0 response.

The results indicated that response strength is inversely related to f0 frequency and rate of f0 change throughout the story. As a result, the male-narrated stories in this study (low and steady f0) evoked stronger f0-tracking compared to female-narrated stories (high and variable f0), for which many responses were not significant. The spatio-temporal analysis revealed that f0-tracking response generators were not fixed in the brainstem but were voice-dependent as well. Voices with high and variable f0 evoked subcortically-dominated responses with a latency between 7 and 12 ms. Voices with low and steady f0 evoked responses that are both subcortically (latency of 13-15 ms) and cortically (latency of 23-26 ms) generated, with the right primary auditory cortex as a likely cortical source. Finally, additional experiments revealed that response strength greatly improves for voices with strong higher harmonics, which is particularly useful to boost the small responses evoked by voices with high f0.

sentences: Dual mode of PGRP-LE-dependent NF-κB pathway activation in bacteria infected guts Some of the interactions between prokaryotes and eukaryotes are mediated by a molecular dialogue between microbial MAMPs and host PRRs. Bacteria-derived peptidoglycan, which possesses all the characteristics of a MAMP, is detected by membrane-bound or cytosolic PRRs belonging to various families of proteins in both animals and plants (PGRP, Nod, Lys-M...). If the identity and the epistatic relationship between the downstream components of the signaling cascades activated upon PGN/PRR interactions are well characterized, little is known about the subcellular events requires to translate these early sensing steps into downstream target gene transcription. Using a model of Drosophila enteric infection, we show that gut-associated bacteria can induce PGRP-LE intracellular aggregation. Observed in both enterocytes and entero-endocrine cells, these aggregates were found to co-localize with the early endosome marker Rab5. In vivo functional analysis further demonstrates that, whereas some PGRP-LE target genes, such as antimicrobial peptides, can be activated independently of Rab5, other such as the PGRP-SC1 amidase, need the combine action of PGRP-LE and Rab5 to be transcribed. These results demonstrate how by using different intracellular signaling routes, the same ligand/receptor complex can activate different target genes in the same cell.

sentences: VirClust, a tool for hierarchical clustering, core gene detection and annotation of (prokaryotic) viruses Recent years have seen major changes in the classification criteria and taxonomy of viruses. The current classification scheme, also called "megataxonomy of viruses", recognizes five different viral realms, defined based on the presence of viral hallmark genes. Within the realms, viruses are classified into hierarchical taxons, ideally defined by their shared genes. Therefore, there is currently a need for virus classification tools based on such shared genes / proteins. Here, VirClust is presented - a novel tool capable of performing i) hierarchical clustering of viruses based on intergenomic distances calculated from their protein cluster content, ii) identification of core proteins and iii) annotation of viral proteins. VirClust groups proteins into clusters both based on BLASTP sequence similarity, which identifies more related proteins, and also based on hidden markow models (HMM), which identifies more distantly related proteins. Furthermore, VirClust provides an integrated visualization of the hierarchical clustering tree and of the distribution of the protein content, which allows the identification of the genomic features responsible for the respective clustering. By using different intergenomic distances, the hierarchical trees produced by VirClust can be split into viral genome clusters of different taxonomic ranks. VirClust is freely available, as web-service (virclust.icbm.de) and stand-alone tool.

sentences: A nanopore ion source delivers single amino acid and peptide ions directly into the gas phase A technology for sequencing single proteins would expand our understanding of biology and improve the detection and treatment of disease [1]. Approaches based on fluorosequencing [2], nanopores [3, 4], and tunneling spectroscopy [5] are under development and show promise. However, only mass spectrometry (MS) has demonstrated an ability to identify amino acids with minimal degeneracy [6]. We envision sequencing a protein by fragmenting it and delivering its constituent amino acids into a mass spectrometer in sequential order, but existing ion sources employ a background gas that scrambles the spatial ordering of ions and degrades their transmission. Here we report an ion source comprising a glass capillary with a sub-100 nm diameter pore that emits amino acid ions from aqueous solution directly into vacuum. Emitted ions travel collision-less trajectories before striking a single-ion detector. We measured unsolvated ions of 16 different amino acids as well as glutathione and two of its post-translationally modified variants.

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Discovery of an Antarctic ascidian-associated uncultivated Verrucomicrobia that encodes antimelanoma palmerolide biosynthetic capacity The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially-produced hybrid non-ribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74 Kbp candidate BGC encoding the multi-modular enzymatic machinery (hybrid Type I-trans-AT polyketide synthase-non-ribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide-gene targets and a single 16S rRNA gene variant (R=0.83 - 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a near-complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as Candidatus Synoicihabitans palmerolidicus. The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium.

ImportancePalmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply-branching genus, Candidatus Synoicihabitans palmerolidicus, in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. S. palmerolidicus 4.29 Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways enabling its ascidian-associated lifestyle. The palmerolide-producers genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants.

sentences: Reevaluating claims of ecological speciation in Halichoeres bivittatus Understanding the role of ecological processes in speciation has become one of the most active areas of research in marine population biology in recent decades. The traditional view was that allopatry was the primary driver of speciation in marine taxa, but the geography of the marine environment and the dispersal capabilities of many marine organisms render this view somewhat questionable. One of the earliest and most highly cited empirical examples of ecological speciation with gene flow in marine fishes is that of the slippery dick wrasse, Halichoeres bivittatus. Evidence for this cryptic or incipient speciation event was primarily in the form of a deep north-south divergence in a single mitochondrial locus, combined with a finding that these two haplotypes were associated with different habitat types in the Florida Keys and Bermuda, where they overlap. Here we examine habitat assortment in the Florida Keys using a broader sampling of populations and habitat types than were available for the original study, and find no evidence to support the claim that haplotype frequencies differ between habitat types, and little evidence to support any differences between populations. These results severely undermine claims of ecological speciation with gene flow in Halichoeres bivittatus. We argue that future claims of this type should be supported by multiple lines of evidence that illuminate potential mechanisms and allow researchers to rule out alternative explanations for spatial patterns of genetic differences.

sentences: DynaTMT: A user-friendly tool to process combined SILAC/TMT data The measurement of protein dynamics by proteomics to study cell remodeling has seen increased attention over the last years. This development is largely driven by a number of technological advances in proteomics methods. Pulsed stable isotope labeling in cell culture (SILAC) combined with tandem mass tag (TMT) labeling has evolved as a gold standard for profiling protein synthesis and degradation. While the experimental setup is similar to typical proteomics experiments, the data analysis proves more difficult: After peptide identification through search engines, data extraction requires either custom scripted pipelines or tedious manual table manipulations to extract the TMT-labeled heavy and light peaks of interest. To overcome this limitation, which deters researchers from using protein dynamic proteomics, we developed a user-friendly, browser-based application that allows easy and reproducible data analysis without the need for scripting experience. In addition, we provide a python package that can be implemented in established data analysis pipelines. We anticipate that this tool will ease data analysis and spark further research aimed at monitoring protein translation and degradation by proteomics.

sentences: Microbiome diversity and host immune functions may define the fate of sponge holobionts under future ocean conditions The sponge-associated microbial community contributes to the overall health and adaptive capacity of the sponge holobiont. This community is regulated by the environment, as well as the immune system of the host. However, little is known about the effect of environmental stress on the regulation of host immune functions and how this may, in turn, affect sponge-microbe interactions. In this study, we compared the microbiomes and immune repertoire of two sponge species, the demosponge, Neopetrosia compacta and the calcareous sponge, Leucetta chagosensis, under varying levels of acidification and warming stress. Neopetrosia compacta harbors a diverse bacterial assemblage and possesses a rich repertoire of scavenger receptors while L. chagosensis has a less diverse microbiome and an expanded range of pattern recognition receptors and proteins with immunological domains. Upon exposure to warming and acidification, the microbiome and host transcriptome of N. compacta remained stable, which correlated with high survival. In contrast, the bacterial community of L. chagosensis exhibited drastic restructuring and widespread downregulation of host immune-related pathways, which accompanied tissue necrosis and mortality. Differences in microbiome diversity and immunological repertoire of diverse sponge groups highlight the central role of host-microbe interactions in predicting the fate of sponges under future ocean conditions.

sentences: Impacts of Spaceflight Experience on Human Brain Structure Spaceflight induces widespread changes in human brain morphology. It is unclear if these brain changes differ with varying mission durations or ones history of spaceflight experience (e.g., number of prior missions, time between missions). Here we addressed this issue by quantifying voxelwise post-flight changes in gray matter volume, white matter microstructure, extracellular free water (FW), and ventricular volume in a sample of 28 astronauts. We found that longer missions induced greater ventricular expansion and larger FW displacement at the top of the brain. A greater number of prior missions was associated with white matter microstructure declines in a tract supporting voluntary leg movement. Longer inter-mission intervals were associated with greater ventricle expansion, with compensatory ventricular expansion observed only in those crewmembers with inter-missions intervals of 3 years or longer. Longer missions therefore induce more extensive brain fluid shifts, and the ventricles may require at least 3 years to recover post-flight.

sentences: Ca2+-dependent liquid-liquid phase separation underlies intracellular Ca2+ stores. Endoplasmic/sarcoplasmic reticulum Ca2+ stores are essential to myriad cellular processes, however, the structure of these stores is largely unknown and existing models neither explain observations made in vivo nor sufficiently account for physiological data. We investigate CASQ1 - the major Ca2+ binding protein of skeletal muscle - and discover Ca2+-dependent liquid-liquid phase separation activity. The intrinsic disorder of CASQ1 underlies this activity and is regulated via phosphorylation by the secretory pathway kinase FAM20C. This novel divalent cation driven condensation demonstrates liquid-liquid phase separation occurs within the endoplasmic/sarcoplasmic reticulum, mechanistically explains efficient Ca2+ buffering and storage, and represents a largely unexplored mechanism of divalent-cation driven protein association.

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SARS-CoV-2 variant with higher affinity to ACE2 shows reduced sera neutralization susceptibility BackgroundSeveral new variants of SARS-CoV-2 have emerged since fall 2020 which have multiple mutations in the receptor binding domain (RBD) of the spike protein.

ObjectiveWe aimed to assess how mutations in the SARS-CoV-2 RBD affect receptor affinity to angiotensin-converting enzyme 2 (ACE2) and neutralization by anti-RBD serum antibodies.

MethodsWe produced a SARS-CoV-2 RBD mutant (RBDmut) with key mutations (E484K, K417N, N501Y) from the newly emerged Brazilian variant. Using Biolayer Interferometry, we analyzed the binding of this mutant to ACE2, and the susceptibility to neutralization by sera from vaccinated mice and COVID-19 convalescent patients.

ResultsKinetic profiles showed increased RBDmut - ACE2 affinity compared to RBDwt, and binding of vaccine-elicited or convalescent sera was significantly reduced. Likewise, both sera types showed significantly reduced ability to block RBDmut - ACE2 binding indicating that antibodies induced by RBDwt have reduced capability to neutralize mutant virus.

ConclusionOur physiochemical data show enhanced infectivity and reduced neutralization by polyclonal antibodies of the Brazilian variant of SARS-CoV-2.

Capsule summarySARS-CoV-2 variant with Brazilian RBD mutations shows increased ACE2 affinity and reduced susceptibility to blockage by vaccine-elicited and convalescent sera.

sentences: Lipid-mediated Association of the Slg1 Transmembrane Domains in Yeast Plasma Membranes Clustering of transmembrane proteins underlies a multitude of fundamental biological processes at the plasma membrane (PM) such as receptor activation, lateral domain formation and mechanotransduction. The self-association of the respective transmembrane domains (TMD) has also been suggested to be responsible for the micron-scaled patterns seen for integral membrane proteins in the budding yeast plasma membrane. However, the underlying interplay between local lipid composition and TMD identity is still not mechanistically understood. In this work we combined coarse-grained molecular dynamics (MD) simulations of simplified bilayer systems with high resolution live-cell microscopy to analyze the distribution of a representative helical yeast TMD from the PM sensor Slg1 within different lipid environments. In our simulations we specifically evaluated the effects of acyl chain saturation and anionic lipids head groups on the association of two TMDs. We found that weak lipid-protein interactions significantly affect the configuration of TMD dimers and the free energy of association. Increased amounts of unsaturated phospholipids strongly reduced helix-helix interaction, while the presence of anionic phosphatidylserine (PS) hardly affected dimer formation. We could experimentally confirm this surprising lack of effect of PS using the network factor, a mesoscopic measure of PM pattern formation in yeast cells. Simulations also showed that formation of TMD dimers in turn increased the order parameter of the surrounding lipids and induced long-range perturbations in lipid organization. In summary, our results shed new light on the mechanisms for lipid-mediated dimerization of TMDs in complex lipid mixtures.

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Critical behaviour of the stochastic Wilson-Cowan model Spontaneous brain activity is characterized by bursts and avalanche-like dynamics, with scale-free features typical of critical behaviour. The stochastic version of the celebrated Wilson-Cowan model has been widely studied as a system of spiking neurons reproducing non-trivial features of the neural activity, from avalanche dynamics to oscillatory behaviours. However, to what extent such phenomena are related to the presence of a genuine critical point remains elusive. Here we address this central issue, providing analytical results in the linear approximation and extensive numerical analysis. In particular, we present results supporting the existence of a bona fide critical point, where a second-order-like phase transition occurs, characterized by scale-free avalanche dynamics, scaling with the system size and a diverging relaxation time-scale. Moreover, our study shows that the observed critical behaviour falls within the universality class of the mean-field branching process, where the exponents of the avalanche size and duration distributions are, respectively, -3/2 and -2. We also provide an accurate analysis of the system behaviour as a function of the total number of neurons, focusing on the time correlation functions of the firing rate in a wide range of the parameter space.

Author summaryNetworks of spiking neurons are introduced to describe some features of the brain activity, which are characterized by burst events (avalanches) with power-law distributions of size and duration. The observation of this kind of noisy behaviour in a wide variety of real systems led to the hypothesis that neuronal networks work in the proximity of a critical point. This hypothesis is at the core of an intense debate. At variance with previous claims, here we show that a stochastic version of the Wilson-Cowan model presents a phenomenology in agreement with the existence of a bona fide critical point for a particular choice of the relative synaptic weight between excitatory and inhibitory neurons. The system behaviour at this point shows all features typical of criticality, such as diverging timescales, scaling with the system size and scale-free distributions of avalanche sizes and durations, with exponents corresponding to the mean-field branching process. Our analysis unveils the critical nature of the observed behaviours.

sentences: Learning association for single-cell transcriptomics by integrating profiling of gene expression and alternative polyadenylation Single-cell RNA-sequencing (scRNA-seq) has enabled transcriptome-wide profiling of gene expressions in individual cells. A myriad of computational methods have been proposed to learn cell-cell similarities and/or cluster cells, however, high variability and dropout rate inherent in scRNA-seq confounds reliable quantification of cell-cell associations based on the gene expression profile alone. Lately bioinformatics studies have emerged to capture key transcriptome information on alternative polyadenylation (APA) from standard scRNA-seq and revealed APA dynamics among cell types, suggesting the possibility of discerning cell identities with the APA profile. Complementary information at both layers of APA isoforms and genes creates great potential to develop cost-efficient approaches to dissect cell types based on multiple modalities derived from existing scRNA-seq data without changing experimental technologies. We proposed a toolkit called scLAPA for learning association for single-cell transcriptomics by combing single-cell profiling of gene expression and alternative polyadenylation derived from the same scRNA-seq data. We compared scLAPA with seven similarity metrics and five clustering methods using diverse scRNA-seq datasets. Comparative results showed that scLAPA is more effective and robust for learning cell-cell similarities and clustering cell types than competing methods. Moreover, with scLAPA we found two hidden subpopulations of peripheral blood mononuclear cells that were undetectable using the gene expression data alone. As a comprehensive toolkit, scLAPA provides a unique strategy to learn cell-cell associations, improve cell type clustering and discover novel cell types by augmentation of gene expression profiles with polyadenylation information, which can be incorporated in most existing scRNA-seq pipelines. scLAPA is available at https://github.com/BMILAB/scLAPA.

sentences: Population monitoring of snow leopards using camera trapping in Naryn State Reserve, Kyrgyzstan, between 2016 and 2019 Four field seasons of snow leopard (Panthera uncia) camera trapping inside Naryn State Reserve, performed thanks to citizen science expeditions, allowed detecting a minimal population of five adults, caught every year with an equilibrated sex ratio (1.5:1), and reproduction: five cubs or subadults have been identified from three litters of two different females. Crossings were observed one to three times a year, in front of most camera traps, and several times a month in front of one of them. Overlap of adults territories was observed in front of several camera traps regardless of their sex. Significant snow leopard presence was detected in the buffer area and at Ulan, situated at the reserve border. To avoid poaching on this umbrella species and its preys, extending the more stringent protection measures of the core zone to both the Southern buffer area and land adjacent to Ulan is necessary.

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Nucleation complex behaviour is critical for cortical microtubule array homogeneity and patterning Plant cell walls are versatile materials that can adopt a wide range of mechanical properties through controlled deposition of cellulose fibrils. Wall integrity requires a sufficiently homogeneous fibril distribution to cope effectively with wall stresses. Additionally, specific conditions, such as the negative pressure in water transporting xylem vessels, may require more complex wall patterns, e.g., bands in protoxylem. The orientation and patterning of cellulose fibrils is guided by dynamic cortical microtubules. New microtubules are predominantly nucleated from parent microtubules causing positive feedback on local microtubule density with the potential to yield highly inhomogeneous patterns. Inhomogeneity indeed appears in all current cortical array simulations that include microtubule-based nucleation, suggesting that plant cells must possess an as-yet unknown balancing mechanism to prevent it. Here, in a combined simulation and experimental approach, we show that the naturally limited local recruitment of nucleation complexes to microtubules can counter the positive feedback, whereas local tubulin depletion cannot. We observe that nucleation complexes are preferentially inserted at microtubules. By incorporating our experimental findings in stochastic simulations, we find that the spatial behaviour of nucleation complexes delicately balances the positive feedback, such that differences in local microtubule dynamics - as in developing protoxylem - can quickly turn a homogeneous array into a patterned one. Our results provide insight into how the plant cytoskeleton is wired to meet diverse mechanical requirements and greatly increase the predictive power of computational cell biology studies.

Significance statementThe plant cortical microtubule array is an established model system for self-organisation, with a rich history of complementary experiments, computer simulations, and analytical theory. Understanding how array homogeneity is maintained given that new microtubules nucleate from existing microtubules has been a major hurdle for using mechanistic (simulation) models to predict future wall structures. We overcome this hurdle with detailed observations of the nucleation process from which we derive a more "natural" nucleation algorithm. With this algorithm, we enable various new lines of quantitative, mechanistic research into how cells dynamically control their cell wall properties. At a mechanistic level, moreover, this work relates to the theory on cluster coexistence in Turing-like continuum models and demonstrates its relevance for discrete stochastic entities.

sentences: Belemnite phylogeny and decline during the mid-Cretaceous Belemnites are common fossil coleoid cephalopods of the Mesozoic. They began to diversify in the Triassic-Early Jurassic and maintained this diversity until the early Early Cretaceous. During the mid-Cretaceous, they declined in diversity and distribution, being restricted to only the Boreal and Austral Realm since the Turonian. Here, I present the first cladistic analysis of belemnite phylogeny, spanning taxa representative of the whole diversity and stratigraphic range of the group. This analysis shows that the usually applied subdivision of all belemnites into "Belemnitina" and "Belemnopseina" is not supported. A newly identified clade, the Pseudoalveolata, is suggested here. Pseudoalveolate belemnites represent the last remaining belemnites after the Aptian. Oceanic anoxia and warming are likely the main cause of the mid-Cretaceous belemnite decline, resulting in the Aptian-Albian dominance of the warm-adapted pseudoalveolate genus Neohibolites. The rise of teleost fish diversity during the mid-Cretaceous is discussed and its relevance for belemnite evolution. Some teleosts (e.g., Enchodus) might have taken over the mesopredator niches left by belemnites during the mid-Cretaceous, being better adapted to warming seas. Belemnites were not able to recover their earlier widespread distribution and diversity and the last remaining, disjunctly distributed families, the northern Belemnitellidae and southern Dimitobelidae, became extinct at the K/Pg-boundary.

sentences: Demands of visual processing hierarchy shape laminar compartmentalization of attention modulation of luminance contrast in area V4 Contrast is a key feature of the visual scene that aids object recognition. Attention has been shown to selectively enhance the responses to low contrast stimuli in visual area V4, a critical hub that sends projections both up and down the visual hierarchy. Veridical encoding of contrast information is a key computation in early visual areas, while later stages encode higher level features that benefit from improved sensitivity to low contrast. How area V4 meets these distinct information processing demands in the attentive state is not known. We found that attentional modulation of contrast responses in area V4 is cortical layer and cell-class specific. Putative excitatory neurons in the superficial output layers that project to higher areas show enhanced boosting of low contrast information. On the other hand, putative excitatory neurons of deep output layers that project to early visual areas exhibit contrast-independent scaling. Computational modeling revealed that such layer-wise differences may result from variations in spatial integration extent of inhibitory neurons. These findings reveal that the nature of interactions between attention and contrast in V4 is highly compartmentalized, in alignment with the demands of the visual processing hierarchy.

sentences: 3D genomic capture of regulatory immuno-genetic profiles in COVID-19 patients for prognosis of severe COVID disease outcome Human infection with the SARS-CoV-2 virus leads to coronavirus disease (COVID-19). A striking characteristic of COVID-19 infection in humans is the highly variable host response and the diverse clinical outcomes, ranging from clinically asymptomatic to severe immune reactions leading to hospitalization and death. Here we used a 3D genomic approach to analyse blood samples at the time of COVID diagnosis, from a global cohort of 80 COVID-19 patients, with different degrees of clinical disease outcomes. Using 3D whole genome EpiSwitch(R) arrays to generate over 1 million data points per patient, we identified a distinct and measurable set of differences in genomic organization at immune-related loci that demonstrated prognostic power at baseline to stratify patients with mild forms of illness and those with severe forms that required hospitalization and intensive care unit (ICU) support. Further analysis revealed both well established and new COVID-related dysregulated pathways and loci, including innate and adaptive immunity; ACE2; olfactory, G{beta}{psi}, Ca2+ and nitric oxide (NO) signalling; prostaglandin E2 (PGE2), the acute inflammatory cytokine CCL3, and the T-cell derived chemotactic cytokine CCL5. We identified potential therapeutic agents for mitigation of severe disease outcome, with several already being tested independently, including mTOR inhibitors (rapamycin and tacrolimus) and general immunosuppressants (dexamethasone and hydrocortisone). Machine learning algorithms based on established EpiSwitch(R) methodology further identified a subset of 3D genomic changes that could be used as prognostic molecular biomarker leads for the development of a COVID-19 disease severity test.

sentences: Wireless endovascular nerve stimulation with a millimeter-sized magnetoelectric implant Implanted bioelectronic devices have the potential to treat disorders that are resistant to traditional pharmacological therapies; however, reaching many therapeutic nerve targets requires invasive surgeries and implantation of centimeter-sized devices. Here we show that it is possible to stimulate peripheral nerves from within blood vessels using a millimeter-sized wireless implant. By directing the stimulating leads through the blood vessels we can target specific nerves that are difficult to reach with traditional surgeries. Furthermore, we demonstrate this endovascular nerve stimulation (EVNS) with a millimeter sized wireless stimulator that can be delivered minimally invasively through a percutaneous catheter which would significantly lower the barrier to entry for neuromodulatory treatment approaches because of the reduced risk. This miniaturization is achieved by using magnetoelectric materials to efficiently deliver data and power through tissue to a digitally-programmable 0.8 mm2 CMOS system-on-a-chip. As a proof-of-principle we show wireless stimulation of peripheral nerve targets both directly and from within the blood vessels in rodent and porcine models. The wireless EVNS concept described here provides a path toward minimally invasive bioelectronics where mm-sized implants combined with endovascular stimulation enable access to a number of nerve targets without open surgery or implantation of battery-powered pulse generators.

sentences: Canonical Wnt Signaling Induces Focal Adhesion and Integrin Beta-1 Endocytosis During canonical Wnt signaling, the Lrp6 and Frizzled co-receptors bind to the Wnt growth factor and the complex is endocytosed and sequestered together with Glycogen Synthase Kinase 3 (GSK3), Dishevelled (Dvl), and Axin inside the intraluminal vesicles of late endosomes, known as multivesicular bodies (MVBs). Here we present experiments showing that Wnt causes the endocytosis of focal adhesion (FA) proteins and depletion of Integrin {beta} 1 (ITG{beta}1) from the cell surface. FAs and integrins link the cytoskeleton to the extracellular matrix. Wnt-induced endocytosis caused ITG{beta}1 depletion from the plasma membrane and was accompanied by striking changes in the actin cytoskeleton. In situ protease protection assays in cultured cells showed that ITG{beta}1 was sequestered within membrane-bounded organelles that corresponded to Wnt-induced MVBs containing GSK3 and FA-associated proteins. An in vivo model using Xenopus embryos dorsalized by Wnt8 mRNA showed that ITG{beta}1 depletion decreased Wnt signaling. The finding of a crosstalk between two mayor signaling pathways, canonical Wnt and focal adhesions, should be relevant to human cancer and cell biology.

sentences: Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and non-functional Misfolded protein conformations with decreased functionality can bypass the proteostasis machinery and remain soluble in vivo. This is an unexpected phenomenon as several cellular quality control mechanisms have evolved to rid cells of misfolded proteins. Three questions, then, are: how is it structurally possible for long-lived, soluble, misfolded proteins to bypass the proteostasis machinery and processes? How widespread are these soluble, misfolded states across the proteome? And how long do they persist for? Here, we address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We predict that half of all proteins exhibit subpopulations of misfolded conformations that are likely to bypass molecular chaperones, avoid aggregation, and not be rapidly degraded. These misfolded states may persist for months or longer for some proteins. Structurally characterizing these misfolded states, we observe they have a large amount of native structure, but also contain localized misfolded regions from non-native changes in entanglement, in which a protein segment threads through a loop formed by another portion of the protein that is not found in the native state. The surface properties of these misfolded states are native like, suggesting they may bypass the proteostasis machinery and its regulatory processes to remain soluble, while their entanglements make these states long-lived kinetic traps, as disentanglement requires unfolding of already folded portions of the protein. In terms of function, we predict that one-third of proteins have subpopulations that misfold into less-functional states that have structurally perturbed functional sites yet remain soluble. Data from limited-proteolysis mass spectrometry experiments, which interrogate the misfolded conformations populated by proteins upon unfolding and refolding, are consistent with the structural changes seen in the entangled states of glycerol-3-phosphate dehydrogenase upon misfolding. These results provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass cellular quality controls, and indicate, unexpectedly, this may be a wide-spread phenomenon in proteomes. Such entanglements are observed in many native structures, suggesting the non-native entanglements we observe are plausible. More broadly, these near-native entangled structures suggest a hypothesis for how synonymous mutations can modulate downstream protein structure and function, with these mutations partitioning nascent proteins between these kinetically trapped states.

sentences: Network-based analysis of allele frequency distribution among multiple populations identifies adaptive genomic structural variants Structural variants have a considerable impact on human genomic diversity. However, their evolutionary history remains mostly unexplored. Here, we developed a new method to identify potentially adaptive structural variants based on a network-based analysis that incorporates genotype frequency data from 26 populations simultaneously. Using this method, we analyzed 57,629 structural variants and identified 577 structural variants that show high population distribution. We further showed that 39 and 20 of these putatively adaptive structural variants overlap with coding sequences or are significantly associated with GWAS traits, respectively. Closer inspection of the haplotypic variation associated with these putatively adaptive and functional structural variants reveals deviations from neutral expectations due to (i) population differentiation of rapidly evolving multi-allelic variants, (ii) incomplete sweeps, and (iii) recent population-specific negative selection. Overall, our study provides new methodological insights, documents hundreds of putatively adaptive variants, and introduces evolutionary models that may better explain the complex evolution of structural variants.

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On the relationship between Pathogenic Potential and Infective Inoculum Pathogenic Potential (PP) is a mathematical description of an individual microbe, virus, or parasites ability to cause disease in a host, given the variables of inoculum, symptomology, mortality, and in some instances, median survival time of the host. We investigated the relationship between pathogenic potential (PP) and infective inoculum (I) using two pathogenic fungi in the wax moth Galleria mellonella with mortality as the relevant outcome. Our analysis for C. neoformans infection revealed negative exponential relationship between PP and I. Plotting the log(I) versus the Fraction symptomatic (Fs) over median host survival time (T) revealed a linear relationship, with a slope that varied between the different fungi studied and a y-intercept corresponding to the inoculum that produced no symptoms. The I vs Fs/T slope provided a measure of the pathogenicity of each microbial species, which we call the pathogenicity constant or kPath. The kPath provides a new parameter to quantitatively compare the relative virulence and pathogenicity of microbial species for a given host. In addition, we investigated the PP and Fs/T from values found in preexisting literature. Overall, the relationship between Fs/T and PP versus inoculum varied among microbial species and extrapolation to zero symptoms allowed the calculation of the lowest pathogenic inoculum (LPI) of a microbe. Microbes tended to fall into two groups: those with positive linear relationships between PP and Fs/T vs I, and those that had a negative exponential PP vs I relationship with a positive logarithmic Fs/T vs I relationship. The microbes with linear relationships tended to be bacteria, whereas the exponential-based relationships tended to be fungi or higher order eukaryotes. Differences in the type and sign of the PP vs I and Fs/T vs I relationships for pathogenic microbes suggest fundamental differences in host-microbe interactions leading to disease.

Author SummaryThe ability of a microbe, virus, or parasite to cause disease is dependent on multiple factors, virulence factors. host immune defenses, the infective inoculum, and the type of immune response. For many microbes their capacity for causing disease is highly dependent on the inoculum. The mathematical formula for Pathogenic Potential (PP) is a way to compare the ability of an organism to have a pathogenic effect, as measured by Fraction Symptomatic (Fs), mortality (M), and inoculum (I), and can include the median survival time of the host (T). Increasing inoculum of the fungus Cryptococcus neoformans for a moth host resulted in exponentially smaller pathogenic potential, and the Fs/T versus inoculum plot showed a logarithmic relationship. Together, these relationships show diminishing returns with increasing cryptococcal inoculum, in which each individual fungus plays a smaller role in pathogenicity. Literature data shows that other microbes, mostly bacteria, had linear Fs/T versus inoculum relationships, which indicate that each bacterium contributed an equal amount to pathogenicity. These differences in relationships can point to differences in host-microbe interactions and suggest new ways in which the organism causes disease.

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Somatic mutations in CTCF zinc fingers produce cellular phenotypes explained by structure-function relationships BackgroundHuman cancers commonly contain mutations in transcription factors that lead to aberrant DNA binding or altered effector function at target sites. One such factor significantly mutated in cancer is the evolutionarily-conserved CCCTC-binding factor (CTCF), which has fundamental roles in maintaining chromatin architecture and transcriptional regulation. Numerous cancer genome sequencing and functional studies have revealed CTCFs role as a haploinsufficient tumour suppressor gene. However, to date, structure-function relationships of somatic CTCF mutations have not been examined.

MethodsWe collated somatic CTCF mutations from cancer genome portals and published studies to determine their nature, frequency, distribution and potential functional impact. We undertook an in-depth examination of 5 CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. We performed functional analyses including cell growth, colony-formation, chromatin immunoprecipitation and transcriptional reporter assays. Based on their homology, each ZF mutation was then modelled on CTCFs ZF domain crystal structure and its structural impact analysed using molecular dynamics simulations.

ResultsWe observed an enrichment of somatic missense mutations occurring in the ZF region of CTCF, compared to the unstructured N- and C-termini. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. In silico mutagenesis revealed that L309P had the highest mutation energy and thus most severe impact on protein stability. Molecular docking and molecular dynamics simulations confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding (R339Q, R339W, R377H). Remarkably, R339Q and G420D were stabilised by the formation of new primary DNA bonds. All mutations exhibited some loss or gain of bonds at neighbouring residues, often in adjacent zinc fingers.

ConclusionsOur data confirm the significant negative impact haploinsufficient CTCF ZF mutations have on its tumour suppressor function. A spectrum of loss-, change- and gain-of-function impacts in CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. We have established that diverse cellular phenotypes in CTCF are explained by examining structure-function relationships.

sentences: Seed Germination, Post-fire Plant Growth and Conservation of the Rare Endemic and Endangered Chamaecrista glandulosa var. mirabilis (Fabaceae) Conservation has been challenged by biodiversity loss drivers. Also, fire disturbance can temporarily change ecosystems. Fire effects in soil nutrients and pH, plant abundance, reproduction, seed weight, seed germination, and plant growth were assessed. Four seed starting systems were used for germination and growth under laboratory conditions. We found significant differences in the means of iron, manganese, nickel, soil pH, and plant height, and a significant positive linear relationship between seed weight and plant height. Results indicate that fires increase soil pH and cause changes in micronutrients that can increase plant growth. Large size plants are produced from high weight seeds. Finally, ex situ conservation and species reintroduction were feasible conservation strategies that should be integrated with in situ conservation.

sentences: Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species sweeps and geographical clustering The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on integrative and conjugative elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population sweeps of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 177 MGE that sweep through multiple Bacteroidales species within individual gut microbiomes. We further show that only eight MGE demonstrate multi-species population sweeps in as many human gut microbiomes as the GA1 and GA2 ICE. These data underscore the ubiquity and rapid dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.

sentences: Collagen remodeling dictates pancreatic cancer bioenergetics and outcome through DDR1 activation or degradation Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic, aggressive cancer that frequently progresses by liver metastasis1. Cancer-associated fibroblasts (CAF), extracellular matrix (ECM), and type I collagen (Col I) support2-5 or restrain PDAC progression and may impede blood supply and nutrient availability6-8. The dichotomous role of the stroma in PDAC, and the mechanisms through which it influences patient survival and enables desmoplastic cancers escape nutrient limitation remain poorly understood. Here we show that matrix metalloprotease (MMP)-cleaved or intact Col I (cCol I and iCol I, respectively) exert opposing effects on PDAC bioenergetics, macropinocytosis (MP), tumor growth and liver metastasis. While cCol I activates DDR1 (discoidin domain receptor-1)-NF-{kappa}B-p62-NRF2 signaling to promote PDAC growth, iCol I triggers DDR1 degradation and restrains PDAC growth. Patients whose tumors are enriched in iCol I and low in DDR1 and NRF2 have improved median survival compared to those enriched in cCol I, DDR1 and NRF2. Inhibition of DDR1-stimulated NF-{kappa}B or mitochondrial biogenesis blocked tumorigenesis in wildtype mice but not in mice expressing MMP-resistant Col I. In summary, the diverse effects of tumor stroma on PDAC growth, metastasis, and patient survival are mediated through the Col I-DDR1-NF-{kappa}B-NRF2-mitochondrial biogenesis pathway, presenting multiple new opportunities for PDAC therapy.

sentences: Spotted lanternfly nymphs use multiple righting behaviors during landing Unlike large animals, insects and other very small animals are so unsusceptible to impact-related injuries that they can use falling for dispersal and predator evasion. Reorienting to land upright can mitigate lost access to resources and predation risk. Such behaviors are critical for the spotted lanternfly (SLF) (Lycorma delicatula), an invasive, destructive insect pest spreading rapidly in the US. High-speed video of SLF nymphs released under different conditions showed that these insects self-right using both active midair righting motions previously reported for other insects, and novel post-impact mechanisms that take advantage of their ability to experience near-total energy loss on impact. Unlike during terrestrial self-righting, in which an animal initially at rest on its back uses appendage motions to flip over, SLF nymphs impacted the surface at varying angles and then self-righted during the rebound using coordinated body rotations, foot-substrate adhesion, and active leg motions. These previously-unreported strategies were found to promote disproportionately upright, secure landings on both hard, flat surfaces and tilted, compliant host plant leaves. Our results highlight the importance of examining biomechanical phenomena in ecologically-relevant contexts, and show that, for small animals, the post-impact bounce period can be critical for achieving an upright landing.

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Serological and Molecular Investigation of Foot and Mouth Disease Virus and other animal pathogens at the Interface of Akagera National Park and Surrounding Cattle Farms between 2017 and 2020 BackgroundFoot-and-Mouth Disease Virus (FMDV) is a positive-sense RNA virus of the family of the picornaviridae and responsible for the disease with the highest economic impact, the Foot-and-Mouth Disease (FMD). FMD is endemic in Rwanda but there are gaps in knowing the seroprevalence and molecular epidemiology. This study reports the FMD seroprevalence and molecular characterization of FMDV in Eastern Rwanda. Surveillance in FMDV wild reservoirs, the African buffaloes, was also carried out revealing the presence of other pathogens and commensals.

ResultsThe overall seroprevalence of FMD in the study area is at 9.36% in cattle and 2.65% in goats. We detected FMDV using molecular diagnostic tools such as RT-PCR and RT-LAMP and the phylogenetic analysis of the obtained sequences revealed the presence of serotype SAT 2, lineage II. Sequencing of oropharyngeal fluids collected from African buffaloes revealed the presence of several pathogens and commensals but no FMDV was detected in buffaloes.

The plethora of pathogens identified from the buffalo gut gives an idea of the health challenges faced by cattle keepers in Eastern Rwanda due to possible cross infectivity on wildlife-domestic animals interface regions.

ConclusionsWe recommend further studies to focus on sampling more African buffaloes since the number sampled was statistically insignificant to conclusively exclude the presence or absence of FMDV in Eastern Rwanda buffaloes. The use of RT-PCR alongside RT-LAMP demonstrates that the latter can be adopted in endemic areas such as Rwanda to fill in the gaps in terms of molecular diagnostics. The identification of lineage II of SAT 2 in Rwanda for the first time shows that the pools as previously established are not static over time.

sentences: Cytosolic peptide accumulation activates the NLRP1 and CARD8 inflammasomes NLRP1 and CARD8 are related sensors that form inflammasomes, but the danger signals that they detect are not fully established. These proteins undergo autoproteolysis, generating repressive N-terminal (NT) and inflammatory C-terminal (CT) fragments. The proteasome- mediated degradation of the NT releases the CT from autoinhibition, but the CT is then sequestered in a complex with the full-length sensor and DPP9. Here, we show that cytosolic peptide accumulation activates these inflammasomes. We found that a diverse array of peptides accelerates NT degradation, and those with N-terminal XP sequences also destabilize the ternary complexes. Peptides interfere with many biological processes, including protein folding. We show that unrelated agents that disrupt protein folding also induce NT degradation, but do not cause inflammasome activation because DPP9 sequesters the CT fragments in the absence of XP peptides. Overall, these results indicate that NLRP1 and CARD8 detect protein misfolding that is associated with peptide accumulation.

sentences: Live biotherapeutic throat spray for respiratory virus inhibition and interferon pathway induction Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the selection and formulation of a broad-acting throat spray with live lactobacilli which induce interferon regulatory pathways and are able to inhibit respiratory viruses. Rational selection of Lactobacillaceae strains was based on safety, applicability, and potential antiviral and immunostimulatory efficacy in the URT. Three strains, Lacticaseibacillus casei AMBR2, Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form.

sentences: A novel mechanism of regulation of the transcription factor GLI3 by toll-like receptor signaling The transcription factor GLI3 is a member of the GLI family and has been shown to be regulated by canonical hedgehog (HH) signaling through smoothened (SMO). Little is known about SMO-independent regulation of GLI3. Here, we identify TLR signaling as a novel pathway regulating GLI3 expression. We show that GLI3 expression is induced by LPS/TLR4 in human monocyte cell lines and peripheral blood CD14+ cells. Further analysis identified TRIF, but not MyD88, signaling as the adapter used by TLR4 to regulate GLI3. Using pharmacological and genetic tools, we identified IRF3 as the transcription factor regulating GLI3 downstream of TRIF. Furthermore, using additional TLR ligands that signal exclusively through TRIF such as the TLR4 ligand, MPLA and the TLR3 ligand, Poly(I:C), we confirm the role of TRIF-IRF3 in the regulation of GLI3. We found that IRF3 directly binds to the GLI3 promoter region and this binding was increased upon stimulation of TRIF-IRF3 with Poly(I:C). Furthermore, using IRF3-/- MEFs, we found that Poly(I:C) stimulation no longer induced GLI3 expression. Finally, using macrophages from mice lacking Gli3 expression in myeloid cells (M-Gli3-/-), we found that in the absence of Gli3, LPS stimulated macrophages secrete less CCL2 and TNF- compared with macrophages from wild-type (WT) mice. Taken together, these results identify a novel TLR-TRIF-IRF3 pathway that regulates the expression of GLI3 that regulates inflammatory cytokines and expands our understanding of the non-canonical signaling pathways involved in the regulation of GLI transcription factors.

sentences: Interpersonal synchronization of movement intermittency Most animal species group together and coordinate their behavior in quite sophisticated manners for mating, hunting or defense purposes. In humans, coordination at a macroscopic level (the pacing of movements) is evident both in daily life (e.g., walking) and skilled (e.g., music and dance) behaviors. By examining the fine structure of movement, we here show that interpersonal coordination is established also at a microscopic - sub-movement - level. Natural movements appear as marked by recurrent (2-3 Hz) speed breaks, i.e., submovements, that are traditionally considered the result of intermittency in (visuo)motor control. In a series of interpersonal motor coordination tasks, we demonstrate that submovements are not independent between interacting partners but produced in a tight temporal relation that reflects the directionality in the partners informational coupling. These findings unveil a potential core mechanism for behavioral coordination that is based on between-persons synchronization of the intrinsic dynamics of action-perception cycles.

sentences: Structural Evolution of the Ancient Enzyme, Dissimilatory Sulfite Reductase Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme accompanied diversification of sulfate/sulfite reducing organisms (SRO) into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving SRO lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.

sentences: SMC Complexes Are Guarded by the SUMO Protease Ulp2 Against SUMO-Chain-Mediated Turnover Structural maintenance of chromosomes (SMC) complexes, cohesin, condensin and Smc5/6, are essential for viability and participate in multiple processes, including sister chromatid cohesion, chromosome condensation, and DNA repair. Here we show that SUMO chains target all three SMC complexes and are antagonized by the SUMO protease Ulp2 to prevent their turnover. We uncover that the essential role of the cohesin-associated subunit Pds5 is to counteract SUMO chains jointly with Ulp2. Importantly, fusion of Ulp2 to kleisin Scc1 supports viability of PDS5 null cells and protects cohesin from proteasomal degradation mediated by the SUMO-targeted ubiquitin ligase Slx5/Slx8. The lethality of PDS5 deleted cells can also be bypassed by simultaneous loss of the PCNA unloader, Elg1, and the cohesin releaser, Wpl1, but only when Ulp2 is functional. Condensin and Smc5/6 complex are similarly guarded by Ulp2 against unscheduled SUMO-chain assembly, which we propose to time the availability of SMC complexes on chromatin.

sentences: Heterologous synapsis in C. elegans is regulated by meiotic double-strand breaks and crossovers Alignment of the parental chromosomes during meiotic prophase is key to the formation of genetic exchanges, or crossovers, and consequently to the successful production of gametes. In almost all studied organisms, alignment involves synapsis: the assembly of a conserved inter-chromosomal interface called the synaptonemal complex (SC). While the SC usually synapses homologous sequences, it can assemble between heterologous sequences. However, little is known about the regulation of heterologous synapsis. Here we study the dynamics of heterologous synapsis in the nematode C. elegans. We characterize two experimental scenarios: SC assembly onto a folded-back chromosome that cannot pair with its homologous partner; and synapsis of pseudo-homologs, a fusion chromosome partnering with an unfused chromosome half its size. We observed elevated levels of heterologous synapsis when the number of meiotic double-strand breaks or crossovers were reduced, indicating that the promiscuity of synapsis is regulated by break formation or repair. By manipulating the levels of breaks and crossovers, we infer both chromosome-specific and nucleus-wide regulation on heterologous synapsis. Finally, we identify differences between the two conditions, suggesting that attachment to the nuclear envelope plays a role in regulating heterologous synapsis.

sentences: Perivascular SPP1 Mediates Microglial Engulfment of Synapses in Alzheimers Disease Models Microglia are phagocytes of the brain parenchyma, where they interact with neurons to engulf synapses in a context-dependent manner. Genetic studies in Alzheimers disease (AD) highlight dysfunctional phagocytic signaling in myeloid cells as disease-associated pathway. In AD models, there is a region-specific reactivation of microglia-synapse phagocytosis involving complement; however, what drives microglia-synapse engulfment remains unknown. Here, we show that SPP1 (Osteopontin), a glycoprotein associated with inflammation, is regionally upregulated and modulates microglial synaptic engulfment in AD mouse models. Ultrastructural examination revealed SPP1 expression predominantly by perivascular macrophages, a subtype of border-associated macrophages, in the hippocampus of mice and patient tissues. Cell-cell interaction networks of single-cell transcriptomics data suggested that perivascular SPP1 drives microglial functional states in the hippocampal microenvironment of AD mice. Absence of Spp1 expression resulted in failure of microglia to mediate synaptic phagocytosis. This study suggests a critical role for perivascular SPP1 in neuroimmune crosstalk in AD-relevant context.

sentences: Evidence-based calibration of computational tools for missense variant pathogenicity classification and ClinGen recommendations for clinical use of PP3/BP4 criteria Recommendations from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) for interpreting sequence variants specify the use of computational predictors as Supporting level of evidence for pathogenicity or benignity using criteria PP3 and BP4, respectively. However, score intervals defined by tool developers, and ACMG/AMP recommendations that require the consensus of multiple predictors, lack quantitative support. Previously, we described a probabilistic framework that quantified the strengths of evidence (Supporting, Moderate, Strong, Very Strong) within ACMG/AMP recommendations. We have extended this framework to computational predictors and introduce a new standard that converts a tools scores to PP3 and BP4 evidence strengths. Our approach is based on estimating the local positive predictive value and can calibrate any computational tool or other continuous-scale evidence on any variant type. We estimate thresholds (score intervals) corresponding to each strength of evidence for pathogenicity and benignity for thirteen missense variant interpretation tools, using carefully assembled independent data sets. Most tools achieved Supporting evidence level for both pathogenic and benign classification using newly established thresholds. Multiple tools reached score thresholds justifying Moderate and several reached Strong evidence levels. One tool reached Very Strong evidence level for benign classification on some variants. Based on these findings, we provide recommendations for evidence-based revisions of the PP3 and BP4 ACMG/AMP criteria using individual tools and future assessment of computational methods for clinical interpretation.

sentences: Periodic ethanol supply as a path towards unlimited lifespan of C.elegans dauer larvae The dauer larva is a specialized stage of development optimized for survival under harsh conditions that has been used as a model for stress resistance, metabolic adaptations, and longevity. Recent findings suggest that the dauer larva of C.elegans may utilize external ethanol as an energy source to extend their lifespan. It was shown that while ethanol may serve as an effectively infinite source of energy, some toxic compounds accumulating as byproducts of its metabolism may lead to the damage of mitochondria and thus limit the lifespan of larvae. A minimal mathematical model was proposed to explain the connection between the lifespan of dauer larva and its ethanol metabolism. To explore theoretically if it is possible to extend even further the lifespan of dauer larvae, we incorporated two natural mechanisms describing the recovery of damaged mitochondria and elimination of toxic compounds, which were previously omitted in the model. Numerical simulations of the revised model suggest that while the ethanol concentration is constant, the lifespan still stays limited. However, if ethanol is supplied periodically, with a suitable frequency and amplitude, the dauer could survive as long as we observe the system. Analytical methods further help to explain how the feeding frequency and amplitude affect the lifespan extension. Based on comparison of the model with experimental data for fixed ethanol concentration, we propose the range of feeding protocols that could lead to even longer dauer survival and can be tested experimentally.

sentences: Probing the toxic interactions between the reactive dye Drimaren Red and Human Serum Albumin Azo dyes like Drimaren Red CL-5B (DR, CI Reactive Red 241) represent a class of compounds extensively used in the textile industry and are extremely dangerous to the environment and human health. Therefore, understanding the binding characteristics between such substances and biological macromolecules is essential from a toxic-kinetic perspective. The molecular interaction between DR and Human Serum Albumin (HSA) was investigated through spectroscopic techniques and molecular docking approaches. The results indicate that DR quenches HSA fluorescence following a static mechanism (corroborated by UV-Vis studies) with a moderate interaction (Ka~105 M-1), guided by electrostatic interactions ({Delta}S{degrees}> 0 and {Delta}H{degrees}< 0). DR is 5.52 nm distant from fluorophore residue Trp-214 (according to FRET investigations), and the interaction is mainly related to Tyr residues (as revealed by synchronous fluorescence). The Ellman assay identified a decrease in the content of HSA free thiol. The results of the RLS demonstrate that there are HSA alterations, suggesting damage to the confirmation of the protein. Molecular docking suggests the binding site of DR was located in subdomain IIB HSA, corroborating the experimental properties. Finally, the results suggest a high potential for DR toxicity triggered by contact with key proteins, which affects the biomolecule functionalities.

sentences:
An Oscillatory Mechanism for Multi-level Storage in Short-term Memory Oscillatory activity is commonly observed during the maintenance of information in short-term memory, but its role remains unclear. Non-oscillatory models of short-term memory storage are able to encode stimulus identity through their spatial patterns of activity, but are typically limited to either an all-or-none representation of stimulus amplitude or exhibit a biologically implausible exact-tuning condition. Here, we demonstrate a simple phase-locking mechanism by which oscillatory input enables a circuit to generate persistent or sequential activity patterns that encode information not only in their location but also in their discretely graded amplitudes.

SignificanceA core observation in many memory systems and tasks is the presence of oscillations during memory maintenance. Here, we demonstrate a mechanism for the accumulation and storage of information in short-term memory in which oscillatory activity enables a solution to long-standing challenges in modeling the persistent neural activity underlying working memory. These challenges include the ability to encode information with low firing rates, multi-level storage of stimulus amplitude without extreme fine tuning, and multi-level storage of information in sequential activity. Altogether, this work proposes a new class of models for the storage of information in working memory, a new potential role for brain oscillations, and a novel dynamical mechanism for multi-stability.

sentences: Basic helix-loop-helix pioneer factors interact with the histone octamer to invade nucleosomes and generate nucleosome depleted regions Nucleosomes drastically limit transcription factor (TF) occupancy, while pioneer transcription factors (PFs) somehow circumvent this nucleosome barrier. In this study, we compare nucleosome binding of two conserved S. cerevisiae basic helix-loop-helix (bHLH) TFs, Cbf1 and Pho4. A Cryo-EM structure of Cbf1 in complex with the nucleosome reveals that the Cbf1 HLH region can electrostatically interact with exposed histone residues within a partially unwrapped nucleosome. Single molecule fluorescence studies show that the Cbf1 but not the Pho4 HLH region facilitates efficient nucleosome invasion by slowing its dissociation rate relative to DNA through interactions with histones. In vivo studies show that this enhanced binding provided by the Cbf1 HLH region enables nucleosome invasion and ensuing repositioning. These structural, single molecule, and in vivo studies reveal the mechanistic basis of dissociation rate compensation by pioneer factors and how this translates to facilitating chromatin opening inside cells.

sentences: Evidence for the role of music on the growth and signal response in duckweed Sound vibration, an external mechanical force, has been proved to modulate plant growth and development like rain, wind, and vibration. However, the role of music on plants, especially on signal response, has been usually neglected in research. Herein, we investigated the growth state, gene expression, and signal response in duckweed treated with soft music. The protein content in duckweed after music treatment for 7 days was about 1.6 times that in duckweed without music treatment. Additionally, Fv/Fm in duckweed treated with music was 0.78, which was significantly higher in comparison with the control group (P < 0.01). Interestingly, music promoted the Glu and Ca signaling response. To further explore the global molecular mechanism, we performed transcriptome analysis. A total of 1296 DEGs were found for all these investigated genes in duckweed treated with music compared to the control group. Among these, up-regulation of the expression of metabolism-related genes related to glycolysis, cell wall biosynthesis, oxidative phosphorylation, and pentose phosphate pathways were found. Overall, these results provided a molecular basis to music-triggered signal response, transcriptomic, and growth changes in duckweed, which also highlighted the potential of music as an environmentally friendly stimulus to promote improved protein production in duckweed.

sentences:
Not Quite All in Our Head: Intervention is a Better Predictor of tDCS Mind-Wandering Effects than Subjective Beliefs About Experimental Results BackgroundEstablishing adequate blinding for non-invasive brain stimulation research is a topic of extensive debate, especially regarding the efficacy of sham control methods for transcranial direct current stimulation (tDCS) studies. Fassi and Cohen Kadosh [1] assessed the influence of subjective participant belief regarding stimulation type (active or sham) and dosage on behaviour using data from Filmer et al. [2] who applied five stimulation protocols (anodal 1.0mA, cathodal 1.0mA, cathodal 1.5mA, cathodal 2.0mA and sham) to assess the neural substrates of mind wandering. Fassi and Cohen Kadosh [1] concluded that subjective belief drove the pattern of results observed by Filmer et al. [2].

ObjectiveFassi and Cohen Kadosh [1] did not assess the key contrast between conditions in Filmer et al. (2019) - 2mA vs sham - rather they examined all stimulation conditions. Here, we consider the relationship between objective and subjective intervention in this key contrast.

MethodsWe replicated the analysis and findings of both Filmer et al. [2] and Fassi and Cohen Kadosh [1] before assessing 2mA vs. sham via Bayesian ANOVA on subjective belief regarding stimulation type and dosage.

ResultsOur results support objective intervention as the strongest predictor of stimulation effects on mind-wandering when 2mA vs sham was examined, over and above that of subjective intervention.

ConclusionsThe conclusions made by Filmer et al. [2] are confirmed. However, it is important to control for and understand the possible effects of subjective beliefs in sham-controlled studies. Best practice to prevent these issues remains the inclusion of active control conditions.

sentences:
Interannual climate variability data improves niche estimates in species distribution models AimClimate is an essential element of species niche estimates in many current ecological applications such as species distribution models (SDMs). Climate predictors are often used in the form of long-term mean values. Yet, climate can also be described as spatial or temporal variability for variables like temperature or precipitation. Such variability, spatial or temporal, offers additional insights into niche properties. Here, we test to what degree spatial variability and long-term temporal variability in temperature and precipitation improve SDM predictions globally.

LocationGlobal.

Time period1979-2013

Major taxa studiesMammal, Amphibians, Reptiles

MethodsWe use three different SDM algorithms, and a set of 833 amphibian, 779 reptile, and 2211 mammal species to quantify the effect of spatial and temporal climate variability in SDMs. All SDMs were cross-validated and accessed for their performance using the Area under the Curve (AUC) and the True Skill Statistic (TSS).

ResultsMean performance of SDMs with climatic means as predictors was TSS=0.71 and AUC=0.90. The inclusion of spatial variability offers a significant gain in SDM performance (mean TSS=0.74, mean AUC=0.92), as does the inclusion of temporal variability (mean TSS=0.80, mean AUC=0.94). Including both spatial and temporal variability in SDMs shows similarly high TSS and AUC scores.

Main conclusionsAccounting for temporal rather than spatial variability in climate improved the SDM prediction especially in exotherm groups such as amphibians and reptiles, while for endotermic mammals no such improvement was observed. These results indicate that more detailed information about temporal climate variability offers a highly promising avenue for improving niche estimates and calls for a new set of standard bioclimatic predictors in SDM research.

sentences: In vitro evolution of uracil glycosylase towards DnaKJ and GroEL binding evolves different misfolded states Natural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time-scale and is driven by the given bait. Here, we used directed in vitro evolution of a random library of Uracil glycosylase (eUNG) displayed on yeast surface to select for binding to chaperones GroEL, DnaK+DnaJ+ATP (DnaKJ) or E.coli cell extract (CE). Using binding to the eUNG inhibitor Ugi as probe for native foldedess, the CE selected population was further divided to Ugi binders (+U) (native) or non-binders (-U). We found that GroEL, DnaKJ and CE-U select and enrich for mutations causing eUNG to misfold, with the three being enriched in mutations in buried and conserved positions, with a tendency to increase positive charge. Still, each selection, as well as CE+U and natural evolution of eUNG has its own trajectory. While GroEL and CE-U selected for mutants highly sensitive to protease cleavage, DnaKJ selected for partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. CE+U selected for more neutral mutations than natural evolution. In a more general context, our results show that GroEL has a higher tendency to purge promiscuous misfolded protein mutants from the system, while DnaKJ binds mutants misfolding-prone species that are, upon chaperone release, more likely to natively refold. CE-U shares some of the properties of GroEL and DnaKJ selected populations, while harboring also unique properties, explained by the existence of additional chaperones in CE, such as Tig, HtpG and ClpB.

sentences:
Microtubule-based Transport is Essential to Distribute RNA and Nascent Protein in Skeletal Muscle While the importance of RNA localization in highly differentiated cells is well appreciated, basic principles of RNA localization in skeletal muscle remain poorly characterized. Here, we develop a method to detect single RNA molecules and quantify localization patterns in skeletal myofibers, and we uncover a critical and general role for directed transport of RNPs in muscle. We find that RNAs are localized and translated along cytoskeletal filaments, and we identify the Z-disk as a biological hub for RNA localization and protein synthesis. We show that muscle development triggers complete reliance on the lattice-like microtubule network to transport RNAs and that disruption of microtubules leads to striking accumulation of RNPs and nascent protein around myonuclei. Our observations suggest that active transport may be globally required to distribute RNAs in highly differentiated cells and reveal fundamental mechanisms relevant to myopathies caused by perturbations to RNPs, microtubules, and the nuclear envelope.

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sentences: DLPacker: Deep Learning for Prediction of Amino Acid Side Chain Conformations in Proteins AO_SCPLOWBSTRACTC_SCPLOWPrediction of side chain conformations of amino acids in proteins (also termed  packing) is an important and challenging part of protein structure prediction with many interesting applications in protein design. A variety of methods for packing have been developed but more accurate ones are still needed. Machine learning (ML) methods have recently become a powerful tool for solving various problems in diverse areas of science, including structural biology. In this work we evaluate the potential of Deep Neural Networks (DNNs) for prediction of amino acid side chain conformations. We formulate the problem as image-to-image transformation and train a U-net style DNN to solve the problem. We show that our method outperforms other physics-based methods by a significant margin: reconstruction RMSDs for most amino acids are about 20% smaller compared to SCWRL4 and Rosetta Packer with RMSDs for bulky hydrophobic amino acids Phe, Tyr and Trp being up to 50% smaller.

sentences: Noise in a metabolic pathway leads to persister formation in Mycobacterium tuberculosis Tuberculosis is difficult to treat due to dormant cells in hypoxic granulomas, and stochastically-formed persisters tolerant of antibiotics. Bactericidal antibiotics kill by corrupting their energy-dependent targets. We reasoned that noise in the expression of an energy-generating component will produce rare persister cells. In sorted low ATP M. tuberculosis grown on acetate there is considerable cell-to-cell variation in the level of mRNA coding for AckA, the acetate kinase. Quenching the noise by overexpressing ackA sharply decreases persisters, showing that it acts as the main persister gene under these conditions. This demonstrates that a low energy mechanism is responsible for the formation of M. tuberculosis persisters and suggests that the mechanism of their antibiotic tolerance is similar to that of dormant cells in a granuloma. Entrance into a low energy state driven by stochastic variation in expression of energy-producing enzymes is likely a general mechanism by which bacteria produce persisters.

sentences: 1H NMR chemical exchange techniques reveal local and global effects of oxidized cytosine derivatives 5-carboxycytosine (5caC) is a rare epigenetic modification found in nucleic acids of all domains of life. Despite its sparse genomic abundance, 5caC is presumed to play essential regulatory roles in transcription, maintenance and baseexcision processes in DNA. In this work, we utilize nuclear magnetic resonance (NMR) spectroscopy to address the effects of 5caC incorporation into canonical DNA strands at multiple pH and temperature conditions. Our results demonstrate that 5caC has a pH-dependent global destabilizing and a base-pair mobility enhancing local impact on dsDNA, albeit without any detectable influence on the ground-state B-DNA structure. Measurement of hybridization thermodynamics and kinetics of 5caC-bearing DNA duplexes highlighted how acidic environment (pH 5.8 and 4.7) destabilizes the double-stranded structure by ~10-20 kJ mol-1 at 37 {degrees}C when compared to the same sample at neutral pH. Protonation of 5caC results in a lower activation energy for the dissociation process and a higher barrier for annealing. Studies on conformational exchange on the s time scale regime revealed a sharply localized base-pair motion involving exclusively the modified site and its immediate surroundings. By direct comparison with canonical and 5-formylcytosine (5fC)-edited strands, we were able to address the impact of the two most oxidized naturally occurring cytosine derivatives in the genome. These insights on 5caCs subtle sensitivity to acidic pH contribute to the long standing questions of its capacity as a substrate in base excision repair processes and its purpose as an independent, stable epigenetic mark.

sentences: Drivers of adaptive capacity in wild populations: implications for genetic interventions The unprecedented rate of environmental change in the Anthropocene poses evolutionary challenges for wild populations globally. Active human interventions are being increasingly considered to accelerate natural adaptive processes. Evolutionary models can evaluate how species may fare under future climate, elucidate which evolutionary processes are critical to rapid adaptation, and how active interventions may influence fitness trajectories of organisms. Here we use polygenic metapopulation adaptation models to quantify the relative importance (effect sizes) of different eco-evolutionary parameters on the rates of adaptation in wild populations i) without active interventions, and ii) under a subset of active interventions. We demonstrate that genetic diversity (heterozygosity, He), population connectivity and the effect size of additive genetic variance are the primary drivers of natural adaptation rates. We quantify the effect sizes of these parameters on population fitness across three proposed assisted evolution scenarios and identify critical thresholds for intervention effectiveness and implementation. Specifically, the interventions tested here were most effective at low levels of genetic diversity in target populations (He < 0.2) and when timed during a cold-to-warm phase of an ENSO-like oscillation. Beneficial levels of connectivity were highly dependent on desired outcomes for the meta-population. We also present a global meta-analysis of genetic diversity in tropical reef-building corals as a case study of how thresholds derived from evolutionary models can be used to guide decision making by managers. We find genetic diversity to be highly variable by coral taxon and region, highlighting how thresholds from evolutionary models can be used in conjunction with empirical data to assess intervention needs and priorities. Finally, we highlight the critical knowledge and data gaps to produce the next suite of applied models for conservation management decision-support.

sentences: Polymer physics and machine learning reveal a combinatorial code linking chromatin 3D architecture to 1D epigenetics The mammalian genome has a complex 3D organization, serving vital functional purposes, yet it remains largely unknown how the multitude of specific DNA contacts, e.g., between transcribed and regulatory regions, is orchestrated by chromatin organizers, such as Transcription Factors. Here, we implement a method combining machine learning and polymer physics to infer from only Hi-C data the genomic 1D arrangement of the minimal set of binding sites sufficient to recapitulate, through only physics, 3D contact patterns genome-wide in human and mouse cells. The inferred binding sites are validated by their predictions on how chromatin refolds in a set of duplications at the Sox9 locus against available independent cHi-C data, showing that their different phenotypes originate from distinct enhancer hijackings in their 3D structure. Albeit derived from only Hi-C, our binding sites fall in epigenetic classes that well match chromatin states from epigenetic segmentation studies, such as active, poised and repressed states. However, the inferred binding domains have an overlapping, combinatorial organization along chromosomes, missing in epigenetic segmentations, which is required to explain Hi-C contact specificity with high accuracy. In a reverse approach, the epigenetic profile of binding domains provides a code to derive from only epigenetic marks the DNA binding sites and, hence, the 3D architecture, as validated by successful predictions of Hi-C matrices in an independent set of chromosomes. Overall, our results shed light on how complex 3D architectural information is encrypted in 1D epigenetics via the related, combinatorial arrangement of specific binding sites along the genome.

sentences: Allele mining, evolutionary genetic analysis of TaHKT1;5 gene and evaluation of salinity stress in selected lines of wheat. This study reports the novel allelic diversity in HKT 1;5 gene (High Affinity Potassium Transporter) in bread wheat and its phylogenetic relationship among the paralogs/orthologs of in Triticum aestivum and its wild relatives. HKT 1;5 gene is a known and pivotal gene associated with the salinity tolerance in plants upon the discrimination of K+ over Na+ in leaves without change in Na+ concentration in root. This gene was sequenced in a diverse collection of bread wheat, durum wheat, wild relatives, and ditelosomic lines. Sequence analysis in bread wheat led to the identification of four alleles, which could be distinguished by number of SNPs. Sequence comparison between monocot bread wheat and dicot Arabidopsis thaliana revealed that the HKT1;5 gene is conserved at level of exonic regions; however, the presence of transposable elements especially in intronic regions is further intriguing towards evolutionary relatedness. Two paralogous or major alleles observed in Triticum monoccum and Aegilops tauschii were further categorized as sub-alleles based on their SNPs comparison. This gene was absent in T. urartu in accordance with existing evidence, while it was found in A. speltoides (an allelic variant) with a few base pairs insertion in the exon1 region causing a frameshift mutation with an altered amino acids and genomic database mining unveiled additional alleles in this species. Ditelosomic lines with 4DL and 4DS chromosomes revealed a higher similarity with bread and durum wheat respectively. Phylogenetic studies of HKT1;5 orthologs from different Poaceae species revealed the occurrence of five different ortholog groups with taxonomic consistency. Phenotyping based salinity stress experiment distinguished the unknown lines for salinity tolerance and sensitiveness in comparison with known reference lines and possible allelic comparison was made. The salinity stress analysis further revealed that some known drought/heat tolerance lines showed slightly better salinity tolerance with mean values and variability of traits than known saline tolerant wheat line at controlled ambient.

sentences: Forward Entrainment: Evidence, Controversies, Constraints, and Mechanisms We define forward entrainment as that part of the entrainment process that outlasts the entraining stimulus. In this study, we examine conditions under which one may or may not observe forward entrainment. In part 1, we review and evaluate studies that have observed forward entrainment using a variety of psychophysical methods (detection, discrimination and reaction times), different target stimuli (tones, noise, gaps), different entraining sequences (sinusoidal, rectangular or sawtooth waveforms), a variety of physiological measures (MEG, EEG, ECoG, CSD), in different modalities (auditory and visual), across modalities (audiovisual and auditory-motor), and in different species. In part 2, we review those studies that have failed to observe forward entrainment, with emphasis on evaluating the methodological and stimulus design differences that may clarify the contrasting findings across these two classes of studies. In part 3, we describe those experimental conditions under which we ourselves have failed to observe forward entrainment, and provide new data on use of complex envelope patterns as entraining stimuli, show data on intersubject variability, and provide new findings on psychometric functions that characterize the strength of forward entrainment at different SNRs. In part 4 we theorize on potential mechanisms, describe how neurophysiological and psychophysical studies approach the study of entrainment, and caution against drawing direct causal inferences between the two without compelling evidence beyond correlative measures.

sentences: Lipid distributions and transleaflet cholesterol migration near heterogeneous surfaces in asymmetric bilayers Specific and nonspecific protein-lipid interactions in cell membranes have important roles in an abundance of biological functions. We have used coarse-grained (CG) molecular dynamics (MD) simulations to assess lipid distributions and cholesterol flipping dynamics around surfaces in a model asymmetric plasma membrane containing one of six structurally distinct entities: aquaporin-1 (AQP1), the bacterial {beta}-barrel outer membrane proteins OmpF and OmpX, KcsA potassium channel, WALP23 peptide, and a carbon nanotube (CNT). Our findings revealed varied lipid partitioning and cholesterol flipping times around the different solutes, and putative cholesterol binding sites in AQP1 and KcsA. The results suggest that protein-lipid interactions can be highly variable, and that surface-dependant lipid profiles are effectively manifested in CG simulations with the Martini force field.

sentences: EFHD1 ablation reduces cardiac mitoflash activation and protects cardiomyocytes from ischemia. Altered levels of intracellular calcium (Ca2+) is a highly prevalent feature in different forms of cardiac injury, producing changes in contractility, arrhythmias, and mitochondrial dysfunction. In cardiac ischemia-reperfusion injury, mitochondrial Ca2+ overload leads to pathological production of reactive oxygen species (ROS), activates the permeability transition, and cardiomyocyte death. Here we investigated the cardiac phenotype caused by deletion of EF-hand domain-containing protein D1 (Efhd1-/-), a Ca2+-binding mitochondrial protein whose function is poorly understood. Efhd1-/- mice are viable and have no adverse cardiac phenotypes. They feature reductions in basal ROS levels and mitoflash events, both important precursors for mitochondrial injury, though cardiac mitochondria have normal susceptibility to Ca2+ overload. Notably, we also find that Efhd1-/- mice and their cardiomyocytes are resistant to hypoxic injury.

sentences: Implicit anticipation of probabilistic regularities: Larger CNV emerges for unpredictable events Anticipation of upcoming events plays a crucial role in automatic behaviors. It is, however, still unclear whether the event-related brain potential (ERP) markers of anticipation could track the implicit acquisition of probabilistic regularities that can be considered as building blocks of automatic behaviors. Therefore, in a four-choice reaction time (RT) task performed by young adults (N = 36), the contingent negative variation (CNV) as an ERP marker of anticipation was measured from the onset of a cue stimulus until the presentation of a target stimulus. Due to the probability structure of the task, target stimuli were either predictable or unpredictable, but this was unknown to participants. The cue did not contain predictive information on the upcoming target. Results showed that the CNV amplitude during response preparation was larger before the unpredictable than before the predictable target stimuli. In addition, although RTs increased, the P3 amplitude decreased for the unpredictable as compared with the predictable target stimuli, possibly due to the stronger response preparation that preceded stimulus presentation. These results suggest that enhanced attentional resources are allocated to the implicit anticipation and processing of unpredictable events. This might originate from the formation of internal models on the probabilistic regularities of the stimulus stream, which primarily facilitates the processing of predictable events. Overall, we provide ERP evidence that supports the role of implicit anticipation and predictive processes in the acquisition of probabilistic regularities.

sentences: SHIP164 is a Chorein Motif Containing Lipid Transport Protein that Controls Membrane Dynamics and Traffic at the Endosome-Golgi Interface. Cellular membranes differ in protein and lipid composition as well as in the protein-lipid ratio. Thus, progression of membranous organelles along traffic routes requires mechanisms to control bilayer lipid chemistry and their abundance relative to proteins. The recent structural and functional characterization of VPS13-family proteins has suggested a mechanism through which lipids can be transferred in bulk from one membrane to another at membrane contact sites, and thus independently of vesicular traffic. Here we show that SHIP164 (UHRF1BP1L) shares structural and lipid transfer properties with these proteins and is localized on a subpopulation of vesicle clusters in the early endocytic pathway whose membrane cargo includes the cation-independent mannose-6-phosphate receptor (MPR) and ATG9. Loss of SHIP164 disrupts retrograde traffic of these organelles to the Golgi complex. Our findings raise the possibility that bulk transfer of lipids to endocytic membranes may play a role in their traffic.

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LiPyphilic: A Python toolkit for the analysis of lipid membrane simulations Molecular dynamics simulations are now widely used to study emergent phenomena in lipid membranes with complex compositions. Here, we present LiPyphilic - a fast, fully tested, and easy to install Python package for analysing such simulations. Analysis tools in LiPyphilic include the identification of cholesterol flip-flop events, the classification of local lipid environments, and the degree of interleaflet registration. LiPyphilic is both force field and resolution agnostic, and thanks to the powerful atom selection language of MDAnalysis it can handle membranes with highly complex compositions. LiPyphilic also offers two on-the-fly trajectory transformations to i) fix membranes split across periodic boundaries and ii) perform nojump coordinate unwrapping. Our implementation of nojump unwrapping accounts for fluctuations in box volume under the NPT ensemble -- an issue that most current implementations have overlooked. The full documentation of LiPyphilic, including installation instructions, is available at https://lipyphilic.readthedocs.io/en/latest.

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sentences: The E26 Transformation-Specific-family transcription factor Spi-C is dynamically regulated by external signals in B cells Spi-C is an E26 transformation-specific transcription factor closely related to PU.1 and Spi-B. Spi-C has lineage-instructive functions important in antibody-generating responses, B cell development, and red pulp macrophage generation. Spi-C is inducible by heme- and NF-{kappa}B-dependent pathways in macrophages. The present research aimed to examine the regulation of Spi-C expression in B cells. RT-qPCR analysis revealed that Spic expression was reduced in B cells following addition of lipopolysaccharide, anti-IgM antibodies, CD40L, or cytokines BAFF + IL-4 + IL-5. Cytochalasin treatment partially prevented downregulation of Spic. Unstimulated B cells upregulated Spic over time in culture. To determine the mechanism of Spic regulation, we examined the Spic promoter and upstream regulatory elements. The Spic promoter had unidirectional activity, which was reduced by mutation of an NF-{kappa}B binding site. Spic was repressed by an upstream regulatory region interacting with the heme-binding regulator Bach2. Taken together, these data indicate that Spi-C is dynamically regulated by external signals in B cells and provide insight into the mechanism of regulation.

sentences: CloudReg: Automatic Terabyte-Scale Cross-Modal Brain Volume Registration Quantifying terabyte-scale multi-modal human and animal imaging data requires scalable analysis tools. We developed CloudReg, an open-source, automatic, terabyte-scale, cloud-based image analysis pipeline that pre-processes and registers cross-modal volumetric datasets with artifacts via spatially-varying polynomial intensity transform. CloudReg accurately registers the following datasets to their respective atlases: in vivo human and ex vivo macaque brain magnetic resonance imaging, ex vivo mouse brain micro-computed tomography, and cleared murine brain light-sheet microscopy.

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Impaired dynamics of prefrontal beta-band networks during cognitive action control in Parkinson's disease Among the cognitive symptoms that are associated with Parkinsons disease (PD), alterations in cognitive action control (CAC) are commonly reported in patients. CAC enables the suppression of an automatic action, in favor of a goal-directed one. The implementation of CAC is time-resolved and arguably associated with dynamic changes in functional brain networks. However, the electrophysiological functional networks involved, their dynamic changes, and how these changes are affected by PD, still remain unknown. In this study, to address this gap of knowledge, 21 PD patients and 10 healthy controls (HC) underwent a Simon task while high-density electroencephalography (HD-EEG) was recorded. Source-level dynamic connectivity matrices were estimated using the phase-locking value in the beta (12-25 Hz) and gamma (30-45 Hz) frequency bands. Temporal independent component analyses were used as a dimension reduction tool to isolate the group-specific brain network states that were dominant during the task. Typical microstate metrics were quantified to investigate the presence of these states at the subject-level. Our results first confirmed that PD patients experienced difficulties in inhibiting automatic responses during the task. At the group-level, HC displayed a significant functional network state that involved typical CAC-related prefrontal and cingulate nodes (e.g., inferior frontal cortex). Both group- and subject-level analyses showed that this network was less present in PD to the benefit of other networks involving lateralized temporal and insular components. The presence of this prefrontal network was associated with decreased reaction time. In the gamma band, two networks (fronto-cingulate and fronto-temporal) followed one another in HC, while 3 partially overlapping networks that included fronto-temporal, fronto-occipital and cross-hemispheric temporal connections were found in PD. At the subject-level, differences between PD and HC were less marked. Altogether, this study showed that the functional brain networks observed during CAC and their temporal changes were different in PD patients as compared to HC, and that these differences partially relate to behavioral changes. This study also highlights that task-based dynamic functional connectivity is a promising approach in understanding the cognitive dysfunctions observed in PD and beyond.

HighlightsO_LICognitive action control is associated with dynamic functional networks
C_LIO_LIPrefrontal and cingulate beta connectivity are prominent in healthy controls
C_LIO_LIPD patients have different dynamic networks in which prefrontal nodes are absent
C_LIO_LIThe occurrence of prefrontal beta networks was associated with a decreased reaction time
C_LIO_LIFunctional networks in the gamma band were temporally organized in HC, but overlapping in PD patients
C_LI

sentences: The neural dynamics associated with computational complexity Many everyday tasks require people to solve computationally complex problems. However, little is known about the effects of computational hardness on the neural processes associated with solving such problems. Here, we draw on computational complexity theory to address this issue. We performed an experiment in which participants solved several instances of the 0-1 knapsack problem, a combinatorial optimization problem, while undergoing ultra-high field (7T) functional magnetic resonance imaging (fMRI). Instances varied in two task-independent measures of intrinsic computational hardness: complexity and proof hardness. We characterise a network of brain regions whose activation was correlated with both measures but in distinct ways, including the anterior insula, dorsal anterior cingulate cortex and the intra-parietal sulcus/angular gyrus. Activation and connectivity changed dynamically as a function of complexity and proof hardness, in line with theoretical computational requirements. Overall, our results suggest that computational complexity theory provides a suitable framework to study the effects of computational hardness on the neural processes associated with solving complex cognitive tasks.

sentences: Joint dimension reduction and clustering analysis for single-cell RNA-seq and spatial transcriptomics data Dimension reduction and (spatial) clustering is usually performed sequentially; however, the low-dimensional embeddings estimated in the dimension-reduction step may not be relevant to the class labels inferred in the clustering step. We therefore developed a computation method, Dimension-Reduction Spatial-Clustering (DR-SC), that can simultaneously perform dimension reduction and (spatial) clustering within a unified framework. Joint analysis by DR-SC produces accurate (spatial) clustering results and ensures the effective extraction of biologically informative low-dimensional features. DR-SC is applicable to spatial clustering in spatial transcriptomics that characterizes the spatial organization of the tissue by segregating it into multiple tissue structures. Here, DR-SC relies on a latent hidden Markov random field model to encourage the spatial smoothness of the detected spatial cluster boundaries. Underlying DR-SC is an efficient expectation-maximization algorithm based on an iterative conditional mode. As such, DR-SC is scalable to large sample sizes and can optimize the spatial smoothness parameter in a data-driven manner. With comprehensive simulations and real data applications, we show that DR-SC outperforms existing clustering and spatial clustering methods: it extracts more biologically relevant features than conventional dimension reduction methods, improves clustering performance, and offers improved trajectory inference and visualization for downstream trajectory inference analyses.

sentences: Diisopropylfluorophosphate (DFP) volatizes and cross-contaminates wells in a common 96-well plate zebrafish larvae exposure method Diisopropylfluorophosphate (DFP) is an organophosphate (OP) that is commonly used to study the neurotoxic effects of acutely intoxicating OP exposure. In preliminary studies, we discovered abnormal deaths in DMSO-only exposed larvae housed in the same plate as DFP-exposed larvae, and hypothesized that DFP volatilizes and cross-contaminates wells when using a 96-well plate exposure method for exposing zebrafish larvae. Survivability and acetylcholinesterase activity assays confirmed DFP presence in the tissues of zebrafish ostensibly exposed to DMSO only. These findings indicate DFP cross-contamination, which raises concerns for the experimental design of studies evaluating the toxicity of volatile and semi-volatile substances.

sentences: The ecological consequences and evolution of retron-mediated suicide as a way to protect Escherichia coli from being killed by phage In 1984, retrons were described as DNA sequences that code for a reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA). It would not be until 2020 that a function was shown, when compelling evidence was presented that retrons can protect bacteria from infections by lytic phage via an abortive infection mechanism. When infected with the virulent mutant of the phage lambda, {lambda}VIR, and to a lesser extent other phages, a retron designated Ec48 is activated, the Escherichia coli bearing this element dies and the infecting phage is lost. With the aid of a mathematical model, we explored the a priori conditions under which retrons will protect bacterial populations from invasion by phage and when retron-bearing bacteria will evolve in populations without this element. Using E. coli with and without Ec48, and {lambda}VIR we estimated the parameters of this model and tested the hypotheses generated from our analysis of its properties. Our models and experiments suggest cells expressing retron-mediated abortive infection have no advantage over cells lacking this system as a defense against phage, and we question whether the protection against phage infection is the unique selective force responsible for their evolution in bacteria.

sentences: Control of Leaf Vein Patterning by Regulated Plasmodesma Aperture To form tissue networks, animal cells migrate and interact through proteins protruding from their plasma membranes. Plant cells can do neither, yet plants form vein networks. How plants do so is unclear, but veins are thought to form by the coordinated action of the polar transport and signal transduction of the plant hormone auxin. However, plants inhibited in both pathways still form veins. Patterning of vascular cells into veins is instead prevented in mutants lacking the function of the GNOM (GN) regulator of auxin transport and signaling, suggesting the existence of at least one more GN-dependent vein-patterning pathway. Here we show that pathway depends on the movement of an auxin signal through plasmodesmata (PDs) intercellular channels. PD permeability is high where veins are forming, lowers between veins and nonvascular tissues, but remains high between vein cells. Impaired ability to regulate PD aperture leads to defects in auxin transport and signaling, ultimately leading to vein patterning defects that are enhanced by inhibition of auxin transport or signaling. GN controls PD aperture regulation, and simultaneous inhibition of auxin signaling, auxin transport, and regulated PD aperture phenocopies null gn mutants. Therefore, veins are patterned by the coordinated action of three GN-dependent pathways: auxin signaling, polar auxin transport, and movement of an auxin signal through PDs. We have identified all the key vein-patterning pathways in plants and an unprecedented mechanism of tissue network formation in multicellular organisms.

sentences: Microstructural Impairments in a Topologically Distinct Prefrontal-Habenular Connection in Cocaine Addiction Drug addiction is characterized by neuroadaptations in mesocorticolimbic networks regulating reward and inhibitory control. The habenula (Hb) is central to adaptive reward and aversion-driven behaviors, serving as a hub connecting emotion/cognitive processing regions including the prefrontal cortex (PFC). However, its role in human drug addiction has not been fully explored. Using diffusion tractography, we detailed PFC structural connectivity with three regions, namely the Hb, ventral tegmental area (VTA), and anterior thalamus (AT), and quantified the tract-specific microstructural integrity using diffusion tensor imaging within the anterior limb of the internal capsule (ALIC) in healthy and cocaine-addicted individuals. White matter microstructure in cocaine-addicted individuals was uniquely impaired in PFC-Hb projections in the ALIC, distinguishable from adjacent PFC-VTA and PFC-AT projections, with more pronounced abnormalities in short-term abstinence. These findings extend preclinical evidence of PFC-Hb circuit impairments in addiction and contextualize the plausible existence of a similar PFC-Hb connection in the human brain.

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Taxon-specific shifts in bacterial and archaeal transcription of dissolved organic matter cycling genes in a stratified fjord A considerable fraction of organic matter derived from photosynthesis in the euphotic zone settles into the oceans interior, and under way is degraded by diverse microbial consortia that utilize a suite of extracellular enzymes and membrane transporters. Still, the molecular details that regulate carbon cycling across depths remain little explored. As stratification in fjords has made them attractive models to explore patterns in biological oceanography, we here analyzed bacterial and archaeal transcription in samples from five depth layers in the Gullmar Fjord, Sweden. Transcriptional variation over depth correlated with gradients in chlorophyll a and nutrient concentrations. Differences in transcription between sampling dates (summer and early autumn), were strongly correlated with ammonium concentrations, which potentially was linked with a stronger influence of (micro-)zooplankton grazing in summer. Transcriptional investment in carbohydrate-active enzymes (CAZymes) decreased with depth and shifted toward peptidases, partly a result of elevated CAZyme transcription by Flavobacteriales, Cellvibrionales and Synechococcales at 2-25 m and a dominance of peptidase transcription by Alteromonadales and Rhodobacterales from 50 m and down. In particular, CAZymes for chitin, laminarin, and glycogen were important. High levels of transcription of ammonium transporters by Thaumarchaeota at depth (up to 18% of total transcription), along with the genes for ammonia oxidation and CO2-fixation, indicated that chemolithoautotrophy contributed to the carbon flux in the fjord. The taxon-specific expression of functional genes for processing of the marine DOM pool and nutrients across depths emphasizes the importance of different microbial foraging mechanisms across spatiotemporal scales for shaping biogeochemical cycles.

IMPORTANCEIt is generally recognized that stratification in the ocean strongly influences both the community composition and the distribution of ecological functions of microbial communities, which in turn are expected to shape the biogeochemical cycling of essential elements over depth. Here we used metatranscriptomics analyses to infer molecular detail on the distribution of gene systems central to the utilization of organic matter in a stratified marine system. We thereby uncovered that pronounced shifts in transcription of genes encoding CAZymes, peptidases, and membrane transporters occurred over depth among key prokaryotic orders. This implies that sequential utilization and transformation of organic matter through the water column is a key feature that ultimately influences the efficiency of the biological carbon pump.

sentences: Capturing a crucial 'disorder-to-order transition' at the heart of the coronavirus molecular pathology - triggered by highly persistent, interchangeable salt-bridges The COVID-19 origin debate has greatly been influenced by Genome comparison studies of late, revealing the seemingly sudden emergence of the Furin-Like Cleavage Site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCSSpike) containing its 681PRRAR685 motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a disorder-to-order transition of the FLCSSpike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike-Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCSSpike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seems the other key characteristic features of the Spike-Furin binding in SARS-CoV-2 allowing the system to breathe - a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike-Furin binding events generally in the coronavirus lineage seems to have a preference for ionic bond formation, even with lesser number of cationic residues at their potentially polybasic FLCSSpike patches. The interaction energies are suggestive of a characteristic metastabilities attributed to Spike-Furin interactions generally to the coronavirus lineage - which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. T he current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution but also add substantially to the existing theories of proteolytic cleavages.

sentences: Oligodendrocyte precursor cells engulf synaptic inputs in an experience- and microglia-dependent manner Oligodendrocyte precursor cells (OPCs) are a highly proliferative class of non-neuronal progenitors that largely give rise to myelinating oligodendrocytes. Although OPCs persist across the lifespan, their functions beyond oligodendrogenesis remain to be fully characterized. Here, we show that OPCs contribute to neural circuit remodeling by internalizing presynaptic thalamocortical inputs in both the developing and adult mouse visual cortex. Inputs internalized by OPCs localize to lysosomal compartments, consistent with OPC engulfment of synapses occuring through phagocytosis. We further show that engulfment by OPCs is heightened during experience-dependent plasticity, and that this experience-dependent increase in engulfment requires microglia. These data identify a new function for OPCs beyond the generation of oligodendrocytes and reveal that distinct non-neuronal populations collaborate to modulate synaptic connectivity.

sentences: Neurological mechanism of sensory deficits after developmental exposure to non-dioxin-like polychlorinated biphenyls (PCBs) in zebrafish larvae The most abundant polychlorinated biphenyl (PCB) congeners found in the environment and in humans are neurotoxic. This is of particular concern for early life stages because the exposure of the more vulnerable developing nervous system to chemicals can result in neurobehavioral disorders. To uncover currently unknown links between PCB target mechanisms and neurobehavioral deficits, we investigated the effects of the non-dioxin-like (NDL) congener PCB153 on neuronal morphology and synaptic transmission linked to the proper execution of a sensorimotor response using zebrafish as a vertebrate model. Zebrafish that were exposed during development to concentrations similar to those found in human cord blood and PCB contaminated sites showed a delay in startle response. A similar delay was observed for other NDL congeners but not for the potent dioxin-like congener PCB126. Morphological and biochemical data demonstrate that while exposure to PCB153 induced swelling of afferent sensory neurons, the disruption of dopaminergic and GABAergic signaling is the dominant mechanism associated with motor movement. The effects on important and broadly conserved signaling mechanisms in vertebrates suggest that NDL PCBs may contribute to neurodevelopmental abnormalities in humans and, with the startle response being critical for the survival of fish, to evolutionary adaptation in wildlife.

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ECLIPSER: identifying causal cell types and genes for complex traits through single cell enrichment of e/sQTL-mapped genes in GWAS loci SummaryECLIPSER was developed to identify pathogenic cell types and cell type-specific genes that may affect complex disease susceptibility and trait variation by integrating single cell data with known GWAS loci. ECLIPSER maps genes to GWAS loci for a given complex trait based on expression and splicing quantitative trait loci (e/sQTLs) and other functional data, and tests whether the mapped genes are enriched for cell type-specific expression in particular cell types using single-cell/nucleus RNA-seq data from one or more tissues of interest. A Bayesian Fishers exact test is used to compute fold-enrichment significance. We demonstrate the application of ECLIPSER on various skin diseases and traits using snRNA-seq of healthy human skin samples.

Availability and ImplementationThe source code and documentation for ECLIPSER and a Jupyter notebook for generating output tables and figures are available at https://github.com/segrelabgenomics/ECLIPSER. The source code for GWASvar2gene that maps genes to GWAS loci based on e/sQTLs is available at https://github.com/segrelabgenomics/GWASvar2gene. The analysis presented here used data from GTEx (https://gtexportal.org/home/datasets) and Open Targets Genetics (https://genetics-docs.opentargets.org/data-access/graphql-api), but can also be applied to other GWAS variant lists and QTL studies. Data used to reproduce the results of the paper are available in Supplementary data.

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Phototroph-heterotroph interactions during growth and long-term starvation across Prochlorococcus and Alteromonas diversity Microbial interactions such as those between phytoplankton and bacteria been studied intensively using specific model organisms, due to their potential impact on ecosystems and biogeochemistry. Yet, to what extent interactions differ between closely related organisms, or how these interactions change over time or culture conditions, remains unclear. Here, we characterize the interactions between five strains each of two globally abundant marine microorganisms, Prochlorococcus (a phototroph) and Alteromonas (a heterotroph), from the first encounter between individual strains and over more than a year of repeated cycles of exponential growth and long-term nitrogen starvation. Prochlorococcus-Alteromonas interactions had little effect on traditional growth parameters such as Prochlorococcus growth rate, maximal fluorescence or lag phase, affecting primarily the dynamics of culture decline, which we interpret as representing cell mortality and lysis. The shape of the Prochlorococcus decline curve and the carrying capacity of the co-cultures were determined by the phototroph and not the heterotroph strains involved. Comparing various mathematical models of culture mortality suggests that Prochlorococcus death rate increases over time in mono-cultures but decreases in co-cultures, with cells potentially becoming more resistant to stress. Our results demonstrate intra-species differences in ecologically-relevant co-culture outcomes. These include the recycling efficiency of N and whether the interactions are mutually synergistic or competitive. They also highlight the information-rich growth and death curves as a useful readout of the interaction phenotype.

Significance StatementInteractions between phytoplankton and marine bacteria impact global ecosystems and biogeochemistry. Here, we explore how intra-species variability affects the interactions between Prochlorococcus, a globally abundant photosynthetic cyanobacetrium and Alteromonas, a heterotrophic bacterium that lives off and recycles organic matter. Under nitrogen starvation, Prochlorococcus growing alone increasingly accumulate damage and die, whereas in co-culture with Alteromonas they become increasingly resilient. The specific Prochlorococcus strain had a much larger effect on co-culture behavior than the Alteromonas strain, determining whether the interactions are mutually synergistic or potentially competitive. These results show how ecologically relevant outcomes of interactions may vary between closely related microorganisms, and highlight growth and death curves from laboratory (co)-cultures as information-rich views of microbial growth and death.

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Becoming metrics literate: An analysis of brief videos that teach about the h-index IntroductionAcademia uses scholarly metrics, such as the h-index, to make hiring, promotion, and funding decisions. These high-stakes decisions require that those using scholarly metrics be able to recognize, interpret, critically assess and effectively and ethically use them. This study aimed to characterize educational videos about the h-index to understand available resources and provide recommendations for future educational initiatives.

MethodsThe authors analyzed videos on the h-index posted to YouTube. Videos were identified by searching YouTube and were screened by two authors. To code the videos the authors created a coding sheet, which assessed content and presentation style with a focus on the videos educational quality based on Cognitive Load Theory. Two authors coded each video independently with discrepancies resolved by group consensus.

ResultsThirty-one videos met inclusion criteria. Twenty-one videos (68%) were screencasts and seven used a "talking head" approach. Twenty-six videos defined the h-index (83%) and provided examples of how to calculate and find it. The importance of the h-index in high-stakes decisions was raised in 14 (45%) videos. Sixteen videos (52%) described caveats about using the h-index, with potential disadvantages to early researchers the most prevalent (n=7; 23%). All videos incorporated various educational approaches with potential impact on viewer cognitive load. Most videos (n=21; 68%) displayed amateurish production quality.

DiscussionThe videos featured content with potential to enhance viewers metrics literacies such that many defined the h-index and described its calculation, providing viewers with skills to recognize and interpret the metric. However, less than half described the h-index as an author quality indicator, which has been contested, and caveats about h-index use were inconsistently presented, suggesting room for improvement. While most videos integrated practices to facilitate balancing viewers cognitive load, few (32%) were of professional production quality. Some videos missed opportunities to adopt particular practices that could benefit learning.

sentences: Lineage tracing via associative chromosome/plasmid barcoding with siBar We describe a new method, siBar, which enables simultaneous tracking of plasmids and chromosomal lineages within a bacterial host. siBar involves integration of a linearized plasmid construct carrying a unique combination of two molecular barcodes. Upon recircularization one barcode remains integrated into the host chromosome, while the other remains on the plasmid allowing direct observation of expansion and contraction of adaptive lineages. We also performed a pilot evolution experiment that allowed us to assess the barcode complexity and establish an analytical framework for the analysis of siBar data.

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Predicting Mechanical Ventilation Effects on Six Human Tissue Transcriptomes BackgroundMechanical ventilation (MV) is a lifesaving therapy used for patients with respiratory failure. Nevertheless, MV is associated with numerous complications and increased mortality. The aim of this study is to define the effects of MV on gene expression of direct and peripheral human tissues.

MethodsClassification models were applied to Genotype-Tissue Expression Project (GTEx) gene expression data of six representative tissues- liver, adipose, skin, nerve-tibial, muscle and lung, for performance comparison and feature analysis. We utilized 18 prediction models using the Random Forest (RF), XGBoost (eXtreme Gradient Boosting) decision tree and ANN (Artificial Neural Network) methods to classify ventilation and non-ventilation samples and to compare their prediction performance for the six tissues. In the model comparison, the AUC (area under receiver operating curve), accuracy, precision, recall, and F1 score were used to evaluate the predictive performance of each model. We then conducted feature analysis per each tissue to detect MV marker genes followed by pathway enrichment analysis for these genes.

ResultsXGBoost outperformed the other methods and predicted samples had undergone MV with an average accuracy for the six tissues of 0.951 and average AUC of 0.945. The feature analysis detected a combination of MV marker genes per each tested tissue, some common across several tissues. MV marker genes were mainly related to inflammation and fibrosis as well as cell development and movement regulation. The MV marker genes were significantly enriched in inflammatory and viral pathways.

ConclusionThe XGBoost method demonstrated clear enhanced performance and feature analysis compared to the other models. XGBoost was helpful in detecting the tissue-specific marker genes for identifying transcriptomic changes related to MV. Our results show that MV is associated with reduced development and movement in the tissues and higher inflammation and injury not only in direct tissues such as the lungs but also in peripheral tissues and thus should be carefully considered before being implemented.

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AN INULIN-TYPE FRUCTAN ENRICHED EXCLUSIVE ENTERAL NUTRITION FORMULA SUPPRESSES COLITIS THROUGH GUT MICROBIOME MODULATION AND PROMOTING EXPANSION OF ANTI-INFLAMMATORY T CELL SUBSETS Background & AimsExclusive enteral nutrition (EEN) is used to treat pediatric Crohns disease (CD), but therapeutic benefits are not long lasting. Due to reported lower efficacy EEN is not routinely used to treat pediatric ulcerative colitis (UC). Inulin-type fructans (IN) beneficially modulate the gut microbiome and promote expansion of anti-inflammatory immune cells. We hypothesized that enriching EEN with IN (EENIN) would enhance treatment efficacy. To test this, we examined the effects of EEN-IN on colitis development, the gut microbiome and CD4+ T cells using an adoptive T cell transfer model of colitis.

MethodsTCR-{beta} deficient mice were randomized to one of four groups: 1) Control, 2) Chow, 3) EEN and 4) EEN-IN, and naive CD4+ T cells were adoptively transferred into groups 2-4, after which mice were monitored for 5-weeks prior to experimental endpoint.

ResultsMice fed EEN-IN showed greater colitis protection, with colonic shortening, goblet cell and crypt density loss reduced over that of EEN fed mice and reduced disease activity and immune cell infiltration compared to chow fed mice, and less crypt hyperplasia and higher survival compared to both groups. EENIN mice maintained colonic mucus layer thickness and had increased levels of Foxp3+IL-10+ and Ror{gamma}t+IL- 22+ and reduced levels of Tbet+IFN{gamma}+ and Tbet+TNF+ CD4+ T cells. EEN-IN also lead to higher butyrate, Bifidobacterium spp. and Bacteroides spp. concentrations.

ConclusionThe EEN-IN group showed reduced colitis development as compared to the chow and EEN groups. This highlights the potential benefits of EEN-IN as a novel induction therapy for pediatric CD and UC patients.

SynopsisWe demonstrated that inulin-type fructan enriched exclusive enteral nutrition formula reduced colitis development likely due to butyrate-dependent pathways that helped preserve the mucus layer and promote an anti-inflammatory intestinal environment via expansion of regulatory T cells.

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OGUs enable effective, phylogeny-aware analysis of even shallow metagenome community structures We introduce Operational Genomic Unit (OGU), a metagenome analysis strategy that directly exploits sequence alignment hits to individual reference genomes as the minimum unit for assessing the diversity of microbial communities and their relevance to environmental factors. This approach is independent from taxonomic classification, granting the possibility of maximal resolution of community composition, and organizes features into an accurate hierarchy using a phylogenomic tree. The outputs are suitable for contemporary analytical protocols for community ecology, differential abundance and supervised learning while supporting phylogenetic methods, such as UniFrac and phylofactorization, that are seldomly applied to shotgun metagenomics despite being prevalent in 16S rRNA gene amplicon studies. As demonstrated in one synthetic and two real-world case studies, the OGU method produces biologically meaningful patterns from microbiome datasets. Such patterns further remain detectable at very low metagenomic sequencing depths. Compared with taxonomic unit-based analyses implemented in currently adopted metagenomics tools, and the analysis of 16S rRNA gene amplicon sequence variants, this method shows superiority in informing biologically relevant insights, including stronger correlation with body environment and host sex on the Human Microbiome Project dataset, and more accurate prediction of human age by the gut microbiomes in the Finnish population. We provide Woltka, a bioinformatics tool to implement this method, with full integration with the QIIME 2 package and the Qiita web platform, to facilitate OGU adoption in future metagenomics studies.

ImportanceShotgun metagenomics is a powerful, yet computationally challenging, technique compared to 16S rRNA gene amplicon sequencing for decoding the composition and structure of microbial communities. However, current analyses of metagenomic data are primarily based on taxonomic classification, which is limited in feature resolution compared to 16S rRNA amplicon sequence variant analysis. To solve these challenges, we introduce Operational Genomic Units (OGUs), which are the individual reference genomes derived from sequence alignment results, without further assigning them taxonomy. The OGU method advances current read-based metagenomics in two dimensions: (i) providing maximal resolution of community composition while (ii) permitting use of phylogeny-aware tools. Our analysis of real-world datasets shows several advantages over currently adopted metagenomic analysis methods and the finest-grained 16S rRNA analysis methods in predicting biological traits. We thus propose the adoption of OGU as standard practice in metagenomic studies.

sentences: Is energetics or competition a stronger driver of male smallmouth bass seasonal reproductive timing? Intraspecific competitive ability is often associated with body size and has been shown to influence reproductive timing in many species. However, energetic constraints provide an alternative explanation for size-related differences of reproductive timing. In temperate fishes that experience a winter starvation period, for instance, a negative allometric relationship between body size and winter energy loss might explain why larger males spawn earlier in a season than smaller males, especially in fishes that exhibit paternal care, which is energetically costly and limits parental foraging opportunities. Male smallmouth bass, Micropterus dolomieu, defend nesting territories in which they care for offspring over an extended period. In northern populations, males rely on energy reserves over a winter starvation period and in spring must recoup energy losses before initiating reproduction, making them ideal systems in which to study contributions of competition and energetic allometry on differences of reproductive timing. Here, we harness data on parental male M. dolomieu from a 10-year study and show that larger males required fewer degree days-a measure of thermal energy experienced-in spring before they spawned each year and that the time of peak seasonal reproduction in the population was negatively related to the number of degree days accumulated before reproduction started. Furthermore, we found that growth of individual males between seasons better predicted changes in timing of reproduction than changes in size relative to competitors. Together, these results suggest that timing of reproduction in this population is more strongly influenced by energetic constraints than size-based competition amongst males.

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Integrating structured and unstructured citizen science data to improve wildlife population monitoring Accurate and robust population trend assessments are key to successful biodiversity conservation. Citizen science surveys have provided good evidence of biodiversity declines whilst engaging people with them. Citizen scientists are also collecting opportunistic biodiversity records at unprecedented scales, vastly outnumbering records gathered through structured surveys. Opportunistic records exhibit spatio-temporal biases and heterogeneity in observer effort and skill, but their quantity offers a rich source of information. Data integration, the combination of multiple information sources in a common analytical framework, can potentially improve inferences about populations compared to analysing either in isolation. We combine count data from a structured citizen science survey and detection-nondetection data from an opportunistic citizen science programme. Population trends were modelled using dynamic N-mixture models to integrate both data sources. We applied this approach to two different inferential challenges arising from sparse data: (i) the estimation of population trends for an area smaller than a structured survey stratum, and (ii) the estimation of national population trends for a rare but widespread species. In both cases, data integration yielded population trajectories similar to those estimated from structured survey data alone but had higher precision when the density of opportunistic records was high. In some cases this allowed inferences about population trends where indices derived from single data sources were too uncertain to assess change. However, there were differences in the trend magnitude between the integrated and the standard survey model.

We show that data integration of large-scale structured and unstructured data is feasible and offers potential to improve national and regional wildlife trend estimates, although a need to independently validate trends remains. Smaller gains are achieved in areas where uptake of opportunistic recording is low. The integration of opportunistic records from volunteer-selected locations alone may therefore not adequately address monitoring gaps for management and policy applications. To achieve the latter, scheme organisers should consider providing incentives for achieving representative coverage of target areas in both structured and unstructured recording schemes.

sentences: The BNT162b2 mRNA vaccine induces polyfunctional T cell responses with features of longevity. Vaccination against SARS-CoV-2 infection has shown to be effective in preventing hospitalization for severe COVID-19. However, multiple reports of break-through infections and of waning antibody titers have raised concerns on the durability of the vaccine, and current discussions on vaccination strategies are centered on evaluating the opportunity of a third dose administration. Here, we monitored T cell responses to the Spike protein of SARS-CoV-2 in 71 healthy donors vaccinated with the Pfizer-BioNTech mRNA vaccine (BNT162b2) for up to 6 months after vaccination. We find that vaccination induces the development of a sustained anti-viral memory T cell response which includes both the CD4+ and the CD8+ lymphocyte subsets. These lymphocytes display markers of polyfunctionality, are fit for interaction with cognate cells, show features of memory stemness, and survive in significant numbers the physiological contraction of the immune response. Collectively, this data shows that vaccination with BNT162b2 elicits an immunologically competent and potentially long-lived SARS-CoV-2-specific T cell population. Understanding the immune responses to BNT162b2 provides insights on the immunological basis of the clinical efficacy of the current vaccination campaign and may instruct future vaccination strategies.

sentences: Transiently increased intercommunity regulation characterizes concerted cell phenotypic transition Phenotype transition takes place in many biological processes such as differentiation and reprogramming. A fundamental question is how cells coordinate switching of expressions of clusters of genes. Through analyzing single cell RNA sequencing data in the framework of transition path theory, we studied how such a genome-wide expression program switching proceeds in three different cell transition processes. For each process we reconstructed a reaction coordinate describing the transition progression, and inferred the gene regulation network (GRN) along the reaction coordinate. In all three processes we observed common pattern that the effective number and strength of regulation between different communities increase first and then decrease. The change accompanies with similar change of the GRN frustration, defined as overall confliction between the regulation received by genes and their expression states, and GRN heterogeneity. While studies suggest that biological networks are modularized to contain perturbation effects locally, our analyses reveal a general principle that during a cell phenotypic transition intercommunity interactions increase to concertedly coordinate global gene expression reprogramming, and canalize to specific cell phenotype as Waddington visioned.

sentences: Transmission of one predicts another: Apathogenic proxies for transmission dynamics of a fatal virus Identifying drivers of transmission prior to an epidemic--especially of an emerging pathogen--is a formidable challenge for proactive disease management efforts. We tested a novel approach in the Florida panther, hypothesizing that apathogenic feline immunodeficiency virus (FIV) transmission could predict transmission dynamics for pathogenic feline leukemia virus (FeLV). We derived a transmission network using FIV whole genome sequences, and used exponential random graph models to determine drivers structuring this network. We used these drivers to predict FeLV transmission pathways among panthers and compared predicted outbreak dynamics against empirical FeLV outbreak data. FIV transmission was primarily driven by panther age class and distances between panther home range centroids. Prospective FIV-based modeling predicted FeLV dynamics at least as well as simpler, often retrospective approaches, with evidence that FIV-based predictions captured the spatial structuring of the observed FeLV outbreak. Our finding that an apathogenic agent can predict transmission of an analogously transmitted pathogen is an innovative approach that warrants testing in other host-pathogen systems to determine generalizability. Use of such apathogenic agents holds promise for improving predictions of pathogen transmission in novel host populations, and could thereby provide new strategies for proactive pathogen management in human and animal systems.

sentences: Proteomic portraits reveal evolutionarily conserved and divergent responses to spinal cord injury Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of CSF and serum samples from 111 acute SCI patients and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at six months with an AUC of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response, and establish the CSF abundance of glial fibrillary acidic protein (GFAP) as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.

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The draft chromosome-level genome assembly of tetraploid ground cherry (Prunus fruticosa Pall.) from long reads BackgroundCherries are stone fruits and belong to the economically important plant family of Rosaceae with worldwide cultivation of different species. The ground cherry, Prunus fruticosa Pall. is one ancestor of cultivated sour cherry, an important tetraploid cherry species. Here, we present a long read chromosome-level draft genome assembly and related plastid sequences using the Oxford Nanopore Technology PromethION platform and R10.3 pore type.

FindingThe final assemblies obtained from 117.3 Gb cleaned reads representing 97x coverage of expected 1.2 Gb tetraploid (2n=4x=32) and 0.3 Gb haploid (1n=8) genome sequence of P. fruticosa were calculated. The N50 contig length ranged between 0.3 and 0.5 Mb with the longest contig being [~]6 Mb. BUSCO estimated a completeness between 98.7 % for the 4n and 96.1 % for the 1n datasets.

Using a homology and reference based scaffolding method, we generated a final consensus genome sequence of 366 Mb comprising eight chromosomes. The N50 scaffold was [~]44 Mb with the longest chromosome being 66.5 Mb.

The repeat content was estimated to [~]190 Mb (52 %) and 58,880 protein-coding genes were annotated. The chloroplast and mitochondrial genomes were 158,217 bp and 383,281 bp long, which is in accordance with previously published plastid sequences.

ConclusionThis is the first report of the genome of ground cherry (P. fruticosa) sequenced by long read technology only. The datasets obtained from this study provide a foundation for future breeding, molecular and evolutionary analysis in Prunus studies.

sentences: A two-step metagenomics approach for prey identification from the blood meals of common vampire bats (Desmodus rotundus) The feeding behaviour of the sanguivorous common vampire bat (Desmodus rotundus) facilitates the transmission of pathogens that can impact both human and animal health. To formulate effective strategies in controlling the spread of diseases, there is a need to obtain information on which animals they feed on. One DNA-based approach, shotgun sequencing, can be used to obtain such information. Even though it is costly, shotgun sequencing can be used to simultaneously retrieve prey and vampire bat mitochondrial DNA for population studies within one round of sequencing. However, due to the challenges of analysing shotgun sequenced metagenomic data such as false negatives/positives and typically low proportion of reads mapped to diet items, shotgun sequencing has not been used for the identification of prey from common vampire bat blood meals. To overcome these challenges and generate longer mitochondrial contigs which could be useful for prey population studies, we shotgun sequenced common vampire bat blood meal samples (n=8) and utilised a two-step metagenomic approach based on combining existing bioinformatic workflows (alignment and de novo mtDNA assembly) to identify prey. Further, we validated our results to detections made through metabarcoding. We accurately identified the common vampire bats prey in seven out of eight samples without any false positives. We also generated prey mitochondrial contig lengths between 138bp to 3231bp (mean=985bp, SD=981bp). As we develop more computationally efficient bioinformatics pipelines and reduce sequencing costs, we can expect an uptake in metagenomics dietary studies in the near future.

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Transcriptome Mining Reveals a Spectrum of RNA Viruses in Primitive Plants Current knowledge of plant viruses stems largely from those affecting economically important plants. Yet, plant species in cultivation represent a small and bias subset of the plant kingdom. Here, we describe virus diversity and abundance from a survey of 1079 transcriptomes from species across the breadth of the plant kingdom (Archaeplastida) by analysing open-source data from the One Thousand Plant Transcriptomes Initiative (1KP). We identified 104 potentially novel viruses, of which 40% comprised single-stranded positive-sense RNA viruses across eight orders, including members of the Hepelivirales, Tymovirales, Cryppavirales, Martellivirales and Picornavirales. One-third of the newly described viruses comprised double-stranded RNA viruses from the orders Durnavirales and Ghabrivirales. The remaining were negative-sense RNA viruses from the Rhabdoviridae, Aspiviridae, Yueviridae, Phenuiviridae and the newly proposed Viridisbunyaviridae. Our analysis considerably expands the known host range of 13 virus families to include lower plants (e.g., Benyviridae and Secoviridae) and four virus families to include algae hosts (e.g., Tymoviridae and Chrysoviridae). The discovery of the first 30 kDa movement protein in a non-vascular plant, suggests that the acquisition of plant virus movement proteins occurred prior to the emergence of the plant vascular system. More broadly, however, a co-phylogeny analysis revealed that the evolutionary history of these families is largely driven by cross-species transmission events. Together, these data highlight that numerous RNA virus families are associated with older evolutionary plant lineages than previously thought and that the scarcity of RNA viruses found in lower plants to date likely reflects a lack of investigation rather than their absence.

ImportanceOur knowledge of plant viruses is mainly limited to those infecting economically important host species. In particular, we know little about those viruses infecting primitive plant lineages such as the ferns, lycophytes, bryophytes and charophytes. To expand this understanding, we conducted a broad-scale viral survey of species across the breadth of the plant kingdom. We find that primitive plants harbour a wide diversity of RNA viruses including some that are sufficiently divergent to comprise a new virus family. The primitive plant virome we reveal offers key insights into the evolutionary history of core plant virus gene modules and genome segments. More broadly, this work emphasises that the scarcity of viruses found in these species to date likely reflects the absence of research in this area.

sentences: The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II Transcription Factors (TFs) play a pivotal role in cell fate decision by coordinating distinct gene expression programs. Although most TFs act at the DNA regulatory layer, few TFs can bind RNA and modulate mRNA splicing. Yet, the mechanistic cues underlying TFs function in splicing remain elusive. Focusing on the Drosophila Hox TF Ultrabithorax (Ubx), our work shed light on a novel layer of Ubx function at the RNA level. Transcriptome and genome-wide binding profiles in embryonic mesoderm and Drosophila cells indicate that Ubx regulates mRNA expression and splicing to promote distinct functions in defined cellular contexts. Ubx modulates splicing via its DNA-binding domain, the Homeodomain (HD). Our results demonstrate a new RNA-binding ability of Ubx in cells and in vitro. Notably, the N51 amino acid of the HD, which mediates Ubx-DNA interaction, is non-essential for Ubx-RNA interaction in vitro but is required in vivo. We find that the N51 amino acid is necessary to mediate interaction between Ubx and the active form of the RNA Polymerase II (Pol II S2Phos) in Drosophila cells. By combining molecular and imaging approaches, our results reveal that Ubx mediates elongation-coupled splicing via a dynamic interplay with active Pol II and chromatin binding. Overall, our work uncovered a novel role of the Hox TFs at the mRNA regulatory layer. This could be an essential function for other classes of TFs to control cell diversity.

sentences: LiftPose3D, a deep learning-based approach for transforming 2D to 3D pose in laboratory animals Markerless 3D pose estimation has become an indispensable tool for kinematic studies of laboratory animals. Most current methods recover 3D pose by multi-view triangulation of deep network-based 2D pose estimates. However, triangulation requires multiple, synchronized cameras and elaborate calibration protocols that hinder its widespread adoption in laboratory studies. Here, we describe LiftPose3D, a deep network-based method that overcomes these barriers by reconstructing 3D poses from a single 2D camera view. We illustrate LiftPose3Ds versatility by applying it to multiple experimental systems using flies, mice, rats, and macaque monkeys and in circumstances where 3D triangulation is impractical or impossible. Our framework achieves accurate lifting for stereotyped and non-stereotyped behaviors from different camera angles. Thus, LiftPose3D permits high-quality 3D pose estimation in the absence of complex camera arrays, tedious calibration procedures, and despite occluded body parts in freely behaving animals.

sentences: Transposable element activity in the transcriptomic analysis of mouse pancreatic tumors Transposable elements (TEs) are middle-repeated DNA sequences that can move along chromosomes using internal coding and regulatory regions. By their ability to move and because they are repeated, TEs can promote mutations. Especially they can alter the expression pattern of neighboring genes and have been shown to be involved in the mammalian regulatory network evolution. Human and mouse share more than 95% of their genomes and are affected by comparable diseases, which makes the mouse a perfect model in cancer research. However not much investigation concerning the mouse TE content has been made on this topics. In human cancer condition, a global activation of TEs can been observed which may ask the question of their impact on neighboring gene functioning. In this work, we used RNA sequences of highly aggressive pancreatic tumors from mouse to analyze the gene and TE deregulation happening in this condition compared to pancreas from healthy animals. Our results show that several TE families are deregulated and that the presence of TEs is associated with the expression divergence of genes in the tumor condition. These results illustrate the potential role of TEs in the global deregulation at work in the cancer cells.

sentences: Assessment of CAR-T mediated and targeted cytotoxicity in 3D microfluidic TBNC co-culture models for combination therapy Chimeric antigen receptor (CAR)-T cell therapy is efficacious against many hematological malignancies; however, their therapeutic application to treat solid tumors presents further challenges. A better understanding of how the solid tumor microenvironment (TME) impacts CAR-T anti-tumor effects would enable the selection of effective combination therapies to decipher the optimal course of treatment for patients and to better engineer CAR-Ts. Classical 2D in vitro models do not provide sufficient recapitulation of the native human TME, and in vivo models, such as patient-derived xenografts, are costly, complex and labor intensive. Here, we present a novel 3D, miniaturized assay for the evaluation of EGFR-targeted CAR-T cell cytotoxicity and specificity on tumor-stroma triple-negative breast cancer models in microfluidic devices. CAR-T cells were shown to home towards EGFR-expressing cancer cells to elicit a cytotoxic effect, whilst leaving low EGFR-expressing fibroblasts viable, an effect which was enhanced through combination anti-PD-L1 therapy and carboplatin chemotherapy. Hence, we propose this proof-of-concept immunoassay as a future preclinical screening tool for the development of novel immunotherapeutics and for use in personalized medicine.

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Expression of the F0F1 ATP synthase inhibitor Atp{Theta} depends on small basic DNA-binding proteins and differential mRNA stability F0F1 ATP synthases produce ATP, the universal biological energy source. ATP synthase complexes on cyanobacterial thylakoid membranes use proton gradients generated either by photosynthesis or respiration. Atp{Theta} is an ATP synthase regulator in cyanobacteria which is encoded by the gene atpT. Atp{Theta} inhibits the hydrolysis of ATP (reverse reaction) that otherwise would occur under unfavorable conditions. In the cyanobacterium Synechocystis sp. PCC 6803, Atp{Theta} is expressed maximum in darkness but at very low levels under optimum phototrophic growth conditions or in the presence of glucose. DNA coimmunoprecipitation experiments followed by mass spectrometry identified the binding of the two transcriptional regulators cyAbrB1 and cyAbrB2 to the promoter and the histone-like protein HU to the 5UTR of atpT. Analyses of nucleotide substitutions in the promoter and GFP reporter assays identified a functionally relevant sequence motif resembling the HLR1 element bound by the RpaB transcription factor. Electrophoretic mobility shift assays confirmed interaction of cyAbrB1, cyAbrB2 and RpaB with the promoter DNA. However, overall the effect of transcriptional regulation was comparatively low. In contrast, atpT transcript stabilities differed dramatically, half-lives were 1.6 min in the light, 33 min in the dark and substantial changes were observed if glucose or DCMU were added. These findings show that basic transcriptional control of atpT involves nucleoid-associated DNA-binding proteins, positive regulation through RpaB, while the major effect on the condition-dependent regulation of atpT expression is mediated by controlling mRNA stability, which is related to the cellular redox and energy status.

IMPORTANCEF0F1 ATP synthases are protein complexes that produce ATP, the universal biological energy source in all kinds of organisms. Under unfavorable conditions, ATP synthases can operate in a futile reverse reaction, pumping protons while ATP is used up. Cyanobacteria perform plant-like photosynthesis but they cannot use the same mechanism as plants that inhibit chloroplast ATP synthases entirely during the night because respiratory and photosynthetic complexes are both located in the same membrane system. Atp{Theta} is a small peptide inhibitor of the reverse ATPase function in cyanobacteria encoded by the gene atpT. The production of Atp{Theta} is highly regulated to ensure that it is only synthetized when it is needed. In the here presented work we found that three transcription factors contribute to the regulation of atpT expression. However, we identified the control of mRNA stability as the major regulatory process governing atpT expression. Thus, it is the interplay between transcriptional and posttranscriptional regulation that position the Atp{Theta}-based inhibitory mechanism within the context of the cellular redox and energy balance.

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Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading Xist RNA directs the process of X-chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, enabling us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation, and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a distinct function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique dynamic properties of Xist RNA.

One Sentence SummaryVisualizing Xist RNA dynamics in single cells during X chromosome inactivation

sentences: The SARS-CoV-2 protein NSP2 impairs the microRNA-induced silencing capacity of human cells The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19. Given the absence of effective treatments against SARS-CoV-2, there is an urgent need for a molecular understanding of how the virus influences the machineries of the host cell. The SARS-CoV-2 generates 16 Non-Structural Proteins (NSPs) through proteolytic cleavage of a large precursor protein. In the present study, we focused our attention on the SARS-CoV-2 protein NSP2, whose role in the viral pathogenicity is poorly understood. Recent proteomic studies shed light on the capacity of NSP2 to bind the 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression in human cells. In order to gain a better understanding of the function of NSP2, we attempted to identify the molecular basis of its interaction with 4EHP-GIGYF2. Our data demonstrate that NSP2 physically associates with the endogenous 4EHP-GIGYF2 complex in the cytoplasm. Using co-immunoprecipitation and in vitro interaction assays, we identified both 4EHP and a central segment in GIGYF2 as binding sites for NSP2. We also provide functional evidence that NSP2 impairs the function of GIGYF2 in mediating mRNA silencing using reporter-based assays, thus leading to a reduced activity of microRNAs. Altogether, these data reveal the profound impact of NSP2 on the post-transcriptional silencing of gene expression in human cells, pointing out 4EHP-GIGYF2 targeting as a possible strategy of SARS-CoV-2 to take over the silencing machinery and to suppress host defenses.

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The ability of Pseudomonas aeruginosa to adopt a Small Colony Variant (SCV) phenotype is conserved, and not restricted to clinical isolates A subpopulation of Small Colony Variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolated from cystic fibrosis (CF) lungs biofilms. SCVs have almost exclusively been reported from infected hosts, essentially CF individuals or, by extension, from laboratory cultivation of strains originated from infected hosts. We previously reported the identification of P. aeruginosa SCVs emerging from a non-clinical strain and displaying features shared with clinical SCVs. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to, under laboratory culture conditions, spontaneously adopt a SCV-like smaller alternative morphotype distinguishable from the ancestral parent strain. Unexpectedly, we found that all the P. aeruginosa strains tested have the ability to adopt a SCV morphotype, regardless of their origin. Based on the phenotypes already described for SCVs, the SCV-like morphotypes obtained were clustered in two groups displaying various phenotypic profiles, including one characteristic of already described SCVs. We conclude that the ability to switch to a SCV phenotype is a conserved feature in Pseudomonas aeruginosa.

IMPORTANCEP. aeruginosa is an opportunistic pathogen that thrives in many environments. It is significant public health concern, notably because it is the most prevalent pathogen found in the lungs of people with cystic fibrosis (CF). In infected hosts, its persistence is believed to be related to the emergence of an alternative small colony variant (SCV) phenotype. By reporting the distribution of P. aeruginosa SCVs in various non-clinical environments, this work contributes to understanding a conserved adaptation mechanism used by P. aeruginosa to rapidly adapt in all environments. Counteraction of this strategy could prevent P. aeruginosa persistent infection in the future.

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Explicit expression of mesophyll conductance in the traditional leaf photosynthesis-transpiration coupled model and its physiological significances O_LIAlmost all terrestrial biosphere models (TBMs) still assume infinite mesophyll conductance (gm) to estimate photosynthesis and transpiration. This assumption has caused low accuracy of TBMs to predict leaf gas exchange under certain conditions.
C_LIO_LIIn this study, we developed a photosynthesis-transpiration coupled model that explicitly considers gm and designed an optimized parameterization solution through evaluating four different gm estimation methods in 19 C3 species at 31 experimental treatments.
C_LIO_LIResults indicated that temperature responses of the maximum carboxylation rate (Fcmax) and the electron transport rate (Jmax) estimated by fusing the Bayesian retrieval algorithm and the Sharkey online calculator together with gm temperature response estimated by fusing the chlorophyll fluorescence-gas exchange method and anatomy method predicted leaf gas exchange more accurately. The gm temperature response exhibited activation energy ({Delta}Ha) of 63.13 {+/-} 36.89 kJ mol-1 and entropy ({Delta}S) of 654.49 {+/-} 11.36 J K-1 mol-1. The gm optimal temperature (Topt_gm) explained 58% of variations in photosynthesis optimal temperature (ToptA). The gm explicit expression has equally important effects on photosynthesis and transpiration estimations.
C_LIO_LIResults advanced understandings of better representation of plant photosynthesis and transpiration in TBMs.
C_LI

sentences: Highly efficient healing of critical sized articular cartilage defect in situ using a chemically nucleoside-modified mRNA-enhanced cell therapy Critical sized cartilage defects heal poorly and MSC-based therapies holds promise functional cartilage regeneration either used alone or in combination with growth factors. However, Recombinant protein growth factors were proven to have minimal benefits while to have adverse side effects and high cost. Nonviral mRNA delivery provides a promising, alternative approach to delivering therapeutic proteins within defect lesion for an extended period of time. Despite successful therapeutic outcome in bone and other vascularized tissues, the therapeutic application of mRNA in poorly vascularized tissues such as cartilage is still facing many challenges and rarely studied. We report here using chemically modified messenger RNA encoding TGF-{beta}3(TGF-{beta}3 cmRNA) to enhance the therapeutic efficacy of BMSCs to efficient repair of cartilage defect. Local administration of TGF-{beta}3 cmRNA enhanced BMSCs therapy restored critical-sized cartilage defects in situ in a rat model within 6 weeks with structural and molecular markers similar to its nature counterparts. In addition, the development of osteoarthritis caused by cartilage damage was prevented by this mRNA-enhanced BMSCs therapy evidenced by minimal late-stage OA pharmacological presentations. This novel mRNA enhanced-MSC technology extend the development of new therapeutic approaches for treating functional cartilage repair.

sentences: Shelterin components modulate nucleic acids condensation and phase-separation Telomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, upon changes in their protein composition, telomeres can cluster to form telomere-induced-foci (TIFs) or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.

sentences: Connexin Hemichannels with Prostaglandin Release in Anabolic Function of Bone to Mechanical Loading Mechanical stimulation, such as physical exercise, is essential for bone formation and health. Here, we demonstrate the critical role of osteocytic Cx43 hemichannels in anabolic function of bone in response to mechanical loading. Two transgenic mouse models, R76W and {Delta}130-136, expressing dominant-negative Cx43 mutants in osteocytes were adopted. Mechanical loading of tibial bone increased cortical bone mass and mechanical properties in wild-type and gap junction-impaired R76W mice through increased PGE2, endosteal osteoblast activity, and decreased sclerostin. These anabolic responses were impeded in gap junction/hemichannel-impaired {Delta}130-136 mice and accompanied by increased endosteal osteoclast activity. Specific inhibition of Cx43 hemichannels by Cx43(M1) antibody suppressed PGE2 secretion and impeded loading-induced endosteal osteoblast activity, bone formation and anabolic gene expression. PGE2 administration rescued the osteogenic response to mechanical loading impeded by impaired hemichannels. Together, osteocytic Cx43 hemichannels could be a potential new therapeutic target for treating bone loss and osteoporosis.

sentences: Loss of heterochromatin at endogenous retroviruses creates competition for transcription factor binding The mammalian genome is partitioned into active and inactive regions, broadly termed euchromatin and heterochromatin, respectively. The majority of heterochromatin consists of repetitive elements, including endogenous retroviruses (ERVs). ERVs are enriched in regulatory elements containing transcription factor (TF) binding sites with individual families containing hundreds to thousands of distinct copies scattered throughout the genome. We hypothesized that epigenetic derepression of ERVs (such as that observed during early development) may alter the stoichiometry between TFs and their euchromatic target sites, with ERVs effectively competing for these factors. To test this, we modeled acute heterochromatin loss using inducible deletion of the co-repressor KAP1 in mouse embryonic stem cells (ESCs). Upon KAP1 deletion, we observe clear reductions in chromatin accessibility, histone acetylation, and TF binding at euchromatic regions. To directly test the concept of global binding site competition, we designed exogenous binding site arrays (EBSAs) to introduce upwards of 1500 copies of the OCT4 TF binding motif into ESCs. OCT4 EBSAs specifically reduce chromatin accessibility at POU family motifs and result in reduced transcription of the pluripotency machinery with subsequent differentiation. Overall, these data support a model in which heterochromatin at ERVs promotes euchromatic TF binding and transcriptional homoeostasis. We propose that regulated ERV derepression during pre-implantation may serve as a developmental siphon to weaken the robustness of ongoing transcription programs in favor of the plasticity required for cell fate specification.

sentences: Synaptic Mechanisms of Top-Down Control by The Auditory Cortico-Collicular Pathway Corticofugal projections to evolutionarily ancient, sub-cortical structures are ubiquitous across mammalian sensory systems. These "descending" pathways enable the neocortex to control ascending sensory representations in a predictive or feedback manner, but the underlying cellular mechanisms are poorly understood. Here we combine optogenetic approaches with in vivo and in vitro patch-clamp electrophysiology to study the projection from auditory cortex to the inferior colliculus (IC), a major descending auditory pathway that controls IC neuron feature selectivity, plasticity and auditory perceptual learning. Although individual auditory cortico-collicular synapses were generally weak, IC neurons often integrated inputs from multiple corticofugal axons that generated reliable, tonic depolarizations even during prolonged presynaptic activity. Latency measurements in vivo showed that descending signals reach the IC within 30 ms of sound onset, which in IC neurons corresponded to the peak of synaptic depolarizations evoked by short sounds. Activating ascending and descending pathways at latencies expected in vivo caused a NMDA receptor dependent, supra-linear EPSP summation, indicating that descending signals can non-linearly amplify IC neurons moment-to-moment acoustic responses. Our results shed light upon the synaptic bases of descending sensory control, and imply that heterosynaptic cooperativity contributes to the auditory cortico-collicular pathways role in plasticity and perceptual learning.

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Refining Bulk Segregant Analyses: Ontology-Mediated Discovery of Flowering Time Genes in Brassica oleracea AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSBulk segregant analysis (BSA) can help identify quantitative trait loci (QTLs), but this may result in substantial bycatch of functionally irrelevant genes. Here we develop a Gene Ontology-mediated approach to zoom in on specific markers implicated in flowering time from among QTLs identified by BSA of the giant woody Jersey kale phenotyped in four bulks of flowering onset.

ResultsOur BSA yielded tens of thousands of candidate genes. We reduced this by two orders of magnitude by focusing on genes annotated with terms contained within relevant subgraphs of the Gene Ontology. A further enrichment test led to the pathway for circadian rhythm in plants. The genes that enriched this pathway are attested from previous research as regulating flowering time. Some of these genes were also identified as having functionally significant variation compared to Arabidopsis

ConclusionsWe validated and confirmed our ontology-mediated results through a more targeted, homology-based approach. However, our ontology-mediated approach produced additional genes of putative importance, showing that the approach aids in exploration and discovery. We view our method as potentially applicable to the study of other complex traits and therefore make our workflows available as open-source code and a reusable Docker container.

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Clustering single cell CITE-seq data with a canonical correlation based deep learning method Single cell sequencing examines the sequence information from individual cells with optimized next generation sequencing (NGS) technologies. It provides researchers a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment. CITE-seq, or Cellular Indexing of Transcriptomes and Epitopes by sequencing, is one of the latest innovations in the domain of single cell sequencing. It enables researchers to simultaneously capture RNA and surface protein expression on the same cells so that we can correlate the two data types, identify biomarkers and better characterize cell phenotypes. Although multi-omics sequencing technologies developed rapidly, data analyzing methods tailored for multiomics sequencing data are lacking. Several serious problems have to be faced. An important one is how to integrate the information from different modalities, namely scRNA and protein data, efficiently.

In this paper, we introduce a canonical correlation based deep learning method called scCTClust for clustering analysis over CITE-seq data. We impute and extract the characteristics of the high dimensional RNA part of data with a ZINB model-based autoencoder. A t-kernel distance is introduced to measure the similarity between query cell and cluster centroids. And the protein data rectifies the feature extraction of scRNA data in a canonical correlation analysis(CCA) way. Extensive data experiments shows that scCTClust can precisely recover the dropout values for RNA sequencing data and extract authentic information from both modalities, getting a much better clustering result than state-of-the-art methods, no matter single-omic or multi-omics clustering algorithms.

sentences: Mechanism of the Switch from NO to H2O2 in Endothelium-Dependent Vasodilation in Diabetes Coronary microvascular dysfunction is prevalent among diabetics and is correlated with cardiac mortality. Compromised endothelial-dependent dilation (EDD) is an early event in the progression of diabetes, but the mechanisms remain incompletely understood. Nitric oxide (NO) is the major endothelium-dependent vasodilatory metabolite in the healthy coronary circulation, but switches to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a major risk factor for CAD we hypothesized that a similar switch from NO-to-H2O2 occurs in diabetes. Methods: Vasodilation was measured ex vivo in isolated coronary arteries from wild type (WT) and microRNA-21 (miR-21) null mice fed chow or high fat and sugar diet, and LepR null (db/db) mice using myography. Myocardial blood flow (MBF), blood pressure, and heart rate were measured in vivo using contrast echocardiography and a solid-state pressure sensor catheter. RNA from coronary arteries, endothelial cells and hearts were analyzed via qPCR for gene expression and protein expression was assessed via Western-Blot analyses. Superoxide was detected via electron paramagnetic resonance (EPR). Results: 1) Ex vivo coronary EDD and in vivo MBF was impaired in diabetes. 2) L-NAME (NO-synthase inhibitor) inhibited ex vivo coronary EDD and in vivo MBF in WT, while PEG-catalase (H2O2 scavenger) inhibited diabetic EDD ex vivo and MBF in vivo. 5) miR-21 deficiency blocked the NO-to-H2O2 switch and prevented diabetic vasodilation impairments. 6) Diabetic mice displayed increased serum NO and H2O2, upregulated mRNA expression of Sod1, Sod2, iNos, and Cav-1, and downregulated Pgc-1. Deficiency of miR-21 reversed these changes. 7) miR-21 deficiency increased PGC1, PPAR and eNOS protein and reduced detection of endothelial superoxide. Conclusions: Diabetics exhibit an NO-to-H2O2 switch in the mediator of EDD coronary dilation, which contributed to microvascular dysfunction and is mediated by miR-21. This study represents the first mouse model recapitulating the NO-to-H2O2 switch seen in CAD patients.

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

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Metabolic and Proteomic Defects in Human Hypertrophic Cardiomyopathy RationaleImpaired cardiac energetics in hypertrophic cardiomyopathy (HCM) is thought to result from increased ATP utilization at the sarcomere and is believed to be central to pathophysiology. However, the precise defects in cardiac metabolism and substrate availability in human HCM have not been defined.

ObjectiveThe purpose of this study is to define major disease pathways and determine the pool sizes of intermediary metabolites in human HCM.

Methods and ResultsWe conducted paired proteomic and metabolomic analyses of septal myectomy samples from patients with HCM and compared results to non-failing control human hearts. Increased abundance of extracellular matrix and intermediate filament / Z-disc proteins, and decreased abundance of proteins involved in fatty acid oxidation and cardiac energetics was evident in HCM compared to controls. Acyl carnitines, byproducts of fatty acid oxidation, were markedly depleted in HCM samples. Conversely, the ketone body 3-hydroxybutyrate, lactate, and the 3 branched chain amino acids, were all significantly increased in HCM hearts, suggesting that they may serve as alternate fuel sources for the production of ATP. ATP, nicotinamide adenine dinucleotide (NADH), NADP and NADPH, and acetyl CoA were also severely depleted in HCM hearts. Based on measurements from human skinned muscle fibers, the magnitude of observed reduction in ATP content in the HCM hearts would be expected to decrease the rate of cross-bridge detachment, implying a direct effect of energy depletion on myofilament function that could contribute to diastolic dysfunction.

ConclusionsHCM hearts display profound deficits in cardiac energetics, marked by depletion of fatty acid derivatives and compensatory increases in other metabolites that could serve as alternate fuel sources. These results lend support to the paradigm that energy depletion contributes to the pathophysiology of HCM and also have important therapeutic implications for the future design of metabolic modulators to treat HCM.

sentences: TREM2 is thyroid hormone regulated making the TREM2 pathway druggable with ligands for thyroid hormone receptor Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. We report here that TREM2 is a thyroid hormone regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone. Both endogenous thyroid hormone and sobetirome, a synthetic thyroid hormone agonist drug, suppress pro-inflammatory cytokine production from myeloid cells including macrophages that have been treated with the SARS-CoV-2 spike protein which produces a strong, pro-inflammatory phenotype. Thyroid hormone agonism was also found to induce phagocytic behavior in microglia, a phenotype consistent with activation of the TREM2 pathway. The thyroid hormone antagonist NH-3 blocks the anti-inflammatory effects of thyroid hormone agonists and suppresses microglia phagocytosis. Finally, in a murine experimental autoimmune encephalomyelitis (EAE) multiple sclerosis model, treatment with Sob-AM2, a CNS-penetrating sobetirome prodrug, results in increased Trem2 expression in disease lesion resident myeloid cells which correlates with therapeutic benefit in the EAE clinical score and reduced damage to myelin. Our findings represent the first report of endocrine regulation of TREM2 and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small molecule therapeutic agents.

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Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations.

sentences: Host-specific epibiomes of distinct Acropora cervicornis genotypes persist after field transplantation Microbiome studies across taxa have established the influence of host genotype on microbial recruitment and maintenance. However, research exploring host-specific epibionts in scleractinian corals is scant and the influence of intraspecific differences across environments remains unclear. Here, we studied the epibiome of ten Acropora cervicornis genotypes to investigate the relative roles of host genotype and environment in structuring the epibiome. Coral mucus was sampled in a common garden nursery from replicate ramets of distinct genotypes (T0). Coral fragment replicates (n=3) of each genotype were then transplanted to nine different field sites in the Lower Florida Keys and mucus was again sampled one year later from surviving ramets (T12). 16S rRNA amplicon sequencing was used to assess microbial composition, richness, and beta-diversity. The most abundant and consistent amplicon sequencing variants (ASVs) in all samples belonged to Fokiniaceae (MD3-55 genus) and Cyanobacteria (Synechococccus). The abundances of these bacterial taxa varied consistently between genotypes whereas neither the composition nor taxonomic abundance were significantly different among field sites. Interestingly, several high MD3-55 hosting genotypes showed rapid diversification and an increase in MD3-55 following transplantation. Overall, our results indicate healthy A. cervicornis genotypes retain distinct epibiome signatures through time, suggesting a strong host component. Lastly, our results show that differences in MD3-55 abundances can be consistently detected in the epibiome of distinct host-genotypes of A. cervicornis. As this organism (sensu Aquarickettsia rohweri) has been implicated as a marker of disease resistance, this finding reinforces the potential use of microbial indicators in reef restoration efforts via non-invasive surface/mucus sampling.

sentences: Nematodes can survive in a suspended form of life for indefinite time When environmental conditions are unfavorable, such as the complete absence of water or oxygen, high temperature, freezing or extreme salinity, some organisms can enter suspended animation (cryptobiosis)1. This reversible transition is preceded by execution of complex genetic and biochemical programs (preconditioning)2,3,4. Under laboratory conditions, however, animals have only been maintained in a viable cryptobiotic state for a short time. Here we show that desiccation followed by freezing allows C. elegans dauer larvae to retain full viability over very long periods (around 500 days). Consistent with this finding, recently nematode individuals have been reanimated from the Siberian permafrost5, that according to precise radiocarbon dating shows that they remained in cryptobiosis since the late Pleistocene, for about 46,000 years. Phylogenomic inference based on our high-quality genome assembly and morphological analysis demonstrate that these nematodes belong to a novel parthenogenetic species, which we named Panagrolaimus kolymaensis. Genome analysis revealed that the core of the molecular toolkit for cryptobiosis in P. kolymaensis and C. elegans is orthologous. To survive desiccation and freezing under laboratory conditions these two species display similar biochemical responses. Thus, nematodes possess extraordinarily robust adaptive mechanisms that potentially allow them to remain in suspended animation over geological time scales.

sentences: Pathotyping of Newcastle disease virus: A novel single BsaHI digestion method of detection and differentiation of avirulent strains (lentogenic and mesogenic vaccine strains) from virulent virus We provide a novel single restriction enzyme (RE) (BsaHI) digestion approach for detecting distinct pathotypes of the Newcastle disease virus (NDV). After scanning 4000 F gene nucleotide sequences in the NCBI database, a single RE (BsaHI) digesting site was discovered in the cleavage site. APMV-I "F gene" Class II specific primer-based reverse transcriptase PCR (RT-PCR) was utilized to amplify a 535 bp fragment, which was then digested with a single RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produce 189 and 346 bp fragments, respectively, but the result in velogenic strains remains undigested with 535 bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique had the potential to distinguish between avirulent and virulent strains in a short space of time, making it valuable in NDV surveillance and monitoring research.

sentences: Raman microscopy-based quantification of the physical properties of intracellular lipids The physical properties of lipids, such as viscosity, are homeostatically maintained in cells and are intimately involved in physiological roles. Measurement of the physical properties of plasma membranes has been achieved primarily through chemical or genetically encoded fluorescent probes. However, as most probes target plasma membranes, physical properties of lipids in intracellular organelles, including lipid droplets (LDs) are yet to be analyzed. Here, we present a novel Raman microscopy-based approach for quantifying the physical properties of intracellular lipids under deuterium-labeled fatty acid treatment conditions. Focusing on the fact that Raman spectra of carbon-deuterium vibration are altered depending on the surrounding lipid species, we quantitatively represented the physical properties of lipids as the gauche/trans conformational ratio of the introduced labeled fatty acids, which can be used as an indicator of viscosity. Intracellular Raman imaging revealed that the gauche/trans ratio of cytosolic regions was robustly preserved against perturbations attempting to alter the lipid composition. This was likely due to LDs functioning as a buffer against excess gauche/trans ratio. Our novel approach enables the observation of the physical properties of organelle lipids, which is difficult to perform with conventional probes, and is useful for quantitative assessment of the subcellular lipid environment.

sentences: Slow oscillation-spindle coupling is negatively associated with emotional memoryformation following stress Both stress and sleep enhance emotional memory. They also interact, with the largest effect of sleep on emotional memory being seen when stress occurs shortly before or after encoding. Slow wave sleep (SWS) is critical for long-term episodic memory, facilitated by the temporal coupling of slow oscillations and sleep spindles. Prior work in humans has shown these associations for neutral information in non-stressed participants. Whether coupling interacts with stress to facilitate emotional memory formation is unknown. Here, we addressed this question by reanalyzing an existing dataset of 64 individuals. Participants underwent a psychosocial stressor (32) or comparable control (32) prior to the encoding of 150-line drawings of neutral, positive, and negative images. All participants slept overnight with polysomnography, before being given a surprise memory test the following day. In the stress group, time spent in SWS was positively correlated with memory for images of all valences. Results were driven by those who showed a high cortisol response to the stressor, compared to low responders. The amount of slow oscillation-spindle coupling during SWS was negatively associated with neutral and emotional memory in the stress group only. The association with emotional memory was significantly stronger than for neutral memory within the stress group. These results suggest that stress around the time of initial memory formation impacts the relationship between slow wave sleep and memory.

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A whole-cell recording database of neuromodulatory action in the adult neocortex BackgroundThe recent release of two large intracellular electrophysiological databases now allows high-dimensional systematic analysis of mechanisms of information processing in the neocortex. Here, to complement these efforts, we introduce a freely and publicly available database that provides a comparative insight into the role of various neuromodulatory transmitters in controlling neural information processing.

FindingsA database of in vitro whole-cell patch-clamp recordings from primary somatosensory and motor cortices (layers 2/3) of the adult mice (2-15 months old) from both sexes is introduced. A total of 464 current-clamp experiments from identified excitatory and inhibitory neurons are provided. Experiments include recordings with (i) Step-and-Hold protocol during which the current was transiently held at 10 steps, gradually increasing in amplitude, (ii) "Frozen Noise" injections that model the amplitude and time-varying nature of synaptic inputs to a neuron in biological networks. All experiments follow a within neuron across drug design which includes a vehicle control and a modulation of one of the following targets in the same neuron: dopamine and its receptors D1R, D2R, serotonin 5HT1f receptor, norepinephrine Alpha1, and acetylcholine M1 receptors.

ConclusionsThis dataset is the first to provide a systematic and comparative insight into the role of the selected neuromodulators in controlling cellular excitability. The data will help to mechanistically address how bottom-up information processing can be modulated, providing a reference for studying neural coding characteristics and revealing the contribution of neuromodulation to information processing.

sentences: A unified framework for measuring selection on cellular lineages and traits Intracellular states probed by gene expression profiles and metabolic activities are intrinsically noisy, causing phenotypic variations among cellular lineages. Understanding the adaptive and evolutionary roles of such variations requires clarifying their linkage to population growth rates. Extending a cell lineage statistics framework, here we show that a populations growth rate can be expanded by fitness cumulants of any cell lineage trait. The expansion enables quantifying the contribution of each fitness cumulant, such as variance and skewness, to population growth. We introduce a function that contains all the essential information of cell lineage statistics, including mean lineage fitness and selection strength. We reveal a relation between fitness heterogeneity and population growth rate response to perturbation. We apply the framework to experimental cell lineage data from bacteria to mammalian cells, revealing that third or higher-order cumulants contributions are negligible under constant growth conditions but could be significant in regrowing processes from growth-arrested conditions. Furthermore, we identify cellular populations in which selection leads to an increase of fitness variance among lineages. The framework assumes no particular growth models or environmental conditions, and is thus applicable to various biological phenomena for which phenotypic heterogeneity and cellular proliferation are important.

sentences: Neuropilin-1 Assists SARS-CoV-2 Infection by Stimulating the Separation of Spike Protein Domains S1 and S2 The cell surface receptor Neuropilin-1 (Nrp1) was recently identified as a host factor for SARS-CoV-2 entry. As the Spike protein of SARS-CoV-2 is cleaved into the S1 and the S2 domain by furin protease, Nrp1 binds to the newly created C-terminal RRAR amino acid sequence of the S1 domain. In this study, we model the association of a Nrp1 (a2-b1-b2) protein with the Spike protein computationally and analyze the topological constraints in the SARS-CoV-2 Spike protein for binding with Nrp1 and ACE2. Importantly, we study the exit mechanism of S2 from the S1 domain with the assistance of ACE2 as well as Nrp1 by molecular dynamics pulling simulations. In the presence of Nrp1, by binding the S1 more strongly to the host membrane, there is a high probability of S2 being pulled out, rather than S1 being stretched. Thus, Nrp1 binding could stimulate the exit of S2 from the S1 domain, which will likely increase virus infectivity as the liberated S2 domain mediates the fusion of virus and host membranes. Understanding of such a Nrp1-assisted viral infection opens the gate for the generation of protein-protein inhibitors, such as antibodies, which could attenuate the infection mechanism and protect certain cells in a future combination therapy.

sentences: Cortical actin flow activates an alpha-catenin clutch to assemble adherens junctions Adherens junctions (AJs) fundamentally mediate cell-cell adhesion, yet the mechanisms that determine where or when AJs assemble are not understood. Here we reveal a mechanosensitive clutch that initiates AJ assembly. Before cell-cell contact, -catenin couples surface E-cadherin complexes to retrograde flow of the actin cortex. Cortical flows with opposed orientations persist after contact, applying tension to -catenin within trans-ligated cadherin complexes. Tension unfolds the -catenin actin-binding domain (ABD), which is expected to mediate a catch bond with F-actin. However, catch bond behaviour is not sufficient for AJ assembly in a molecular clutch model. Instead, it is also necessary for the activated ABD to promote cis-clustering of E-cadherin molecules by bundling F-actin. Thus, this -catenin clutch transduces the mechanical signal of cortical flow to assemble AJs.

sentences: Extreme parallel evolution of flagellar motility is facilitated by synonymous sequence Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work we reveal that a powerfully deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observed this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determined that this evolutionary disparity was owed to just 6 synonymous variations within the ntrB locus, which we demonstrated by swapping the sites and observing that we were able to both break (>95% to 0% in AR2) and build (0% to 80% in Pf0-2x) a powerfully deterministic mutational hotspot. Our work reveals a fundamental role for silent genetic variation in determining adaptive outcomes.

sentences: Cloud-enabled Fiji for reproducible, integrated and modular image processing Modern biomedical image analyses workflows contain multiple computational processing tasks giving rise to problems in reproducibility. In addition, image datasets can span both spatial and temporal dimensions, with additional channels for fluorescence and other data, resulting in datasets that are too large to be processed locally on a laptop. For omics analyses, software containers have been shown to enhance reproducibility, facilitate installation and provide access to scalable computational resources on the cloud. However, most image analyses contain steps that are graphical and interactive, features that are not supported by most omics execution engines. We present the containerized and cloud-enabled Biodepot-workflow-builder platform that supports graphics from software containers and has been extended for image analyses. We demonstrate the potential of our modular approach with multi-step workflows that incorporate the popular and open-source Fiji suite for image processing. One of our examples integrates fully interactive Fiji macros with Jupyter notebooks. Our second example illustrates how the complicated cloud setup of an computationally intensive process such as stitching 3D digital pathology datasets using BigStitcher can be automated and simplified. In both examples, users can leverage a form-based graphical interface to execute multi-step workflows with a single click, using the provided sample data and preset input parameters. Alternatively, users can interactively modify the image processing steps in the workflow, apply the workflows to their own data, change the input parameters and macros. By providing interactive graphics support to software containers, our modular platform supports reproducible image analysis workflows, simplified access to cloud resources for analysis of large datasets, and integrated workflows across imaging, genomics and transcriptomics data.

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Effects of arm swing amplitude and lower limb asymmetry on motor variability patterns during treadmill gait Motor variability is a fundamental feature of gait. Altered arm swing and lower limb asymmetry (LLA) may be contributing factors having been shown to affect the magnitude and dynamics of variability in spatiotemporal and trunk motion. However, the effects on lower limb joints remain unclear.

Full-body kinematics of 15 healthy young adults were recorded during treadmill walking using the Computer-Assisted Rehabilitation Environment system. Participants completed six trials, combining three arm swing (AS) amplitude (normal, active, held) and two LLA (symmetrical, asymmetrical) conditions. The mean standard deviation (meanSD), maximum Lyapunov exponent ({lambda}max), detrended fluctuation analysis scaling exponent of range of motion (DFA), and sample entropy (SaEn) were computed for tridimensional trunk, pelvis, and lower limb joint angles, and compared using repeated-measures ANOVAs.

Relative to normal AS, active AS increased meanSD of all joint angles, {lambda}max of frontal plane hip and ankle angles, and SaEn of sagittal plane ankle angles. Active AS, however, did not affect {lambda}max or SaEn of trunk or pelvis angles. LLA increased meanSD of sagittal plane joint angles, {lambda}max of Euclidean norm trunk angle and of lower limb joint angles, and SaEn of ankle dorsiflexion/ plantarflexion, but decreased SaEn of tridimensional trunk angles and hip rotation in the slower moving leg.

Alterations in lower limb variability with active AS and LLA suggest that young adults actively exploit their lower limb redundancies to maintain gait. This appears to preserve trunk stability and regularity during active AS but not during LLA.

sentences: The G2-phase enriched lncRNA SNHG26 is necessary for proper cell cycle progression and proliferation Long noncoding RNAs (lncRNAs) are involved in the regulation of cell cycle, although only a few have been functionally characterized. By combining RNA sequencing and ChIP sequencing of cell cycle synchronized HaCaT cells we have previously identified lncRNAs highly enriched for cell cycle functions. Based on a cyclic expression profile and an overall high correlation to histone 3 lysine 4 trimethylation (H3K4me3) and RNA polymerase II (Pol II) signals, the lncRNA SNHG26 was identified as a top candidate. In the present study we report that downregulation of SNHG26 affects mitochondrial stress, proliferation, cell cycle phase distribution, and gene expression in cis- and in trans, and that this effect is reversed by upregulation of SNHG26. We also find that the effect on cell cycle phase distribution is cell type specific and stable over time. Results indicate an oncogenic role of SNHG26, possibly by affecting cell cycle progression through the regulation of downstream MYC-responsive genes.

sentences: Implicitly Learned Higher Order Associations Differentiates Recent and Remote Retrieval of Temporal Order Memory Memory of an ordered sequence of distinct events requires encoding the temporal order as well as the intervals that separates these events. In this study, using order place association task where the animal learns to associate the location of the food pellet to the order of entry into the event arena, we probe the nature of temporal order memory in mice. In our task, individual trials, become distinct events, as the animal is trained to form unique association between entry order and a correct location. The inter-trial intervals (> 30 mins) are chosen deliberately to minimise the working memory contributions. We develop this paradigm initially using 4 order place associates and later extend it to 5 paired associates. Our results show that animals not only acquire these explicit (entry order to place) associations but also higher order associations that can only be inferred implicitly from the temporal order of these events. As an indicator of such higher order learning during the probe trail the mice exhibit predominantly prospective errors that declines proportionally with temporal distance. On the other hand, prior to acquiring the sequence the retrospective errors are dominant. Additionally, we also tested the nature of such acquisitions when temporal order CS is presented along with flavour as a compound stimulus comprising of order and flavour both simultaneously being paired with location. Results from these experiments indicate that the animal learns both order-place and flavour-place associations. Comparing with pure order place training, we find that the additional flavour in compound training did not interfere with the ability of the animals to acquire the order place associations. When tested remotely, pure order place associations could be retrieved only after a reminder training. Further higher order associations representing the temporal relationship between the events is markedly absent in the remote time.

sentences: Non-identifiability and the Blessings of Misspecification in Models of Molecular Fitness and Phylogeny Understanding the consequences of mutation for molecular fitness and function is a fundamental problem in biology. Recently, generative probabilistic models have emerged as a powerful tool for estimating fitness from evolutionary sequence data, with accuracy sufficient to predict both laboratory measurements of function and disease risk in humans, and to design novel functional proteins. Existing techniques rest on an assumed relationship between density estimation and fitness estimation, a relationship that we interrogate in this article. We prove that fitness is not identifiable from observational sequence data alone, placing fundamental limits on our ability to disentangle fitness landscapes from phylogenetic history. We show on real datasets that perfect density estimation in the limit of infinite data would, with high confidence, result in poor fitness estimation; current models perform accurate fitness estimation because of, not despite, misspecification. Our results challenge the conventional wisdom that bigger models trained on bigger datasets will inevitably lead to better fitness estimation, and suggest novel estimation strategies going forward.

sentences: Neutralizing antibody-independent immunity to SARS-CoV-2 in hamsters and hACE-2 transgenic mice immunized with a RBD/Nucleocapsid fusion protein The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFN{gamma} response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies. Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. Thus, the N protein should be considered to induce T-cell-based immunity to improve SARS-CoV-2 vaccines, and eventually to circumvent the immune scape by variants.

sentences: Fungal communities from geothermal soils in Yellowstone National Park Geothermal soils offer unique insight into the way extreme environmental factors shape communities of organisms. However, little is known about the fungi growing in these environments and in particular how localized steep abiotic gradients affect fungal diversity. We used metabarcoding to characterize soil fungi surrounding a hot spring-fed thermal creek with water up to 84 {degrees}C and pH 10 in Yellowstone National Park. We found a significant association between fungal communities and soil variable principal components, and we identify the key trends in co-varying soil variables that explain the variation in fungal community. Saprotrophic and ectomycorrhizal fungi community profiles followed, and were significantly associated with, different soil variable principal components, highlighting potential differences in the factors that structure these different fungal trophic guilds. In addition, in vitro growth experiments in four target fungal species revealed a wide range of tolerances to pH levels but not to heat. Overall, our results documenting turnover in fungal species within a few hundred meters suggest many co-varying environmental factors structure the diverse fungal communities found in the soils of Yellowstone National Park.

sentences: Towards Understanding Comprehensive Morphometric Changes and Its Correlation with Cognition and Exposure to Fighting in Active Professional Boxers Professional athletes exposed to repetitive head impacts are at increased risk for developing a progressive neurological syndrome known as traumatic encephalopathy syndrome and neuropathology seen on autopsy called chronic traumatic encephalopathy (CTE). The early identification of individuals at increased risk for CTE is important and the search for biomarkers is underway. In this study, we utilized data from a large cohort study to compare differences in regional brain volumes, cortical thickness, voxel-based morphometric (VBM)-derived measures, and graph-theoretical measures derived from large-scale topographical maps in active professional boxers. We compared the above morphometric measures between active professional boxers with low cognitive scores (impaired boxers) and active professional boxers with intact cognitive scores (nonimpaired boxers). The cognitive scores were evaluated through neuropsychological evaluation. As an exploratory analysis, we also examined the power of various machine-learning algorithms to identify impaired and nonimpaired boxers using both group-level regression-driven analysis and previously identified hypothesis-driven cortical thickness and volumetric measures. We found significant group-level differences between impaired and nonimpaired boxers in cortical thickness in a single brain region (right precuneus), differences in VBM-derived gray matter density encompassing the caudate, putamen, and thalamus; and white matter density encompassing the right paracentral lobule, but no differences in any graph-theoretical network properties. Additionally, we found that a priori hypothesis-driven T1-derived cortical thickness and volumetric analysis performed better than traditional regression-based analysis. Overall, this study suggests that neuroanatomical differences exist between impaired and nonimpaired active professional boxers, and that hypothesis-driven techniques are likely necessary to become reliable biomarkers.

sentences: CaV1 and CaV2 calcium channels mediate the release of distinct pools of synaptic vesicles Activation of voltage-gated calcium channels at synapses leads to local increases in calcium and the fusion of synaptic vesicles. However, presynaptic output will be determined by the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at C. elegans neuromuscular junctions, CaV2, and CaV1 mediate the release of two distinct pools of synaptic vesicles. Superresolution microscopy demonstrates that CaV2 channels are concentrated in densely packed clusters [~]300 nm in diameter with active zone proteins Neurexin, -Liprin, SYDE, ELKS, RIMB, -Catulin, and MAGI. The CaV2 channels mediate the fusion of vesicles docked within 100 nm of the dense projection and is colocalized with to the synaptic vesicle priming protein UNC-13L. By contrast, CaV1 channels are dispersed in the synaptic varicosity and are coupled to internal calcium stores via the ryanodine receptor. The CaV1 and ryanodine receptor mediate the fusion of vesicles docked broadly in the synaptic varicosity and are colocalized with the vesicle priming protein UNC-13S. These distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.

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Evaluating features of scientific conferences: A call for improvements Scientific conferences provide valuable opportunities for researchers across career stages and disciplines to present their latest work, and to network with their peers. These meetings have largely been held in-person with rapid proliferation in the number of meetings and attendees. Yet the format and quality of their organization lag behind what is possible and as a result, the current experience of attending conferences in many disciplines remains unchanged in many respects. We created a database of 270 national and international academic conferences held in-person during 2018-2019 in various disciplines and examined them for their features, costs and impact on the community. We found that many meetings could still be improved significantly in terms of diversity, inclusivity, promoting early career researcher (ECR) networking and career development, venue accessibility, and importantly, reducing the meetings carbon footprint. It is important to accelerate and mandate efforts to improve conferences so that researchers in all disciplines, in particular ECRs, consistently benefit from scientific gatherings, for years to come. We discuss a combination of approaches and recommendations to make conferences more modern, effective, equitable and intellectually productive for the research community and environmentally sustainable for our planet.

"They always say time changes things, but you actually have to change them yourself."

-- Andy Warhol

sentences: Optogenetic activation of the serotonergic neural circuit between dorsal raphe nucleus and the pre-Botzinger complex contributes to inhibition of the seizure-induced respiratory arrest in the DBA/1 mouse SUDEP model Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death among epilepsy patients, occurring even more frequently in cases with anti-epileptic drug resistance. However, the underlying mechanism of SUDEP remains elusive. Our previous study demonstrated that enhancement of serotonin (5-HT) synthesis by intraperitoneal (IP) injection of 5-hydroxytryptophan (5-HTP) significantly reduced the incidence of seizure-induced respiratory arrest (S-IRA) in a DBA/1 mouse SUDEP model. Given that the 5-HT2A receptor (5-HT2AR) plays an important role in mediating the respiration system in the brain, we hypothesized that 5-HT2AR plays a key role in S-IRA and SUDEP. To test this hypothesis, we examined whether the decreased incidence of S-IRA evoked by either acoustic stimulation or pentylenetetrazole (PTZ) injection following 5-HTP administration will be blocked by treatment with ketanserin (KET), a selective antagonist of 5HT2AR, in the DBA/1 mouse SUDEP model. We observed that the reduction in S-IRA by 5-HTP was significantly reversed by IP or intracerebroventricular injection of KET. Considering the localization of 5-HT2AR in the pre-Botzinger complex (PBC), which plays a key role in regulating respiratory rhythm, we next examined whether KET acts on 5-HT2AR in the PBC. To test this hypothesis, we activated the neural circuit between the dorsal raphe nucleus (DR) and PBC using optogenetics technology. We observed that stimulation of TPH2-ChETA-expressing neurons in the DR reduced the incidence of S-IRA evoked by PTZ, and this suppressant effect was significantly reversed by administration of KET in the bilateral PBC with no changes in electroencephalogram activity. The neural circuit between the DR and PBC was confirmed by injection of cholera toxin subunit B555 (CTB-555), a nerve tracer, in the DR or PBC separately. Calcium signaling evoked by PTZ within neurons of the PBC during seizures was significantly reduced by photostimulation of the DR. Taken together, our findings suggest that 5-HT2AR plays a critical role in regulating S-IRA and targeting the serotonergic neural circuit between the DR and PBC is a promising approach to preventing SUDEP.

sentences: An intranasal nanoparticle STING agonist has broad protective immunity against respiratory viruses and variants Respiratory viral infections, especially Influenza (endemic) or SARS-CoV-2 (pandemic since 2020), cause morbidity and mortality worldwide. Despite remarkable progress in the development and deployment of vaccines, they are clearly impacted by the rapid emergence of viral variants. The development of an off-the-shelf, effective, safe, and low-cost drug for prophylaxis against respiratory viral infections is a major unmet medical need. Here, we developed NanoSTING, a liposomally encapsulated formulation of the endogenous STING agonist, 2-3 cGAMP, to function as an immunoantiviral. NanoSTING rapidly activates the bodys innate immune system to facilitate a broad-spectrum antiviral response against SARS-CoV-2 and influenza variants in hamsters and mice. We demonstrate that a single intranasal dose of NanoSTING can: (1) treat infections throughout the respiratory system and minimize clinical symptoms, (2) protect against highly pathogenic strains of SARS-CoV-2 (alpha and delta), (3) provide durable protection against reinfection from the same strains without the need for retreatment, (4) prevent transmission of the highly infectious SARS-CoV-2 Omicron strain, and (5) provide protection against both oseltamivir-sensitive and resistant strains of influenza. Mechanistically, administration of NanoSTING rapidly upregulated interferon-stimulated and antiviral pathways in both the nasal turbinates and lung. Our results support using NanoSTING as a thermostable, immunoantiviral with broad-spectrum antiviral properties making it appealing as a therapeutic for prophylactic or early post-exposure treatment.

sentences: Geometry of inter-areal interactions in mouse visual cortex The response of a set of neurons in an area is the result of the sensory input, the interaction of the neurons within the area as well as the long range interactions between areas. We aimed to study the relation between interactions among multiple areas, and if they are fixed or dynamic. The structural connectivity provides a substrate for these interactions, but anatomical connectivity is not known in sufficient detail and it only gives us a static picture. Using the Allen Brain Observatory Visual Coding Neuropixels dataset, which includes simultaneous recordings of spiking activity from up to 6 hierarchically organized mouse cortical visual areas, we estimate the functional connectivity between neurons using a linear model of responses to flashed static grating stimuli. We characterize functional connectivity between populations via interaction subspaces. We find that distinct subspaces of a source area mediate interactions with distinct target areas, supporting the notion that cortical areas use distinct channels to communicate. Most importantly, using a piecewise linear model for activity within each trial, we find that these interactions evolve dynamically over tens of milliseconds following a stimulus presentation. Inter-areal subspaces become more aligned with the intra-areal subspaces during epochs in which a feedforward wave of activity propagates through visual cortical areas. When the short-term dynamics are averaged over, we find that the interaction subspaces are stable over multiple stimulus blocks. These findings have important implications for understanding how information flows through biological neural networks composed of interconnected modules, each of which may have a distinct functional specialization.

sentences: Removal Of Environmental Pollutants (Lead, Chromium And Cadmium) Using Root And Leaf Tissues Of Indian Mustard, Rice And Wheat Plants The environment is polluted day by day and it is very much expensive to remediate the environmental pollutants by physicochemical process. Therefore, using plants as a process to remediate pollutants is essential. In this study, phytoremediation of root and leaf tissues of Indian mustard (Brassica juncea), rice (Oryza sativa) and wheat (Triticum aestivum) against three most environment pollutants viz. lead (Pb), chromium (Cr) and cadmium (Cd) of Buriganga riverbank soil of Dhaka city, Bangladesh were assessed. The highest amount of Pb was found in the leaf (11.6755 {+/-}1.9860 mg/kg dry weight) and root (51.4251 {+/-}5.0320 mg/kg dry weight) of B. juncea. However, the highest amount of Cr was found in the leaf (5.9871{+/-}0.9032 mg/kg dry weight) of B. juncea; and in the root (46.4739{+/-}2.2920 mg/kg dry weight) of O. sativa respectively. Although the highest amount (0.9624{+/-}0.0920 mg/kg dry weight) of Cd was found in the leaf of B. juncea; the amount of Cd in the root was approximately same in all the three plants. This research also found more amounts of heavy metals compared to other studies using Indian mustard, rice and wheat against the three most environmental pollutants viz., Pb, Cr and Cd. Moreover, this study revealed that B. juncea is the highest hyperaccumulator species regarding Pb, Cr and Cd accumulation and can be used to clean up the polluted Buriganga riverbank soil.

sentences: Broad-scale variation in human genetic diversity levels is predicted by purifying selection on coding and non-coding elements Analyses of genetic variation in many taxa have established that neutral genetic diversity is shaped by natural selection at linked sites. Whether the mode of selection is primarily the fixation of strongly beneficial alleles (selective sweeps) or purifying selection on deleterious mutations (background selection) remains unknown, however. We address this question in humans by fitting a model of the joint effects of selective sweeps and background selection to autosomal polymorphism data from the 1000 Genomes Project. After controlling for variation in mutation rates along the genome, a model of background selection alone explains [~]60% of the variance in diversity levels at the megabase scale. Adding the effects of selective sweeps driven by adaptive substitutions to the model does not improve the fit, and when both modes of selection are considered jointly, selective sweeps are estimated to have had little or no effect on linked neutral diversity. The regions under purifying selection are best predicted by phylogenetic conservation, with [~]80% of the deleterious mutations affecting neutral diversity occurring in non-exonic regions. Thus, background selection is the dominant mode of linked selection in humans, with marked effects on diversity levels throughout autosomes.

sentences: Idiopathic chronic diarrhea in rhesus macaques is not associated with enteric viral infections While recent changes in treatment have reduced the lethality of idiopathic chronic diarrhea (ICD), this condition remains one of the most common causes of rhesus macaque deaths in non-human primate research centers. We compared the eukaryotic viromes in fecal swabs from 52 animals with ICD and 41 healthy animals. Viral metagenomics targeting virus-like particles was used to identify viruses shed by each animal. Five viruses belonging to the Picornaviridae, one to the Caliciviridae, one to the Parvoviridae, and one to the Adenoviridae families were identified. The fraction of reads matching each viral species was then used to estimate and compare viral loads in ICD cases versus healthy controls. None of the eukaryotic viruses detected in fecal swabs were strongly associated with ICD. Other potential causes of ICD are discussed.

sentences: The isoforms of pyruvate kinase act as nutrient sensors for the β-cell KATP channel Insulin release from pancreatic {beta}-cells is triggered by the metabolism-dependent closure of ATP-sensitive K+ (KATP) channels. Here, we used isoform-specific deletion of pyruvate kinase (PK) to identify the unique roles of PKm1 and PKm2 in the {beta}-cell nutrient response. In the absence of PK activity, cytosolic ATP/ADP was insufficient to close KATP channels. Both PK isoforms were found on the plasma membrane, where they endow KATP channels with nutrient sensitivity and specificity. Although PKm1 accounts for >90% of cellular activity, PKm2 controls glucosedependent KATP closure. Amino acids, which generate mitochondrial phosphoenolpyruvate (PEP) without allosterically activating PKm2, depend on PKm1 to raise ATP/ADP and close KATP channels. Shifting the {beta}-cell from PKm2 to PKm1 correspondingly increased amino acid sensitivity and boosted secretory output. In addition to these membrane depolarizing "on-switches," {beta}-cell deletion of PCK2 exposed a mitochondrial PEP-dependent "off-switch" that facilitates Ca2+ extrusion. Restricting PCK2 prolonged Ca2+ influx and increased insulin secretion. In summary, it is not the KATP channel alone, but the PK-KATP microcompartment that confers nutrient sensitivity to the {beta}-cell plasma membrane. The differential response of the PK isoforms to the glycolytic and mitochondrial sources of PEP influences {beta}-cell nutrient preference, and controls the oscillatory cycle regulating insulin secretion.

sentences: Identification of SUMO targets required to maintain human stem cells in the pluripotent state. To determine the role of SUMO modification in the maintenance of pluripotent stem cells we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 initiated changes associated with loss of pluripotency such as reduced expression of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created 3 networks of proteins with functions in regulation of gene expression, ribosome biogenesis and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The remainder have roles in transcription and chromatin structure regulation. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centred on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO is involved in the maintenance of the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

sentences: A genome-scale antibiotic screen in Serratia marcescens identifies YdgH as a conserved modifier of cephalosporin and detergent susceptibility Serratia marcescens, a member of the order Enterobacterales, is adept at colonizing healthcare environments and an important cause of invasive infections. Antibiotic resistance is a daunting problem in S. marcescens because in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility in S. marcescens, a high-density transposon insertion library was subjected to sub-minimal inhibitory concentrations of two cephalosporins, cefoxitin and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and chose ydgH, a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of ydgH in S. marcescens resulted in decreased susceptibility to multiple 3rd generation cephalosporins, and in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes in Escherichia coli O157:H7 and Enterobacter cloacae mutants. YdgH is predicted to localize to the periplasm and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance in S. marcescens and will serve as a resource for further investigations of this important pathogen.

sentences: Anxiety attenuates learning advantages conferred by statistical stability and induces loss of volatility-attuning in brain activity Anxiety can alter an individuals perception of their external sensory environment. Previous studies suggest that anxiety can increase the magnitude of neural responses to unexpected (or surprising) stimuli. Additionally, surprise responses are reported to be boosted during stable compared to volatile environments. Few studies, however, have examined how learning is impacted by both threat and volatility. To investigate these effects, we used threat-of-shock to transiently increase subjective anxiety in healthy adults during an auditory oddball task, in which the regularity could be stable or volatile, while undergoing functional Magnetic Resonance Imaging (fMRI) scanning. We then used Bayesian Model Selection (BMS) mapping to pinpoint the brain areas where different models of anxiety displayed the highest evidence. Behaviourally, we found that threat-of-shock eliminated the accuracy advantage conferred by environmental stability over volatility in the task at hand. Neurally, we found that threat-of-shock led to both attenuation and loss of volatility-attuning of neural activity evoked by surprising sounds across most subcortical and limbic brain regions including the thalamus, basal ganglia, claustrum, insula, anterior cingulate, hippocampal gyrus and also the superior temporal gyrus. Conversely, within two small clusters in the left medial frontal gyrus and extrastriate area, threat-of-shock boosted the neural activity (relative to the safe and volatile condition) to the levels observed during the safe and stable condition, while also inducing a loss of volatility-attuning. Taken together, our findings suggest that threat eliminates the learning advantage conferred by statistical stability compared to volatility. Thus, we propose that anxiety disrupts behavioural adaptation to environmental statistics, and that multiple subcortical and limbic regions are implicated in this process.

sentences: Quantitative genetic basis of floral design in a natural plant population The amount of genetic variation of floral traits and the degree to which they are genetically correlated are important parameters for the study of plant evolution. Estimates of these parameters can reveal the effect of historical selection relative to neutral processes such as mutation and drift, and allow us to predict the short-term evolutionary trajectory of a population under various selective regimes. Here, we assess the heritability and genetic correlation of the floral design of a native N. American tetraploid plant, Silene stellata (Caryophyllaceae), in a natural population. Specifically, we use a linear mixed model to estimate the genetic parameters based on a genealogy reconstructed from highly variable molecular markers. Overall, we found significant heritabilities in five out of nine studied traits. The level of heritability was intermediate (0.027 - 0.441). Interestingly, the floral trait showing the highest level of genetic variation was previously shown to be under strong sexually conflicting selection, which may serve as a mechanism for maintaining the observed genetic variation. Additionally, we also found prevalent positive genetic correlations between floral traits. Our results suggest that S. stellata is capable of responding to phenotypic selection on its floral design, while the abundant positive genetic correlations could also constrain the evolutionary trajectories to certain directions. Furthermore, this study demonstrates the utility and feasibility of marker-based approaches for estimating genetic parameters in natural populations.

sentences: The ATF6β-calreticulin axis protects hippocampal neurons against endoplasmic reticulum stress and excitotoxicity While ATF6 plays a central role in the endoplasmic reticulum (ER) stress response, the function of ATF6{beta} is largely unknown. Here, we demonstrate that ATF6{beta} is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin, a molecular chaperone in the ER with a high Ca2+-binding capacity. Calreticulin expression was reduced to ~50% in the central nervous system of Atf6b-/- mice, and restored by ATF6{beta}. Analysis using cultured hippocampal neurons revealed that ATF6{beta} deficiency reduced Ca2+ stores in the ER and enhanced ER stress-induced death, which was rescued by ATF6{beta}, calreticulin, Ca2+-modulating reagents such as BAPTA-AM and 2-APB, and ER stress inhibitor salubrinal. In vivo, kainate-induced neuronal death was enhanced in hippocampi of Atf6b-/- and Calr+/- mice, and restored by 2-APB and salubrinal. These results suggest that the ATF6{beta}-calreticulin axis plays a critical role in the neuronal survival by improving Ca2+ homeostasis under ER stress.

sentences: Estimating cell-type-specific gene co-expression networks from bulk gene expression data with an application to Alzheimer's disease Inferring and characterizing gene co-expression networks has led to important insights on the molecular mechanisms of complex diseases. Most co-expression analyses to date have been performed on gene expression data collected from bulk tissues with different cell type compositions across samples. As a result, the co-expression estimates only offer an aggregate view of the underlying gene regulations and can be confounded by heterogeneity in cell type compositions, failing to reveal gene coordination that may be distinct across different cell types. In this paper, we describe a flexible framework for estimating cell-type-specific gene co-expression networks from bulk sample data, without making specific assumptions on the distributions of gene expression profiles in different cell types. We develop a novel sparse least squares estimator, referred to as CSNet, that is efficient to implement and has good theoretical properties. Using CSNet, we analyzed the bulk gene expression data from a cohort study on Alzheimers disease and identified previously unknown cell-type-specific co-expressions among Alzheimers disease risk genes, suggesting cell-type-specific disease pathology for Alzheimers disease.

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MMMVI: Detecting SARS-CoV-2 Variants of Concern in Metagenomic Samples MotivationSARS-CoV-2 is the causative agent of the COVID-19 pandemic. Variants of Concern (VOCs) and Variants of Interest (VOIs) are lineages that represent a greater risk to public health, and can be differentiated from the wildtype virus based on unique profiles of signature mutations. Metagenomic sequence analysis of wastewater represents an emerging form of surveillance that can capture early signal for these variants in a community prior to detection through public health testing or genomic surveillance activities. However, because multiple viral genomes are likely to be present in a metagenomic sample, additional analytical scrutiny of the sequencing reads beyond variant calling is needed to increase confidence in diagnostic determinations.

ResultsWhere multiple signature mutations are present on a given read, they can be used as enhanced biomarkers to confirm the presence of a VOC/VOI in the sample. We have developed MMMVI, a tool to aggregate and report on the likely presence of a VOC/VOI in a set of metagenomic reads based on the detection of reads bearing multiple signature mutations.

AvailabilityMMMVI is implemented in Python, and is available under the MIT licence from https://github.com/dorbarker/voc-identify/

Contactdillon.barker@canada.ca

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Constrained evolutionary paths to macrolide resistance in a Neisseria commensal converge on ribosomal genes through sequence duplication Neisseria commensals are an indisputable source of resistance for their pathogenic relatives; however, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance ([≤] 2 g/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n=7/16), we find mutations encoding resistance converge on the gene encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through duplication events. Importantly, one of the laboratory evolved mutations in rpmH is identical, and two nearly identical, to those recently reported to confer high-level resistance to azithromycin in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Finally, we assessed the potential of horizontal transfer of derived resistance mutations into multiple strains of N. gonorrhoeae. Though we were unable to transform N. gonorrhoeae in this case, studies like this will be critical for predicting commensal alleles that are at risk of rapid dissemination into pathogen populations.

ImportanceCommensal bacterial populations have been increasingly recognized for their importance as sources of resistance for pathogens, however the collection of antimicrobial resistance (AMR) mechanisms within these communities are often understudied. The risk of reduced antibiotic susceptibility as a result of horizontal gene transfer (HGT) is amplified in highly recombinogenic genera, such as the Neisseria. Indeed, there have been multiple documented cases of macrolide and beta-lactam resistance acquisition in the pathogen N. gonorrhoeae from close commensal relatives. This work uncovers multiple novel azithromycin resistance-conferring mutations in a Neisseria commensal through experimental evolution, investigates their fitness impacts, and explores the possibility of transfer to N. gonorrhoeae. Ultimately these types of studies will illuminate those resistance mutations that may rapidly be acquired across species boundaries.

sentences: Pulsations and flows in tissues: two collective dynamics with simple cellular rules Collective motions of epithelial cells in vivo are essential for morphogenesis in developmental biology. Tissues elongate, contract, flow, and oscillate, sculpting embryos. These tissue level dynamics are known, but their physical mechanisms at the cellular level are unclear, with various behaviors that depend on the tissues and species. Moreover, investigations on in vitro tissue behavior usually focus on only one type of cell dynamics and use diverse theoretical approaches, making systematic comparisons between studies challenging. Here, we show that the very same epithelial monolayer of Madine Darby Canine Kidney (MDCK) cells can exhibit two types of tissue kinematics, pulsations, and coherent flows. We also report that these distinct motions can be controlled with internal and external cues by using quantitative live imaging of the acto-myosin cytoskeleton, specific inhibitors, microfabrication, and friction modulation of the substrate. We further demonstrate with a unified vertex model that both behaviors depend on the interplay between velocity alignment and random diffusion of cell polarization. When alignment and diffusion are comparable, a pulsatile flow emerges, whereas the tissue undergoes long-range flows when velocity alignment dominates. We propose that environmental friction, acto-myosin distributions, and cell polarization kinetics serve as the essential factors regulating the dynamics of tissue morphogenesis.

sentences: APOK3, a pollen killer antidote in Arabidopsis thaliana According to the principles of heredity, each parental allele of hybrids equally participates in the progeny. At some loci, however, it happens that one allele is favored to the expense of the other. Gamete killers are genetic systems where one allele (the killer) triggers the death of the gametes carrying the other (killed) allele. They have been found in many organisms, and are of major interest to understand mechanisms of evolution and speciation. Gamete killers are particularly prevalent in plants, where they can compromise crop breeding. Here, we deciphered a pollen killer in Arabidopsis thaliana by exploiting natural variation, de novo genomic sequencing and mutants, and analyzing segregations in crosses. We found that the killer allele carries an antidote gene flanked by two elements mandatory for the killing activity. We identified the gene encoding the antidote, a chimeric protein addressed to mitochondria. This gene appeared in the species by association of domains recruited from other genes, and it recently underwent duplications within a highly variable locus, particularly in the killer genotypes. Exploring the species diversity, we identified sequence polymorphisms correlated with the antidote activity.

sentences: Honey bees can store and retrieve independent memory traces after complex experiences that combine appetitive and aversive associations Real-world experiences do often mix appetitive and aversive events. Understanding the ability of animals to extract, store and use this information is an important issue in neurobiology. We used honey bees as model to study learning and memory after a differential conditioning that combines appetitive and aversive training trials. First of all, we describe an aversive conditioning paradigm that constitutes a clear opposite of the well known appetitive olfactory conditioning of the proboscis extension response. A neutral odour is presented paired with the bitter substance quinine. Aversive memory is evidenced later as an odour-specific impairment in appetitive conditioning. Then we tested the effect of mixing appetitive and aversive conditioning trials distributed along the same training session. Differential conditioning protocols like this were used before to study the ability to discriminate odours, however they were not focused on whether appetitive and aversive memories are formed. We found that after a differential conditioning, honey bees establish independent appetitive and aversive memories that do not interfere with each other during acquisition or storage. Finally, we moved the question forward to retrieval and memory expression to evaluate what happens when appetitive and the aversive learned odours are mixed during test. Interestingly, opposite memories compete in a way that they do not cancel each other out. Honey bees showed the ability to switch from expressing appetitive to aversive memory depending on their satiation level.

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Deciphering the genetic links between NAFLD and co-occurring conditions using a liver gene regulatory network Background & AimsNon-alcoholic fatty liver disease (NAFLD) is a multi-system metabolic disease that co-occurs with various hepatic and extra-hepatic diseases. The phenotypic manifestation of NAFLD is primarily observed in the liver. Therefore, identifying liver-specific gene regulatory interactions between variants associated with NAFLD and multimorbid conditions may help to improve our understanding of underlying shared aetiology.

MethodsHere, we constructed a liver-specific gene regulatory network (LGRN) consisting of genome-wide spatially constrained expression quantitative trait loci (eQTLs) and their target genes. The LGRN was used to identify regulatory interactions involving NAFLD-associated genetic modifiers and their inter-relationships to other complex traits.

Results and ConclusionsWe demonstrate that MBOAT7 and IL32, which are associated with NAFLD progression, are regulated by spatially constrained eQTLs that are enriched for an association with liver enzyme levels. MBOAT7 transcript levels are also linked to eQTLs associated with cirrhosis, and other traits that commonly co-occur with NAFLD. In addition, genes that encode interacting partners of NAFLD-candidate genes within the liver-specific protein-protein interaction network were affected by eQTLs enriched for phenotypes relevant to NAFLD (e.g. IgG glycosylation patterns, OSA). Furthermore, we identified distinct gene regulatory networks formed by the NAFLD-associated eQTLs in normal versus diseased liver, consistent with the context-specificity of the eQTLs effects. Interestingly, genes targeted by NAFLD-associated eQTLs within the LGRN were also affected by eQTLs associated with NAFLD-related traits (e.g. obesity and body fat percentage). Overall, the genetic links identified between these traits expand our understanding of shared regulatory mechanisms underlying NAFLD multimorbidities.

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Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

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sentences: Multiple genes in cis mediate the effects of a single chromatin accessibility variant on aberrant synaptic development and function in human neurons Despite hundreds of risk loci from genome-wide association studies of neuropsychiatric disorders, causal variants/genes remain largely unknown. Here, in NEUROG2-induced human neurons, we identified 31 risk SNPs in 26 schizophrenia (SZ) risk loci that displayed allele-specific open chromatin (ASoC) and were likely to be functional. Editing the strongest ASoC SNP rs2027349 near vacuolar protein sorting 45 homolog (VPS45) altered the expression of VPS45, lncRNA AC244033.2, and a distal gene, C1orf54, in human neurons. Notably, the global gene expression changes in neurons were enriched for SZ risk and correlated with post-mortem brain gene expression signatures of neuropsychiatric disorders. Neurons carrying the risk allele exhibited increased dendritic complexity, synaptic puncta density, and hyperactivity, which were reversed by knocking-down distinct cis-regulated genes (VPS45, AC244033.2, or C1orf54), suggesting a phenotypic contribution from all three genes. Interestingly, transcriptomic analysis of knockdown cells suggested a non-additive effects of these genes. Our study reveals a compound effect of multiple genes at a single SZ locus on synaptic development and function, providing a mechanistic link between a non-coding SZ risk variant and disease-related cellular phenotypes.

sentences: Thermophoresis of Molecules and Structures of different Sizes in Self-assembled Biomatrices Upon subjecting biomolecules to non-equilibrium conditions, many biochemical and biophysical features such as biomolecular diffusion, protein folding, interaction kinetics, as well as enzyme-catalyzed reactions can be characterized in an aqueous solution. However, most assays under non-equilibrium conditions cannot be performed in complex self-assembled biomatrices (e.g. extracellular matrices) due to the limitations associated with sample handling, reaction design, and optical detection. Herein, we report the study of biomolecular thermodiffusion in non-covalently assembled synthetic or naturally derived hydrogels. This approach has been demonstrated with a large variety of analytes, including small molecules, polysaccharides, DNAs, DNA origami, and proteins in various polymer networks. The in-biomatrix method has also shown advantages over in-solution measurements: First, it allows us to analyze biomolecules in 3D matrices in a high-throughput fashion. Second, the aggregation of analytes can be remarkably prevented. Although the underlying physics of thermodiffusion is still not well-understood, we demonstrated that the thermodiffusion of surrounding networks will enhance the thermodiffusion of the analyte, an effect counteracting the hindered movement by the polymer network.

sentences: Oscillatory tracking of pseudo-rhythmic speech is constrained by linguistic predictions Neuronal oscillations putatively track speech in order to optimize sensory processing. However, it is unclear how isochronous brain oscillations can track pseudo-rhythmic speech input. Here we propose that oscillations can track pseudo-rhythmic speech when considering that speech time is dependent on predictions flowing from internal language models. We show that the temporal dynamics of speech are dependent on the predictability of words in a sentence. A computational model including oscillations, feedback, and inhibition is able to track the natural pseudo-rhythmic speech input. As the model processes, it generates temporal phase codes, which are a candidate mechanism for carrying information forward in time. The model is optimally sensitive to the natural temporal speech dynamics and can explain empirical data on temporal speech illusions. Our results reveal that speech tracking does not only rely on the input acoustics but instead entails an interaction between oscillations and constraints flowing from internal language models.

sentences: Lipid type doping of the sponge (L3) mesophase The polymorphism of lipid aggregates has long attracted detailed study due to the myriad factors that determine the final mesophase observed. This study is driven by the need to understand mesophase behaviour for a number of applications, such as drug delivery and membrane protein crystallography. In the case of the latter, the role of the so-called  sponge (L3) mesophase has been often noted, but not extensively studied by itself. The L3 mesophase can be formed in monoolein/water systems on the addition of butanediol to water, which partitions the headgroup region of the membrane, and decreases its elastic moduli. Like cubic mesophases, it is bicontinuous, but unlike them, has no long-range translational symmetry. In our present study, we show that the formation of the L3 phase can delicately depend on the addition of dopant lipids to the mesophase. While electrostatically neutral molecules similar in shape to monoolein (DOPE, cholesterol) have little effect on the general mesophase behaviour, others (DOPC, DDM) significantly reduce the region in which it can form. Additionally, we show that by combining cholesterol with the anionic lipid DOPG, it is possible to form the largest stable L3 mesophases observed to date, with correlation lengths over 220 [A].

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Regulation of osteoblast autophagy based on PI3K/AKT/mTOR signaling pathway study on the effect of β-ecdysterone on fracture healing PurposeTo investigate the effects of {beta}-ecdysterone on fracture healing and the underlying mechanism.

MethodsMTT assay was used to detect the cell viability and alkaline phosphatase (ALP) activity was measured using a commercial kit. AO/PI and flow cytometry assays were used to determine the state of apoptosis of osteoblasts. The expression level of RunX2, ATG7 and LC3 was evaluated by qRT-PCR and Western blot assays. X-ray and HE staining were conducted on the fractured femur to evaluate the pathological state. Immunohistochemical assay was used to detect the expression level of Beclin-1 and immunofluorescence assay was used to measure the expression level of LC3 in the fractured femurs. Western blot was utilized to determine the expression level of PI3K, p-AKT1, AKT1, p-mTOR, mTOR, p-p70S6K, and p70S6K.

ResultsThe ALP activity and expression of RunX2 in fractured osteoblasts were significantly suppressed by 3-methyladenine and elevated by rapamycin, 60, and 80 M {beta}-ecdysterone. The apoptotic state of fractured osteoblasts was enhanced by 3-methyladenine and alleviated by rapamycin, 60, and 80 M {beta}-ecdysterone. The state of autophagy both in fractured osteoblasts and femurs was inhibited by 3-methyladenine and facilitated by rapamycin and {beta}-ecdysterone. Compared to control, Garrett score in 3-methyladenine group was significantly decreased and promoted in rapamycin and {beta}-ecdysterone groups, accompanied by ameliorated pathological state. Lastly, the PI3K/AKT/mTOR pathway both in fractured osteoblasts and femurs was activated by 3-methyladenine and inhibited by rapamycin and {beta}-ecdysterone.

Conclusion{beta}-ecdysterone might facilitate fracture healing by activating autophagy through suppressing PI3K/AKT/mTOR signal pathway.

sentences: Neural defensive circuits underlie helping under threat in humans Empathy for others distress has long been considered the driving force of helping. However, when deciding to help others in danger, one must consider not only their distress, but also the risk to oneself. Whereas the role of self-defence in helping has been overlooked in human research, studies in other animals indicate defensive responses are necessary for the protection of conspecifics. In this pre-registered study (N=49), we demonstrate that human defensive neural circuits are implicated in helping others under threat. Participants underwent fMRI scanning while deciding whether to help another participant avoid aversive electrical shocks, at the risk of also being shocked. We found that higher engagement of neural circuits that coordinate fast escape from self-directed danger (including the insula, PAG and ACC) facilitated decisions to help others. Importantly, using Representational Similarity Analysis, we found that the strength with which the amygdala and insula uniquely represented the threat to oneself (and not the others distress) predicted helping. Our findings indicate that in humans, as other mammals, defensive mechanisms play a greater role in helping behaviour than previously understood.

sentences: Collagen polarization provides a structural memory for the elongation of epithelial anlage. Epithelial networks are commonly generated by processes where multicellular aggregates elongate and branch. Here we focus on understanding cellular mechanisms for elongation, using an organotypic culture system as a model of mammary epithelial anlage. Isotropic cell aggregates broke symmetry and slowly elongated when transplanted into collagen 1 gels. The elongating regions of aggregates displayed enhanced cell proliferation that was necessary for elongation to occur. Strikingly, this loco-regional increase in cell proliferation occurred where collagen 1 fibrils reorganized into bundles which were polarized with the elongating aggregates. Applying external stretch as a cell-independent way to reorganize the ECM, we found that collagen polarization stimulated regional cell proliferation to precipitate symmetry-breaking and elongation. This required {beta}1-integrin and ERK signaling. We propose that collagen polarization supports epithelial anlagen elongation by stimulating loco-regional cell proliferation. This could provide a long-lasting structural memory of the initial axis that is generated when anlage break symmetry.

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Restructured mitochondrial-nuclear interaction in Plasmodium falciparum dormancy and persister survival after artemisinin exposure Artemisinin and its semi-synthetic derivatives (ART) are fast acting, potent antimalarials; however, their use in malaria treatment is frequently confounded by recrudescences from bloodstream Plasmodium parasites that enter into and later reactivate from a dormant persister state. Here we provide evidence that the mitochondria of dihydroartemisinin (DHA)-exposed persisters are dramatically altered and enlarged relative to the mitochondria of young, actively replicating ring forms. Restructured mitochondrial-nuclear associations and an altered metabolic state are consistent with stress from reactive oxygen species. New contacts between the mitochondria and nuclei may support communication pathways of mitochondrial retrograde signaling, resulting in transcriptional changes in the nucleus as a survival response. Further characterization of the organelle communication and metabolic dependencies of persisters may suggest strategies to combat recrudescences of malaria after treatment.

IMPORTANCEThe major first-line treatment for malaria, especially the deadliest form caused by Plasmodium falciparum, is combination therapy with an artemisinin-based drug (ART) plus a partner drug to assure complete cure. Without an effective partner drug, ART administration alone can fail because of the ability of small populations of blood-stage malaria parasites to enter into a dormant state and survive repeated treatments for a week or more. Understanding the nature of parasites in dormancy (persisters), and their ability to wake and reestablish actively-propagating parasitemias (recrudesce) after ART exposure, may suggest strategies to improve treatment outcomes and counter the threats posed by parasites that develop resistance to partner drugs. Here we show that persisters have dramatically altered mitochondria and mitochondrial-nuclear interactions associated with features of metabolic quiescence. Restructured associations between the mitochondria and nuclei may support signaling pathways that enable the ART survival responses of dormancy.

sentences: Identification of enriched hyperthermophilic microbial communities from a deep-sea hydrothermal vent chimney under electrolithoautotrophic culture conditions. Deep-sea hydrothermal vents are extreme and complex ecosystems based on a trophic chain. We are still unsure of the identities of the first colonizers of these environments and their metabolism, but they are thought to be (hyper)thermophilic autotrophs. Here we investigate whether the electric potential observed across hydrothermal chimneys could serve as an energy source for these first colonizers. Experiments were performed in a two-chamber microbial electrochemical system inoculated with deep-sea hydrothermal chimney samples, with a cathode as sole electron donor, CO2 as sole carbon source, and nitrate, sulfate, or oxygen as electron acceptors. After a few days of culture, all three experiments showed growth of electrotrophic biofilms consuming the electrons (directly or indirectly) and producing organic compounds including acetate, glycerol, and pyruvate. Within the biofilms, the only known autotroph species retrieved were members of Archaeoglobales. Various heterotrophic phyla also grew through trophic interactions, with Thermococcales growing in all three experiments as well as other bacterial groups specific to each electron acceptor. This electrotrophic metabolism as energy source driving initial microbial colonization of conductive hydrothermal chimneys is discussed.

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Immunity vs Sociality: Adaptive evolution tests suggest social lifestyle exerts greater selection pressures than host-pathogen coevolution in the bees Hosts and their parasites and pathogens are locked in antagonistic co-evolution. The genetic consequence of this can be seen in the rates of adaptive evolution in immunologically important loci in many taxa. As the risk of disease transmission increases we might also expect to see greater rates of adaptive evolution on genes of immune function. The evolution of sociality and its elaborations in insects represent enormous shift in disease transmission risk. Here, we examine whether sociality in the bees corresponds to changes in the rate of adaptive evolution in both classical canonical immune genes, and genes with putative immune functions identified from meta-analyses of honey-bee transcriptomic responses to infection. We find that measures of gene-wide adaptive evolution do not differ among canonical immune, non-canonical candidate immune, and background gene sets, but that branch-site adaptive evolution does increase with sociality regardless of gene category. Solitary species have greater rates of adaptive evolution in canonical immune genes than background genes, supporting the suggestion that social immune mechanisms may instead be the site of host-pathogen co-evolution in social species. We identify three genes with putative roles in immunity that warrant further attention (Vitel-logenin Vg, disks large 1 tumour suppressor, and the uncharacterised protein LOC100577972). There are more gene family changes after the origin of sociality across all gene classes, with contractions occur-ring after the elaboration of sociality to complex eusociality. There are few genes or functions under adaptive selection that appear to be shared outside of specific lineages, suggesting that evolution of the immune system may be specific to individual species and their pathogen interactions.

SignificanceInfectious disease drives rapid evolution of immune genes, but infection risk should be much higher in social species. To examine whether greater sociality drives faster immune system evolution we compared the rate of immune gene evolution in solitary, social, and highly eusocial bees. To account for possible novel immune genes in bees, we analysed classical immune genes alongside candidate immune genes inferred from other studies. Surprisingly, we find that solitary bees have the highest rate of immune gene evolution relative to background genes but that sociality is associated with rapid evolution across the whole genome. These findings suggest that 1) accelerated immune gene evolution is not universal, 2) immune gene evolution is moderated by sociality in that solitary species invest more into immune gene change, and 3) that social genomes are highly dynamic, which may obscure evolution at immunological loci. The types of immune genes and functions appear mostly lineage-specific, regardless of sociality, suggesting individual evolutionary his-tories exert more selection pressure than general patterns of greater pathogen exposure introduced by social living.

sentences: Multisite imaging of neural activity using a genetically encoded calcium sensor in the honey bee Apis mellifera Sociality is classified as one of the major transitions in evolution, and the most advanced level of sociality is found in eusocial insects such as the honey bees. This sophisticated social behavior seems to be associated with higher cognitive abilities in these insects, however the study of their neural bases has been limited by the lack of tools that allow measuring neuronal activity simultaneously in different brain regions of the honey bee brain. Here, we developed the first pan-neuronal genetic driver in a Hymenopteran model organism, the honey bee, and expressed the calcium indicator GCaMP6f under the control of the honey bee synapsin promoter. We show that GCaMP6f is widely expressed in the honey bee brain, allowing to record neural activity from multiple brain regions simultaneously, including from poorly investigated brain regions such as the mushroom body calyces or the lateral horn. Our recordings show that while odorant quality (chemical structure) and quantity are faithfully encoded in the honeybee antennal lobe, odor coding in the lateral horn departs from this simple physico-chemical coding, in line with the role of this structure in coding the biological value of odorants. These brain recordings represent the first use of a neurogenetic tool for recording neural activity in a eusocial insect.

sentences: Visuospatial attention updates the cortical representation of sound under reduced sensory uncertainty Selective attentional biases arising from one sensory modality may manifest in another. The effects of visuospatial attention, often considered a foundation for visual object perception, are unclear in the auditory domain during audiovisual (AV) scene processing. This study investigates temporal and spatial factors that facilitate such cross-modal bias transfer at the neural level. Auditory encoding of random tone pips in AV scenes was investigated via a temporal response function model (TRF) of the participants electroencephalogram (N=30). The spatially uninformative pips were associated with spatially distributed visual contrast reversals ( flips) through asynchronous, probabilistic AV temporal onset distributions. Participants deployed visuospatial selection on these AV stimuli to perform a task. A late (~300 ms) cross-modal transfer of the unimodal attentional bias was found on the neural representation of pips. Transfer depended on the selected visual input being (i) presented during or shortly after a related sound in a relatively limited temporal window (<165 ms); and (ii) positioned across limited (1:4) visual foreground to background ratios. In addition, the magnitude of attentional enhancement was proportional to the proximity of flips to the foreground area. The results indicate that ongoing neural representations of sounds can incorporate relevant visuospatial attributes for auditory stream segregation.

sentences: Left Hemisphere Dominance for Bilateral Kinematic Encoding in the Human Brain Neurophysiological studies in humans and non-human primates have revealed movement representations in both the contralateral and ipsilateral hemisphere. Inspired by clinical observations, we ask if this bilateral representation differs for the left and right hemispheres. Electrocorticography (ECoG) was recorded in human participants during an instructed-delay reaching task, with movements produced with either the contralateral or ipsilateral arm. Using a cross-validated kinematic encoding model, we found stronger bilateral encoding in the left hemisphere, an effect that was present during preparation and was amplified during execution. Consistent with this asymmetry, we also observed better across-arm generalization in the left hemisphere, indicating similar neural representations for right and left arm movements. Notably, these left hemisphere electrodes were centered over premotor and parietal regions. The more extensive bilateral encoding in the left hemisphere adds a new perspective to the pervasive neuropsychological finding that the left hemisphere plays a dominant role in praxis.

sentences: Airway Basal Cells Show Regionally Distinct Potential to Undergo Metaplastic Differentiation Basal cells are multipotent stem cells of a variety of organs, and in the lung are known as crucial components of the airway epithelium. However, it remains unclear how diverse basal cells are and whether distinct subpopulations respond differently to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the murine trachea. Among these we found two major subpopulations comprising the most stem-like progenitor compartment, but with distinct signatures and ability to self-renew and differentiate. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to initiate an aberrant program of differentiation in response to environmental perturbations in primary cultures and in injury mouse models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of cartilage-deficient mouse mutants. Lastly, we show that key features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide critical insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.

sentences: Differences in gait stability, trunk and foot accelerations between healthy young and older women Our aim was to evaluate differences in gait acceleration intensity, variability and stability of feet and trunk between older females and young females using inertial sensors. Twenty older females (OF; mean age 68.4, SD 4.1 years) and eighteen young females (YF; mean age 22.3, SD 1.7 years) were asked to walk straight for 100 meters at their preferred speed, while wearing inertial sensors on heels and lower back. We calculated spatiotemporal measures, foot and trunk acceleration characteristics and their variability, as well as trunk stability using the local divergence exponent (LDE). Two-way analysis of variance (including the factors foot and age), Students t-test, and Mann-Whitney U test were used to compare statistical differences of measures between groups. Cohens d effects were calculated for each variable. Foot maximum vertical acceleration and amplitude, trunk-foot vertical acceleration attenuation, as well as their variability were significantly smaller in OF than in YF. In contrast, trunk mediolateral acceleration amplitude, maximum vertical acceleration, and amplitude, as well as their variability were significantly larger in OF than in YF. Moreover, OF showed lower stability (i.e., higher LDE values) in mediolateral acceleration, mediolateral and vertical angular velocity of the trunk. Even though we measured healthy older females, these participants showed lower vertical foot accelerations with higher vertical trunk acceleration, lower trunk-foot vertical acceleration attenuation, less gait stability, and more variability of the trunk, and hence, were more likely to fall. These findings suggest that instrumented gait measurements may help for early detection of changes or impairments in gait performance, even before this can be observed by clinical eye or gait speed.

sentences: Cold adaptation drives population genomic divergence in the ecological specialist, Drosophila montana Detecting signatures of ecological adaptation in comparative genomics is challenging, but analysing population samples with characterised geographic distributions, such as clinal variation, can help identify genes showing covariation with important ecological variation. Here we analysed patterns of geographic variation in the cold-adapted species Drosophila montana across phenotypes, genotypes and environmental conditions and tested for signatures of cold adaptation in population genomic divergence. We first derived the climatic variables associated with the geographic distribution of 24 populations across two continents to trace the scale of environmental variation experienced by the species, and measured variation in the cold tolerance of the flies of six populations from different geographic contexts. We then performed pooled whole genome sequencing of these six populations, and used Bayesian methods to identify SNPs where genetic differentiation is associated with both climatic variables and the population phenotypic measurements, while controlling for effects of demography and population structure. The top candidate SNPs were enriched on the X and 4th chromosomes, and they also lay near genes implicated in other studies of cold tolerance and population divergence in this species and its close relatives. We conclude that ecological adaptation has contributed to the divergence of D. montana populations throughout the genome and in particular on the X and 4th chromosomes, which also showed highest interpopulation FST. This study demonstrates that ecological selection can drive genomic divergence at different scales, from candidate genes to chromosome-wide effects.

sentences: A selective bottleneck during host entry drives the evolution of new legume symbionts During the emergence of new host-microbe symbioses, multiple selective pressures-acting at the different steps of the microbial life cycle-shape the phenotypic traits that jointly determine microbial fitness. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts are still poorly known. Here we characterized the dynamics of phenotypic adaptation and its underlying genetic bases during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly driven by selection acting on competitiveness for host entry, which outweighed selection acting on within-host proliferation. Whole-population sequencing of evolved bacteria revealed that phenotypic adaptation was supported by the continuous accumulation of new mutations and the sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that all of them improved competitiveness for host entry, while only a subset of those also improved within host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.

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VisVariant: A java program to visualise genetic variants in next-generation sequencing data SummaryExtremely large datasets are impossible or very difficult for humans to comprehend by standard mental approaches. Intuitive visualization of genetic variants in genomic sequencing data could help in the review and confirmation process of variants called by automated variant calling programs. To help facilitate interpretation of genetic variant next-generation sequencing (NGS) data we developed VisVariant, a customizable visualization tool that creates a figure showing the overlapping sequence information of thousands of individual reads including the variant and flanking regions.

Availability and implementationDetailed information on how to download, install and run VisVariant together with an example is available on our github website [https://github.com/hugging-biorxiv/visvariant].

sentences: Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and among the most significant epigenetic modifications. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in the CpG context allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. For this method, a CpG methyltransferase was used to transfer an azide to cytosines from a synthetic S-adenosyl-l-methionine cofactor analog. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased two-fold by the addition of a nucleosidase that degrades the inactive by-product of the azide-cofactor after labeling, and prevents its inhibitory effect. We first used the method to determine the decline in global DNA methylation in chronic lymphocytic leukemia patients and then performed whole genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published methylation maps produced by bisulfite sequencing. Although mapping resolution is limited by optical detection to 500-1000 base pairs, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

sentences: Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging Intermediate filaments (IF) are involved in key cellular functions including polarization, migration, and protection against large deformations. These functions are related to their remarkable ability to extend without breaking, a capacity that should be determined by the molecular organization of subunits within filaments. However, this structure-mechanics relationship remains poorly understood at the molecular level. Here, using super-resolution microscopy (SRM), we show that vimentin filaments exhibit a ~49 nm axial repeat both in cells and in vitro. As unit-length-filaments (ULFs) were measured at ~59 nm, this demonstrates a partial overlap of ULFs during filament assembly. Using an SRM-compatible stretching device, we also provide evidence that the extensibility of vimentin is due to the unfolding of its subunits and not to their sliding, thus establishing a direct link between the structural organization and its mechanical properties. Overall, our results pave the way for future studies of IF assembly, mechanical and structural properties in cells.

sentences: Cortical Surface-Informed Volumetric Spatial Smoothing of fMRI Data via Graph Signal Processing Conventionally, as a preprocessing step, functional MRI (fMRI) data are spatially smoothed before further analysis, be it for activation mapping on task-based fMRI or functional connectivity analysis on resting-state fMRI data. When images are smoothed volumetrically, however, isotropic Gaussian kernels are generally used, which do not adapt to the underlying brain structure. Alternatively, cortical surface smoothing procedures provide the benefit of adapting the smoothing process to the underlying morphology, but require projecting volumetric data on to the surface. In this paper, leveraging principles from graph signal processing, we propose a volumetric spatial smoothing method that takes advantage of the gray-white and pial cortical surfaces, and as such, adapts the filtering process to the underlying morphological details at each point in the cortex.

sentences: Dynamics of the habitat use of three predatory freshwater fish in a lentic ecosystem To understand the conditions of coexistence in multiple-species predator community, we studied longitudinal and vertical movement of pike (Esox lucius), pikeperch (Sander lucioperca) and catfish (Silurus glanis) in the [R]imov Reservoir, using an autonomous telemetry system for 11 months. We found significant differences among these three species in movement and depth that varied considerably in time, with the greatest differences between warm (late spring and early autumn) and cold season (late autumn to early spring). Preference for different sections of the reservoir was stable for pike, while pikeperch and catfish frequently visited tributary during the warm season, and moved closer to the dam during the cold season. Pike longitudinal activity was highest in the cold season, pikeperch in the warm season, and catfish activity peaked in both the warm and cold seasons. Overlap in the depth used among species was higher in the warm season, when all species used the upper layer of the water column, and lower in cold season, when pikeperch and catfish used deeper areas. These results demonstrated ability of predators to actively inspect a large portion of the reservoir in both longitudinal and vertical dimensions, although differing in the timing of their habitat use and activity.

sentences: Attentional modulation of intrinsic timescales in visual cortex and spatial networks Neural activity fluctuates endogenously on timescales varying across the neocortex. The variation in these intrinsic timescales relates to the functional specialization of cortical areas and their involvement in the temporal integration of information. Yet, it is unknown whether the timescales can adjust rapidly and selectively to the demands of a cognitive task. We measured intrinsic timescales of local spiking activity within columns of area V4 while monkeys performed spatial attention tasks. The ongoing spiking activity unfolded across at least two distinct timescales--fast and slow--and the slow timescale increased when monkeys attended to the receptive fields location. A recurrent network model shows that multiple timescales in local dynamics arise from spatial connectivity mimicking vertical and horizontal interactions in visual cortex and that slow timescales increase with the efficacy of recurrent interactions. Our results reveal that targeted neural populations integrate information over variable timescales following the demands of a cognitive task and propose an underlying network mechanism.

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Mesenchymal Lineage Heterogeneity Underlies Non-Redundant Functions of Pancreatic Cancer-Associated Fibroblasts Cancer-associated fibroblast (CAF) heterogeneity is increasingly appreciated, but the origins and functions of distinct CAF subtypes remain poorly understood. The abundant and transcriptionally diverse CAF population in pancreatic ductal adenocarcinoma (PDAC) is thought to arise from a common cell of origin, pancreatic stellate cells (PSCs), with diversification resulting from cytokine and growth factor gradients within the tumor microenvironment. Here we analyzed the differentiation and function of PSCs during tumor progression in vivo. Contrary to expectations, we found that PSCs give rise to a numerically minor subset of PDAC CAFs. Targeted ablation of PSC-derived CAFs within their host tissue revealed non-redundant functions for this defined CAF population in shaping the PDAC microenvironment, including production of specific components of the extracellular matrix. Together, these findings link stromal evolution from distinct cells of origin to transcriptional heterogeneity among PDAC CAFs, and demonstrate unique functions for CAFs of a defined cellular origin.

Statement of significanceBy tracking and ablating a specific CAF population, we find that a numerically minor CAF subtype from a defined cell of origin plays unique roles in establishing the pancreatic tumor microenvironment. Together with prior studies, this work suggests that mesenchymal lineage heterogeneity as well as signaling gradients diversify PDAC CAFs.

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Increased Functional Connectivity Characterises the Prodromal Phase of the Migraine Cycle Episodic migraine is reflected by cyclic changes in behaviour and cortical processing. In this study, we aimed to identify how functional connectivities change over the entire migraine cycle.

By using longitudinal neuroimaging and a whole-brain analysis approach, we tested 12 episodic migraine patients across 82 fMRI recordings during spontaneous headache attacks, with follow-up measurements over the pain-free interval without any external stimulation.

We focussed on two different trajectories with a linear increase of connectivity and a peak either during the prodromal phase or during the headache attack. We found cycle-related connectivity changes for the anterior insula and the posterior orbitofrontal cortex. Both regions showed the highest connections with sensory, motor, and cingulate areas in the prodromal phase. The connectivity between the hypothalamus and the locus coeruleus was strongest during the headache attack.

The peak connectivity during the prodromal phase and its collapse during the headache can be regarded as a mechanism of normalising cortical processing. The increase of synchronicity of functional connections during the ictal phase of the migraine cycle may contribute to the variety of symptoms of migraine attacks, such as headache, hypersensitivity to sensory modalities, and autonomous symptoms. We speculate about a malfunction at the molecular level in agranular frontal and insular brain regions, which needs to be followed up upon in subsequent studies.

Abbreviated SummaryEpisodic migraine is a cyclic disease. We investigated functional connectivities over an entire migraine cycle. We found cycle-related connectivity changes for two proximate agranular regions. The prodromal increase and the collapse of connectivity during the headache may reflect normalising cortical processing.

sentences: Delimiting the cryptic diversity and host preferences of Sycophila parasitoid wasps associated with oak galls using phylogenomic data Cryptic species diversity is a major challenge for the species-rich community of parasitoids attacking oak gall wasps due to a high degree of sexual dimorphism, morphological plasticity, small size, and poorly known biology. As such, we know very little about the number of species present, nor the evolutionary forces responsible for generating this diversity. One hypothesis is that trait diversity in the gall wasps, including the morphology of the galls they induce, have evolved in response to selection imposed by the parasitoid community, with reciprocal selection driving diversification of the parasitoids. Using a rare continental scale data set of Sycophila parasitoid wasps reared from 44 species of cynipid galls from 18 species of oak across the US, we combined mitochondrial DNA barcodes, Ultraconserved Elements (UCEs), morphological, and natural history data to delimit putative species. We also generate the first large-scale assessment of ecological specialization and host association in this species-rich group with implications for evolutionary ecology and biocontrol. We find most Sycophila target multiple gall wasp species with similar gall morphologies and are generally associated with smaller galls. This result suggests that parasitoid wasps such as Sycophila have adaptations allowing them to exploit particular host trait combinations, while hosts with contrasting traits are resistant to attack. With our congruence-based method of species delimitation, our study has nearly doubled the number of Sycophila wasps among the lineages studied, which highlights the need for taxonomic revision of Sycophila, as many species cannot be identified morphologically or by using molecular delimitation methods.

sentences: Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR Mucopolysaccharidosis type VII (MPS VII) is a recessively inherited lysosomal storage disorder caused due to {beta}-glucuronidase ({beta}-GUS) enzyme deficiency. Prominent clinical symptoms include hydrops fetalis, musculoskeletal deformities, neurodegeneration and hepatosplenomegaly leading to premature death in most cases. Apart from these, MPS VII is also characterized as adipose storage deficiency disorder although the underlying mechanism of this lean phenotype in the patients or {beta}-GUS-deficient mice still remains a mystery. We addressed this issue using our recently developed Drosophila model of MPS VII (the CG2135-/- fly), which also exhibited a significant loss of adiposity. We report here that the lean phenotype of the CG2135-/- fly is due to fewer number of adipocytes, smaller lipid droplets and reduced adipogenesis. Our data further revealed that there is an abnormal accumulation of autophagosomes in the CG2135-/- larvae due to autophagosome-lysosome fusion defect. Decreased lysosome-mediated turnover also led to attenuated mTOR activity in the CG2135-/- flies. Interestingly, treatment of the CG2135-/- larvae with mTOR stimulators, 3BDO or glucose, led to the restoration of mTOR activity with simultaneous correction of the autophagy defect and adipose storage deficiency. Our finding thus established a hitherto unknown mechanistic link between autophagy dysfunction, mTOR downregulation and reduced adiposity in MPS VII.

sentences: Call count surveys as a tool to monitor habitats occupied by Cheer Pheasants (Catreus wallichii) in Shimla, Himachal Pradesh The vulnerable Cheer Pheasant (Catreus wallichii) is found in the western Himalayas. It prefers successional grasslands which are maintained by residents near villages. Therefore, it is prone to anthropogenic pressures which have led to local extinctions and a decline in population. We conducted five call count surveys at dusk and dawn from 15 April - 15 May 2020 in grasslands of Dharbhog panchayat, Shimla, Himachal Pradesh to assess presence of Cheer Pheasants in different habitat types. We used frequency of occurrence (number of encounters / number of visits) to determine presence or absence of Cheer Pheasant in an area of 10.5 km2 with ten established call count stations. We encountered Cheer Pheasants at six call count stations and the frequency of occurrence of the species ranged from 0 - 0.60. This survey provides an index of occupancy of the species and can be used to assess the change in habitat occupied by the species with time. The local forest department can also use these results to regulate over grazing and burning of grasslands during the breeding season in areas where we recorded the species.

sentences: Molecular properties of Ankrd26 affected by mutations linked to leukemia and carcinoma formation Derailed signaling originating from the plasma membrane is associated with many types of cancer. Mutations in ANKRD26 have been linked to different human cancers and to thrombocytopenia. These mutations include an N-terminal truncation associated with acute myeloid leukemia (AML). We conducted an initial characterization of Ankrd26s protein properties and unveil that an N-terminal amphipathic structure of Ankrd26 lacking in the AML-associated mutant is critical for membrane binding and for Ankrd26s ability to bend membranes by partial membrane insertion. Another Ankrd26 mutation linked to papillary thyroid carcinoma includes truncation and fusion with the kinase domain of the protooncogene RET. Our characterization of Ankrd26 properties reveals that the Ankrd26 part in the Ankrd26-RET fusion mutant is able to anchor the RET kinase domain to the plasma membrane and to mediate self-association - a function we demonstrate to be mediated by Ankrd26s coiled coil domain. Ankrd26-RET fusion led to a massive increase in RET autophosphorylation, i.e. to aberrant RET signaling. Understanding the molecular mechanistic properties of Ankrd26 thus provides important insights into Ankrd26-associated pathomechanisms in human cancer patients.

sentences: Complete sequences of six Major Histocompatibility Complex haplotypes, including all the major MHC class II structures Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina and ultra-long Nanopore sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (B38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.32% - 0.69% in a 1000 Genomes Project read re-mapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.

sentences: Semaphorin3F Drives Dendritic Spine Pruning through Rho-GTPase Signaling Dendritic spines of cortical pyramidal neurons are initially overproduced then remodeled substantially in the adolescent brain to achieve appropriate excitatory balance in mature circuits. Here we investigated the molecular mechanism of developmental spine pruning by Semaphorin 3F (Sema3F) and its holoreceptor complex, which consists of immunoglobulin-class adhesion molecule NrCAM, Neuropilin-2 (Npn2), and PlexinA3 (PlexA3) signaling subunits. Structure-function studies of the NrCAM-Npn2 interface showed that NrCAM stabilizes binding between Npn2 and PlexA3 necessary for Sema3F-induced spine pruning. Using a mouse neuronal culture system, we identified a dual signaling pathway for Sema3F-induced pruning, which involves activation of Tiam1-Rac1-PAK1-3 -LIMK1/2-Cofilin1 and RhoA-ROCK1/2-Myosin II in dendritic spines. Inhibitors of actin remodeling impaired spine collapse in the cortical neurons. Elucidation of these pathways expands our understanding of critical events that sculpt neuronal networks and may provide insight into how interruptions to these pathways could lead to spine dysgenesis in diseases such as autism, bipolar disorder, and schizophrenia.

sentences: MKP-1 modulates Ubiquitination/Phosphorylation of TLR signaling Ubiquitination and phosphorylation are reversible post-translational protein modifications regulating physiological and pathological processes. MAPK phosphatase (MKP)-1 regulates innate and adaptive immunity. The multifaceted roles of MKP-1 were attributed to dephosphorylation of p38 and JNK mitogen activated kinases (MAPKs). We show that the lack of MKP-1 modulates the landscape of ubiquitin ligases and deubiquitinase enzymes (DUBs). MKP-1 deficient mice showed an aberrant regulation of several DUBs and increased expression of proteins and genes involved in IL-1/TLR signaling upstream of MAPK, including IL-1R1, IRAK1, TRAF6, phosphorylated TAK1 and an increased K63-polyubiquitination on TRAF6. Increased K63-polyubiquitination on TRAF6 was associated with an enhanced phosphorylated form of A20. Among abundant DUBs, Ubiquitin-specific-protease-13 (USP13), which cleaves polyubiquitin- chains on client proteins, was substantially enhanced in murine MKP-1 deficient BMDMs. An inhibitor of USP13 decreased the K63-polyubiquitination on TRAF6, TAK1-phosphorylation, IL-1{beta} and TNF- induction in response to LPS in BMDMs. Our data show for the first time that MKP-1 modulates the ligase activity of TRAF6 through modulation of specific DUBs.

sentences: Checks and balances of the RTG pathway under arginine deprivation and canavanine exposure in Saccharomyces cerevisiae Decades of research in Saccharomyces cerevisiae underlie the current dogma of mitochondrial retrograde (RTG) signaling: Rtg2-dependent translocation of the heterodimer Rtg1/Rtg3 from the cytoplasm to the nucleus induces the transcription of RTG-target genes under glutamate starvation or loss of respiration. We previously found that RTG mutants show severe growth inhibition from arginine deprivation and are highly sensitive to canavanine when grown on glucose. Here, we show that on solid media, RTG mutants are also sensitive to thialysine, a toxic lysine analog, although lysine deprivation causes a milder growth defect. Growth on an alternative carbon source restores RTG mutants ability to grow without arginine or lysine and improves their tolerance of toxic analogs; deletion of MIG1 affords a similar rescue on glucose and improves canavanine tolerance, except for in rtg2{Delta}. It is well known that the target of rapamycin (TOR) signaling pathway inhibits the RTG pathway. Batch growth experiments with or without TOR inhibition reveal phenotypic and regulatory differences between RTG mutants. rtg1{Delta} can sustain simultaneous canavanine exposure and TOR inhibition via rapamycin, but rtg2{Delta} and rtg3{Delta} cannot. Surprisingly, our data show that under fermentative lifestyle and arginine deprivation, both RTG signaling and TOR activity are required. This expands the universe of TOR and RTG signaling, suggesting bilateral communication rather than unidirectional RTG regulation by TOR. To the best of our knowledge, this work shows for the first time that Rtg3 activity can be separate from its role as a heterodimer with Rtg1. This work also strongly suggests a specific role for Rtg2 in canavanine tolerance.

sentences: Apolipoprotein E4 has extensive conformational heterogeneity in lipid free and bound forms The {varepsilon}4-allele variant of Apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimers disease, though it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. We apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically-detailed model of monomeric ApoE4 and probe the effect of lipid association. Our data reveal that ApoE4 is far more disordered than previously thought and retains significant conformational heterogeneity after binding lipids. In particular, the behavior of the hinge region and C-terminal domain of ApoE4 differs substantially from that proposed in previous models and provides a crucial foundation for understanding how ApoE4 differs from non-pathogenic and protective variants of the protein.

sentences: Cucumeropsis mannii seed oil (CMSO) ameliorates adipokines dysfunction and dyslipidemia in male Wistar rats exposed to Bisphenol-A Bisphenol-A (BPA) and its analog are extensively utilized in the production of plastics which are rather ubiquitous in our environment. At high temperatures, BPA is leached into water and food packed in plastic containers. This research investigated the ameliorative effects of CMSO on adipokines dysfunction and dyslipidemia in male Wistar rats exposed to Bisphenol-A. thirty-six (36) albino rats weighing 100 - 200 g were randomly assigned into six (6) different experimental groups of controls (1, 2, and 3) and the tests (4, 5, and 6). Group 1 was given only 1 ml of olive oil, group 2 received 100 mg/Kg body weight (b.w) of BPA, group 3 was given 7.5 ml/Kg b.w of CMSO, groups 4, 5, and 6 received 100 mg/Kg b.w of BPA and 7.5, 5 and 2.5 mg/Kg b.w of CMSO respectively. CMSO and BPA were concurrently administered via oral intubation for periods of 42 days. Lipid profile and adipokines levels were determined in plasma and adipose tissue. BPA in male rats significantly (p<0.05) elevated the levels of cholesterol, triglycerides, LDL-C, liptin, and coronary and atherogenic risk indices in plasma and adipose tissue with reductions in HDL-C and adiponectin levels. BPA plus CMSO in male rats significantly (p<0.05) decreased the levels of cholesterol, triglycerides, LDL-C, liptin, and coronary and atherogenic risk indices with an elevation of HDL-C and adiponectin levels in both plasma and adipose tissue. These results suggest that CMSO could be useful in the management of cardiovascular-related diseases induced by BPA.

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Rapid and accurate identification of Escherichia coli STEC O157:H7 by mass spectrometry, artificial intelligence and detection of specific biomarkers peaks. The different pathotypes of Escherichia can produce a large number of human diseases. Surveillance becomes complex since their differentiation are not easy.

Particularly, the detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 consists of stool culture of a diarrheal sample in enrichment and/or selective media, identification of presumptive colonies and confirmation by Multiplex PCR technique for the genotypic characterization of serogroup O157 and Shiga toxins (stx1 and stx2), in addition to the traditional biochemical identification.

All of these procedures are laborious, require a certain level of training, are time consuming and expensive. Among the currently most widely used methodologies, MALDI-TOF MS mass spectrometry (matrix-assisted laser desorption/ionization with time-of-flight mass detection), allows a quick and easy way to obtain a protein spectrum of a microorganism, not only in order to identify the genus and species, but also the discovery of potential biomarker peaks of a certain characteristic. In the present work, the information obtained from 60 clinical isolates was used to detect peptide fingerprints of STEC O157:H7 and other diarrheagenic E. coli. The differences found in the protein profiles of the different pathotypes established the foundations for the development and evaluation of classification models through automated training.

The application of the Biomarkers in combination with the predictive models on a new set of samples (n=142), achieved 99.3% of correct classifications, allowing the distinction between STEC O157:H7 isolates from the other diarrheal Escherichia coli.

Therefore, given that STEC O157:H7 is the main causal agent of haemolytic uremic syndrome and based on the performance values obtained in the present work (Sensitivity=98.5% and Specificity=100%), this development could be a useful tool for diagnosis of the disease in clinical microbiology laboratories.

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A non-conventional archaeal fluorinase identified by in silico mining catalyzes the fastest natural nucleophilic fluorination Fluorinases, the only enzymes known to catalyze the transfer of fluorine to an organic molecule, are essential biocatalysts for the sustainable synthesis of valuable organofluorines. However, the few fluorinases identified so far have low turnover rates that hamper biotechnological applications. Here, we isolated and characterized putative fluorinases retrieved from systematic in silico mining and identified a non-conventional archaeal enzyme from Methanosaeta sp. that mediates the highest fluorination rate reported to date. Furthermore, we demonstrate enhanced production of fluoronucleotides in vivo in a bacterial host engineered with this archaeal fluorinase, paving the way towards synthetic metabolism for efficient biohalogenations.

Fastest biological SN2 fluorination

sentences: The role of antifreeze glycopeptides (AFGP) and polyvinyl alcohol/polyglycerol (X/Z-1000) cocktails as ice modulators during partial freezing of rat livers. The current liver organ shortage has pushed the field of liver transplantation to develop new methods to prolong the preservation time of livers from the current clinical standard of static cold storage. Our approach, termed partial freezing, aims to induce a thermodynamically stable frozen state at deeper storage temperatures (-10{degrees}C to -15{degrees}C) than can be achieved with supercooling, while simultaneously maintaining a sufficient unfrozen fraction to limit dehydration and ice damage. This research first demonstrated that partially frozen glycerol treated rat livers were functionally similar after thawing from either -10 or -15{degrees}C with respect to subnormothermic machine perfusion metrics and histology. Next, we assessed the effect of adding either of two ice modulators, antifreeze glycoprotein (AFGP) and a polyvinyl alcohol/polyglycerol combination (X/Z-1000), on the viability and structural integrity of partially frozen rat livers compared to glycerol-only control livers. Results showed that AFGP livers had high levels of ATP and the least edema but suffered from significant endothelial cell damage. X/Z-1000 livers had the highest levels of ATP and energy charge (EC) but also demonstrated endothelial damage and post-thaw edema. Glycerol-only control livers exhibited the least DNA damage on Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining but also had the lowest levels of ATP and EC. Further research is necessary to optimize the ideal ice modulator cocktail for our partial-freezing protocol. Modifications to cryoprotective agent (CPA) combinations, as well as improvements to machine perfusion CPA loading and unloading, can help improve the viability of these partially frozen organs.

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Defining the Sensitivity Landscape of 74,389 EGFR Variants to Tyrosine Kinase Inhibitors BackgroundTyrosine kinase inhibitors (TKIs) therapy is a standard treatment for patients with advanced non-small-cell lung carcinoma (NSCLC) when activating epidermal growth factor receptor (EGFR) mutations are detected. However, except for the well-studied EGFR mutations, most EGFR mutations lack treatment regimens.

MethodsWe constructed two EGFR variant libraries containing substitutions, deletions, or insertions using the saturation mutagenesis method. All the variants were located in the EGFR mutation hotspot (exons 18-21). The sensitivity of these variants to afatinib, erlotinib, gefitinib, icotinib, and osimertinib was systematically studied by determining their enrichment in massively parallel cytotoxicity assays using an endogenous EGFR-depleted cell line, PC9.

ResultsA total of 3,914 and 70,475 variants were detected in the constructed EGFR Substitution-Deletion (Sub-Del) and exon 20 Insertion (Ins) libraries, accounting for 99.3% and 55.8% of the designed variants, respectively. Of the 3,914 Sub-Del variants, 813 were highly enriched in the reversible TKI (erlotinib, gefitinib, icotinib) cytotoxicity assays and 51 were enriched in the irreversible TKI (afatinib, osimertinib) cytotoxicity assays. For the 70,475 Ins variants, insertions at amino acid positions 770-774 were highly enriched in all the five TKI cytotoxicity assays. Moreover, the top 5% of the enriched insertion variants included a glycine or serine insertion at high frequency.

ConclusionsWe present a comprehensive reference for the sensitivity of EGFR variants to five commonly used TKIs. The approach used here should be applicable to other genes and targeted drugs.

sentences: Structural and Functional Diversity among Agonist-Bound States of the GLP-1 Receptor Recent advances in G protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multi-dimensional signal propagation mediated by these receptors is, in part, dependent on their conformational mobility. However, the relationship between receptor function and static structures determined via crystallography or cryo-electron microscopy is not always clear. This study examines the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide-1 receptor (GLP-1R), an important clinical target. We employ variants of the peptides GLP-1 and exendin-4 to explore the interplay between helical propensity near the agonist N-terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an exendin-4 analogue, the GLP-1R and Gs protein revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.

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Tracing the Indian Population Ancestry by cis-linked Mutations in HBB Gene BackgroundHuman left their genetic footprints during the time of migration throughout the different countries all over the world. Human evolution was studied through various markers. India is a country of rich heritage and cultural diversity. The modern Indian population is derived from two ancestral groups, viz.-Ancestral North Indians (ANI) and Ancestral South Indians (ASI).

AimFinding out the migratory route of the modern Indian population by studying  cis acting mutations of human beta-globin (HBB) genes.

Subjects and methodsA total of 120 thalassemia subjects were enrolled. DNA sequencing was done for mutation detection in the HBB gene. Some previous literature reviews were gone through for tracing mutations, all over the world and in the Indian subcontinent.

ResultsNine thalassemia patients were found where HBB:c.92G>C and HBB:c.-92C>G mutations co-exist together in  cis condition. Only one patient had HBB:c.51delC and HBB:c.33C>A. The pedigree analysis confirmed the presence of these mutations in  cis condition and vertical transmission from one generation to the next. Literature reviews also reassure the co-existence of these mutations from different countries.

ConclusionThe co-existence of these  cis acting mutations helps to point out the possible migratory route of ANI population after venturing out of Africa.

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Cost-effectiveness of quadrivalent versus trivalent inactivated influenza vaccines for the Portuguese elderly population BackgroundQuadrivalent Inactivated Vaccine (QIV) is expected to replace Trivalent Inactivated Vaccine (TIV) over time. In Portugal, TIV is free of charge for risk groups, which include older adults. In its turn, QIV - which provides broader protection as it includes an additional lineage B strain - was introduced in Portugal in October 2018, but only from the influenza season 2019/20 was provided free of charge for the risk groups. This study evaluates the cost-effectiveness of switching from TIV to QIV from the National Health Service perspective in the Portuguese elderly mainland population ([≥] 65 years old).

MethodsA decision tree model was developed to compare TIV and QIV, based on Portuguese hospitalization data for the 2015/16 influenza season. The primary health economic outcome under consideration was the Incremental Cost-Effectiveness Ratio (ICER). In addition, a one-Way Sensitivity Analysis was performed to evaluate the impact of model parameters on the ICER; Probabilistic Sensitivity Analysis was also carried out to analyze the robustness of the base case results.

ResultsThe high cost of QIV (approximately three times the cost of TIV) would lead to a total increment of 5,283,047 {euro}, and the resulting ICER would be 26,403,007{euro}/QALY, mainly above the usual willingness-to-pay threshold.

ConclusionsFrom the National Health Service perspective, our findings reveal that QIV is not cost-effective for the Portuguese elderly population due to the high cost. If the QIV costs were the same as the TIV, then QIV would be cost-effective.

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Gestationally-Dependent Immune Organization at the Maternal-Fetal Interface The immune system and placenta have a dynamic relationship across gestation to accommodate fetal growth and development. High-resolution characterization of this maternal- fetal interface is necessary to better understand the immunology of pregnancy and its complications. We developed a single-cell framework to simultaneously immuno-phenotype circulating, endovascular, and tissue-resident cells at the maternal-fetal interface throughout gestation, discriminating maternal and fetal contributions. Our data reveal distinct immune profiles across the endovascular and tissue compartments with tractable dynamics throughout gestation that respond to a systemic immune challenge in a gestationally-dependent manner. We uncover that mononuclear phagocytes and neutrophils drive the temporal immune composition of the placenta with remarkably diverse populations, including PD-L1-expressing subsets having compartmental and early gestational bias. Our approach and accompanying datasets provide a resource for additional investigations into gestational immunology and evoke a more significant role for the innate immune system in establishing the microenvironment of early pregnancy.

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sentences: Dual origin and multiple neuropeptidergic trajectories of hypothalamic POMC progenitors revealed by developmental single-cell transcriptomics Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential to regulate food intake and energy balance. However, the ontogenetic transcriptional programs that specify the identity and functioning of these neurons are poorly understood. Here, we use scRNAseq to define the transcriptomes characterizing POMC progenitors in the developing hypothalamus and TRAP-seq to analyze the subsequent translatomes of mature POMC neurons. Our data show that Pomc-expressing neurons originate from multiple developmental pathways expressing distinct combinations of transcription factors. The predominant cluster, featured by high levels of Pomc and Prdm12 transcripts represents the precursor of canonical arcuate POMC neurons. Additional clusters of progenitors expressing lower levels of Pomc mature into different neuronal phenotypes characterized by distinct combinations of transcription factors, neuropeptides, processing enzymes, cell surface and nuclear receptors. We conclude that the genetic programs specifying the identity and differentiation of arcuate POMC neurons are diverse and generate a heterogeneous repertoire of neuronal phenotypes early in development that continue to evolve postnatally.

sentences: Fine-scale quantification of GC-biased gene conversion intensity in mammals. GC-biased gene conversion (gBGC) is a molecular evolutionary force that favours GC over AT alleles irrespective of their fitness effect. Quantifying the variation in time and across genomes of its intensity is key to properly interpret patterns of molecular evolution. In particular, the existing literature is unclear regarding the relationship between gBGC strength and species effective population size, Ne. Here we analysed the nucleotide substitution pattern in coding sequences of closely related species of mammals, thus accessing a high resolution map of the intensity of gBGC. Our maximum likelihood approach shows that gBGC is pervasive, highly variable among species and genes, and of strength positively correlated with Ne in mammals. We estimate that gBGC explains up to 60% of the total amount of synonymous AT[->]GC substitutions. We show that the fine-scale analysis of gBGC-induced nucleotide substitutions has the potential to inform on various aspects of molecular evolution, such as the distribution of fitness effects of mutations and the dynamics of recombination hotspots.

sentences: α-Synuclein pre-formed fibrils induce prion-like protein aggregation and neurotoxicity in C. elegans models of Parkinson's disease Parkinsons disease (PD) is a debilitating neurodegenerative disorder characterized by progressive motor decline and the aggregation of -synuclein protein. Growing evidence suggests that -synuclein aggregates may spread from neurons of the digestive tract to the brain in a prion-like manner. While rodent models have recapitulated gut-to-brain -synuclein transmission, animal models that are amenable to high-throughput investigations are needed to facilitate the discovery of disease mechanisms. Here we describe the first C. elegans models in which feeding with -synuclein pre-formed fibrils (PFFs) induced prion-like dopamine neuron degeneration and seeding of aggregation of human -synuclein expressed in the host. PFF acceleration of -synuclein aggregation in C. elegans muscle cells was associated with a progressive motor deficit, whereas feeding with -synuclein monomer produced much milder effects. RNAi-mediated knockdown of the C. elegans syndecan sdn-1, and enzymes involved in heparan sulfate proteoglycan biosynthesis, afforded protection from PFF-induced seeding of aggregation and toxicity, as well as dopaminergic neurodegeneration. This work offers new models by which to investigate gut-derived -synuclein spreading and propagation of disease.

sentences: Emergent Diversity and Persistent Turnover in Evolving Microbial Cross-Feeding Networks A distinguishing feature of many ecological networks in the microbial realm is the diversity of substrates that could potentially serve as energy sources for microbial consumers. The microorganisms are themselves the agents of compound diversification via metabolite excretion or overflow metabolism. It has been suggested that the emerging richness of different substrates is an important condition for the immense biological diversity in microbial ecosystems. In this work, we study how complex cross-feeding networks (CFN) of microbial species may develop from a simple initial community given some elemental evolutionary mechanisms of resource-dependent speciation and extinctions using a network flow model. We report results of several numerical experiments and report an in-depth analysis of the evolutionary dynamics. We find that even in stable environments, the system is subject to persisting turnover, indicating an ongoing co-evolution. Further, we compare the impact of different parameters, such as the ratio of mineralization, as well as the metabolic versatility and variability on the evolving community structure. The results imply that high microbial and molecular diversity is an emergent property of evolution in cross-feeding networks, which affects transformation and accumulation of substrates in natural systems, such as soils and oceans, with potential relevance to biotechnological applications.

sentences: A marked enhancement of a BLOC-1 gene, pallidin, associated with somnolent mouse models deficient in histamine transmission Histamine (HA) and orexin (Ox, or hypocretin) neurons act distinctly and synergistically in wake control. A double knock out mouse genotype lacking both HA and Ox shows all sleep disorders of human narcolepsy. We identified in this mouse brain a sharp upregulation of a BLOC-1 gene, pallidin that is associated with dramatic changes in the balance of cholinergic and aminergic systems in mice and an enhanced sleep in drosophila. This study demonstrates potential sleep disorders-associated compensatory mechanisms with pallid as a novel biomarker.

sentences: The Impact of Neuronal Structure on Cortical Network Architecture It has become increasingly clear that the neurons in the cerebral cortex are not randomly interconnected. This wiring specificity can result from synapse formation mechanisms that interconnect neurons depending on their activity or genetically defined identity. Here we report that in addition to these synapse formation mechanisms, the structural composition of the neuropil provides a third prominent source by which wiring specificity emerges in cortical networks. This structurally determined wiring specificity reflects the packing density, morphological diversity and similarity of the dendritic and axonal processes. The higher these three factors are, the more recurrent the topology of the networks. Conversely, low density, diversity and similarity yield feedforward networks. These principles predict connectivity patterns from subcellular to network scales that are remarkably consistent with empirical observations from a rich body of literature. Thus, cortical network architectures reflect the specific morphological properties of their constituents to much larger degrees than previously thought.

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Oral Glucosamine Ameliorates Aggravated Neurological Phenotype in Mucopolysaccharidosis III Type C Mouse Model Expressing Misfolded HGSNAT Variant ObjectiveOver 55% of mucopolysaccharidosis IIIC (MPS IIIC) patients have at least one allelic missense variant responsible for misfolding of heparan sulfate acetyl-CoA:-glucosaminide N- acetyltransferase (HGSNAT). These variants are potentially treatable with pharmacological chaperones, such as a competitive HGSNAT inhibitor, glucosamine. Since the constitutive HGSNAT knockout mice, we generated previously cannot be used to test such strategy in vivo, we generated a novel model, the HgsnatP304L strain, expressing misfolded mutant HGSNAT with human missense mutation Pro311Leu (Pro304Leu in the mouse enzyme).

ResultsHgsnatP304L mice present deficits in short-term (novel object recognition test) and working/spatial (Y-maze test) memory at 4 months of age, 2-4 months earlier than previously described gene-targeted Hgsnat-Geo mice, which lack HGSNAT protein. HgsnatP304L mice also show increased severity of synaptic deficits in CA1 neurons, and accelerated course of CNS pathology including neuronal storage of heparan sulfate, accumulation of misfolded proteins, increase of simple gangliosides, and neuroinflammation as compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat- Geo neurons aggravated reduction of synaptic proteins. Memory deficits and majority of pathological changes in the brain were rescued in mice receiving daily doses of oral glucosamine.

InterpretationAltogether, our data for the first time demonstrate dominant-negative effects of the misfolded HGSNAT Pro304Leu variant and show that these effects are treatable by oral administration of glucosamine, suggesting that patients, affected with missense mutations preventing normal folding of the enzyme, could benefit from chaperone therapy.

sentences: WIN 55,212-2 shows anti-inflammatory and survival properties in human iPSC-derived cardiomyocytes infected with SARS-CoV-2 Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that cause damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the "cytokine storm", strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins 6, 8, 18 and tumor necrosis factor-alpha (TNF-) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.

sentences: In vitro evaluation of the activity of terpenes and cannabidiol against Human Coronavirus E229 The activity of a new, terpene-based formulation, code-named NT-VRL-1, against Human Coronavirus (HCoV) strain 229E was evaluated in human lung fibroblasts (MRC-5 cells), with and without the addition of cannabidiol (CBD). The tested formulation exhibited an antiviral effect when it was pre-incubated with the host cells prior to virus infection. The combination of NT-VRL-1 with CBD potentiated the antiviral effect better than the positive controls pyrazofurin and glycyrrhizin. There was a strong correlation between the quantitative results from a cell-viability assay and the cytopathic effect seen under the microscope after 72 h. To the best of our knowledge, this is the first report of activity of a combination of terpenes and CBD against a coronavirus.

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Habitat complexity in organic olive orchards modulates the abundance of natural enemies but not the attraction to plant species Semi-natural habitat complexity and organic management could affect the abundance and diversity of natural enemies and pollinators in olive orchards. Nonetheless, in such agroecosystems the effect of plant structure, plant richness, and plant attraction on the arthropod fauna has been poorly documented. Here we evaluate the influence of those effects jointly as an expression of arthropod abundance and richness in olive trees, ground cover, and adjacent vegetation within organic olive orchards. For this, we used generalized linear models and non-metric multidimensional scaling (NMDS) integrating generalized additive models. Our results suggest that natural enemies and pollinators are mainly attracted to A. radiatus, D. catholica, and L. longirrostris within ground cover and G. cinerea speciosa, Q. rotundifolia, R. officinalis, T. zygis gracilis, and U. parviflorus within adjacent vegetation. Accordingly, habitat complexity showed a positive relationship with the abundance of key families of natural enemies and pollinators but not with the number of taxa. NMDS showed that plant richness and plant arrangement and scattering affected the key families differently, suggesting that each key family responds to their individual needs for plant resources but forming groups modulated by complexity. This pattern was especially seeing in predators and omnivores. Our findings support that the higher the plant richness and structure of a semi natural-habitat within an olive orchard, the higher the abundance and richness of a given arthropod community (a pattern found in natural ecosystems). The information presented here can be used by producers and technicians to increase the presence and abundance of natural enemies and pollinators within organic olive orchards, and thus improve the ecosystem services provided by semi-natural habitats.

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Dissecting indirect genetic effects from peers in laboratory mice The phenotype of one individual can be affected not only by the individuals own genotypes (direct genetic effects, DGE) but also by genotypes of interacting partners (indirect genetic effects, IGE). IGE have been detected using polygenic models in multiple species, including laboratory mice and humans. However, the underlying mechanisms remain largely unknown. Genome-wide association studies of IGE (igeGWAS) can point to IGE genes, but have not yet been applied to non-familial IGE arising from "peers" and affecting biomedical phenotypes. In addition, the extent to which igeGWAS will identify loci not identified by dgeGWAS remains an open question. Finally, findings from igeGWAS have not been confirmed by experimental manipulation.

We leveraged a dataset of 170 behavioural, physiological and morphological phenotypes measured in 1,812 genetically heterogeneous laboratory mice to study IGE arising between same-sex, adult, unrelated laboratory mice housed in the same cage. We developed methods for igeGWAS in this context and identified 24 significant IGE loci for 17 phenotypes (FDR < 10%). There was no overlap between IGE loci and DGE loci for the same phenotype, which was consistent with the moderate genetic correlations between DGE and IGE for the same phenotype estimated using polygenic models. Finally, we fine-mapped seven significant IGE loci to individual genes and confirmed, in an experiment with a knockout model, that Epha4 gives rise to IGE on stress-coping strategy and wound healing.

Our results demonstrate the potential for igeGWAS to identify IGE genes and shed some light into the mechanisms of peer influence.

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Global change affects differently at local scales animal populations: why Starlings still expands to the south while other temperate species go to the north? Global change, which regroups global warming, landscape transformations and other anthropic modifications of ecosystems, has effects on populations and communities and produces modifications in the expansion area of species. While some species disappear, other ones are beneficiated by the new conditions and some of them evolve in new adapted forms or leave their ancient distribution area.

As climate change tends to increase the temperature in several regions of the world, some species have been seen to leave areas in equatorial regions in order to join colder areas either towards the north of the northern hemisphere or towards the south of the southern one.

Many birds as have moved geographically in direction to the poles and in many cases they have anticipated their laying dates. Actually, two tit species that use to lay their eggs in a period that their fledging dates synchronize with the emerging dates of caterpillars are now evolving to reproductive in periods earlier than before the climate change. Several species are reacting like that and other ones are moving to the north in Europe for example. Nevertheless, and very curiously, European starling, Sturnus vulgaris, populations are behaving on the contrary: their laying dates are moving towards later spring and their distribution area is moving towards the south. In this study we explore and discuss about different factors that may explain this difference from other birds.

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Brain virtual histology with X-ray phase-contrast tomography Part II: 3D morphologies of amyloid-β plaques in Alzheimer's disease models While numerous transgenic mouse strains have been produced to model the formation of amyloid-{beta} (A{beta}) plaques in the brain, efficient methods for whole-brain 3D analysis of A{beta} deposits are lacking. Moreover, standard immunohistochemistry performed on brain slices precludes any shape analysis of A{beta} plaques. The present study shows how in-line (propagation-based) X-ray phase-contrast tomography (XPCT) combined with ethanol-induced brain sample dehydration enables hippocampus-wide detection and morphometric analysis of A{beta} plaques. Performed in three distinct Alzheimer mouse strains, the proposed workflow identified differences in signal intensity and 3D shape parameters: 3xTg displayed a different type of A{beta} plaques, with a larger volume and area, greater elongation, flatness and mean breadth, and more intense average signal than J20 and APP/PS1. As a label-free non-destructive technique, XPCT can be combined with standard immunohistochemistry. XPCT virtual histology could thus become instrumental in quantifying the 3D spreading and the morphological impact of seeding when studying prion-like properties of A{beta} aggregates in animal models of Alzheimers disease. This is Part II of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part I shows how in-line XPCT enables 3D myelin mapping in the whole rodent brain and in human autopsy brain tissue.

HighlightsO_LIX-ray phase-contrast tomography (XPCT) enables whole brain detection of A{beta} plaques
C_LIO_LIMorphometric parameters of A{beta} plaques may be readily retrieved from XPCT data
C_LIO_LINew shape parameters were successfully extracted from three Alzheimers disease models
C_LIO_LIA Fiji-based "biologist-friendly" analysis workflow is proposed and shared
C_LIO_LIXPCT is a powerful virtual histology tool that requires minimal sample preparation
C_LI

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sentences: Population receptive fields in non-human primates from whole-brain fMRI and large-scale neurophysiology in visual cortex Population receptive field (pRF) modeling is a popular fMRI method to map the retinotopic organization of the human brain. While fMRI-based pRF-maps are qualitatively similar to invasively recorded single-cell receptive fields in animals, it remains unclear what neuronal signal they represent. We addressed this question in awake non-human primates comparing whole-brain fMRI and large-scale neurophysiological recordings in areas V1 and V4 of the visual cortex. We examined the fits of several pRF-models based on the fMRI BOLD-signal, multi-unit spiking activity (MUA) and local field potential (LFP) power in different frequency bands. We found that pRFs derived from BOLD-fMRI were most similar to MUA-pRFs in V1 and V4, while pRFs based on LFP gamma power also gave a good approximation. FMRI-based pRFs thus reliably reflect neuronal receptive field properties in the primate brain. In addition to our results in V1 and V4, the whole-brain fMRI measurements revealed retinotopic tuning in many other cortical and subcortical areas with a consistent increase in pRF-size with increasing eccentricity, as well as a retinotopically specific deactivation of default-mode network nodes similar to previous observations in humans.

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Genome-wide, genic, and site-specific changes associated with the convergent evolution of sociality in spiders Sociality is a striking phenotypic innovation that independently evolved dozens of times across animals. Sociogenomic approaches have begun to elucidate the molecular underpinnings of social life in social insects and vertebrates, but the degree to which the convergent evolution of sociality involves convergent molecular evolution remains controversial and largely unknown. Spiders are a powerful system for identifying the genomic causes and consequences of social life because sociality is estimated to have independently evolved 15 times, and each origin likely occurred recently, within the past few million years. To determine if there are statistically supported genomic signatures of protein-coding sequence evolution associated with the convergent evolution of sociality in spiders, we compared the genomes or transcriptomes of 24 spider species that vary in social organization and represent at least seven independent origins of sociality. We identified hundreds of genes that experienced shifts in patterns of molecular evolution during the convergent evolution of sociality, and these genes were enriched for several annotated functions, including neural function, neurogenesis, and behavior, as well as immune function, growth, and metabolism. We also found evidence that directional selection for specific substitutions repeatedly occurred in social species for several genes, in particular the calcium channel gene TPC1. Finally, supporting previous results, we found elevated genome-wide rates of molecular evolution in social species, resulting mainly from relaxation of selection. Altogether, we identify genome-wide, genic, and site-specific changes that repeatedly occurred during the evolution of sociality in spiders.

Significance StatementThe transition from solitary to social life is a major transition in evolution that repeatedly occurred, but the genetic underpinnings are largely unknown. To identify genomic changes associated with sociality, we compared the genomes of 24 spider species, representing seven recent and independent origins of sociality. We identified hundreds of genes and many functional classes of genes that tended to experience shifts in molecular evolution, including genes that have previously been implicated in animal social behavior and human behavioral disorders. Our study shows that while the precise genetic details vary, the repeated evolution of sociality in spiders predictably leaves genome-wide signatures and involves sets of genes with conserved functions that may play general roles in the evolution of social behavior.

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Advanced paternal age diversifies individual trajectories of vocalization patterns in neonatal mice Infant crying is an innate communicative behavior that is frequently impaired in certain neurodevelopmental disorders (NDDs). Since advanced paternal age is a reported risk factor for NDDs in offspring, we evaluated the impact of a fathers age on early vocal development in C57BL/6J mice. We recorded and applied a unique combination of computational analyses to ultrasonic vocalizations (USVs) emitted by mouse pups sired by young and aged fathers. Our data showed that advanced paternal age reduced the number and duration of USVs, and altered the syllable composition in pups. Moreover, pups born to young fathers showed convergent vocal characteristics with a rich repertoire during postnatal development, while those born to aged fathers exhibited more divergent vocal patterns with limited repertoire. Principal component analysis in conjunction with clustering analysis demonstrated that pups from aged fathers deviated from typical trajectories of vocal development, which were considered as atypical individuals. Thus, our study indicates that advanced paternal age has a significant effect on offsprings early vocal development. It is suggested that the trajectories of vocal development could be a useful marker of the NDD-like phenotype associated with the advanced paternal age. In addition, our comprehensive computational analysis described here is an effective approach to characterize the altered individual diversity relevant to neurodevelopmental disorders.

One Sentence SummaryAdvanced paternal age affects vocal development in early postnatal mice, with more pups showing atypical developmental trajectories.

sentences: High axial-resolution optical stimulation of neurons in vivo via two-photon optogenetics with speckle-free beaded-ring pattern Two-photon optogenetics has become an indispensable technology in neuroscience, due to its capability in precise and specific manipulation of neural activities. Scanless holographic approach is generally adopted to meet the requirement of stimulating neural emsembles simultaneously. However, the commonly used disk patterns fail in achieving single-neuron resolution, especially in axial dimension, and its inherent speckles decrease the stimulation efficiency. Here, we propose a novel speckle-free, beaded-ring pattern for high axial-resolution optical stimulation of neurons in vivo. Using a dye pool and a fluorescent thin-film as samples, we verify that, compared to those with disk patterns, higher axial resolution and better localization ability can be achieved with beaded-ring patterns. Furthermore, we perform two-photon based all-optical physiology with neurons in mouse S1 cortex in vivo, and demonstrate that the axial resolution obtained by beaded-ring patterns can be improved by 24% when stimulating multiple neurons, compared with that of disk patterns.

sentences: Engineering site-selective incorporation of fluorine into natural product analogs While bioactive compounds are commonly derived both by human design as well as from living organisms, man-made and natural products typically display very different structural characteristics. As such, a longstanding goal in the discovery of new molecular function is to develop approaches to incorporate the advantageous elements of both groups of molecules, thereby expanding the molecular space accessible for this purpose. In this work, we report the engineering a fluorine-selective enzyme that can complement mutated acyltransferase (AT) domains of a modular polyketide synthase, which are the main determinants of the identity and location of substituents on polyketides, to produce different fluorinated regioisomers of the erythromycin precursor in vitro. We further show that by engineering cell uptake of fluorinated building blocks, we can control fluorine selectivity in vivo to produce selectively fluorinated polyketides using engineered E. coli. These results demonstrate that it is possible to introduce fluorine, a key synthetic design element for drug development, selectively into the scaffold of a complex natural product and produce these analogs by microbial fermentation.

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metapredict: a fast, accurate, and easy-to-use predictor of consensus disorder and structure Intrinsically disordered proteins and protein regions make up a substantial fraction of many proteomes where they play a wide variety of essential roles. A critical first step in understanding the role of disordered protein regions in biological function is to identify those disordered regions correctly. Computational methods for disorder prediction have emerged as a core set of tools to guide experiments, interpret results, and develop hypotheses. Given the multiple different predictors available, consensus scores have emerged as a popular approach to mitigate biases or limitations of any single method. Consensus scores integrate the outcome of multiple independent disorder predictors and provide a per-residue value that reflects the number of tools that predict a residue to be disordered. Although consensus scores help mitigate the inherent problems of using any single disorder predictor, they are computationally expensive to generate. They also necessitate the installation of multiple different software tools, which can be prohibitively difficult. To address this challenge, we developed a deep-learning-based predictor of consensus disorder scores. Our predictor, metapredict, utilizes a bidirectional recurrent neural network trained on the consensus disorder scores from 12 proteomes. By benchmarking metapredict using two orthogonal approaches, we found that metapredict is among the most accurate disorder predictors currently available. Metapredict is also remarkably fast, enabling proteome-scale disorder prediction in minutes. Importantly, metapredict is fully open source and is distributed as a Python package, a collection of command-line tools, and a web server, maximizing the potential practical utility of the predictor. We believe metapredict offers a convenient, accessible, accurate, and high-performance predictor for single-proteins and proteomes alike.

Statement of SignificanceIntrinsically disordered regions are found across all kingdoms of life where they play a variety of essential roles. Being able to accurately and quickly identify disordered regions in proteins using just the amino acid sequence is critical for the appropriate design and interpretation of experiments. Despite this, performing large-scale disorder prediction on thousands of sequences is challenging using extant disorder predictors due to various difficulties including general installation and computational requirements. We have developed an accurate, high-performance and easy-to-use predictor of protein disorder and structure. Our predictor, metapredict, was designed for both proteome-scale analysis and individual sequence predictions alike. Metapredict is implemented as a collection of local tools and an online web server, and is appropriate for both seasoned computational biologists and novices alike.

sentences: PEPSDI: Scalable and flexible inference framework for stochastic dynamic single-cell models Mathematical modelling is an invaluable tool to describe dynamic cellular processes and to rationalise cell-to-cell variability within the population. This requires statistical methods to infer unknown model parameters from dynamic, multi-individual data accounting for heterogeneity caused by both intrinsic and extrinsic noise. Here we present PEPSDI, a scalable and flexible framework for Bayesian inference in state-space mixed-effects stochastic dynamic single-cell models. Unlike previous frameworks, PEPSDI imposes a few modelling assumptions when inferring unknown model parameters from time-lapse data. Specifically, it can infer model parameters when intrinsic noise is modelled by either exact or approximate stochastic simulators, and when extrinsic noise is modelled by either time-varying, or time-constant parameters that vary between cells. This allowed us to identify hexokinase activity as a source of extrinsic noise, and to deduce that sugar availability dictates cell-to-cell variability in the budding yeast Saccharomyces cerevisiae SNF1 nutrient sensing pathway.

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Mutagenesis-visualization: analysis of site saturation mutagenesis datasets in Python SummarySite-saturation mutagenesis experiments have been transformative in our study of protein function. Despite the rich data generated from such experiments, current tools for processing, analyzing, and visualizing the data offer only a limited set of static visualization tools that are difficult to customize. Furthermore, usage of the tools requires extensive experience and programming knowledge, slowing the research process for those in the biological field who are unfamiliar with programming. Here, we introduce mutagenesis-visualization, a Python package for creating publication-quality figures for site-saturation mutagenesis datasets without the need for prior Python or statistics experience, where each of the graphs is generated with a one-line command. The plots can be rendered as native Matplotlib objects (easy to stylize) or Plotly objects (interactive graphs). Additionally, the software offers the possibility to visualize the datasets on Pymol.

Availability and implementationThe software can be installed from PyPI or GitHub using the pip package manager and is compatible with Python [≥] 3.8. The documentation can be found at readthedocs and the source code on GitHub.

sentences: BubR1 recruitment to the kinetochore via Bub1 enhances Spindle Assembly Checkpoint signaling During mitosis, unattached kinetochores in a dividing cell activate the Spindle Assembly Checkpoint (SAC) and delay anaphase onset by generating the anaphase-inhibitory Mitotic Checkpoint Complex (MCC). These kinetochores generate the MCC by recruiting MCC constituent proteins, including BubR1. In principle, BubR1 recruitment to signaling kinetochores should increase its local concentration and promote MCC formation. However, in human cells BubR1 is mainly thought to sensitize the SAC to silencing; whether BubR1 localization to signaling kinetochores per se enhances SAC signaling activity remains unknown. Therefore, we used ectopic SAC activation systems (eSAC) to isolate two molecules that recruit BubR1 to the kinetochore: the checkpoint protein Bub1 and the KI and MELT motifs in the kinetochore protein KNL1, and observed their contribution to eSAC signaling. Our quantitative analyses and mathematical modeling show that the Bub1-mediated BubR1 recruitment to the human kinetochore promotes SAC signaling and highlight BubR1s dual role of directly strengthening the SAC and indirectly silencing it.

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EEG microstate dynamics indicate a U-shaped path to propofol-induced loss of consciousness It is commonly believed that the stream of consciousness is not continuous but parsed into transient brain states manifesting themselves as discrete spatiotemporal patterns of global neuronal activity. Electroencephalographical (EEG) microstates are proposed as the neurophysiological correlates of these transiently stable brain states that last for fractions of seconds. To further understand the link between EEG microstate dynamics and consciousness, we continuously recorded high-density EEG in 23 surgical patients from their awake state to unconsciousness, induced by step-wise increasing concentrations of the intravenous anesthetic propofol. Besides the conventional parameters of microstate dynamics, we introduce a new method that estimates the complexity of microstate sequences. The brain activity under the surgical anesthesia showed a decreased sequence complexity of the stereotypical microstates, which became sparser and longer-lasting. However, we observed an initial increase in microstates temporal dynamics and complexity with increasing depth of sedation leading to a distinctive "U-shape" that may be linked to the paradoxical excitation induced by moderate levels of propofol. Our results support the idea that the brain is in a metastable state under normal conditions, balancing between order and chaos in order to flexibly switch from one state to another. The temporal dynamics of EEG microstates indicate changes of this critical balance between stability and transition that lead to altered states of consciousness.

HighlightsO_LIEEG microstates capture discrete spatiotemporal patterns of global neuronal activity
C_LIO_LIWe studied their temporal dynamics in relation to different states of consciousness
C_LIO_LIWe introduce a new method to estimate the complexity of microstates sequences
C_LIO_LIWith moderate sedation complexity increases then decreases with full sedation
C_LIO_LIComplexity of microstate sequences is sensitive to altered states of consciousness
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sentences: Characterization of Quantum Dots with Hyperspectral Fluorescence Microscopy for Multiplexed Optical Imaging of Biomolecules The optical properties of quantum dots were extensively characterized using a hyperspectral fluorescence microscopy system. The system provides a single excitation wavelength in the ultraviolet and 311 emission wavelength channels in the visible. This allows detection of multiple fluorophores (e.g. different quantum dots) with a high spectral resolution in one go which is not achievable with a conventional fluorescence microscope where different filter sets have to be used. A spectral library was established based on the spectral profiles of six types of quantum dots. Notably, a slight spectral shift was observed for all samples while the sample was drying. Subsequently, two quantum dot mixture samples were studied. Using the spectral unmixing approach, the relative proportions of each quantum dot within a homogeneous mixture and the spatial distribution of each quantum dot within a heterogeneous mixture were calculated. The calculated values match well with the theoretical predictions. Hence, the analysis method presented here can be used for simultaneous imaging of multiple fluorophores using hyperspectral imaging technology. The results provide valuable information for the realization of real-time multi-channel in vivo fluorescent imaging of biomolecules.

sentences: CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3{zeta} and NKG2D/2B4+CD3{zeta}) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

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Cellular calcium levels influenced by NCA-2 impact circadian period determination in Neurospora Intracellular calcium signaling has been implicated in control of a variety of circadian processes in animals and plants but its role in microbial clocks has remained largely cryptic. To examine the role of intracellular Ca2+ in the Neurospora clock we screened knockouts of calcium transporter genes and identified a gene encoding a calcium exporter, nca-2, uniquely as having significant period effects. Loss of NCA-2 results in an increase in cytosolic calcium level, and this leads to hyper-phosphorylation of core clock components, FRQ and WC-1, and a short period as measured by both the core oscillator and overt clock. Genetic analyses showed that mutations in certain frq phospho-sites, and in Ca2+-calmodulin-dependent kinase (camk-2), are epistatic to nca-2 in controlling the pace of the oscillator. These data are consistent with a model in which elevated intracellular Ca+2 leads to increased activity of CAMK-2 leading to enhanced FRQ phosphorylation, accelerated closure of the circadian feedback loop, and a shortened circadian period length. At a mechanistic level some CAMKs undergo more auto-phosphorylations in {Delta}nca-2, consistent with high calcium in the {Delta}nca-2 mutant influencing the enzymatic activity of CAMKs. NCA-2 interacts with multiple proteins including CSP-6, a protein known to be required for circadian output. Most importantly, expression of nca-2 is circadian clock-controlled at both the transcriptional and translational levels, and this in combination with the period effects seen in strains lacking NCA-2, firmly places calcium signaling within the larger circadian system where it acts as both an input to and output from the core clock.

ImportanceCircadian rhythms are based on cell-autonomous, auto-regulatory, feedback loops formed by interlocked positive and negative arms, and they regulate myriad molecular and cellular processes in most eukaryotes including fungi. Intracellular calcium signaling is also a process that impacts a broad range of biological events in most eukaryotes. Clues have suggested that calcium signaling can influence circadian oscillators through multiple pathways; however, mechanistic details have been lacking in microorganisms. Building on prior work describing calcium transporters in the fungus Neurospora, one such transporter, NCA-2, was identified as a regulator of circadian period length. Increased intracellular calcium levels caused by loss of NCA-2 results in over-activation of calcium-responsive protein kinases, in turn leading to a shortened circadian period length. Importantly, expression of NCA-2 is itself controlled by the molecular clock. In this way calcium signaling can be seen as providing both input to and output from the circadian system.

sentences: A Chemical Mutagenesis Approach to Insert Post-Translational Modifications in Aggregation-Prone Proteins Neurodegenerative diseases are a class of disorders linked to the formation in the nervous system of fibrillar protein aggregates called amyloids. This aggregation process is affected by a variety of post-translational modifications, whose specific mechanisms are not fully understood yet. Emerging chemical mutagenesis technology is currently striving to address the challenge of introducing protein post-translational modifications, while maintaining proteins stable and soluble during the modification reaction. Several amyloidogenic proteins are highly aggregation-prone, and current modification procedures lead to unexpected precipitation of these proteins, affecting their yield and downstream characterization. Here, we present a method for maintaining amyloidogenic proteins soluble during chemical mutagenesis. As proof-of-principle, we applied our method to mimic the phosphorylation of the serine 26 and the acetylation of the lysine 28 of the 40-residue long variant of amyloid-{beta} peptide, whose aggregation is linked to Alzheimers disease.

sentences: Evaluation of the sensitivity and specificity of three diagnostic tests for Coxiella burnetii infection in cattle and buffaloes in Punjab (India) using Bayesian latent class analysis Q Fever is a zoonotic disease of significant animal and public health concern, caused by Coxiella burnetii (C. burnetii), an obligate intracellular bacterium. This study was done to evaluate the sensitivity (Se) and specificity (Sp) of three diagnostic methods to diagnose C. burnetii infection in cattle and buffaloes in Punjab, India: an indirect ELISA method applied in serum samples and a trans-Polymerase Chain Reaction (trans-PCR) technique applied in milk samples and genital swabs. Bayesian Latent Class Models were developed following the STARD-BLCM reporting guidelines. Conditional independence was assumed between the tests, given (i) the different biological principle of ELISA and trans-PCR and (ii) the fact that the trans-PCR was performed on different tissues. The ELISA method in the serum samples showed the highest Se of 0.97 (95% Probability Intervals (PIs): 0.93; 0.99) compared to the trans-PCR method applied in milk samples 0.76 (0.62; 0.87) and genital swabs 0.7 (0.55; 0.82). The Sps of all tests were high, with trans-PCR in genital swabs recording the highest Sp of 0.99 (0.98; 1), while the Sp of trans-PCR in milk samples and ELISA in serum samples were 0.98 (0.96; 0.99) and 0.95 (0.93; 0.97) respectively. The study results show that none of the applied tests are perfect, therefore, a testing regimen based on the diagnostic characteristic of the tests may be considered for diagnosis of C. burnetii.

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Neural cell injury pathology due to high-rate mechanical loading Successful detection and prevention of brain injuries relies on the quantitative identification of cellular injury thresholds associated with the underlying pathology. Here, by combining a recently developed inertial microcavitation rheology technique with a 3D in vitro neural tissue model, we quantify and resolve the structural pathology and critical injury strain thresholds of neural cells occurring at high loading rates such as encountered in blast, cavitation or directed energy exposures. We find that neuronal dendritic spines characterized by MAP2 displayed the lowest physical failure strain at 7.3%, whereas microtubules and filamentous actin were able to tolerate appreciably higher strains (14%) prior to injury. Interestingly, while these critical injury thresholds were similar to previous literature values reported for moderate and lower strain rates (< 100 1/s), the pathology of primary injury reported here was distinctly different by being purely physical in nature as compared to biochemical activation during apoptosis or necrosis.

TeaserControlled microcavitation enables quantitative identification of injury thresholds in neural cells.

sentences: Anti-arthralgic activity of the n-hexane extract (HTp) of yellow oleander seeds;Thevetia peruviana (Pers.) K. Schum Thevetia peruviana (Pers.) K.Schum. (Apocynaceae) seeds are known to possess cardioactive glycosides such as thevetin A, thevetin B, nerifolin etc. They are also used locally for general pain relief for which there is no scientific evidence to our knowledge. Arthralgia is regarded generally as pain without inflammation. It is endemic in the society and sufferers continue to imbibe pain relieving drugs in their tons all over the world. Analgesic activity test was carried out using the formalin-induced pain models, at 0.1g, 0.2g and 0.3g/kg doses of n-hexane extracts of Thevetia peruviana seeds (HTp) in Wistar mice. Diclofenac was used as positive control. Acute toxicity test was carried out at doses of 1000, 2500 and 5000 mg/kg weight of test subject. It was observed that HTp at concentrations of 0.1g, 0.2g and 0.3g/kg showed significant analgesic effect at compared to the control. The percentage inhibition observed was 29.60%, 44.80% and 50.72% for the early pain phase and 100% for the late pain phase respectively, indicating HTps NSAID-like property. HTp showed the highest percentage inhibition at 300 mg/kg (50.72 %) and significant (P<0.005) pain reduction. HTp did not produce any toxicity up to a dose of 5000 mg/kg weight which is very interesting as the seeds are known for their toxicity due to the cardiac glycoside presence. The results of the study suggest that HTp does indeed relieve pain significantly in a dose dependent manner, thus justifying its use in management of arthralgia.

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Colon Innervating TRPA1 Positive Nociceptors Influence Mucosal Health In Mice IntroductionTransient receptor potential ankyrin-1 positive (TRPA1+ve) nociceptors, primarily present as peptidergic neuronal afferents in the colon are sensors of disturbance in lower gastrointestinal tract including pain induced by different pathologies. Their therapeutic role in the alleviation of chronic pain (receptor antagonism and receptor desensitization) associated with inflammatory bowel diseases (IBD) is reported. However, there is limited literature available about their role in formation and sustenance of the mucosal layer, and its interaction with host physiology as well as luminal microbial community. The aim of this study focuses on the effects of nociceptive TRPA1 channel desensitization on colonic mucus production and gut health.

MethodsTRPA1+ve nociceptors were desensitized by rectal administration of capsazepine. Ileum, colon was harvested and cecum content was collected. We performed morphological/histological analysis, gut permeability alteration, gene expression changes, colon metabolite profiling, and gut microbial abundance in these animals.

ResultsWe found that presence of TRPA1-positive nociceptors is required for mucus layer integrity, using an intra-rectal capsazepine-induced TRPA1 desensitization model. Desensitization of TRPA1 positive nociceptors resulted in damaged mucosal lining, resultant increase in gut permeability and altered transcriptional profile of genes for goblet cell markers, mucus regulation, immune response and tight junction proteins. The damage to mucosal lining prevented its role in enterosyne (short chain fatty acids) actions.

ConclusionThese results suggest that caution may need to be exercised before employing TRPA1 desensitization as a therapeutic option to alleviate pain caused due to IBD.

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Proteomic analysis of mouse kidney tissue associates peroxisomal dysfunction with early diabetic kidney disease BackgroundThe absence of efficient inhibitors for DKD progression reflects the gaps in our understanding of DKD molecular pathogenesis. A comprehensive proteomic analysis was performed on glomeruli and kidney cortex of diabetic mice with subsequent validation of findings in human biopsies and - omics datasets aiming to better understand the underlying molecular biology of early DKD development and progression.

MethodsLC-MS/MS was employed to analyze the kidney proteome of DKD mouse models: Glomeruli of Ins2Akita mice 2 month and 4 month old, and cortex of db/db mice 6 month old. Following label-free quantification, the abundance of detected proteins were correlated with existing kidney datasets and functionally annotated. Tissue sections from 16 DKD patients were analyzed by IHC.

ResultsPathway analysis of differentially expressed proteins in the early and late DKD versus controls predicted dysregulation in DKD hallmarks (such as peroxisomal lipid metabolism, {beta}-oxidation and TCA cycle) supporting the functional relevance of the findings. Comparing the observed protein changes in early and late DKD, consistent upregulation of 21 and downregulation of 18 proteins was detected. Among these were downregulated peroxisomal proteins such as NUDT19, ACOX1, and AMACR and upregulated mitochondrial proteins related to aminoacid metabolism including GLS, GLDC, and GCAT. Several of these changes were also observed in the kidney cortex proteome of db/db mice. IHC of human kidney further confirmed the differential expression of NUDT19, AGPS, AMACR and CAT proteins in DKD.

ConclusionsOur study shows an extensive differential expression of peroxisomal proteins in the early stages of DKD that persists regardless of the disease severity. These proteins therefore represent potential markers of early DKD pathogenesis. Collectively, essential pathways associated with peroxisomes such as lipid {beta}-oxidation, plasmalogen synthesis, aminoacid metabolism and response to oxidative stress are downregulated in early DKD, providing new perspectives and potential markers of diabetic kidney dysfunction.

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Mechanical forces impair antigen discrimination by reducing differences in T cell receptor off-rates T cells use their T cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains unclear. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds), and this was confirmed by molecular dynamic simulations. Consistent with these findings, we show that the best characterized catch-bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination and we suggest this new force-shielding role for the adhesion receptors CD2 and LFA-1.

One sentence summaryMechanical forces disproportionately accelerate the off-rates of higher-affinity antigens reducing T cell antigen discrimination

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Ancient dental calculus preserves signatures of biofilm succession and inter-individual variation independent of dental pathology Dental calculus preserves oral microbes, enabling comparative studies of the oral microbiome and health through time. However, small sample sizes and limited dental health metadata have hindered health-focused investigations to date. Here we investigate the relationship between tobacco pipe smoking and dental calculus microbiomes. Dental calculus from 75 individuals from the 19th century Middenbeemster skeletal collection (Netherlands) were analyzed by metagenomics. Demographic and dental health parameters were systematically recorded, including the presence/number of pipe notches. Comparative data sets from European populations before and after the introduction of tobacco were also analyzed. Calculus species profiles were compared with oral pathology to examine associations between microbiome community, smoking behavior, and oral health status. The Middenbeemster individuals exhibited relatively poor oral health, with a high prevalence of periodontal disease, caries, heavy calculus deposits, and antemortem tooth loss. No associations between pipe notches and dental pathologies, or microbial species composition, were found. Calculus samples before and after the introduction of tobacco showed highly similar species profiles. Observed inter-individual microbiome differences were consistent with previously described variation in human populations from the Upper Paleolithic to the present. Dental calculus may not preserve microbial indicators of health and disease status as distinctly as dental plaque.

Research HighlightsO_LINo associations between calculus species profiles and oral health metrics were detected in a single large population
C_LIO_LIA minority of individuals have a dental calculus species profile characterized by low levels of Streptococcus and high levels of anaerobic taxa
C_LI

sentences: Prediction-error neurons in circuits with multiple neuron types: Formation, refinement and functional implications Predictable sensory stimuli do not evoke significant responses in a subset of cortical excitatory neurons. Some of those neurons, however, change their activity upon mismatches between actual and predicted stimuli. Different variants of these prediction-error neurons exist and they differ in their responses to unexpected sensory stimuli. However, it is unclear how these variants can develop and co-exist in the same recurrent network, and how they are simultaneously shaped by the astonishing diversity of inhibitory interneurons. Here, we study these questions in a computational network model with three types of inhibitory interneurons. We find that balancing excitation and inhibition in multiple pathways gives rise to heterogeneous prediction-error circuits. Dependent on the networks initial connectivity and distribution of actual and predicted sensory inputs, these circuits can form different variants of prediction-error neurons that are robust to network perturbations and generalize to stimuli not seen during learning. These variants can be learned simultaneously via homeostatic inhibitory plasticity with low baseline firing rates. Finally, we demonstrate that prediction-error neurons can support biased perception, we illustrate a number of functional implications, and we discuss testable predictions.

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Chemically-induced epimutagenesis allows bypassing reproductive barriers in hybrid seeds The "triploid block" prevents interploidy hybridizations in flowering plants, and is characterized by failure in endosperm development, arrest in embryogenesis, and seed collapse. Many genetic components of triploid seed lethality have been successfully identified in the model plant Arabidopsis thaliana, most notably the paternally expressed imprinted genes (PEGs) that are up-regulated in the tetraploid endosperm with paternal excess. Previous studies have shown that the paternal epigenome is a key determinant of the triploid block response, as the loss of DNA methylation in diploid pollen suppresses the triploid block almost completely. Here, we demonstrate that triploid seed collapse is bypassed in Arabidopsis plants treated with the DNA methyltransferase inhibitor 5-Azacytidine during seed germination and early growth. We have identified strong suppressor lines showing stable transgenerational inheritance of hypomethylation in CG context, as well as normalized expression of PEGs in triploid seeds. Importantly, differentially methylated loci segregate in the progeny of "epimutagenized" plants, which may allow the identification of epialleles involved in the triploid block response in future studies. Finally, we demonstrate that chemically-induced epimutagenesis allows bypassing hybridization barriers in crosses between different Capsella species, thus potentially emerging as a novel strategy for producing triploids and interspecific hybrids with high agronomical interest.

One sentence summaryGenome-wide loss of DNA methylation induced by 5-Azacytidine allows bypassing interploidy and interspecific hybridization barriers in Arabidopsis and Capsella.

sentences: Linear and partially linear models of behavioural trait variation using admixture regression Admixture regression methodology exploits the natural experiment of random mating between individuals with different ancestral backgrounds to infer the environmental and genetic components to trait variation across racial and ethnic groups. This paper provides a statistical framework for admixture regression based on the linear polygenic index model and applies it to neuropsychological performance data from the Adolescent Brain Cognitive Development (ABCD) database. We develop and apply a new test of the differential impact of multi-racial identities on trait variation, an orthogonalization procedure for added explanatory variables, and a partially linear semiparametric functional form. We find a statistically significant genetic component to neuropsychological performance differences across racial identities, and find some possible evidence of nonlinearity in the link between admixture and neuropsychological performance scores in the ABCD data.

sentences: Cut-and-Paste DNA Insertion with Engineered Type V-K CRISPR-associated Transposases CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. Type V-K CASTs offer distinct technological advantages over type I CASTs given their smaller coding size, fewer components, and unidirectional insertions. However, the utility of type V-K CASTs is hindered by a replicative transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid co-integrate products. Here, we overcome this limitation by engineering new CASTs with dramatically improved product purity. To do so, we compensate for the absence of the TnsA subunit in multiple type V-K CASTs by engineering a Homing Endonuclease-assisted Large-sequence Integrating CAST compleX, or HELIX system. HELIX utilizes a nicking homing endonuclease (nHE) fused to TnsB to restore the 5 " nicking capability needed for dual-nicking of the DNA donor. By leveraging distinct features of both type V-K and type I systems, HELIX enables cut-and-paste DNA insertion with up to 99.3% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. Furthermore, we demonstrate the versatility of this approach by generating HELIX systems for other CAST orthologs. We also establish the feasibility of creating a minimal, 3-component HELIX, simplifying the number of proteins that must be expressed. Together, HELIX streamlines and improves the application of CRISPR-based transposition technologies, eliminating barriers for efficient and specific RNA-guided DNA insertions.

sentences: SparsePro: an efficient genome-wide fine-mapping method integrating summary statistics and functional annotations Identifying causal variants from genome-wide association studies (GWASs) is challenging due to widespread linkage disequilibrium (LD). Functional annotations of the genome may help prioritize variants that are biologically relevant and thus improve fine-mapping of GWAS results. However, classical fine-mapping methods have a high computational cost, particularly when the underlying genetic architecture and LD patterns are complex. Here, we propose a novel approach, SparsePro, to efficiently conduct functionally informed statistical fine-mapping. Our method enjoys two major innovations: First, by creating a sparse low-dimensional projection of the high-dimensional genotype data, we enable a linear search of causal variants instead of a combinatorial search of causal configurations used in most existing methods; Second, we adopt a probabilistic framework with a highly efficient variational expectation-maximization algorithm to integrate statistical associations and functional priors. We evaluate SparsePro through extensive simulations using resources from the UK Biobank. Compared to state-of-the-art methods, SparsePro achieved more accurate and well-calibrated posterior inference with greatly reduced computation time. We demonstrate the utility of SparsePro by investigating the genetic architecture of five functional biomarkers of vital organs. We show that, compared to other methods, the causal variants identified by SparsePro are highly enriched for expression quantitative trait loci and explain a larger proportion of trait heritability. We also identify potential causal variants contributing to the genetically encoded coordination mechanisms between vital organs, and pinpoint target genes with potential pleiotropic effects. In summary, we have developed an efficient genome-wide fine-mapping method with the ability to integrate functional annotations. Our method may have wide utility in understanding the genetics of complex traits as well as in increasing the yield of functional follow-up studies of GWASs. SparsePro software is available on GitHub at https://github.com/zhwm/SparsePro.

sentences: Molecular and cognitive signatures of ageing partially restored through synthetic delivery of IL2 to the brain Cognitive decline is a common pathological outcome during aging, with an ill-defined cellular or molecular basis. Among the cellular changes observed with age are alterations to neuronal plasticity, changes in the glial compartment and the decline of the neurogenic niche. In the recent years, the concept of inflammaging, defined as a low-grade inflammation increasing with age, has emerged as a nexus for age-related diseases. This increase of basal inflammation is also observed in the central nervous system. While not classically considered a neurological cell type, infiltrating T cells increase in the brain with age, and may be responsible for amplification of inflammatory cascades and disruptions to the neurogenic niche. Recently, a small resident population of regulatory T cells has been identified in the brain, and the capacity of IL2-mediated expansion of this population to counter neuroinflammatory disease has been demonstrated. Here we test a brain-specific IL2 delivery system for the prevention of neurological decline in aging mice. We identify the molecular hallmarks of aging in the brain glial compartments, and identify partial restoration of this signature through IL2 treatment. At a behavioral level, brain IL2 delivery prevented the age-induced defect in spatial learning, without improving the general decline in motor skill or arousal. These results identify immune modulation as a potential path to preserving cognitive function for healthy ageing.

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RGS14 modulates locomotor behavior and ERK signaling induced by environmental novelty and cocaine within discrete limbic structures RationaleIn rodents, exposure to novel environments or psychostimulants promotes locomotor activity. Indeed, locomotor reactivity to novelty strongly predicts behavioral responses to psychostimulants in animal models of addiction. RGS14 is a plasticity restricting protein with unique functional domains that enable it to suppress ERK-dependent signaling as well as regulate G protein activity. Although recent studies show that RGS14 is expressed in multiple limbic regions implicated in psychostimulant- and novelty-induced hyperlocomotion, its function has been studied almost entirely in the context of hippocampal physiology and hippocampusdependent behaviors.

ObjectiveWe sought to determine whether RGS14 modulates novelty- and psychostimulant-induced locomotion and neuronal activity.

MethodsWe assessed Rgs14 knockout (RGS14 KO) mice and wild-type (WT) littermate controls using novelty-induced locomotion (NIL) and cocaine-induced locomotion (CIL) behavioral tests with subsequent quantification of c-fos and phosphorylated ERK (pERK) induction in limbic regions that express RGS14.

ResultsCompared to WT controls, RGS14 KO mice exhibited attenuated locomotor responses in the NIL test, driven by avoidance of the center of the novel environment. By contrast, RGS14 KO mice demonstrated augmented peripheral locomotion in the CIL test conducted in either a familiar or novel environment. The absence of RGS14 enhanced induction of c-fos and pERK in the central amygdala and hippocampus (areas CA1 and CA2) when cocaine was administered in a novel environment.

ConclusionsRGS14 regulates novelty- and psychostimulant-induced hyperlocomotion, particularly with respect to thigmotaxis. Further, our findings suggest RGS14 may reduce neuronal activity in discrete limbic subregions by inhibiting ERK-dependent signaling and transcription.

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Metagenomic data for Halichondria panicea from Illumina and Nanopore sequencing and preliminary genome assemblies for the sponge and two microbial symbionts. ObjectivesThese data were collected to generate a novel reference metagenome for the sponge Halichondria panicea and its microbiome for subsequent differential expression analyses.

Data descriptionThese data include raw sequences from four separate sequencing runs of the metagenome of a single individual of Halichondria panicea - one Illumina MiSeq (2x300 bp, paired-end) run and three Oxford Nanopore Technologies (ONT) long-read sequencing runs, generating 53.8 and 7.42 Gbp respectively. Comparing assemblies of Illumina, ONT and an Illumina-ONT hybrid revealed the hybrid to be the  best assembly, comprising 163 Mbp in 63,555 scaffolds (N50: 3,084). This assembly, however, was still highly fragmented and only contained 52% of core metazoan genes (with 77.9% partial genes), so it was also not complete.

However, this sponge is an emerging model species for field and laboratory work, and there is considerable interest in genomic sequencing of this species. Although the resultant assemblies from the data presented here are suboptimal, this data note can inform future studies by providing an estimated genome size and coverage requirements for future sequencing, sharing additional data to potentially improve other suboptimal assemblies of this species, and outlining potential limitations and pitfalls of the combined Illumina and ONT approach to novel genome sequencing.

sentences: Endo-lysosomal assembly variations among Human Leukocyte Antigen class I (HLA-I) allotypes The extreme polymorphisms of HLA-I proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes (CTL). The canonical endoplasmic reticulum (ER) HLA-I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA-I molecules. We hypothesized variable influences of HLA-I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA-I are variable. For example, in moDCs, compared with HLA-B*08:01, HLA-B*35:01 displays reduced cell-surface stability and greater receptivity to exogenous peptide. Perturbations of endo-lysosomal pH negatively affect the surface expression of moDC HLA-B*35:01 but not HLA-B*08:01, causing HLA-B*35:01 accumulation in LAMP1+ compartments. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for HLA-B*35:01. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA-I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA-I expression and increase the efficiency of cross-presentation.

sentences: Exit from quiescence couples epithelial stress amplification to fluidization Cellular quiescence is a state of reversible cell cycle arrest that is associated with tissue dormancy. Timely regulated entry into and exit from quiescence is important for processes such as tissue homeostasis, tissue repair, stem cell maintenance, developmental processes and immunity. Here we show that quiescent human keratinocyte monolayers contain an actinomyosin-based system that facilitates global viscoelastic flow upon serum-stimulated exit from quiescence. Mechanistically, serum exposure causes rapid amplification of pre-existing contractile sites leading to a burst in monolayer stress that subsequently drives monolayer fluidization. The stress magnitude after quiescence exit correlates with quiescence depth, and a critical stress level must be reached to overcome the cell sheet displacement barrier. The study shows that static quiescent cell monolayers are mechanically poised for motility and identifies global stress amplification as a mechanism for tissue fluidization.

sentences: cGAS recruitment to micronuclei is dictated by pre-existing nuclear chromatin status. Micronuclei (MN) are aberrant cytosolic compartments containing broken genomic fragments or whole lagging chromosomes. MN envelopes irreversibly rupture, allowing the viral receptor cGAS to localize to MN and initiate an inflammatory signalling cascade. Here, we demonstrate that MN envelope rupture is not sufficient for cGAS localization. Unlike MN that arise following ionizing radiation (IR), ruptured MN generated from acute transcription stressors DRB or siSRSF1 are refractory to cGAS localization. Recruitment of cGAS to MN is blocked by inhibiting the histone methyltransferase DOT1L prior to IR exposure, demonstrating that cGAS recruitment to MN is dictated by nuclear chromatin organization at the time of DNA damage. Loss of cGAS+ MN, caused either by acute transcription stressors or by preventing DOT1L-deposited histone methylation, corresponded to significantly decreased cGAS-dependent inflammatory signalling. These results implicate nuclear chromatin organization in micronuclear composition and activity, influencing the ability of damage-induced MN to retain cytosolic proteins upon rupture.

sentences: Chemical control and herbicide resistance of hairy fleabane  in Jordan Two field experiments were conducted to evaluate the effectiveness of 12 herbicides in controlling hairy fleabane [Conyza bonariensis (L.) Cronquist] in a date palm orchard located in the central Jordan valley during the spring of 2017. Results showed that C. bonariensis resists paraquat (2.5, 5 and 7.5kgha-1), oxadiazon (5kgha-1) and oxyflourfen (3.3kgha-1) herbicides applied at normal or higher than the recommended rates. None of the three herbicides was significantly effective against the weed and treated plants continued growing normally similar to those of untreated control. Higher rates (10-fold of the recommended rates) of the same herbicides failed to control the weed. The effect of other tested herbicides on the weed was varied with bromoxynil plus MCPA (buctril(R)M), 2,4-D-iso-octyl ester, glyphosate, glyphosate trimesium and triclopyr were most effective and completely controlled the weed at recommended rates of application. Testing paraquat, oxadiazon and oxyflourfen using the normal recommended and 10-fold higher rates on two populations of C. bonariensis grown from seeds of the date palm and al-Twal (another site in the Jordan Valley) weed populations and grown in pots under glasshouse conditions showed that Date palm population was resistant to the three herbicides at both application rates while al-Twal site population was highly susceptible and completely controlled at normal and high rates of the three herbicides. It is concluded that certain populations of C. bonariensis developed resistance to paraquat, oxadiazon and oxyflourfen but control of this weed was possible using other herbicides of different mechanism of action. Herbicide rotation or other nonchemical weed control methods have been suggested to prevent or reduce the buildup and spread of resistant populations of this weed species. These results represent the first report on herbicide resistance of C. bonariensis in Jordan.

sentences: Changes in splicing and neuromodulatory gene expression programs in sensory neurons with pheromone signaling and social experience Social experience and pheromone signaling in ORNs affect pheromone responses and male courtship behaviors in Drosophila, however, the molecular mechanisms underlying this circuit-level neuromodulation remain less clear. Previous studies identified social experience and pheromone signaling-dependent modulation of chromatin around behavioral switch gene fruitless, which encodes a transcription factor necessary and sufficient for male behaviors. To identify the molecular mechanisms driving social experience-dependent neuromodulation, we performed RNA-seq from antennal samples of mutant fruit flies in pheromone receptors and fruitless, as well as grouped or isolated wild-type males. We found that loss of pheromone detection differentially alters the levels of fruitless exons suggesting changes in splicing patterns. In addition, many Fruitless target neuromodulatory genes, such as neurotransmitter receptors, ion channels, and ion transporters, are differentially regulated by social context and pheromone signaling. Our results suggest that modulation of circuit activity and behaviors in response to social experience and pheromone signaling arise due to changes in transcriptional programs for neuromodulators downstream of behavioral switch gene function.

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Assessing Myf5 and Lbx1 contribution to carapace development by reproducing their turtle-specific signatures in mouse embryos BackgroundThe turtle carapace is an evolutionary novelty resulting from changes in the processes that build ribs and their associated muscles in most tetrapod species. Turtle embryos have several unique features that might play a role in this process, including the carapacial ridge, a Myf5 gene with shorter coding region that generates an alternative splice variant lacking exon 2, and unusual expression patterns of Lbx1 and HGF.

ResultsWe generated Myf5 alleles reproducing the Myf5 turtle expression features. At mid gestation, mouse embryos expressing Myf5 lacking exon 2 reproduced some early properties of turtle somites, but still developed into viable and fertile mice. Extending Lbx1 expression into the hypaxial dermomyotomal lip of trunk somites to mimic the turtle Lbx1 expression pattern, produced fusions in the distal part of the ribs.

ConclusionsTurtle-like Myf5 activity might generate a plastic state in developing trunk somites under which they can either enter carapace morphogenetic routes, possibly triggered by signals from the carapacial ridge, or still engage in the development of a standard tetrapod ribcage in the absence of those signals. In addition, trunk Lbx1 expression might play a later role in the formation of the lateral border of the carapace.

sentences: Liquid-liquid phase separation of florigen activation complex induces flowering Floral transition, regulated by the systemic action of the mobile florigen protein FLOWERING LOCUS T (FT), is essential for successful plant reproduction1. How FT controls downstream gene expression remains incompletely understood, although it relies on the florigen activation complex (FAC), a core component of FT function2-4. The FAC is a nucleus-localized transcriptional activator of genes encoding MADS-box transcription factors critical to reproductive development and consists of florigen FT; a scaffold 14-3-3 protein that is a key component for complex assembly; and FD, a basic leucine-zipper protein that recruits the FAC to DNA. Here we report that the FAC exhibits phase separation. In rice shoot apical meristem cells, rice florigen Heading date 3a (Hd3a) fused to the green fluorescent protein formed speckles in the nucleus. The FAC speckle is formed in a FAC-dependent manner in tobacco cells. Recombinant Hd3a, but not OsFD1, phase-separated in vitro, and this effect was enhanced in the presence of 14-3-3 protein. Furthermore, mutations affecting functionally important residues in the pocket region or C-terminal disordered region of Hd3a affected FAC phase separation, providing a biochemical framework for the proteins effect on flowering. The ability to form condensates via phase transition represents a previously unknown mechanism for gene activation by the FAC.

sentences: THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO complex, and Trf4, a key component of the TRAMP complex involved in nuclear RNA degradation, are necessary to prevent CAG fragility and repeat contractions in a S. cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and genome-wide transcription-replication conflicts (TRCs), implicating impairment of transcription elongation as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility and TRCs suggests that co-transcriptional R-loops are the main cause of CAG fragility in the thp2{Delta} mutants. In contrast, CAG fragility and TRCs in the trf4{Delta} mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

sentences: Deep RNN with Pseudo Loss Objective for Forecasting Stop-over Decisions of Wild Migratory Birds. Forecasting stop-over decisions and mapping the stop-over sites of wild migratory birds is fast becoming important in light of recent developments affecting global health. Migratory wild birds stop at sites with access to food resources so they can rest before continuing with their journey. Unfortunately, these sites offer opportunities for these birds to spread pathogens and viruses by interacting with the ecosystem. While previous work has focused on predicting stop-over sites using historical information, we emphasize that this is not useful for any planning efforts by health authorities and instead offer a new perspective by proposing an approach that can forecast the duration of stop-over. In this work, first we cast this problem as a classification task and show how pseudo labels and losses in a Bi-directional recurrent neural network can help improve performance given the presence of significantly underrepresented class. We use dataset of Turkey vulture (avian pox vector) movement over several years for the forecasting task where we compare our approach with a variety of baselines and show that it outperforms them. We also use this dataset and the White Fronted Geese (avian flu vector) movement dataset to understand the nature of the habitats used for stop-over using a publicly available model pre-trained on more than half a million land cover images. By knowing the preferred stop-over habitats and the time spent in and between stop-overs using our model, we can help relevant authorities come up with efficient intervention measures.

sentences: Visualization of bacterial type 3 secretion system components down the molecular level by MINFLUX nanoscopy Type 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.

sentences: Differential neural circuitry behind autism subtypes with imbalanced social-communicative and restricted repetitive behavior symptoms Social-communication (SC) and restricted repetitive behaviors (RRB) are autism diagnostic symptom domains. SC and RRB severity can markedly differ within and between individuals and may be underpinned by different neural circuitry and genetic mechanisms. Modeling SC-RRB balance could help identify how neural circuitry and genetic mechanisms map onto such phenotypic heterogeneity. Here we developed a phenotypic stratification model that makes highly accurate (97-99%) out-of-sample SC=RRB, SC>RRB, and RRB>SC subtype predictions. Applying this model to resting state fMRI data from the EU-AIMS LEAP dataset (n=509), we find that while the phenotypic subtypes share many commonalities in terms of intrinsic functional connectivity, they also show replicable differences within some networks compared to a typically-developing group (TD). Specifically, the somatomotor network is hypoconnected with perisylvian circuitry in SC>RRB and visual association circuitry in SC=RRB. The SC=RRB subtype show hyperconnectivity between medial motor and anterior salience circuitry. Genes that are highly expressed within these networks show a differential enrichment pattern with known autism-associated genes, indicating that such circuits are affected by differing autism-associated genomic mechanisms. These results suggest that SC-RRB imbalance subtypes share many commonalities, but also express subtle differences in functional neural circuitry and the genomic underpinnings behind such circuitry.

sentences: Connectional Asymmetry of the Inferior Parietal Lobule Shapes Hemispheric Specialization in Humans, Chimpanzees, and Rhesus Macaques The inferior parietal lobule (IPL) is one of the most expanded cortical regions in humans relative to other primates. It is also among the most structurally and functionally asymmetric regions in the human cerebral cortex. Whether the structural and connectional asymmetries of IPL subdivisions differ across primate species and how this relates to functional asymmetries remain unclear. We identified IPL subregions that exhibited positive allometric in both hemispheres, scaling across rhesus macaque monkeys, chimpanzees, and humans. The patterns of IPL subregions asymmetry were similar in chimpanzees and humans, but no IPL asymmetries were evident in macaques. Among the comparative sample of primates, humans showed the most widespread asymmetric connections in the frontal, parietal, and temporal cortices, constituting leftward asymmetric networks that may provide an anatomical basis for language and tool use. Unique human asymmetric connectivity between the IPL and primary motor cortex might be related to handedness. These findings suggest that structural and connectional asymmetries may underlie hemispheric specialization of the human brain.

sentences: Log-order improved in trans hammerhead ribozyme turnover rates: reevaluating therapeutic space for small catalytic RNAs We discovered an enhanced functionality hammerhead ribozyme (EhhRz), designed to act in trans against human rod opsin (RHO) mRNA, with turnover activity >300 nM min-1 under substrate-excess conditions and physiological Mg2+ levels (1 mM). We developed a real-time moderate-throughput fluorescence quantitative hhRz kinetic assay, which is linear with substrate and product moles and supported by gel-based measures. The EhhRz targets a CUC{downarrow} cleavage site in a substrate with no predicted secondary/tertiary structure and demonstrates classic Michaelis-Menten turnover behavior when the substrate is in 10-fold excess (Vmax/Km up to 1.60 x 108 min-1 M-1), which is comparable to RNase A. EhhRzs show cooperative titration with a Kd of 0.73 {+/-} 0.02 mM at cellular Mg2+ concentrations and a Hill coefficient of 1.73 {+/-} 0.07. The upstream EhhRz antisense flank (bound to a downstream substrate flank) interacts with stem-loop II, and examinations of different variants revealed that a U7 residue in the downstream flank of the substrate is not essential for enhanced activity. Under single-turnover conditions with substrate pre-annealed to enzyme, reaction rates exceeded 1,000 min-1. These findings show that RNA catalysis approaches the efficiency of the ribosome and suggests EhhRz in trans is a druggable nucleic acid therapeutic.

sentences: IL1B loss is associated with increased AR activity in castration-resistant prostate cancer Castration-resistant prostate cancer represents a continuum of phenotypes, including tumors with high levels of androgen receptor (AR) expression and activity and those which do not express AR and rely on alternative pathways for survival. The process by which AR-positive prostate cancer cells and tumors lose AR expression and acquire neuroendocrine features is referred to as neuroendocrine differentiation. Numerous therapies and exposures have been demonstrated to induce neuroendocrine differentiation in vitro, including the pro-inflammatory cytokine, interleukin 1 beta (IL-1{beta}), encoded by the gene IL1B. The purpose of our studies was to determine the relationship between the expression and activity of AR in relationship to IL-1{beta} and IL1B in prostate cancer. We performed analysis of de-identified human clinical data and generated prostate cancer cell lines with overexpression or knockout of IL1B. In primary prostate cancer, higher expression of IL1B predicts longer time to biochemical recurrence. In metastatic castration-resistant prostate cancer, IL1B expression is decreased and inversely correlates with AR and AR-target gene expression and AR activity, while positively correlating with the neuroendocrine prostate cancer (NEPC) score and neuroendocrine marker gene expression. In vitro, we report that AR-positive castration-resistant prostate cancer cells (C4-2B, 22Rv1) secrete IL-1{beta}, and knockout of IL1B in these cells results in increased AR activity, in the presence and absence of dihydrotestosterone (DHT). Importantly, knockout of IL1B prevented AR attrition during androgen-deprivation. Taken together, our studies demonstrate that loss of IL1B in AR-positive castration-resistant prostate cancer cells can increase and maintain AR activity in the absence of androgens, suggesting another potential mechanism of high AR activity in castration-resistant prostate cancer.

sentences: Informeasure: an R/Bioconductor package to quantify nonlinear dependence between variables in biological networks from an information theory perspective Using information measures to infer biological regulatory networks can observe nonlinear relationship between variables, but it is computationally challenging and there is currently no convenient tool available. We here describe an information theory R package named Informeasure that devotes to quantifying nonlinear dependence between variables in biological regulatory networks from an information theory perspective. This package compiles most of the information measures currently available: mutual information, conditional mutual information, interaction information, partial information decomposition and part mutual information. The first estimator is used to infer bivariate networks while the last four estimators are dedicated to analysis of trivariate networks. The base installation of this turn-key package allows users to approach these information measures out of the box. Informeasure is implemented in R program and is available as an R/Bioconductor package at https://bioconductor.org/packages/Informeasure.

sentences: Drug combination prediction for cancer treatment using disease-specific drug response profiles and single-cell transcriptional signatures Developing novel cancer treatments is a challenging task that can benefit from computational techniques matching transcriptional signatures to large-scale drug response data. Here, we present  retriever, a tool that extracts robust disease-specific transcriptional drug response profiles based on cellular response profiles to hundreds of compounds from the LINCS-L1000 project. We used retriever to extract transcriptional drug response signatures of triple-negative breast cancer (TNBC) cell lines and combined these with a single-cell RNA-seq breast cancer atlas to predict drug combinations that antagonize TNBC-specific disease signatures. After systematically testing 152 drug response profiles and 11,476 drug combinations, we identified the combination of kinase inhibitors QL-XII-47 and GSK-690693 as the topmost promising candidate for TNBC treatment. Our new computational approach allows the identification of drugs and drug combinations targeting specific tumor cell types and subpopulations in individual patients. It is, therefore, highly suitable for the development of new personalized cancer treatment strategies.

sentences: Pore-forming protein 	βγ-CAT promptly responses to fasting with capacity to deliver macromolecular nutrients During animal fasting, the nutrient supply and metabolism switch from carbohydrates to a new reliance on the catabolism of energy-dense lipid stores. Assembled under tight regulation, {beta}{gamma}-CAT is a pore-forming protein and trefoil factor complex identified in toad Bombina maxima. Here, we determined that this protein complex is a constitutive component in toad blood, that actively responds to the animal fasting. The protein complex was able to promote cellular albumin and albumin-bound fatty acid uptake in a variety of epithelial and endothelial cells, and the effects were attenuated by a macropinocytosis inhibitor. Endothelial cell-derived exosomes containing largely enriched albumin and fatty acids, called nutrisomes, were released in the presence of {beta}{gamma}-CAT. These specific nutrient vesicles were readily taken by starved muscle cells to support their survival. The results uncovered that pore-forming protein {beta}{gamma}-CAT is a fasting responsive element able to drive cell vesicular import and export of macromolecular nutrients.

sentences: Monitoring promoter activity by RNA editing based reporter Traditional methods monitoring the promoter activity require the insertion of reporter protein (e.g. fluorescent protein) downstream of a targeted promoter. These approaches suffer from low sensitivity and potential interference to endogenous transcripts especially when the targeted transcript is a noncoding RNA. Here, we develop a mechanistically different reporter system to monitor promoter activity based on ribozyme processed ADAR engaging RNA directed editing (REDDIT). We show that REDDIT can be used to monitor the promoter activity of protein coding, long noncoding RNA, and microRNA (miRNA) genes. Furthermore, REDDIT reporter can also be adapted to use bioluminescence imaging to monitor the promoter activity, which is more suitable for in vivo live imaging. Finally, the reporter sensitivity may be further increased by the circularization of ADAR recruiting RNA. REDDIT provides a powerful platform that is promising in a variety of applications such as monitoring the promoter activity, cell lineage tracing, and processing and manipulating genetic information for synthetic biology applications.

sentences: LXR agonist Prevents Peripheral Neuropathy and modifies PNS immune cells in Aged Mice Peripheral neuropathy is a common and progressive disorder in the elderly that interferes with daily activities and increases the risk of injury. It is of importance to find efficient treatments to treat or delay this age-related neurodegeneration. We previously demonstrated that activation of the cholesterol sensor Liver X receptor (LXR) with the potent agonist GW3965, alleviates pain in a diet-induced obesity model. Because cholesterol had also been linked to neuropathy during aging, we sought to test whether LXR activation may improve neuropathy and pain in aged mice by treating 21-month-old mice for 3 months with GW3965. Treatment resulted in a significant increase in nerve fibers of the sub-basal plexus, accompanied by a change in polarization, metabolism, and cholesterol content of macrophages in the sciatic nerve. These results suggest that activation of the LXR may block the progression of neuropathy associated with aging by modifying nerve-immune cell cholesterol, thereby providing new pathways to target in efforts to delay neuropathy during aging.

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Upregulation of Somatostatin Receptor Type 2 in a Receptor-Deficient In Vivo Pancreatic Neuroendocrine Tumor Model Improves Tumor Response to Targeted 177Lu-DOTATATE PurposeThe goal of this study was to test whether histone deacetylase inhibitors (HDACis) restore somatostatin receptor type 2 (SSTR2) expression in models of high-grade pancreatic neuroendocrine tumors (PNETs), thereby facilitating effective treatment with 177Lu-DOTATATE therapy.

MethodsTo assess tumor grade correlation with SSTR2 expression, we assessed human SSTR2 promoter methylation and expression levels in 96 NIH patient samples and merged the GSE149395 and GSE117852 datasets. We used three NET cell lines (QGP-1, BON-1, GOT-1) characterized by variable SSTR2 expression profiles for functional in vitro studies using HDACis. Finally, the QGP-1 xenograft mouse model, with low basal SSTR2 expression, was used to analyze the therapeutic efficacy of combined HDACi and 177Lu-DOTATATE therapies.

ResultsHuman PNET SSTR2 promoter methylation showed a significant positive correlation with higher tumor grades (P = 0.000014). We also found a significant negative correlation (P < 0.0001) between SSTR2 promoter methylation and SSTR2 expression in three NET cell lines. In vitro, SSTR2 expression increased significantly in BON-1 and QGP-1 cells at 48 and 72 hours in a dose-dependent fashion using two different HDACis, valproic acid and CI-994. In vivo studies demonstrated a significant increase in 177Lu-DOTATATE tumor uptake in QGP-1-engrafted mice after 10 days of CI-994 pretreatment (P = 0.0175). Treatment with 177Lu-DOTATATE reduced tumor size in mice pretreated with CI-994 compared to 177Lu-DOTATATE alone (at 15 days, P = 0.0028).

ConclusionHDACis increase SSTR2 surface expression in models of high-grade, SSTR2-deficient PNETs. This approach has the potential to improve tumor response to targeted therapy with 177 Lu-DOTATATE in patients with receptor-negative, metastatic PNETs.

Translational Relevance StatementPancreatic neuroendocrine tumors (PNETs) express high levels of somatostatin receptor type 2 (SSTR2), a unique target for both tumor imaging and therapy. Unfortunately, high-grade PNETs lose SSTR2 surface expression and thus become ineligible for SSTR2-targeted 177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT). Restoring SSTR2 expression through the reversal of inhibitory epigenetic gene silencing mechanisms has the potential for improving tumor responsiveness to PRRT. We demonstrate that histone deacetylase inhibitors (HDACis) upregulate SSTR2 surface expression in three NET cell lines in vitro. In an in vivo PNET xenograft model with low basal SSTR2 expression, our studies validate a significantly higher tumor uptake of SSTR2-targeted 177Lu-DOTATATE in animals pretreated with HDACis compared to controls. Furthermore, we show that this higher tumor uptake results in significant anti-tumor response when compared to standard PRRT alone. Our preclinical results thus provide a rationale for utilizing HDACi pretreatment to improve targeted radionuclide therapy in patients with SSTR2-negative, metastatic PNETs.

sentences: Mitogenomic analyses provide further evidence for multiple miniaturization events during the evolution of Seychelles caecilian amphibians Whole mitochondrial genomes have been helpful in estimating phylogenetic relationships in many organismal groups, including caecilian amphibians. Despite the increasing ease of obtaining mitochondrial genome sequences from high-throughput sequencing, several species of caecilian lack this important molecular resource. As part of a targeted-sequence capture project of nuclear ultraconserved elements for a small but substantially diverse radiation of caecilian amphibians found on the granitic Seychelles, we examined off-target sequences to determine if we captured enough mitochondrial fragments to reconstruct mitogenomes. We reconstructed (near-)complete mitogenomes for six of the eight species of Seychelles caecilians and completed 14 independent phylogenetic analyses (Bayesian inference and maximum likelihood) on different mitochondrial datasets assembled using different alignment techniques. As with other studies, we were unable to fully resolve internal phylogenetic relationships of the group. However, we found strong support in most analyses that a recently described miniaturized species, Hypogeophis pti, and another similarly-sized miniaturized species, H. brevis are not sister taxa. Our study suggests that miniature species of caecilians likely evolved at least twice on the Seychelles and highlights the need to revise genus-level taxonomy of Seychelles caecilians while providing further evidence that off-target sequences often contain enough mitochondrial fragments to reconstruct mitogenomes.

sentences: Nanoscale architecture and coordination of actin cores within the sealing zone of human osteoclasts Osteoclasts are unique in their capacity to degrade bone tissue. To achieve this process, osteoclasts form a specific structure called the sealing zone, which creates a close contact with bone and confines the release of protons and hydrolases for bone degradation. The sealing zone is composed of actin structures called podosomes nested in a dense actin network. The organization of these actin structures inside the sealing zone at the nano scale is still unknown. Here, we combine cutting-edge microscopy methods to reveal the nanoscale architecture and dynamics of the sealing zone formed by human osteoclasts on bone surface. Random illumination microscopy allowed the identification and live imaging of densely packed actin cores within the sealing zone. A cross-correlation analysis of the fluctuations of actin content at these cores indicates that they are locally synchronized. Further examination shows that the sealing zone is composed of groups of synchronized cores linked by -actinin1 positive filaments, and encircled by adhesion complexes. Thus, we propose that the confinement of bone degradation mediators is achieved through the coordination of islets of actin cores and not by the global coordination of all podosomal subunits forming the sealing zone.

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Interstitial and cerebrospinal fluid exchanging process revealed by phase alternate labeling with null recovery MRI PurposeTo develop Phase Alternate LAbeling with Null recovery (PALAN) MRI methods for the quantification of interstitial to cerebrospinal fluid flow (ICF) and cerebrospinal to interstitial fluid flow (CIF) in the brain.

MethodIn both T1-PALAN and apparent diffusion coefficient (ADC)-PALAN MRI methods, the cerebrospinal fluid (CSF) signal was nulled, while the residual interstitial fluid (ISF) was labeled by alternating the phase of pulses. ICF was extracted from the difference between the recovery curves of CSF with and without labeling. Similarly, CIF was measured by the T2-PALAN MRI method by labeling CSF, which took advance of the significant T2 difference between CSF and parenchyma.

ResultsBoth T1-PALAN and ADC-PALAN observed a rapid occurrence of ICF at 67{+/-}56 ms and 13{+/-}2 ms interstitial fluid transit times, respectively. ICF signal peaked at 1.5 s for both methods. ICF was 1153{+/-}270 ml/100ml/min with T1-PALAN in the third and lateral ventricles, which was higher than 891{+/-}60 ml/100ml/min obtained by ADC-PALAN. The results of the T2-PALAN suggested the ISF exchanging from ependymal layer to the parenchyma was extremely slow.

ConclusionThe PALAN methods are suitable tools to study ISF and CSF flow kinetics in the brain.

sentences: The PPLD has advantages over conventional regression methods in application to moderately sized genome-wide association studies In earlier work, we have developed and evaluated an alternative approach to the analysis of GWAS data, based on a statistic called the PPLD. More recently, motivated by a GWAS for genetic modifiers of the X-linked Mendelian disorder Duchenne Muscular Dystrophy (DMD), we adapted the PPLD for application to time-to-event (TE) phenotypes. Because DMD itself is relatively rare, this is a setting in which the very large sample sizes generally assembled for GWAS are simply not attainable. For this reason, statistical methods specially adapted for use in small data sets are required. Here we explore the behavior of the TE-PPLD via simulations, comparing the TE-PPLD with Cox Proportional Hazards analysis in the context of small to moderate sample sizes. Our results will help to inform our approach to the DMD study going forward, and they illustrate several respects in which the TE-PPLD, and by extension the original PPLD, offer advantages over regression-based approaches to GWAS in this context.

sentences: Inhibition of HSP90 reversed STAT3 mediated muscle wasting induced by cancer cachexia Cancer cachexia is one of the most common causes of death among cancer patients, no effective anti-cachectic treatment is currently available. In experimental cachectic models, aberrant activation of STAT3 in skeletal muscle has been found to contribute to muscle wasting. However, its clinical association, the factors regulating STAT3 activation, and the molecular mechanisms of STAT3-induced muscle atrophy in cancer cachexia remain incompletely understood. Here, we show that an enhanced interaction between STAT3 and HSP90, which causes the persistent STAT3 activation in the skeletal muscle of cancer cachexia patients, is the crucial event for the development of cachectic muscle wasting. Administration of HSP90 inhibitors alleviated the muscle wasting in C26 tumor-bearing cachetic mice model or C26 conditional medium induced C2C12 myotube atrophy. A mechanistic study indicated that in cachectic skeletal muscle, prolonged STAT3 activation triggered muscle wasting in a FOXO1-dependent manner, STAT3 activated FOXO1 by binding directly to its promoter. Our results provide key insights into the role of the HSP90/STAT3/FOXO1 axis in cachectic muscle wasting, which shows promising therapeutic potential as a target for the treatment of cancer cachexia.

sentences: A cerebellar-prepontine circuit for tonic immobility triggered by inescapable threat Sudden changes in the sensory environment are frequently perceived as threats and may provoke defensive behavioral states. One such state is tonic immobility, a conserved defensive strategy characterized by a powerful suppression of movement and motor reflexes. Tonic immobility has been associated with multiple brainstem regions and cell types, but the underlying circuit is not known. Here, we demonstrate that a strong vibratory stimulus evokes tonic immobility in larval zebrafish defined by suppression of exploratory locomotion and sensorimotor responses. Using a circuit-breaking screen and targeted neuron ablations, we show that cerebellar granule cells and a cluster of glutamatergic ventral prepontine neurons (vPPNs) that express key stress-associated neuropeptides are critical components of the circuit that suppresses movement. The complete sensorimotor circuit transmits information from primary sensory neurons through the cerebellum to vPPNs to regulate reticulospinal premotor neurons. These results show that cerebellar regulation of a neuropeptide-rich prepontine structure governs a conserved and ancestral defensive behavior that is triggered by inescapable threat.

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Corticostriatal dynamics underlying components of binge-like eating in mice Binge eating (BE) is a maladaptive repetitive feeding behavior present across nearly all eating disorder diagnoses. Despite the substantial negative impact of BE on psychological and physiological health, its underlying neural mechanisms are largely unknown. Other repetitive behavior disorders (e.g., obsessive compulsive disorder) show dysfunction within corticostriatal circuitry. Additionally, previous pre-clinical and clinical work has highlighted an imbalance between goal-directed and habitual responding in BE. The aim of the current study was to longitudinally examine in vivo neural activity within corticostriatal regions associated with habitual behavior- the infralimbic cortex (IL) and dorsolateral striatum (DLS)- in a robust pre-clinical model for BE. Female C57BL/6 mice (N=32) were randomized to receive: 1) intermittent (daily, 2-hour) binge-like access to palatable food (BE mice), or 2) continuous, non-intermittent (24-hour) access to palatable food (non-BE mice). In vivo calcium imaging was performed via fiber photometry at baseline and after chronic (4 weeks) engagement in the model for BE. Feeding behaviors (feeding bout onset/offset) during the recordings were captured using lickometers which generated TTL outputs for precise alignment of behavior to neural data. IL showed no specific changes in neural activity related to BE. However, BE animals showed decreased DLS activity at feeding onset and offset at the chronic timepoint when compared to baseline. Additionally, BE mice had significantly lower DLS activity at feeding onset and offset at the chronic timepoint compared to non-BE mice. These results point to a role for DLS hypofunction in chronic BE, highlighting a potential target for future treatment intervention.

Significance StatementBinge eating is a chronic and repetitive eating behavior that is associated with poor physiological and psychosocial outcomes. Despite the negative impact of binge eating, little is known about the neurobiological mechanisms contributing to the chronic course and persistence of the behavior. To investigate potential neural mechanisms underlying binge eating, we are using approaches developed to monitor neural activity in rodents. This study is the first to identify longitudinal changes in neural activity within regions of the prefrontal cortex and dorsal striatum during binge-like eating behavior in mice. Findings from this work could inform targeted biological treatments for binge eating.

sentences: Long-range morphogen gradient formation by cell-to-cell signal propagation Morphogen gradients are a central concept in developmental biology. Their formation often involves the secretion of morphogens from a local source, that spread by diffusion in the cell field, where molecules eventually get degraded. This implies limits to both the time and length scales over which morphogen gradients can form which are set by diffusion coefficients and degradation rates. Towards the goal of identifying plausible mechanisms capable of extending the gradient range, we here use theory to explore properties of a cell-to-cell signaling relay. Inspired by the millimeter-scale Wnt-expression and signaling gradients in flatworms, we consider morphogen-mediated morphogen production in the cell field. We show that such a relay can generate stable morphogen and signaling gradients that emanate from a source of morphogen at a boundary. This gradient formation can be related to an effective diffusion and an effective degradation that result from morphogen production due to signaling relay. If the secretion of morphogen produced in response to the relay is polarized, it further gives rise to an effective drift. We find that signaling relay can generate long-ranged gradients in relevant times without relying on extreme choices of diffusion coefficients or degradation rates, thus exceeding the limits set by physiological diffusion coefficients and degradation rates. A signaling relay is hence an attractive principle to conceptualize long-ranged gradient formation by slowly diffusing morphogens that are relevant for patterning in adult contexts such as regeneration and tissue turn-over.

sentences: BNP facilitates NMB-mediated histaminergic itch via NPRC-NMBR crosstalk B-type natriuretic peptide (BNP) binds to its two cognate receptors NPRA and NPRC, encoded by Npr1 and Npr3, respectively, with equal potency and both are expressed in the spinal cord. Moreover, natriuretic peptides (NP) signal through the inhibitory cGMP pathway, raising the question of how BNP may transmit itch information. We report that Npr3 is highly restricted to laminae I-II of the dorsal horn, and partially overlaps with neuromedin B receptor (NMBR) that encodes histaminergic itch. Functional studies indicate that NPRC is required for itch evoked by histamine but not chloroquine (CQ), a nonhistaminergic pruritogen. Importantly, BNP significantly facilitates scratching behaviors mediated by NMB, but not gastrin releasing peptide (GRP) that encodes nonhistaminergic itch. Consistently, BNP evoked Ca2+ response in NMBR/NPRC HEK 293 cells and BNP-saporin that ablated both Npr1 and Npr3 neurons impaired histamine-, but not CQ-evoked, itch. These results reveal a previously unknown mechanism by which BNP changes its inhibitory mode of action to the facilitation of itch through a novel NPRC-NMBR cross-talk. Our studies suggest that neuropeptides encode histaminergic and nonhistaminergic itch not only through distinct modes but also in synergy.

sentences: Encapsulated actomyosin patterns drive cell-like membrane shape changes Cell shape changes from locomotion to cytokinesis are, to a large extent, driven by myosin-driven remodeling of cortical actin patterns. Passive crosslinkers such as -actinin and fascin as well actin nucleator Arp2/3 complex largely determine the architecture and connectivity of actin network patterns; consequently, they regulate network remodeling and membrane shape changes. Membrane constriction in animal cell cytokinesis proceeds by assembly and contraction of a contractile ring pattern rich in -actinin and myosin at the equator of the cell cortex, with which the ring is contiguous. Here we reconstitute actomyosin networks inside cell-sized lipid bilayer vesicles and show that, depending on vesicle size and concentrations of -actinin and fascin, actomyosin networks assemble into ring and aster-like patterns. Anchoring actin to the membrane enhances the interaction of the contractile networks with lipid membrane but does not change the architecture of the patterns. A membrane-bound actomyosin ring exerts force and constricts the membrane. An Arp2/3 complex-mediated actomyosin cortex is shown to assemble a ring-like pattern at the equatorial cortex and contribute to myosin-driven clustering of the cortex and consequently membrane deformation. An active gel theory unifies a model for the observed membrane constriction and protrusion induced by the membrane-bound actomyosin networks.

sentences: Experimental microevolution of Trypanosoma cruzi reveals hybridization and clonal mechanisms driving rapid diversification of genome sequence and structure. Protozoa and fungi are known to have extraordinarily diverse mechanisms of genetic exchange. However, the presence and epidemiological relevance of genetic exchange in Trypanosoma cruzi, the agent of Chagas disease, has been controversial and debated for many years. Field studies have identified both predominantly clonal and sexually recombining natural populations. Two of six natural T. cruzi lineages (TcV and TcVI) show hybrid mosaicism, using analysis of single-gene locus markers. The formation of hybrid strains in vitro has been achieved and this provides a framework to study the mechanisms and adaptive significance of genetic exchange. Using whole genome sequencing of a set of experimental hybrids strains, we have confirmed that hybrid formation initially results in tetraploid parasites. The hybrid progeny showed novel mutations that were not attributable to either (diploid) parent showing an increase in amino acid changes. In long-term culture, up to 800 generations, there was progressive, gradual erosion of progeny genomes towards triploidy, yet retention of elevated copy number was observed at several core housekeeping loci. Our findings indicate hybrid formation by fusion of diploid T. cruzi, followed by sporadic genome erosion, but with substantial potential for adaptive evolution, as has been described as a genetic feature of other organisms, such as some fungi.

sentences: Dissecting errors of the exogenously-driven and endogenously-driven saccadic tasks in the common marmosets Various saccadic tasks used traditionally in oculomotor research, including both exogenously-driven and endogenously-driven saccades, have been proposed as clinical diagnostic for human movement disorders. Recently, common marmosets have been proven to be a good primate model for these movement disorders. However, whether similar saccadic measurements can be used for marmosets was not tested. Here, we trained three marmosets on the gap task, an exogenously-driven saccadic task, and the oculomotor delayed response (ODR) task, an endogenously-driven saccadic task. We demonstrated that with long-term training, they were able to learn and switch between the two tasks. The marmosets showed undershooting tendency in the gap and both under- and overshooting tendency in the ODR task when they made saccades to the target. We also categorized the error trails into distractive, impulsive, and visuomotor errors, depending on when a false saccade happened concerning the task progression, and interpreted possible causes of errors based on the saccade response time and the task history. Each error category may indicate specific failures in a particular aspect of cognitive or sensorimotor processes. Finally, and critically, we found that saccades from successful trials exhibited the highest saccade peak velocity and the shortest saccade duration comparing to the same saccade amplitude from error trials. Taken together, we showed the potential of training the same marmoset on conflicting oculomotor tasks simultaneously. We documented the baseline performance and compared successful and error trials in both gap and ODR tasks from the same animals. We propose that analyzing the error trials in sync with successful trials could provide a further understanding of the marmosets cognitive and sensorimotor processes in both healthy and disease conditions.

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Cerebrovascular response to exercise interacts with individual genotype and amyloid-beta deposition to influence response inhibition with aging The etiology of cognitive dysfunction associated with Alzheimers disease (AD) and dementia is multifactorial. Yet, mechanistic interactions among key neurobiological factors linked to AD pathology are unclear. This study tested the effect of interactions between cerebrovascular function, individual genotype, and structural brain pathology on response inhibition performance, an early and sensitive indicator of cognitive executive dysfunction with aging.

We quantified cerebrovascular response (CVR) to moderate-intensity aerobic exercise using transcranial doppler ultrasound and global amyloid-beta (A{beta}) deposition using positron emission tomography in a group of cognitively normal older adults genotyped as APOE4 carriers and noncarriers. We quantified response inhibition during a cognitive Stroop test.

Individuals with blunted CVR possessed greater A{beta} deposition. There was CVR-by-carrier status-by-A{beta} interaction on response inhibition. Blunted CVR was associated with impaired response inhibition specifically in carriers. Despite having greater A{beta} deposition, carriers with higher CVR demonstrated better response inhibition.

Cerebrovascular interactions with individual genotype and structural brain pathology may provide a physiologically-informed target for precision-medicine approaches for early treatment and prevention of cognitive dysfunction with aging.

HighlightsO_LINeurobiological interactions between CVR, APOE genotype, and A{beta} are behaviorally significant.
C_LIO_LIBlunted CVR to exercise is associated with impaired response inhibition specifically in APOE4 carriers.
C_LIO_LIAPOE4 carriers with more robust CVR have higher response inhibition performance, despite having greater A{beta} deposition.
C_LIO_LIAssessment of multifactorial neurobiological variables offers an early and sensitive biomarker of cognitive behavioral dysfunction with aging.
C_LI

sentences: DeepSCM: an efficient convolutional neural network surrogate model for the screening of therapeutic antibody viscosity Predicting high concentration antibody viscosity is essential for developing subcutaneous administration. Computer simulations provide promising tools to reach this aim. One such model is the spatial charge map (SCM) proposed by Agrawal and coworkers (mAbs. 2015, 8(1):43-48). SCM applies molecular dynamics simulations to calculate a score for the screening of antibody viscosity at high concentrations. However, molecular dynamics simulations are computationally costly and require structural information, a significant application bottleneck. In this work, high throughput computing was performed to calculate the SCM scores for 6596 nonredundant antibody variable regions. A convolutional neural network surrogate model, DeepSCM, requiring only sequence information, was then developed based on this dataset. The linear correlation coefficient of the DeepSCM and SCM scores achieved 0.9 on the test set (N=1320). The DeepSCM model was applied to screen the viscosity of 38 therapeutic antibodies that SCM correctly classified and resulted in only one misclassification. The DeepSCM model will facilitate high concentration antibody viscosity screening. The code and parameters are freely available at https://github.com/Lailabcode/DeepSCM.

sentences: SHOOT: phylogenetic gene search and ortholog inference Determining the evolutionary relationships between genes is fundamental to comparative biological research. Here we present the phylogenetic search, SHOOT. SHOOT searches a user query sequence against a database of phylogenetic trees and returns a tree with the query sequence correctly placed within it. We show that SHOOT performs this analysis with comparable speed to a BLAST search. We demonstrate that SHOOT phylogenetic placements are as accurate as conventional tree inference and it can identify orthologs with high accuracy. In summary, SHOOT is a fast and accurate tool for phylogenetic analyses of novel query sequences. It is available online at www.shoot.bio.

sentences: Nearly maximal information gain due to time integration in central dogma reactions Living cells process information about their environment through the central dogma processes of transcription and translation, which drive the cellular response to stimuli. Here, we study the transfer of information from environmental input to the transcript and protein expression levels. Evaluation of both experimental and analogous simulation data reveals that transcription and translation are not two simple information channels connected in series. Instead, we show that the central dogma reactions often create a time-integrating information channel, where the translation channel receives and integrates multiple outputs from the transcription channel. This information channel model of the central dogma provides new information-theoretic selection criteria for the central dogma rate constants. Using the data for four well-studied species we show that their central dogma rate constants achieve information gain due to time integration while also keeping the loss due to stochasticity in translation relatively low (< 0.5 bits).

sentences: The methylation of SDC2 and TFPI2 defined three methylator phenotypes of colorectal cancer Methylation-based noninvasive molecular diagnostics are easy and feasible tools for the early detection of colorectal cancer (CRC). However, many of them have the limitation of low sensitivity with some CRCs detection failed in clinical practice. In this study, the clinical and pathological characteristics, as well as molecular features of three methylator-groups, defined by the promoter methylation status of SDC2 and TFPI2, were investigated in order to improve the performance of CRC detection. The Illumina Infinium 450k Human DNA methylation data and clinical information of CRCs were collected from The Cancer Genome Atlas (TCGA) project and Gene Expression Omnibus (GEO) database. CRC samples were divided into three groups, HH (dual-positive), HL (single positive) and LL (dual-negative) according to the methylation status of SDC2 and TFPI2 promoters. Differences in age, tumor location, microsatellite instable status and differentially expressed genes (DEGs) were evaluated among the three groups and these findings were then confirmed in our inner CRC dataset. The combination of methylated SDC2 and TFPI2 showed a superior performance of distinguishing CRCs from normal controls than each alone. Samples of HL group were more often originated from left-side CRCs whereas very few of them were from right-side (P < 0.05). HH grouped CRCs showed a higher level of microsatellite instability and mutation load than other two groups (mean nonsynonymous mutations for HH/HL/LL: 10.55/3.91/7.02, P = 0.0055). All mutations of BRAF, one of the five typical CpG island methylator phenotype (CIMP) related genes, were found in HH group (HH/HL/LL: 51/0/0, P = 0.018). Also there was a significantly older patient age at the diagnosis in HH group. Gene expression analysis identified 37, 84 and 22 group-specific DEGs for HH, HL and LL, respectively. Functional enrichment analysis suggested that HH specific DEGs were mainly related to the regulation of transcription and other processes, while LL specific DEGs were enriched in the biological processes of extracellular matrix interaction and cell migration. The three defined mathylator groups showed great difference in tumor location, patient age, MSI and ECM biological process, which could facilitate the development of more effective biomarkers for CRC detection.

sentences: Mobius Assembly for Plant Systems highlights promoter-terminator interaction in gene regulation Plant synthetic biology is a fast-evolving field that employs engineering principles to empower research and bioproduction in plant systems. Nevertheless, in the whole synthetic biology landscape, plant systems lag compared to microbial and mammalian systems. When it comes to multigene delivery to plants, the predictability of the outcome is decreased since it depends on three different chassis: E. coli, Agrobacterium, and the plant species. Here we aimed to develop standardised and streamlined tools for genetic engineering in plant synthetic biology. We have devised Mobius Assembly for Plant Systems (MAPS), a user-friendly Golden Gate Assembly system for fast and easy generation of complex DNA constructs. MAPS is based on a new group of small plant binary vectors (pMAPs) that contains an origin of replication from a cryptic plasmid of Paracoccus pantotrophus. The functionality of the pMAP vectors was confirmed by transforming the MM1 cell culture, demonstrating for the first time that plant transformation is dependent on the Agrobacterium strains and plasmids; plasmid stability was highly dependent on the plasmid and bacterial strain. We made a library of new short promoters and terminators and characterised them using a high-throughput protoplast expression assay. Our results underscored the strong influence of terminators in gene expression, and they altered the strength of promoters in some combinations and indicated the presence of synergistic interactions between promoters and terminators. Overall this work will further facilitate plant synthetic biology and contribute to improving its predictability, which is challenged by combinatorial interactions among the genetic parts, vectors, and chassis.

sentences: An evolutionarily conserved cis-regulatory element of Nkx3.2 contributes to early jaw joint morphology in zebrafish The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element of Nkx3.2 that is deeply conserved in gnathostomes but undetectable in the jawless hagfish. This enhancer is active in the developing jaw joint region of the zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 leads to a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

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CcLBD25 functions as a key regulator of haustorium development in Cuscuta campestris Parasitic plants reduce yield of crops worldwide. Cuscuta campestris is a stem parasite that attaches to its host, using haustoria to extract nutrients and water. We analyzed the transcriptome of six C. campestris tissues and identified a key gene, CcLBD25, as highly expressed in prehaustoria and haustoria. Our gene co-expression networks (GCN) from different tissue types and laser-capture microdissection (LCM) RNA-Seq data indicate that CcLBD25 could be essential for regulating cell wall loosening and organogenesis. We employed host-induced gene silencing (HIGS) by generating transgenic tomato hosts that express hairpin RNAs to target and down-regulate CcLBD25 in the parasite. Our results showed that C. campestris growing on CcLBD25 RNAi transgenic tomatoes transited to the flowering stage earlier and had less biomass compared with C. campestris growing on wild type host. This suggests that the parasites growing on the transgenic plants were stressed due to insufficient nutrient acquisition. Anatomy of C. campestris haustoria growing on CcLBD25 RNAi tomatoes showed reduced pectin digestion and lack of searching hyphae, which interfered with haustorium penetration and the formation of vascular connections. We developed an in vitro haustorium (IVH) system to assay the number of prehaustoria produced on strands from C. campestris. When C. campestris was grown on CcLBD25 RNAi tomatoes or wild type tomatoes, the former produce fewer prehaustoria than the latter, indicating that down-regulating CcLBD25 may affect haustorium initiation. The results of this study shed light on the role of CcLBD25 in haustorium development and might help to develop a parasite-resistant system in crops.

One-sentence summaryCcLBD25 plays a pivotal role in haustorium initiation, regulating pectin digestion, and searching hyphae development during the haustorium penetration process.

sentences: Diffusion Fails to Make a Stink In this work we consider the question of whether a simple diffusive model can explain the scent tracking behaviors found in nature. For such tracking to occur, both the concentration of a scent and its gradient must be above some threshold. Applying these conditions to the solutions of various diffusion equations, we find that a purely diffusive model cannot simultaneously satisfy the tracking conditions when parameters are in the experimentally observed range. This demonstrates the necessity of modelling odor dispersal with full fluid dynamics, where non-linear phenomena such as turbulence play a critical role.

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A Guide to Pre-Processing High-Throughput Animal Tracking Data O_LIModern, high-throughput animal tracking studies collect increasingly large volumes of data at very fine temporal scales. At these scales, location error can exceed the animals step size, leading to mis-estimation of key movement metrics such as speed.  Cleaning the data to reduce location errors prior to analyses is one of the main ways movement ecologists deal with noisy data, and has the advantage of being more scalable to massive datasets than more complex methods. Though data cleaning is widely recommended, and ecologists routinely consider cleaned data to be the ground-truth, inclusive uniform guidance on this crucial step, and on how to organise the cleaning of massive datasets, is still rather scarce.
C_LIO_LIA pipeline for cleaning massive high-throughput datasets must balance ease of use and computationally efficient signal vs. noise screening, in which location errors are rejected without discarding valid animal movements. Another useful feature of a pre-processing pipeline is efficiently segmenting and clustering location data for statistical methods, while also being scalable to large datasets and robust to imperfect sampling. Manual methods being prohibitively time consuming, and to boost reproducibility, a robust pre-processing pipeline must be automated.
C_LIO_LIIn this article we provide guidance on building pipelines for pre-processing high-throughput animal tracking data in order to prepare it for subsequent analysis. Our recommended pipeline, consisting of removing outliers, smoothing the filtered result, and thinning it to a uniform sampling interval, is applicable to many massive tracking datasets. We apply this pipeline to simulated movement data with location errors, and also show a case study of how large volumes of cleaned data can be transformed into biologically meaningful  residence patches, for quick biological inference on animal space use. We use calibration data to illustrate how pre-processing improves its quality, and to verify that the residence patch synthesis accurately captures animal space use. Finally, turning to tracking data from Egyptian fruit bats (Rousettus aegyptiacus), we demonstrate the pre-processing pipeline and residence patch method in a fully worked out example.
C_LIO_LITo help with fast implementation of standardised methods, we developed the R package atlastools, which we also introduce here. Our pre-processing pipeline and atlastools can be used with any high-throughput animal movement data in which the high data-volume combined with knowledge of the tracked individuals movement capacity can be used to reduce location errors. The atlastools function is easy to use for beginners, while providing a template for further development. The use of common pre-processing steps that are simple yet robust promotes standardised methods in the field of movement ecology and leads to better inferences from data.
C_LI

sentences: cFos ensembles in the dentate gyrus rapidly segregate over time and do not form a stable map of space Transgenic cFos reporter mice are used to identify and manipulate neurons that store contextual information during fear learning. It is not clear, however, how spatial information acquired over several training days is integrated in the hippocampus. Using a water maze task, we observed that cFos expression patterns in the dentate gyrus are temporally unstable and shift daily. Surprisingly, cFos patterns did not get more stable with increasing spatial memory precision. Despite the fact that cFos was no longer expressed, optogenetic inhibition of neurons that expressed cFos on the first training day affected performance days later. Triggered by training, {Delta}FosB accumulates and provides a negative feedback mechanism that makes the cFos ensemble in the dentate gyrus dependent on the history of activity. Shifting cFos expression to a different set of granule cells every day may aid the formation of episodic memories.

sentences: Transcriptomic Analysis of Human Sensory Neurons in Painful Diabetic Neuropathy Reveals Inflammation and Neuronal Loss Pathological sensations caused by peripheral painful neuropathy occurring in Type 2 diabetes mellitus (T2DM) are often described as  sharp and  burning and are commonly spontaneous in origin. Proposed etiologies implicate dysfunction of nociceptive sensory neurons in dorsal root ganglia (DRG) induced by generation of reactive oxygen species, microvascular defects, and ongoing axonal degeneration and regeneration. To investigate the molecular mechanisms contributing to diabetic pain, DRGs were acquired postmortem from patients who had been experiencing painful diabetic peripheral neuropathy (DPN) and subjected to transcriptome analyses to identify genes contributing to pathological processes and neuropathic pain. DPN occurs in distal extremities resulting in the characteristic "glove and stocking" pattern. Accordingly, the L4 and L5 DRGs, which contain the perikarya of primary afferent neurons innervating the foot, were analyzed from five DPN patients and compared with seven controls. Transcriptom e analyses identified 844 differentially expressed genes. We observed increases in levels of inflammation-associated genes from macrophages in DPN patients that may contribute to increased pain hypersensitivity and, conversely, there were frequent decreases in neuronally-related genes. The elevated inflammatory gene profile and the accompanying downregulation of multiple neuronal genes provide new insights into intraganglionic pathology and mechanisms causing neuropathic pain in DPN patients with T2DM.

sentences: Global genomic epidemiology, resistome, virulome and plasmidome of the Extraintestinal Pathogenic Escherichia coli (ExPEC) ST38 lineage Most of the extraintestinal human infections worldwide are determined by some specific Extraintestinal Pathogenic Escherichia coli (ExPEC) lineages, which also present a zoonotic character. One of these lineages belongs to ST38, which is considered a uropathogenic/enteroaggregative E. coli hybrid and a high-risk globally disseminated ExPEC. To get insights on the aspects of ST38 epidemiology and evolution as a multidrug resistant and pathogenic lineage, this study performed a global comparative genomic analysis on ST38 genomes. A total of 376 genomes recovered from environments, humans, livestock, wild and domestic animals in all continents throughout three decades were analyzed considering the ST38 phylogenomics, resistome, plasmidome, and virulome. In general, our analyses revealed that, independently of host sources and geographic origin, these genomes were distributed in two clusters comprising clonal and non-clonal genomes. Moreover, the ST38 accessory genome was not strictly associated with clusters and sub-clusters. Interestingly, the High Pathogenicity Island (HPI) prevailed in the major cluster (cluster 2), which comprised most of the genomes from human origin recovered worldwide (2000 to 2020). In addition, the ExPEC ST38 harbors a huge and diverse plasmidome that might contribute to the evolution of this lineage, reflecting in ST38 ability to live in a diversity of niches as a commensal or pathogenic organism.

sentences: Nonlinear collision between anisotropic propagating waves in mouse somatosensory cortex How does cellular organization shape the spatio-temporal patterns of activity in the cortex while processing sensory information? After measuring the propagation of activity in the mouse primary somatosensory cortex (S1) in response to single whisker deflections with Voltage Sensitive Dye (VSD) imaging, we developed a two dimensional mean field model of S1. We observed that, for strong enough excitatory cortical interactions, whisker deflections generate a propagating wave in S1. We developed an inversion method that reconstructs model parameters from VSD data, revealing that a spatially heterogeneous organization of synaptic strengths between pyramidal neurons in S1 is likely to be responsible for the anisotropic spatio-temporal patterns of activity measured experimentally. Finally, we report that two consecutive stimuli activating different spatial locations in S1 generate two waves which collide sub-linearly. In the model, such sub-linear interaction is explained by a lower sensitivity to external perturbations of neural networks during activated states.

sentences: Widespread nociceptive maps in the human neonatal somatosensory cortex Topographic cortical maps are essential for spatial localisation of sensory stimulation and generation of appropriate task-related motor responses. Somatosensation and nociception are finely mapped and aligned in the adult somatosensory (S1) cortex, but in infancy, when pain behaviour is disorganised and poorly directed, nociceptive maps may be less refined. We compared the topographic pattern of S1 activation following noxious (clinically required heel lance) and innocuous (touch) mechanical stimulation of the same skin region in newborn infants (n=32) using multi-optode functional near-infrared spectroscopy (fNIRS). Signal to noise ratio and overall activation area did not differ with stimulus modality. Within S1 cortex, touch and lance of the heel elicit localised, partially overlapping increases in oxygenated haemoglobin (HbO), but while touch activation was restricted to the heel area, lance activation extended into cortical hand regions. The data reveals a widespread cortical nociceptive map in infant S1, consistent with their poorly directed pain behaviour.

sentences: Homology between the flagellar export apparatus and ATP synthetase: evidence from synteny predating the Last Universal Common Ancestor Evidence of homology between proteins in the ATP synthetase and the bacterial flagellar motor (BFM) has been accumulating since the 1980s. Specifically, the BFMs Type 3 Secretion System (T3SS) export apparatus FliH, FliI, and FliJ are considered homologous to FO-b + F1-{delta}, F1-/{beta}, and F1-{gamma}, and have similar structure and interactions. We review the discoveries that advanced the homology hypothesis and then conduct a further test by examining gene order in the two systems and their relatives. Conservation of gene order, or synteny, is often observed between closely related prokaryote species, but usually degrades with phylogenetic distance. As a result, observed conservation of synteny over vast phylogenetic distances can be evidence of shared ancestral coexpression, interaction, and function. We constructed a gene order dataset by examining the order of fliH, fliI, and fliJ genes across the phylogenetic breadth of flagellar and nonflagellar T3SS. We compared this to published surveys of gene order in the F1FO-ATP synthetase, its N-ATPase relatives, and the bacterial/archaeal V- and A-type ATPases. Strikingly, the fliHIJ gene order was deeply conserved, with the few exceptions appearing derived, and exactly matching the widely conserved F-ATPase gene order atpFHAG, coding for subunits b-{delta}--{gamma}. The V/A-type ATPases have a similar conserved gene order shared for homologous components. Our results further strengthen the argument for homology between these systems, and suggest a rare case of synteny conserved over billions of years, dating back to well before the Last Universal Common Ancestor (LUCA).

sentences: Intron-mediated induction of phenotypic heterogeneity Introns are universally present in the nuclear genomes of eukaryotes1. The budding yeast, an otherwise intron-poor species, preserves two sets of ribosomal protein (RP) genes differing primarily in their introns2-4. Despite recent findings on the role of RP introns under stress and starvation5-7, understanding the contribution of introns to ribosome regulation remains challenging. Here, combining isogrowth profiling8 with single-cell protein measurements9, we found that introns can mediate inducible phenotypic heterogeneity conferring a clear fitness advantage. Osmotic stress leads to bimodal expression of the small ribosomal subunit protein Rps22B, mediated by an intron in the 5 untranslated region of its transcript. The two resulting yeast subpopulations differ in their ability to cope with starvation. Low Rps22B protein levels resulted in prolonged survival under sustained starvation, while high Rps22B levels enabled cells to grow faster after transient starvation. Further, yeast growing at high sugar concentrations - similar to those in ripe grapes - exhibit bimodal Rps22B expression when approaching stationary phase. Differential intron-mediated regulation of RP genes thus provides a way to diversify the population when starvation looms in natural environments. Our findings reveal a new role for introns in inducing phenotypic heterogeneity in changing environments and suggest that duplicated RP genes in yeast contribute to resolving the evolutionary conflict between precise expression control and environmental responsiveness10.

sentences: Chemical inhibition of prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming Endothelial cell (EC) metabolism has emerged as a driver of angiogenesis. While hypoxia inactivates prolyl-4 hydroxylase domain containing proteins 1-3 (PHD1-3) and stabilizes hypoxia inducible factors (HIFs) stimulating angiogenesis, the effects of PHDs on EC functions remain unclear. Here, we investigated the impact of PHD inhibition by dimethyloxalylglycine (DMOG) on angiogenic competence and metabolism of human vascular ECs. PHD inhibition reduced EC proliferation, migration, and tube formation capacities. Furthermore, transcriptomic and metabolomic analyses revealed an unfavorable metabolic reprogramming for angiogenesis following treatment with DMOG. Despite the induction of glycolytic genes and high levels of lactate, multiple genes encoding sub-units of mitochondrial complex I were suppressed with concurrent decline in nicotinamide adenine dinucleotide (NAD+) levels. Importantly, defective EC migration due to DMOG could be partially restored by augmenting NAD+ levels. Combined, our data provide metabolic insights into the mechanism by which chemical PHD inhibition impairs angiogenic competence of human vascular ECs.

sentences: How synonymous mutations alter enzyme structure andfunction over long time scales The specific activity of enzymes can be altered over long time scales in cells by synonymous mutations, which change an mRNA molecules sequence but not the encoded proteins primary structure. How this happens at the molecular level is unknown. Here, we investigate this issue by applying multiscale modeling to three E. coli enzymes - type III chloramphenicol acetyltransferase, D-alanine-D-alanine ligase B, and dihydrofolate reductase. This modeling involves coarse-grained simulations of protein synthesis and post-translational behavior, all-atom simulations as a test of robustness, and QM/MM calculations to characterize enzymatic function. We first demonstrate that our model predicts experimentally measured changes in specific activity due to synonymous mutations. Then, we show that changes in codon translation rates induced by synonymous mutations cause shifts in co-translational and post-translational folding pathways that kinetically partition molecules into subpopulations that very slowly interconvert to the native, functional state. These long-lived states exhibit reduced catalytic activity, as demonstrated by their increased activation energies for the reactions they carry out. Structurally, these states resemble the native state, with localized misfolding near the active sites of the enzymes. The localized misfolding involves noncovalent lasso entanglements - a topology in which the protein backbone forms a loop closed by noncovalent native contacts which is then threaded by another portion of the protein. Such entanglements are often kinetic traps, as they can require a large proportion of the protein to unfold, which is energetically unfavorable, before they can disentangle and attain the native state. The near-native structures of these misfolded states allow them to bypass the proteostasis machinery and remain soluble, as they exhibit similar hydrophobic surface areas as the native state. These entangled structures persist in all-atom simulations as well, indicating that these conclusions are independent of model resolution. Moreover, the structures of long-lived entangled states are supported by agreement with limited-proteolysis mass spectrometry results. Thus, synonymous mutations cause shifts in the co- and post-translational structural ensemble of proteins, whose altered subpopulations lead to long-term changes in the specific activities of some enzymes. The formation of entangled subpopulations is a plausible mechanism through which changes in translation elongation rate alter ensemble-averaged specific activities, which can ultimately affect the efficiency of biochemical pathways and phenotypic traits.

sentences: Food interactions observed in a pharmacokinetic investigation comparing two marketed cold preparations (BNO1016 and ELOM-080) after administration to male beagle dogs The cold remedies Sinupret extract (BNO 1016) and Gelomyrtol forte (ELOM-080) represent the two top-selling cold remedies in Germany nowadays. Whereas BNO 1016 is a typical immediate release coated tablet, ELOM-080 is an enteric-coated soft gelatin capsule. The latter formulation, however, is at risk of pharmacokinetic interactions affecting absorption especially in the case of concomitant food intake. In the present pilot study, we investigated the risk of a possible food effect on BNO 1016 in comparison to ELOM-080 in three male beagle dogs. Single doses of BNO 1016 and ELOM-80 at 80 mg/kg and 160 mg/kg, respectively, were administered under fasting and fed conditions according to a 4-period, 4-treatment within-design. Blood sampling took place until up to 30 h p.a. and plasma concentrations of gentiopicroside, verbenalin and from a further marker analyte (BNO 1016 analytes) as well as 1,8-cineole, limonene and perillic acid (ELOM-080 analytes) were determined. Pharmacokinetic parameters focusing on rate and extent of absorption were derived. BNO 1016 analytes demonstrated a homogenous course in all animals in both, the fasted and fed state. Pharmacokinetic characteristics of a typical immediate release drug formulation were observed for all analytes and a food effect could be ruled out. ELOM-080 analytes also showed a homogeneous picture in the fasted state. However, lag-times (tlag) of up to 2 h p.a. with corresponding tmax values of 3 to 4 h were observed, reflecting a longer gastric residence time of the formulation. In the fed state, ELOM-080 showed significant pharmacokinetic characteristics suggesting a clear food effect. A major observation was a double peak phenomenon that could be observed in two out of three dogs. Furthermore, lag-times of some analytes up to 3-4 h and corresponding tmax values of up to 6-8 h occurred. In contrast to BNO 1016, these findings suggest that, as with other enteric-coated formulations, there may be a significant risk for food effects with ELOM-080 also in humans.

sentences: Spatially Interacting Phosphorylation Sites and Mutations in Cancer Advances in mass-spectrometry have generated increasingly large-scale proteomics datasets containing tens of thousands of phosphorylation sites (phosphosites) that require prioritization. We develop a bioinformatics tool called HotPho and systematically discover 3D co-clustering of phosphosites and cancer mutations on protein structures. HotPho identifies 474 such hybrid clusters containing 1,255 co-clustering phosphosites, including RET p.S904/Y928, the conserved HRAS/KRAS p.Y96, and IDH1 p.Y139/IDH2 p.Y179 that are adjacent to recurrent mutations on protein structures not found by linear proximity approaches. Hybrid clusters, enriched in histone and kinase domains, frequently include expression-associated mutations experimentally shown as activating and conferring genetic dependency. Approximately 300 co-clustering phosphosites are verified in patient samples of 5 cancer types or previously implicated in cancer, including CTNNB1 p.S29/Y30, EGFR p.S720, MAPK1 p.S142, and PTPN12 p.S275. In summary, systematic 3D clustering analysis highlights nearly 3,000 likely functional mutations and over 1,000 cancer phosphosites for downstream investigation and evaluation of potential clinical relevance.

sentences: Novel and known transcriptional targets of ALS/FTD protein TDP-43: Meta-analysis and interactive graphical databases TDP-43 proteinopathy is the major pathological hallmark of amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal dementia (FTD). Mounting evidence implicates a loss of normal TDP-43 function in neurodegeneration, either resultant from or independent of TDP-43 aggregation. TDP-43 knockdown is therefore a common paradigm for modelling ALS and FTD. However, because TDP-43 can interact directly with thousands of mRNA targets and regulate the function of other RNA binding proteins, the phenotype of TDP-43 depletion is likely to differ depending on the proteomic and transcriptomic profile of the model cell type. Here, we conducted a meta-analysis of publicly available RNA-sequencing datasets that utilized TDP-43 knockdown to model ALS or FTD, and validated these against RNA-sequencing data from TDP-43-immunonegative neuronal nuclei from ALS/FTD brain. We present these analyses as easy-to-use interactive graphical databases. Of 9 TDP-43-knockdown datasets identified, 4 showed significant depletion of TARDBP (human HeLa and SH-SY5Y cell lines, induced human motor neurons, and mouse striatal tissue). There was little overlap in differentially expressed genes between TDP-43-knockdown model cell types, but PFKP, RANBP1, KIAA1324, ELAVL3, and STMN2 were among the common TDP-43 targets. Similarly, there were few genes that showed common patterns of differential exon usage between cell types and which validated in TDP-43-immunonegative neurons, but these included well-known targets POLDIP3, RANBP1, STMN2, and UNC13A, and novel targets EXD3, CEP290, KPNA4, and MMAB. Enrichment analysis showed that TDP-43 knockdown in different cell types affected a unique range of biological pathways. Together, these data identify novel TDP-43 targets, validate known TDP-43 targets, and show that TDP-43 plays both conserved and cell-type-specific roles in the regulation of gene expression and splicing. Identification of cell-type-specific TDP-43 targets will enable sensitive mapping of cell-autonomous TDP-43 dysfunction beyond just neurons, while shared TDP-43 targets are likely to have therapeutic value across myriad cell types.

sentences: Inflammatory signals from fatty bone marrow supports the early stages of DNMT3a driven clonal hematopoiesis Age related cancer is not only due to the random accumulation of mutations, but also how phenotypes are selected by the aging environment. While fatty bone marrow (FBM), is one of the hallmarks of bone marrow ageing, it is unknown whether FBM can modify the evolution of the early stages of leukemia and clonal hematopoiesis (CH). To address this question, we established FBM mice models and transplanted both human and mice preleukemic hematopoietic stem cells (PreL-HSCs) carrying DNMT3A mutations. We demonstrate that castration which models age related andropenia result in FBM. A significant increase in self-renewal was found when DNMT3AMut - preL-HSPCs were exposed to FBM. To better understand the mechanisms of the FBM-preL-HSPCs interaction, we performed single cell RNA-sequencing on HSPCs three days after FBM exposure. A 20-50 fold increase in DNMT3AMut-preL-HSCs was observed under FBM conditions in comparison to other conditions. PreL-HSPCs exposed to FBM exhibited an activated inflammatory signaling (IL-6 and INF{gamma}). Cytokine analysis of BM fluid demonstrated increased IL-6 levels under FBM conditions. Anti-IL-6 neutralizing antibodies significantly reduced the selective advantage of DNMT3AMut-preL-HSPCs exposed to FBM. Overall, age related paracrine FBM inflammatory signals promote DNMT3A-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 receptor.

sentences: Strong immobilization of C. elegans en masse on cultivation plates by thermoelectric cooling for high-throughput submicron-resolution microscopy Despite its profound impact on biology, most high-resolution in vivo microscopy approaches remain low throughput because current immobilization techniques require significant manual effort. We greatly accelerate imaging of the nematode Caenorhabditis elegans by implementing a simple cooling approach to easily immobilize entire populations directly on their cultivation plates. We optimize and characterize cooling immobilization. Counterintuitively, relatively warmer temperatures immobilize animals significantly more effectively than colder temperatures utilized in prior studies. This enhanced immobilization enables clear submicron-resolution fluorescence imaging, which is challenging to achieve with most current immobilization techniques. We demonstrate 64x magnification 3D imaging and timelapse recording of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. By obviating individual animal manipulation, our approach could also empower automated imaging of large C. elegans populations within standard experimental setups and workflows.

sentences: Deep learning tools and modeling to estimate the temporal expression of cell cycle proteins from 2D still images Automatic characterization of fluorescent labeling in intact mammalian tissues remains a challenge due to the lack of quantifying techniques capable of segregating densely packed nuclei and intricate tissue patterns. Here, we describe a powerful deep learning-based approach that couples remarkably precise nuclear segmentation with quantitation of fluorescent labeling intensity within segmented nuclei, and then apply it to the analysis of cell cycle dependent protein concentration in mouse tissues using 2D fluorescent still images. First, several existing deep learning-based methods were evaluated to accurately segment nuclei using different imaging modalities with a small training dataset. Next, we developed a deep learning-based approach to identify and measure fluorescent labels within segmented nuclei, and created an ImageJ plugin to allow for efficient manual correction of nuclear segmentation and label identification. Lastly, using fluorescence intensity as a readout for protein concentration, a three-step global estimation method was applied to the characterization of the cell cycle dependent expression of E2F proteins in the developing mouse intestine.

sentences: Preclinical establishment of a divalent vaccine against SARS-CoV-2 First-generation vaccines against SARS-CoV-2 have been administered to more than 60% of the population in developed countries. However, the monovalent vaccines currently available in Europe do not confer adequate and durable immune protection. To satisfy the need for a novel vaccine, we engineered a divalent gene construct consisting of the receptor binding domain (RBD, 300-685 aa) of the spike protein and the immunodominant region of the nucleocapsid (100-300 aa). This fusion protein was cloned into a pET-30a plasmid and expressed either in Escherichia coli or in a recombinant baculovirus in insect cells. Following purification via its His-tag, the fusion protein was mixed with adjuvant, and administered to mice in a prime-booster-mode. Upon testing for IgG antibody response against nucleocapsid and RBD, a titer of 10-4 - 10-5 was demonstrated 14 days after the first booster injection in 72% of the animals, which could be increased to 100% by a second booster. Notably, comparable IgG responses were detected against the delta, gamma and omicron variants of the RBD region. Durability testing revealed the presence of IgG beyond 90 days. In addition, granzyme A and perforin mRNA expression (cytolytic effector cell molecules) was increased in cytotoxic lymphocytes isolated from peripheral blood. Ex vivo stimulation of T-cells by nucleocapsid and RBD peptides showed antigen-specific upregulation of CD44 in vaccinated mice among their CD4+ and CD8+ T-cells. No side-effect was documented in the central nervous system, be it either endothelial inflammation or neuronal damage. Cumulatively, the combined induction of B-cell and T-cell response by a bivalent protein-based vaccine directed against two structural SARS-CoV-2 proteins represents a proof-of-principle approach alternative to existing mRNA vaccination strategies, which could confer long-lasting immunity against all known viral strains.

sentences: Mechanotaxis directs Pseudomonas aeruginosa twitching motility. The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by retractile extracellular filaments called type IV pili. Single cells twitch by successive pili extension, attachment and retraction. However, whether and how single cells control twitching migration remains unclear. We discovered that P. aeruginosa actively directs twitching in the direction of mechanical input from type IV pili, in a process we call mechanotaxis. The Chp chemotaxis-like system controls the balance of forward and reverse twitching migration of single cells in response to the mechanical signal. On surfaces, Chp senses type IV pili attachment at one pole thereby sensing a spatially-resolved signal. As a result, the Chp response regulators PilG and PilH control the polarization of the extension motor PilB. PilG stimulates polarization favoring forward migration, while PilH inhibits polarization inducing reversal. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. This distinct localization of response regulators establishes a signaling landscape known as local-excitation, global-inhibition in higher order organisms, identifying a conserved strategy to transduce spatially-resolved signals. Our discovery finally resolves the function of the Chp system and expands our view of the signals regulating motility.

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hC9ORF78 localizes to kinetochores and is required for proper chromosome segregation. C9ORF78 is a poorly characterized protein found in diverse eukaryotes. Previous work indicated overexpression of hC9ORF78 (aka HCA59) in malignant tissues indicating a possible involvement in growth regulatory pathways. Additional studies in fission yeast and humans uncover a potential function in regulating the spliceosome. In studies of GFP-tagged hC9ORF78 we observed a dramatic reduction in protein abundance in cells grown to confluence and/or deprived of serum growth factors. Serum stimulation induced synchronous re-expression of the protein in HeLa cells. This effect was also observed with the endogenous protein. Overexpressing either E2F1 or N-Myc resulted in elevated hC9ORF78 expression potentially explaining the serum-dependent upregulation of the protein. Immunofluorescence analysis indicates that hC9ORF78 localizes to nuclei in interphase but does not appear to concentrate in speckles as would be expected for a splicing protein. Surprisingly, a subpopulation of hC9ORF78 co-localizes with ACA, Mad1 and Hec1 in mitotic cells suggesting that this protein may associate with kinetochores or centromeres. Furthermore, knocking-down hC9ORF78 caused mis-alignment of chromosomes in mitosis. These studies uncover novel mitotic function and subcellular localization of cancer antigen hC9ORF78.

SUMMARY STATEMENThC9ORF78 regulates chromosome segregation.

sentences: Role of RALBP1 in Oxidative Stress and Mitochondrial Dysfunction in Alzheimer's Disease The purpose of our study is to understand the role of the Ralbp1 gene in oxidative stress (OS), mitochondrial dysfunction and cognition in Alzheimers disease (AD) pathogenesis. The Ralbp1 gene encodes the 76 kDa protein Rlip (aka RLIP76). Previous studies have revealed its role in OS-related cancer. However, Rlip is transcriptionally regulated by EP300, a CREB-binding protein that is important for synaptic plasticity in the brain. Rlip functions as a stress-responsive/protective transporter of glutathione conjugates (GS-E) and xenobiotic toxins. OS causes rapid cellular accumulation of Rlip and its translocation from a tubulin-bound complex to the plasma membrane, mitochondria and nucleus. Therefore, Rlip may play an important role in maintaining cognitive function in the face of OS-related injury. This study is aimed to determine whether Rlip deficiency in mice is associated with AD-like cognitive and mitochondrial dysfunction. Brain tissue obtained from cohorts of wildtype and Rlip+/- mice were analyzed for OS markers, expression of genes that regulate mitochondrial fission/fusion, and synaptic integrity. We also examined mitochondrial ultrastructure in mouse brains obtained from these mice and further analyzed the impact of Rlip deficiency on gene networks of AD, aging, inhibition of stress-activated gene expression, mitochondrial function, and CREB signaling. Our studies revealed a significant increase in the levels of OS markers and alterations in the expression of genes and proteins involved in mitochondrial biogenesis, dynamics and synapses in brain tissues of these mice. Furthermore, we compared the cognitive function of wildtype and Rlip+/- mice. Behavioral, basic motor and sensory function tests in Rlip+/- mice revealed cognitive decline, similar to AD. Gene network analysis indicated dysregulation of stress-activated gene expression, mitochondrial function, and CREB signaling genes in the Rlip+/- mouse liver. Our results suggest that the Rlip deficiency-associated increase in OS and mitochondrial dysfunction could contribute to the development of OS-related AD processes. Therefore, the restoration of Rlip activity and endogenous cytoprotective mechanisms by pharmacological interventions is a novel approach to protect against AD.

sentences: The carbon footprint of bioinformatics Bioinformatic research relies on large-scale computational infrastructures which have a non-zero carbon footprint. So far, no study has quantified the environmental costs of bioinformatic tools and commonly run analyses. In this study, we estimate the bioinformatic carbon footprint (in kilograms of CO2 equivalent units, kgCO2e) using the freely available Green Algorithms calculator (www.green-algorithms.org). We assess (i) bioinformatic approaches in genome-wide association studies (GWAS), RNA sequencing, genome assembly, metagenomics, phylogenetics and molecular simulations, as well as (ii) computation strategies, such as parallelisation, CPU (central processing unit) vs GPU (graphics processing unit), cloud vs. local computing infrastructure and geography. In particular, for GWAS, we found that biobank-scale analyses emitted substantial kgCO2e and simple software upgrades could make GWAS greener, e.g. upgrading from BOLT-LMM v1 to v2.3 reduced carbon footprint by 73%. Switching from the average data centre to a more efficient data centres can reduce carbon footprint by ~34%. Memory over-allocation can be a substantial contributor to an algorithms carbon footprint. The use of faster processors or greater parallelisation reduces run time but can lead to, sometimes substantially, greater carbon footprint. Finally, we provide guidance on how researchers can reduce power consumption and minimise kgCO2e. Overall, this work elucidates the carbon footprint of common analyses in bioinformatics and provides solutions which empower a move toward greener research.

sentences: Sensory prediction errors increase coding efficiency in mouse visual cortex through gain amplification The response of cortical neurons to sensory stimuli is shaped both by past events (adaptation) and the expectation of future events (prediction). Here we employed a visual stimulus paradigm with different levels of predictability to characterise how expectation influences orientation selectivity in the primary visual cortex (V1) of mice. We recorded neuronal activity using two-photon calcium imaging (GCaMP6f) while animals viewed sequences of grating stimuli which either varied randomly in their orientations or rotated predictably with occasional transitions to an unexpected orientation. For single neurons and the population, there was significant enhancement in the gain of orientation-selective responses to unexpected gratings. This gain-enhancement for unexpected stimuli was prominent in both awake and anaesthetised mice. We implemented a computational model to demonstrate how trial-to-trial variability in neuronal responses were best characterised when adaptation and expectation effects were combined.

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Transmission of Klebsiella strains and plasmids within and between Grey-headed flying fox colonies The Grey-headed flying fox (Pteropus poliocephalus) is an endemic Australian fruit bat, known to carry pathogens with zoonotic potential. We recently showed these bats harbour the bacterial pathogens Klebsiella pneumoniae and closely related species in the K. pneumoniae species complex (KpSC). However, the dynamics of Klebsiella transmission and gene flow within flying fox colonies were not explored and remain poorly understood.

Here we report a high-resolution genomic comparison of 39 KpSC isolates from Greyheaded flying foxes. Illumina whole genome sequences (n=39) were assembled de novo and the Kleborate genotyping tool was used to infer sequence types (STs). Oxford Nanopore sequences were generated for 13 isolates (one for each distinct ST) in order to generate high-quality completed reference genomes. Read mapping and variant calling was used to identify single nucleotide variants (SNVs) within each ST, using the relevant reference genome. In silico genome-scale metabolic models were generated to predict and compare substrate usage to 59 previously published KpSC models for isolates from human and environmental sources, which indicated no distinction on the basis of metabolic capabilities.

High-resolution genome comparisons identified five putative strain transmission clusters (four intra- and one inter-colony, n=2-15 isolates each, [≤]25 pairwise SNVs). Inter-colony transmission of Klebsiella africana was found between two flying fox populations located within flying distance. The 13 completed genomes harboured 11 plasmids, all of which showed 37-98% coverage (mean 73%) and [≥]95% identity to those previously reported from human-associated KpSC. Comparison of plasmids from different flying fox associated KpSC indicated an interspecies horizontal plasmid transmission between K. pneumoniae and K. africana for a 98 kbp plasmid, pFF1003.

These data indicate that KpSC are able to transmit directly via flying fox populations or indirectly via a common source, and that these isolates can harbour plasmids with similarity to those found in human derived KpSC, indicating gene flow is occurring between isolates from Grey-headed flying fox KpSC and human clinical isolates.

sentences: Dynamic integration and segregation of amygdala subregional functional circuits linking to physiological arousal The dynamical organization of brain networks is essential to support human cognition and emotion for rapid adaption to ever-changing environment. As the core nodes of emotion-related brain circuitry, the basolateral amygdala (BLA) and centromedial amygdala (CMA) as two major amygdalar nuclei, are recognized to play distinct roles in affective functions and internal states, via their unique connections with cortical and subcortical structures in rodents. However, little is known how the dynamical organization of emotion-related brain circuitry reflects internal autonomic responses in humans. Using resting-state functional magnetic resonance imaging (fMRI) with K-means clustering approach in a total of 79 young healthy individuals (cohort 1: 42; cohort 2: 37), we identified two distinct states of BLA- and CMA-based intrinsic connectivity patterns, with one state (integration) showing generally stronger BLA- and CMA-based intrinsic connectivity with multiple brain networks, while the other (segregation) exhibiting weaker yet dissociable connectivity patterns. In an independent cohort 2 of fMRI data with concurrent recording of skin conductance, we replicated two similar dynamic states and further found higher skin conductance level in the integration than segregation state. Moreover, machine learning-based Elastic-net regression analyses revealed that time-varying BLA and CMA intrinsic connectivity with distinct network configurations yield higher predictive values for spontaneous fluctuations of skin conductance level in the integration than segregation state. Our findings highlight dynamic functional organization of emotion-related amygdala nuclei circuits and networks and its links to spontaneous autonomic arousal in humans.

sentences: Lipid production from Zygosaccharomyces siamensis AP1 using glycerol as a carbon source Yeasts are considered as potential lipid producer because they are easy to cultivate, able to grow in high cell densities and can produce high concentrations of lipids. Previously we had isolated a promising lipid producing yeast, Zygosaccharomyces siamensis AP1, from wild honey of Central Sulawesi, Indonesia. The strain was able to produce around 19% (w/w biomass) of lipid on glucose. However, its ability to produce lipid on other types of carbon source is still not studied. In this study, we compared the strain ability to produce lipid on glycerol and glucose using different C/N ratio. We also investigated optimum temperature, pH, and growth time for lipid production on glycerol. We found that growing the strain on glycerol with C/N ratio 120 gave the highest lipid content (29.7%). We also found that optimum growth temperature and pH are 30{degrees}C and 5, respectively. Maximum lipid biomass, 0.28 g/L, was produced when the strain was grown in the optimum conditions for 72 hours. In conclusion, our results suggest that glycerol is a potential cheap carbon source for lipid production from Z. siamensis AP1. We suggest further study to increase lipid yield either by fermentation or strain improvement.

sentences: ROMO1 is essential for glucose coupling in the pancreatic beta cell of male mice Reactive oxygen species modulator 1 (ROMO1) is a highly conserved inner mitochondrial membrane protein that senses ROS and regulates mitochondrial dynamics 1. ROMO1 is required for mitochondrial fusion in vitro, and silencing ROMO1 increases sensitivity to cell death stimuli. How ROMO1 promotes mitochondrial fusion and its physiological role remain unclear. Here we show that ROMO1 is essential for embryonic development, as ROMO1-null mice die before embryonic day 8.5, earlier than GTPases OPA1 or MFN1/2 that catalyze mitochondrial membrane fusion. Knockout of ROMO1 in adult pancreatic {beta} cells results in impaired glucose homeostasis in male mice due to an insulin secretion defect resulting from impaired glucose sensing. Mitochondria in ROMO1 {beta} cell KO cells were swollen and fragmented, consistent with a role for ROMO1 in mitochondrial fusion in vivo. While basal respiration was normal in ROMO1{beta} cell KO islets, spare respiratory capacity was lost. Taken together, our data indicate that ROMO1 is required for nutrient coupling in the {beta} cell and point to a critical role for spare respiratory capacity in the maintenance of euglycemia in males.

sentences: Cell cycle exit and stem cell differentiation are coupled through regulation of mitochondrial activity in the Drosophila testis Stem cells maintain tissue homeostasis by proliferating to replace cells lost to damage or natural turnover. Whereas stem and progenitor cells proliferate, fully differentiated cells exit the cell cycle. How cell identity and cell cycle state are coordinated during this process is still poorly understood. The Drosophila testis niche supports germline stem cells and somatic cyst stem cells (CySCs), which are the only proliferating somatic cells in the testis. CySCs give rise to post-mitotic cyst cells and therefore provide a tractable model to ask how stem cell identity is linked to proliferation. We show that the G1/S cyclin, Cyclin E, is required for CySC self-renewal; however, its canonical transcriptional regulator, a complex of the E2f1 and Dp transcription factors is dispensable for self-renewal and cell cycle progression. Nevertheless, we demonstrate that E2f1/Dp activity must be silenced to allow CySCs to differentiate. We show that E2f1/Dp activity inhibits the expression of genes important for mitochondrial activity. Furthermore, promoting mitochondrial activity or biogenesis is sufficient to rescue the differentiation of CySCs with ectopic E2f1/Dp activity but not their exit from the cell cycle. Our findings together indicate that E2f1/Dp coordinates cell cycle progression with stem cell identity by regulating the metabolic state of CySCs.

sentences: Clinical and immunological signatures of severe COVID-19 in previously healthy patients with clonal hematopoiesis Identifying additional risk factors for COVID-19 severity in numerous previously healthy patients without canonical clinical risk factors remains challenging. In this study, we investigate whether clonal hematopoiesis of indeterminate potential (CHIP), a common aging-related process that predisposes various inflammatory responses, may exert COVID-19 severity. We examine the clinical impact of CHIP in 143 laboratory-confirmed COVID-19 patients. Both stratified analyses and logistic regression including the interaction between canonical risk factors and CHIP show that CHIP is an independent risk factor for severe COVID-19, especially in previously healthy patients. Analyses of 60,310 single-cell immune transcriptome profiles identify distinct immunological signatures for CHIP (+) severe COVID-19 patients, particularly in classical monocytes, with a marked increase in pro-inflammatory cytokine responses and potent IFN-{gamma} mediated hyperinflammation signature. We further demonstrate that the enhanced expression of CHIP (+) specific IFN-{gamma} response genes is attributed to the CHIP mutation-dependent epigenetic reprogramming of poised or bivalent cis-regulatory elements. Our results highlight a unique immunopathogenic mechanism of CHIP in the progression of severe COVID-19, which could be extended to elucidate how CHIP contributes to a variety of human infectious diseases.

sentences: Decision formation in parietal cortex transcends a fixed frame of reference Neurons in the lateral intraparietal cortex (area LIP) represent the formation of a decision when it is linked to a specific action, such as an eye movement to a choice target. However, these neurons should be unable to represent a decision that transpires across actions that would disrupt this linkage. We investigated this limitation by recording simultaneously from many neurons. While intervening actions disrupt the representation by single neurons, the ensemble achieves continuity of the decision process by passing information from currently active neurons to neurons that will become active after the action. In this way, the representation of an evolving decision can be generalized across actions and transcends the frame of reference that specifies the neural response fields. The finding extends previous observations of receptive field remapping, thought to support the stability of perception across eye movements, to the continuity of a thought process, such as a decision.

sentences: LYRUS: A Machine Learning Model for Predicting the Pathogenicity of Missense Variants Single amino acid variations (SAVs) are a primary contributor to variations in the human genome. Identifying pathogenic SAVs can aid in the diagnosis and understanding of the genetic architecture of complex diseases, such as cancer. Most approaches for predicting the functional effects or pathogenicity of SAVs rely on either sequence or structural information. Nevertheless, previous analyses have shown that methods that depend on only sequence or structural information may have limited accuracy. Recently, researchers have attempted to increase the accuracy of their predictions by incorporating protein dynamics into pathogenicity predictions. This study presents < Lai Yang Rubenstein Uzun Sarkar > (LYRUS), a machine learning method that uses an XGBoost classifier selected by TPOT to predict the pathogenicity of SAVs. LYRUS incorporates five sequence-based features, six structure-based features, and four dynamics-based features. Uniquely, LYRUS includes a newly-proposed sequence co-evolution feature called variation number. LYRUSs performance was evaluated using a dataset that contains 4,363 protein structures corresponding to 20,307 SAVs based on human genetic variant data from the ClinVar database. Based on our dataset, the LYRUS classifier has a higher accuracy, specificity, F-measure, and Matthews correlation coefficient (MCC) than alternative methods including PolyPhen2, PROVEAN, SIFT, Rhapsody, EVMutation, MutationAssessor, SuSPect, FATHMM, and MVP. Variation numbers used within LYRUS differ greatly between pathogenic and neutral SAVs, and have a high feature weight in the XGBoost classifier employed by this method. Applications of the method to PTEN and TP53 further corroborate LYRUSs strong performance. LYRUS is freely available and the source code can be found at https://github.com/jiaying2508/LYRUS.

sentences: Slow waves form expanding, memory-rich mesostates steered by local excitability in fading anesthesia During sleep and anesthesia, large groups of neurons throughout the entire cortex activate rhythmically producing wavefronts of activity referred to as slow-wave activity (SWA). In the arousal process, the brain restores its integrative and complex activity. The network mechanisms underlying this global state transition remain however to be elucidated. Here we investigated the network features shaping the SWA under fading anesthesia. Using electrocorticographical recordings of wide cortical areas of the mouse brain, we developed a quantitative measure of the anesthesia level based on slow-wave frequency and complexity. At deep anesthesia, we document a stringent alternation of posterior-anterior-posterior modes of propagation. With fading anesthesia, SWA evolves to produce a plethora of metastable spatiotemporal patterns. A network model of spiking neurons reproduced the data using short-range connectivity, subcortical input and a local activity-dependent adaptation. The emergence from deep anesthesia does not require modifying the connectivity, but small changes in the local excitability of cortical cell assemblies, further supporting the hypothesis of a tight bound between scales in the brain.

sentences: Green synthesis of silver nanoparticles using psychrotolerant strains of Tulasnella albida from South Orkney Islands (Antarctica) Nanoparticles are widely studied due to their possible uses in biological and technological systems. Four psychrotolerant strains of Tulasnella albida isolated from Antarctica were tested and compared in their ability to synthesize silver nanoparticles. The four strains were capable of synthesizing silver nanoparticles with the addition of AgNO3 (final concentration of 0.5 mM), showing similar results under the same conditions: 28{degrees}C, 200 rpm, pH 9. Additionally, we registered the synthesis of nanoparticles at 6{degrees}C using biomass generated at the same temperature. For the characterization of synthesized nanoparticles, TEM and SEM microscopy analyses were performed. The images obtained showed the existence of spherically shaped silver nanoparticles with a log-normal size distribution centered at 2 nm diameter for 28{degrees}C. The largest ones showed a capping shell around them, appearing associated with the formation of small silver nanoparticles. Theoretical calculations of optical absorption based on core-shell Ag-Ag2O nanoparticles were used to characterize the experimental absorption spectra of silver nanoparticles colloids. This work contributes to expanding the studies and possible technological applications of psychrotolerant organisms in the industry, particularly in the green synthesis of nanoparticles at suboptimal conditions.

sentences: Rye: genetic ancestry inference at biobank scale Biobank projects around the world are generating genomic data for many thousands and even millions of individuals. Computational methods are needed to handle these massive data sets, including tools for genetic ancestry (GA) inference. Current methods for GA inference are generally accurate, but they are slow and do not scale to biobank-size genomic datasets. Here we present Rye - a new algorithm for GA inference at biobank scale. We compare the accuracy and runtime performance of Rye to the widely used RFMix and ADMIXTURE programs, and we apply it to a dataset of 488,221 genome-wide variant samples from the UK Biobank. Rye infers GA based on principal component analysis (PCA) of genomic variant samples from ancestral reference populations and query individuals. The algorithms accuracy is powered by Metropolis-Hastings optimization and its speed is provided by non-negative least squares (NNLS) regression. Rye produces highly accurate GA estimates for three-way admixed populations - African, European, and Native American - compared to RFMix and ADMIXTURE (R2 = 0.998 - 1.00), and shows 50x runtime improvement compared to ADMIXTURE on the UK Biobank dataset. Rye analysis of UK Biobank samples demonstrates how it can be used to infer GA at different levels of relatedness. We discuss user consideration and options for the use of Rye; the program and its documentation are distributed on the GitHub repository: https://github.com/healthdisparities/rye.

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Sensory nerves enhance triple-negative breast cancer migration and metastasis via the axon guidance molecule PlexinB3 In breast cancer, nerve presence has been correlated with more invasive disease and worse prognosis, yet the mechanisms by which different types of peripheral nerves drive tumor progression remain poorly understood. In this study, we identified sensory nerves as more abundant in human triple-negative breast cancer (TNBC) tumors. Coinjection of sensory neurons isolated from the dorsal root ganglia (DRG) of adult female mice with human TNBC cells in immunocompromised mice increased the number of lung metastases. Direct in vitro co-culture of human TNBC cells with the dorsal root ganglia (DRG) of adult female mice revealed that TNBC cells adhere to sensory neuron fibers leading to an increase in migration speed. Species-specific RNA sequencing revealed that co-culture of TNBC cells with sensory nerves upregulates the expression of genes associated with cell migration and adhesion in cancer cells. We demonstrate that the axon guidance molecule Plexin B3 mediates cancer cell adhesion to and migration on sensory nerves. Together, our results identify a novel mechanism by which nerves contribute to breast cancer migration and metastasis by inducing a shift in TNBC cell gene expression and support the rationale for disrupting neuron-cancer cell interactions to target metastasis.

SignificanceThe presence of nerves in breast tumors has been associated with poor outcome. Understanding the mechanisms by which nerves contribute to tumor progression could help identify novel strategies to target metastatic disease.

sentences: Allele-resolved single-cell multi-omics uncovers the dynamics and transcriptional kinetics of X-chromosome upregulation X-chromosome inactivation (XCI) and upregulation (XCU) are the major opposing chromosome-wide modes of gene regulation that collectively achieve dosage compensation in mammals, but the regulatory link between the two remains elusive. Here, we use allele-resolved single-cell RNA-seq combined with chromatin accessibility profiling to finely dissect the separate effects of XCI and XCU on RNA levels during mouse development. We uncover that balanced X dosage is flexibly attained through expression tuning by XCU in a sex- and lineage-specific manner along varying degrees of XCI and across developmental and cellular states. Male blastomeres achieve XCU upon zygotic genome activation while females experience two distinct waves of XCU, upon imprinted- and random XCI, and ablation of Xist impedes female XCU. Contrary to widely established models of mammalian dosage compensation, naive female embryonic cells carrying two active X chromosomes do not exhibit upregulation but express both alleles at basal level, yet collectively exceeding the RNA output of a single hyperactive allele. We show, in vivo and in vitro, that XCU is kinetically driven by X-specific modulation of transcriptional burst frequency, coinciding with increased compartmentalization of the hyperactive allele. Altogether, our data provide unprecedented insights into the dynamics of mammalian XCU, prompting a revised model of the chain in events of allelic regulation by XCU and XCI in unitedly achieving stable cellular levels of X-chromosome transcripts.

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Hebbian learning with elasticity explains how the spontaneous motor tempo affects music performance synchronization Music has a tempo (or frequency of the underlying beat) that musicians maintain throughout a performance. Musicians can maintain this tempo on their own or paced by a metronome. Behavioral studies have found that each musician shows a spontaneous rate of movement, called spontaneous motor tempo (SMT), which can be measured when a musician spontaneously plays a simple melody. Data shows that a musicians SMT systematically influences how actions affect tempo and synchronization. In this study we present a model that captures this phenomenon. To develop our model, we review the results from three musical performance settings that have been previously published: (1) solo musical performance with a pacing metronome tempo that is different from the SMT, (2) solo musical performance without a metronome at a tempo that is faster or slower than the SMT, and (3) duet musical performance between musician pairs with matching or mismatching SMTs. In the first setting, the asynchrony between the pacing metronome and the musicians tempo grew as a function of the difference between the metronome tempo and the musicians SMT. In the second setting, musicians drifted away from the initial spontaneous tempo toward the SMT. And in the third setting, the absolute asynchronies between performing musicians were smaller if their SMTs matched compared to when they did not. Based on these observations, we hypothesize that, while musicians can perform musical actions at a tempo different from their SMT, the SMT constantly acts as a pulling force. We developed a model to test our hypothesis. The model is an oscillatory dynamical system with Hebbian and elastic tempo learning that simulates music performance. We model SMT as the dynamical systems natural frequency. Hebbian learning lets the systems frequency adapt to match the stimulus frequency. The pulling force is modeled as an elasticity term that pulls the learned frequency toward the systems natural frequency. We used this model to simulate the three music performance settings, replicating behavioral results. Our model also lets us make predictions about performance settings not yet tested. The present study offers a dynamical explanation of how an individuals SMT affects adaptive synchronization in realistic musical performance.

Author summaryIndividuals can keep a musical tempo on their own or timed by another individual or a metronome. Experiments show that individuals show a specific spontaneous rate of periodic action, for example walking, blinking, or singing. Moreover, in a simple metronome synchronization task, an individuals spontaneous rate determines that the individual will tend to anticipate a metronome that is slower, and lag a metronome that is faster. Researchers have hypothesized the mechanisms explaining how spontaneous rates affect synchronization, but no hypothesis can account for all observations yet. Our hypothesis is that individuals rely on elastic Hebbian frequency learning during synchronization tasks to adapt the rate of their movements and match another individuals actions or metronome tempo. Elastic Hebbian frequency learning also explains why an individuals spontaneous rate persists after carrying out a musical synchronization task. We define a new model with elastic Hebbian frequency learning and use it to simulate existing empirical data. Not only can our model explain the empirical data, but it can also make testable predictions. Our results support the theory that the brains endogenous rhythms give rise to spontaneous rates of movement, and that learning dynamics interact with such brain rhythms to allow for flexible synchronization.

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Bacterial Infection of the Placenta Induces Sex-Specific Responses in the Fetal Brain BACKGROUNDEpidemiological data indicate that prenatal infection is associated with an increased risk of several neurodevelopmental disorders in the progeny. These disorders display sex differences in presentation. The role of the placenta, which is a target of prenatal infection, in the sex-specificity of neurodevelopmental abnormalities is unknown. We used an imaging-based animal model of the bacterial pathogen Listeria monocytogenes to identify sex-specific effects of placental infection on neurodevelopment of the fetus.

METHODSPregnant CD1 mice were infected with a bioluminescent strain of Listeria on embryonic day 14.5 (E14.5). Excised fetuses were imaged on E18.5 to identify the infected placentas. The associated fetal brains were analyzed for gene expression and altered brain structure due to infection. The behavior of adult offspring affected by prenatal Listeria infection was analyzed.

RESULTSPlacental infection induced sex-specific alteration of gene expression patterns in the fetal brain and resulted in abnormal cortical development correlated with placental infection levels. Furthermore, male offspring exhibited abnormal social interaction, whereas females exhibited elevated anxiety.

CONCLUSIONPlacental infection by Listeria induced sex-specific abnormalities in neurodevelopment of the fetus. Prenatal infection also affected the behavior of the offspring in a sex-specific manner.

ImpactO_LIPlacental infection with Listeria monocytogenes induces sexually dichotomous gene expression patterns in the fetal brain.
C_LIO_LIAbnormal cortical lamination is correlated with placental infection levels.
C_LIO_LIPlacental infection results in autism related behavior in male offspring and heightened anxiety level in female offspring.
C_LI

sentences: Parallel genetic changes underlie integrated craniofacial traits in an adaptive radiation of trophic specialist pupfishes Many factors such as divergence time, shared standing genetic variation, frequency of introgression, and mutation rates can influence the likelihood of whether populations adapt to similar environments via parallel or non-parallel genetic changes. However, the frequency of parallel vs non-parallel genetic changes resulting in parallel phenotypic evolution is still unknown. In this study, we used a QTL mapping approach to investigate the genetic basis of highly divergent craniofacial traits between scale- and snail-eating trophic specialist species across similar hypersaline lake environments in an adaptive radiation of pupfishes endemic to San Salvador Island, Bahamas. We raised F2 intercrosses of scale- and snail-eaters from two different lake populations of sympatric specialists, estimated linkage maps, scanned for significant QTL for 30 skeletal and craniofacial traits, and compared the location of QTL between lakes to quantify parallel and non-parallel genetic changes. We found strong support for parallel genetic changes in both lakes for five traits in which we detected a significant QTL in at least one lake. However, many of these shared QTL affected different, but highly correlated craniofacial traits in each lake, suggesting that pleiotropy and trait integration should not be neglected when estimating rates of parallel evolution. We further observed a 23-52% increase in adaptive introgression within shared QTL, suggesting that introgression may be important for parallel evolution. Overall, our results suggest that the same genomic regions contribute to parallel integrated craniofacial phenotypes across lakes. We also highlight the need for more expansive searches for shared QTL when testing for parallel evolution.

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Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment revealed by Cryo-EM and hydrogen/deuterium exchange mass spectrometry Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible.

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Prolonged β-Adrenergic Stimulation Disperses Ryanodine Receptor Clusters in Cardiomyocytes: Implications for Heart Failure Ryanodine Receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that "dispersion" of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged {beta}-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion, and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hour triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of CaMKII reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of {beta}-adrenergic stimulation over the longer term, and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.

Significance statementThe heartbeat is triggered by the release of Ca2+ from Ryanodine Receptors (RyRs) within cardiomyocytes. Recent data indicate RyR arrangement is highly malleable. However, mechanisms controlling RyR reorganisation and the subsequent impact on Ca2+ homeostasis remain unclear. Here, we show that prolonged {beta}-adrenergic stimulation causes RyR clusters to disperse, drastically altering the frequency and kinetics of Ca2+ release events called "Ca2+ sparks" in a process that is dependent on CaMKII and PKA. In healthy cells, these compensatory effects protect against arrhythmogenic Ca2+ over-activity. However, during heart failure, RyR hyper-phosphorylation and dispersion impairs Ca2+ release and cardiac performance. Thus, RyR localization and function are intimately linked via channel phosphorylation which, while finely tuned in health, underlies impaired cardiac function during pathology.

sentences: Almost nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an olfactory chemoattractant for the grass puffer Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this unknown function TTX analog as an olfactory chemoattractant.

sentences: Rational Designed Hybrid Peptides Show up to a 6-fold Increase in Antimicrobial Activity and Demonstrate Different Ultrastructural Changes as the Parental Peptides measured by BioSAXS Antimicrobial peptides (AMPs) are a promising class of compounds being developed against multi-drug resistant bacteria. Hybridization has been reported to increase antimicrobial activity. Here, two proline-rich peptides (consP1: VRKPPYLPRPRPRPL-CONH2 and Bac5-v291: RWRRPIRRRPIRPPFWR-CONH2) were combined with two arginine-isoleucine-rich peptides (optP1: KIILRIRWR-CONH2 and optP7: KRRVRWIIW-CONH2). Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit the bacterial ribosome, shown also for Bac5-v291, whereas it is hypothesized a "dirty drug" model for the arginine-isoleucine-rich peptides. That hypothesis was underpinned by transmission electron microscopy and biological small-angle X-ray scattering (BioSAXS). The hybrid peptides showed a stronger antimicrobial activity compared to the proline-rich peptides, except when compared to Bac5-v291 against E. coli. The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold, however, it was not a general increase but was dependent on the combination of peptides and bacteria. BioSAXS experiments revealed that proline-rich peptides and arginine-isoleucine-rich peptides induce very different ultrastructural changes in E. coli, whereas a hybrid peptide (hyP7B5GK) shows changes, different to both parental peptides and the untreated control. These different ultrastructural changes indicated that the mode of action of the parental peptides is different from each other as well as from the hybrid peptide hyP7B5GK. All peptides showed very low haemolytic activity, some of them showed a 100-fold or larger therapeutic window, demonstrating the potential for further drug development.

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24-nt phasiRNAs move from tapetal to meiotic cells in maize anthers O_LIIn maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known.
C_LIO_LIUsing laser capture microdissection, we analyzed tapetal cells, meiocytes, and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products.
C_LIO_LIBy integrating RNA and small RNA profiling plus single-molecule and small RNA FISH (smFISH or sRNA-FISH) spatial detection, we demonstrate that the tapetum is the primary site of 24-PHAS precursor and Dcl5 transcripts and the resulting 24-nt phasiRNAs. Interestingly, 24-nt phasiRNAs accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum.
C_LIO_LIOur data support the conclusion that 24-nt phasiRNAs are mobile from tapetum to meiocytes and to other somatic cells. We discuss possible roles for 24-nt phasiRNAs in anther cell types.
C_LI

sentences: The Influence of Phosphorus and Transplanting Date on Onion (Allium Cepa L.) Quality Parameters and yield of bulb Under Irrigation at Adami Tulu Jedo Kombolcha Woreda, East showa zone, Oromia Region. A field experiment was showed to decide the influence of phosphorus level and transplanting date on quality parameters of onion at Adami Tulu Jedo Kombolcha Woreda, during 2017/18 season. The treatments consisted of four phosphorus levels (0, 20, 40 and 60 kg P ha-1) and three transplanting date (42, 49 and 56 days). The experiment was laid out in randomized complete block design with four replications. The result showed that phosphorus level and transplanting date significantly influenced bulb dry matter content, marketable bulb yield, medium size bulb yield. Among these parameters, marketable bulb yield was also significantly influenced by the interaction of phosphorus level and transplanting date. On the other hand, small size bulb yield, large size bulb yield, over size bulb yield and under size bulb yield were only influenced by the effect of phosphorus levels. In this study, fertilized 60 kg P ha-1 with transplanting at 56 days of transplanting date recorded the highest marketable bulb yield, but no significant difference was showed with that obtained at 40 kg P ha-1 with the same transplanting date. Treatment combinations of no P (control) and transplanting date at 42 days produced the lowest amounts of marketable bulb yield. The economic analysis revealed that the highest net benefit with low cost of production was obtained in response to the application of 40 kg P ha-1 and the transplanting age of 56 days. The marginal rate of return for this treatment was 11983% which is found to be economically feasible for producing bulb in the districts.

sentences: Proteochemometric method for pIC50 prediction of Flaviviridae Viruses remain an area of concern despite constant development of antiviral drugs and therapies. One of the contributors among others is the flaviviridae family of viruses. Like other spaces, antiviral peptides (AVP) are gaining importance for studying flaviviridae family. Along with antiviral properties of peptides, information about bioactivity takes it even closer to accurate predictions of peptide capabilities. Experimental identification of bioactivity of each potential peptide is an expensive and time consuming task. Computational methods like Proteochemometric modelling (PCM) are promising for prediction of bioactivity based on peptide and target sequence. The additional edge PCM methods bring in is the aspect of considering both peptide and target properties instead of only looking at peptide properties. In this study, we propose prediction of pIC50 for AVP against flaviviridae family target proteins. The target proteins were manually curated from literature. Here we utilize the PCM descriptors as peptide descriptors, target descriptors and cross term descriptors. We observe taking peptide and target information improves the results qualitatively and gives better pIC50 predictions. The R2 and MAPE values are 0.85 and 8.44 % respectively

sentences: Structured Modulations of Ongoing Variability by Task and Development In carefully designed experiments, cognitive scientists interpret the mean event-related potentials (ERP) in terms of cognitive operations. However, the huge signal variability from one trial to the next, questions the representability of such mean events. We explored here whether this variability is an unwanted noise, or an informative part of the neural response. We took advantage of the rapid changes in the visual system during human infancy and analyzed the variability of visual responses to central and lateralized faces in 2-to 6-month-old infants and adults using high-density electroencephalography (EEG). We observed that neural trajectories of individual trials always remain very far from ERP components, only moderately bending their direction with a substantial temporal jitter across trials. However, single trial trajectories displayed characteristic patterns of acceleration and deceleration when approaching ERP components, as if they were under the active influence of steering forces causing transient attraction and stabilization. These dynamic events could only partly be accounted for by induced microstate transitions or phase reset phenomena. Furthermore, these structured modulations of response variability, both between and within trials, had a rich sequential organization, which, in infants, was modulated by the task difficulty. Our approaches to characterize Event Related Variability (ERV) expand and reinterpret classic ERP analyses, making them compliant with pervasive neural variability and providing a more faithful description of neural events following stimulus presentation.

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The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Ongoing work has identified several transcriptional regulators of nitrate signaling, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN 7 (NLP7), but additional regulators likely remain to be identified. Here, we characterized Arabidopsis NLP2 as a master upstream transcriptional regulator of early nitrate responses that interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, a DNA binding motif, and some target genes with NLP7. Additional genetic, genomic and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multi-tiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production.

One-sentence summaryNLP2 and NLP7 orchestrate plant responses to nitrate supply and control nitrate- dependent regulation of carbon and energy metabolism.

sentences: ATP biphasically modulates LLPS of TDP-43 PLD by specifically binding arginine residues Mysteriously neurons maintain ATP concentrations of [~]3 mM but whether ATP modulates TDP-43 LLPS remains completely unexplored. Here we characterized the effect of ATP on LLPS of TDP-43 PLD and seven mutants by DIC and NMR. The results revealed: 1) ATP induces and subsequently dissolves LLPS of TDP-43 PLD by specifically binding Arg saturated at 1:100. 2) ATP modifies the conformation-specific electrostatic property beyond just imposing screening effect. 3) Reversibility of LLPS of TDP-43 PLD and further exaggeration into aggregation appear to be controlled by a delicate network composed of both attractive and inhibitory interactions. Results together establish that ATP might be a universal but specific regulator for most, if not all, R-containing intrinsically-disordered regions by altering physicochemical properties, conformations, dynamics, LLPS and aggregation. Under physiological conditions, TDP-43 is highly bound with ATP and thus inhibited for LLPS, highlighting a central role of ATP in cell physiology, pathology and aging.

sentences: Analyzing Biomarker Discovery: Estimating the Reproducibility of Biomarker Sets Many researchers try to understand a biological condition by identifying biomarkers. This is typically done using univariate hypothesis testing over a labeled dataset, declaring a feature to be a biomarker if there is a significant statistical difference between its values for the subjects with different outcomes. However, such sets of proposed biomarkers are often not reproducible - subsequent studies often fail to identify the same sets. Indeed, there is often a very small overlap between the biomarkers proposed in pairs of related studies that explore the same phenotypes over the same distribution of subjects. This paper first defines the Reproducibility Score for a labeled dataset as a measure (taking values between 0 and 1) of the reproducibility of the results produced by an arbitrary fixed biomarker discovery process for a given distribution of subjects. We then provide ways to reliably estimate this score by defining algorithms that produce an over-bound and an under-bound for this score for a given dataset and biomarker discovery process, for the case of univariate hypothesis testing on dichotomous groups. We confirm that these approximations are meaningful by providing empirical results on a large number of datasets and show that these predictions match known reproducibility results. We have also created a publicly available website, hosted at https://biomarker.shinyapps.io/BiomarkerReprod/, that produces these Reproducibility Score approximations for any given dataset (with continuous or discrete features and binary class labels).

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ENHANCED GRAVITROPISM 2 encodes a STERILE ALPHA MOTIVE containing protein that controls root growth angle in barley and wheat The root growth angle defines how roots grow towards the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience and as a consequence yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin independent higher responsiveness to gravity compared to wild type plants. We cloned the EGT2 gene by a combination of bulked segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. Subcellular localization experiments revealed that EGT2 localizes to the nucleus and cytoplasm. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA-seq, we observed that seven expansin genes were transcriptionally downregulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.

Significance StatementTo date the potential of utilizing root traits in plant breeding remains largely untapped. In this study we cloned and characterized the ENHANCED GRAVITROPISM2 (EGT2) gene of barley that encodes a STERILE ALPHA MOTIF domain containing protein. We demonstrated that EGT2 is a key gene of root growth angle regulation in response to gravity which is conserved in barley and wheat and could be a promising target for crop improvement in cereals.

sentences: Conditions under which distributions of edge length ratios on phylogenetic trees can be used to order evolutionary events Two recent high profile studies have attempted to use edge (branch) length ratios from large sets of phylogenetic trees to determine the relative ages of genes of different origins in the evolution of eukaryotic cells. This approach can be straightforwardly justified if substitution rates are constant over the tree for a given protein. However, such strict molecular clock assumptions are not expected to hold on the billion-year timescale. Here we propose an alternative set of conditions under which comparisons of edge length distributions from multiple sets of phylogenies of proteins with different origins can be validly used to discern the order of their origins. We also point out scenarios where these conditions are not expected to hold and caution is warranted.

sentences: Effect of SARS-CoV-2 spike mutations on animal ACE2 usage and in vitro neutralization sensitivity The emergence of SARS-CoV-2 variants poses greater challenges to the control of COVID-19 pandemic. Here, we parallelly investigated three important characteristics of seven SARS-CoV-2 variants, including two mink-associated variants, the B.1.617.1 variant, and the four WHO-designated variants of concerns (B.1.1.7, B.1.351, P.1, and B.1.617.2). We first investigated the ability of these variants to bind and use animal ACE2 orthologs as entry receptor. We found that, in contrast to a prototype variant, the B.1.1.7, B.1.351, and P.1 variants had significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins, suggesting increased susceptibility of these species to these SARS-CoV-2 variants. We then evaluated in vitro neutralization sensitivity of these variants to four monoclonal antibodies in clinical use. We observed that all the variants were partially or completely resistant against at least one of the four tested antibodies, with B.1.351 and P.1 showing significant resistance to three of them. As ACE2-Ig is a broad-spectrum anti-SARS-CoV-2 drug candidate, we then evaluated in vitro neutralization sensitivity of these variants to eight ACE2-Ig constructs previously described in three different studies. All the SARS-CoV-2 variants were efficiently neutralized by these ACE2-Ig constructs. Interestingly, compared to the prototype variant, most tested variants including the variants of concern B.1.1.7, B.1.351, P.1, and B.1.617.2 showed significantly increased (up to [~]15-fold) neutralization sensitivity to ACE2-Ig constructs that are not heavily mutated in the spike-binding interface of the soluble ACE2 domain, suggesting that SARS-CoV-2 evolves toward better utilizing ACE2, and that ACE2-Ig is an attractive drug candidate for coping with SARS-CoV-2 mutations.

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Stimulation of the Dorsal Root Ganglion using an Injectrode ObjectiveThe goal of this work was to compare afferent fiber recruitment by dorsal root ganglion (DRG) stimulation using an injectable polymer electrode (Injectrode(R)) and a more traditional cylindrical metal electrode.

ApproachWe exposed the L6 and L7 DRG in four cats via a partial laminectomy or burr hole. We stimulated the DRG using an Injectrode or a stainless steel electrode using biphasic pulses at three different pulse widths (80, 150, 300 s) and pulse amplitudes spanning the range used for clinical DRG stimulation. We recorded antidromic evoked compound action potentials (ECAPs) in the sciatic, tibial, and common peroneal nerves using nerve cuffs. We calculated the conduction velocity of the ECAPs and determined the charge-thresholds and recruitment rates for ECAPs from A, A{beta}, and A{delta} fibers. We also performed electrochemical impedance spectroscopy measurements for both electrode types.

Main ResultsThe ECAP thresholds for the Injectrode did not differ from the stainless steel electrode across all primary afferents (A, A{beta}, A{delta}) and pulse widths; charge-thresholds increased with wider pulse widths. Thresholds for generating ECAPs from A{beta} fibers were 100.0 {+/-} 32.3 nC using the stainless steel electrode, and 90.9 {+/-} 42.9 nC using the Injectrode. The ECAP thresholds from the Injectrode were consistent over several hours of stimulation. The rate of recruitment was similar between the Injectrodes and stainless steel electrode and decreased with wider pulse widths.

SignificanceThe Injectrode can effectively excite primary afferents when used for DRG stimulation within the range of parameters used for clinical DRG stimulation. The Injectrode can be implanted through minimally invasive techniques while achieving similar neural activation to conventional electrodes, making it an excellent candidate for future DRG stimulation and neuroprosthetic applications.

sentences: Complete chloroplast genomes and phylogeny in three Euterpe palms (E. edulis, E. oleraceae and E. precatoria) from different Brazilian biomes The Brazilian palm fruits and hearts-of-palm of Euterpe edulis, E. oleracea and E. precatoria are an important source for agro-industrial production, but overexploitation requires conservation strategies to maintain genetic diversity. Chloroplast genomes have conserved sequences, which are useful to explore evolutionary questions. Besides the plastid DNA, genome skimming allows the identification of other genomic resources, such as single nucleotide polymorphisms (SNPs), providing information about the genetic diversity of species. We sequenced the chloroplast genome and identified gene content in the three Euterpe species. We performed comparative analyses, described the polymorphisms among the chloroplast genome sequences (repeats, indels and SNPs) and performed a phylogenomic inference based on 55 palm species chloroplast genomes. Finally, using the remaining data from genome skimming, the nuclear and mitochondrial reads, we identified SNPs and estimated the genetic diversity among these Euterpe species. The Euterpe chloroplast genomes varied from 159,232 to 159,275 bp and presented a conserved quadripartite structure with high synteny with other palms. In a pairwise comparison, we found a greater number of insertions/deletions (indels = 93 and 103) and SNPs (284 and 254) between E. edulis/E. oleracea and E. edulis/E. precatoria when compared to E. oleracea/E. precatoria (58 indels and 114 SNPs). Also, the phylogeny indicated a closer relationship between E. oleracea/E. precatoria. The nuclear and mitochondrial genome analyses identified 1,077 SNPs and high divergence among species (FST = 0.77), especially between E. edulis and E. precatoria (FST = 0.86). These results showed that, despite the few structural differences among the chloroplast genomes of these Euterpe palms, a differentiation between E. edulis and the other Euterpe species can be identified by point mutations. This study not only brings new knowledge about the evolution of Euterpe chloroplast genomes, but also these new resources open the way for future phylogenomic inferences and comparative analyses within Arecaceae.

sentences: A Novel Cytoskeletal Action of Xylosides Proteoglycan glycosaminoglycan (GAG) chains are attached to a serine residue in the protein through a linkage series of sugars, the first of which is xylose. Xylosides are chemicals which compete with the xylose at the enzyme xylosyl transferase to prevent the attachment of GAG chains to proteins. These compounds have been employed at concentrations in the millimolar range as tools to study the role of GAG chains in proteoglycan function. In the course of our studies with xylosides, we conducted a dose-response curve for xyloside actions on neural cells. To our surprise, we found that concentrations of xylosides in the nanomolar to micromolar range had major effects on cell morphology. These effects are due to changes in cytoskeletal dynamics. Concentrations of xylosides which were effective in altering morphology did not alter GAG chain synthesis rates, nor did they produce any changes in gene expression as determined by RNAseq of treated cells. These observations support a novel action of xylosides on neuronal cells.

sentences: Amino acid availability determines plant immune homeostasis in the rhizosphere microbiome Microbes possess conserved microbe-associated molecular patterns (MAMPs) such as flagellin that are recognized by plant receptors to induce immunity. Despite containing the same MAMPs as pathogens, commensals thrive in the plant rhizosphere microbiome indicating they must suppress or evade host immunity. The beneficial bacteria Pseudomonas capeferrum WCS358 can suppress Arabidopsis root immunity via acidification by secreting gluconic acid. While gluconic acid is sufficient to suppress immunity, we found that it is not necessary in a second beneficial strain Pseudomonas simiae WCS417, which produces more gluconic acid than WCS358. To uncover mechanisms that contribute to the suppression of Arabidopsis immunity, we performed a forward genetic screen in EMS-mutagenized P. simiae WCS417 using a flagellin-inducible CYP71A12pro:GUS reporter as an Arabidopsis immune readout. We identified a mutant that cannot suppress flagellin-elicited CYP71A12pro:GUS expression or acidify the rhizosphere. Next generation sequencing revealed a mutation in the catabolic site of an ornithine carbamoyltransferase argF, which is required for arginine biosynthesis. The mutant could be complemented by expression of argF from a plasmid, and a {Delta}argF mutant could not suppress immunity. Fungal pathogens can use alkalization through production of ammonia and glutamate, the arginine biosynthetic precursors, to promote their own growth and virulence. Therefore, we hypothesized that the biosynthesis of specific amino acids may be necessary to reduce levels of ammonia and glutamate to prevent rhizosphere alkalization and bacterial overgrowth. Genetically blocking arginine, glutamine, or proline biosynthesis, or by adding corresponding exogenous amino acids, resulted in rhizosphere alkalization. Interestingly, exogenous amino acids caused bacterial overgrowth in a gluconic acid-deficient mutants. Our findings show that bacterial amino acid biosynthesis contributes to acidification by preventing accumulation of glutamate precursors and the resulting alkalization. Collectively this work shows that by regulating nutrient availability, plants have the potential to regulate their immune homeostasis in the rhizosphere microbiome.

sentences: Inbreeding and learning affect fitness and colonization of new host plants, a behavioral innovation in the spider mite Tetranychus urticae Habitat fragmentation increases the isolation of natural populations resulting in reduced genetic variability and increased species extinction risk. Behavioral innovation through learning, i.e., the expression of a new learned behavior in a novel context, can help animals colonize new suitable and increasingly fragmented habitats. It has remained unclear, however, how reduced genetic variability affects learning for colonizing more or less suitable habitats. Here, we show that inbreeding in a subsocial invertebrate, the spider mite Tetranychus urticae, reduces novel host plant colonization and reproductive fitness. When provided with the possibility to learn from previous experience with a host plant species, outbred mites showed aversive learning ability, but inbred mites did not adapt their behavior. We further found a putative general cost of learning in both inbred and outbred mites. Our results reveal that inbreeding affects the learning component of behavioral innovation for host plant colonization.

sentences: Effects of gestational age at birth on perinatal structural brain development in healthy term-born babies Multiple studies have demonstrated less favourable childhood outcomes in infants born in early term (37-38 weeks gestation) compared to those born at full term (40-41 weeks gestation). While this could be due to higher perinatal morbidity, gestational age at birth may also have a direct effect on the brain and subsequent neurodevelopment in term-born babies. Here we characterise structural brain correlates of gestational age at birth in healthy term-born neonates and their relationship to later neurodevelopmental outcome. We used T2 and diffusion weighted Magnetic Resonance Images acquired in the neonatal period from a cohort (n=454) of healthy babies born at term age (>37 weeks gestation) and scanned between 1 and 41 days after birth. Images were analysed using tensor based morphometry (TBM) and tract based spatial statistics (TBSS). Neurodevelopment was subsequently assessed at age 18 months using the Bayley-III Scales of Infant and Toddler Development, and the effects of gestational age at birth and related neuroimaging findings on outcome were analysed with linear regression. Infants born earlier had areas of higher relative ventricular volume, and lower relative brain volume in the basal ganglia, cerebellum and brainstem. Earlier birth was also associated with lower fractional anisotropy, higher mean, axial and radial diffusivity in major white matter tracts. Gestational age at birth was positively associated with all Bayley-III subscales at age 18 months. Linear regression models predicting outcome from gestational age at birth were significantly improved by adding neuroimaging features associated with gestational age at birth. This work adds to the growing body of evidence of the impact of early term birth and highlights the importance of considering the effect of gestational age at birth in future neuroimaging studies including term-born babies.

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Massive genome decay and expansion of insertion sequences drive the evolution of a novel host-restricted bacterial pathogen BackgroundThe emergence of new pathogens is a major threat to public and veterinary health. Changes in bacterial habitat such as those associated with a switch in host or disease tropism are often accompanied by genetic adaptation. Staphylococcus aureus is a multi-host bacterial species comprising strains with distinct tropisms for human and livestock species. A microaerophilic subspecies, Staphylococcus aureus subsp. anaerobius, is responsible for outbreaks of Morels disease, a lymphadenitis in small ruminants. However, the evolutionary history of S. aureus subsp. anaerobius and its relatedness to S. aureus are unknown.

ResultsEvolutionary genomic analyses of clinical S. aureus subsp. anaerobius isolates revealed a highly conserved clone that descended from a S. aureus progenitor about 1000 years ago before differentiating into distinct lineages representing African and European isolates. S. aureus subsp. anaerobius has undergone limited clonal expansion, with a restricted population size, and an evolutionary rate 10-fold slower than S. aureus. The transition to its current restricted ecological niche involved acquisition of a pathogenicity island encoding a ruminant host-specific effector of abscess formation, several large chromosomal re-arrangements, and the accumulation of at least 205 pseudogenes resulting in a highly fastidious metabolism. Importantly, expansion of [~]87 insertion sequences (IS) located largely in intergenic regions provided distinct mechanisms for the control of expression of flanking genes, representing a novel concept of the IS regulon.

ConclusionsOur findings reveal the remarkable evolutionary trajectory of a host-restricted bacterial pathogen that resulted from extensive remodelling of the S. aureus genome through an array of parallel mechanisms.

sentences: Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uterus of birds, such as Japanese quail, Coturnix japonica, resulting in brown-speckled eggshells. The molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis and performed genetic linkage mapping to identify the causative genomic region of the white eggshell phenotype. We showed that 11 genes, including the 5-aminolevulinic acid synthase 1 (ALAS1) and ferroxidase hephaestin-like 1 (HEPHL1) genes, were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, and their expression was downregulated in these cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail.

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Burkholderia from fungus gardens of fungus-growing ants produce antifungals that inhibit the specialized parasite Escovopsis Within animal associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus Leucoagaricus spp. converting plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus Escovopsis. Here, we examine the potential role of Burkholderia spp. that occur within ant fungus gardens in inhibiting Escovopsis. We isolated members of the bacterial genera Burkholderia spp. and Paraburkholderia spp. from 50% of the 52 colonies sampled, indicating that the family Burkholderiaceae are common fungus garden inhabitants of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one Escovopsis strain with a zone of inhibition greater than 1 cm. Genomic assessment of Burkholderiaceae isolates indicated that isolates with strong inhibition all belonged to the genus Burkholderia and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed these compounds as responsible for antifungal activity that inhibit Escovopsis but, at low concentrations, not Leucoagaricus spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of the fungus-farming ant colony.

IMPORTANCEMany organisms partner with microbes to defend themselves against parasites and pathogens. Fungus-growing ants must protect Leucoagaricus spp., the fungal mutualist that provides sustenance for the ants, from a specialized fungal parasite, Escovopsis spp. The ants take multiple approaches, including weeding their fungus gardens to remove Escovopsis spores, as well as harboring Pseudonocardia that produce antifungals that inhibit Escovopsis. In addition, a genus of bacteria commonly found in fungus gardens, Burkholderia spp., is known to produce secondary metabolites that inhibit Escovopsis spp. In this study, we isolated Burkholderia spp. from fungus-growing ants, assessed the isolates ability to inhibit Escovopsis spp., and identified two compounds responsible for inhibition. Our findings suggest that Burkholderia spp. are often found in fungus gardens, adding another possible mechanism within the fungus-growing ant system to suppress the growth of the specialized parasite Escovopsis.

sentences: Self-consistent solution to the Flory-Huggins model and applications in protein liquid-liquid phase separation A self-consistent analytical solution for binodal concentrations of the 2-component Flory-Huggins model is derived. We show that this form extends the validity of the Ginzburg-Landau expansion away from the critical point to cover the whole phase space, from which an exponential scaling law of the dilute phase binodal concentration emerges. This approach allows us to fit experimental protein liquid-liquid phase separation data and extract parameters including the effective chain length and the solute-solvent interaction energy. We demonstrate explicitly the power of this approach by quantifying the interaction energy contributions of individual amino acids. This analytical framework can serve as a new way to decode the protein sequence grammar for liquid-liquid phase separation.

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The host factor ANP32A is required for influenza A virus vRNA and cRNA synthesis Influenza A viruses are negative-sense RNA viruses that rely on their own viral replication machinery to replicate and transcribe their segmented single-stranded RNA genome. The viral ribonucleoprotein complexes in which viral RNA is replicated consist of a nucleoprotein scaffold around which the RNA genome is bound, and a heterotrimeric RNA-dependent RNA polymerase that catalyzes viral replication. The RNA polymerase copies the viral RNA (vRNA) via a replicative intermediate, called the complementary RNA (cRNA), and subsequently uses this cRNA to make more vRNA copies. To ensure that new cRNA and vRNA molecules are associated with ribonucleoproteins in which they can be amplified, the active RNA polymerase recruits a second polymerase to encapsidate the cRNA or vRNA. Host factor ANP32A has been shown to be essential for viral replication and to facilitate the formation of a dimer between viral RNA polymerases and differences between mammalian and avian ANP32A proteins are sufficient to restrict viral replication. It has been proposed that ANP32A is only required for the synthesis of vRNA molecules from a cRNA, but not vice versa. However, this view does not match recent molecular evidence. Here we use minigenome assays, virus infections, and viral promoter mutations to demonstrate that ANP32A is essential for both vRNA and cRNA synthesis. Moreover, we show that ANP32 is not only needed for the actively replicating polymerase, but also for the polymerase that is encapsidating nascent viral RNA products. Overall, these results provide new insights into influenza A virus replication and host adaptation.

IMPORTANCEZoonotic avian influenza A viruses pose a constant threat to global health and they have the potential to cause highly pathogenic pandemic outbreaks. Species variations in host factor ANP32A play a key role in supporting the activity of avian influenza A virus RNA polymerases in mammalian hosts. Here we show that ANP32A acts at two stages in the influenza A virus replication cycle, supporting recent structural experiments and in line with its essential role. Understanding how ANP32A supports viral RNA polymerase activity and how it supports avian polymerase function in mammalian hosts is important for understanding influenza A virus replication and the development of antiviral strategies against influenza A viruses.

sentences: Fine-scale Population Structure of North American  Arabidopsis thaliana  Reveals Multiple Sources of Introduction from Across Eurasia Large-scale movement of organisms across their habitable range, or migration, is an important evolutionary process that can contribute to observed patterns of genetic diversity and our understanding of the adaptive spread of alleles. While human migrations have been studied in great detail with modern and ancient genomes, recent anthropogenic influence on reducing the biogeographical constraints on the migration of non-native species has presented opportunities in several study systems to ask the questions about how repeated introductions shape genetic diversity in the introduced range. We present here the most comprehensive view of population structure of North American Arabidopsis thaliana by studying a set of 500 (whole-genome sequenced) and over 2800 (RAD-seq genotyped) individuals in the context of global diversity represented by Afro-Eurasian genomes. We use haplotype-sharing, phylogenetic modeling and rare-allele sharing based methods to identify putative sources of introductions of extant N. American A. thaliana from the native range of Afro-Eurasia. We find evidence of admixture among the introduced lineages that has resulted in the increased haplotype diversity and reduced mutational load. Further, we also present signals of selection in the immune-system related genes that impart qualitative disease resistance to pathogens of bacterial and oomycete origins. Thus, multiple introductions to a non-native range can quickly increase adaptive potential of a colonizing species by increasing haplotypic diversity through admixture. The results presented here lay the foundation for further investigations into the functional significance of admixture.

sentences: Immunogenicity and efficacy of Heat Inactivated Whole-Cell Vaccine to increase murine survival after Extensively drug-resistant Acinetobacter baumannii infection. Due to the rapid emergence of extensively drug-resistant (XDR) strains worldwide there is a necessity for greater consideration for the role of preventive vaccines to combat these pathogens. The study reported the effectiveness of a heat-inactivated whole-cell vaccine (HI-WCV) against A. baumannii to produce protective immunity with evaluation of the bactericidal antibody responses in mice after intramuscular inoculation with different XDR strains of A. baumannii. Six XDR A. baumannii strains emulsified with complete freunds adjuvant (CFA) were used for intramuscular inoculation in the experimental group of mice (n=4) in three different phases on 14 days interval whereas placebo-controlled mice (n=4) received phosphate-buffered saline emulsified with CFA in same the schedule. Serum was collected from tail blood of each mouse on 10th day after each inoculation and by cardiac puncture on 14th day after lethal dose from the experimental group and after death of the placebo-controlled group. Mice inoculated with heat-inactivated whole-cell vaccine survived and showed a higher neutralizing (IgG) antibody response after 2nd (>7-fold titre) and 3rd inoculation (>12-fold titre) whereas all mice from placebo-controlled group did not survived. Sera from experimental group of mice collected (38th day) after 3rd inoculation resulted in higher serum bactericidal killing index (42.12) after three hours incubation with an XDR A. baumannii over sera collected after 1st and 2nd inoculation (50% cutoff value=14.5). These results suggest that intramuscular vaccination with heat-inactivated whole-cell vaccine stimulated IgG antibody responses that can increase murine survival after XDR A. baumannii infection.

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Effect of paclitaxel treatment on cellular mechanics and morphology of human oesophageal squamous cell carcinoma of South African origin in 2D and 3D environments During chemotherapy, structural and mechanical changes in malignant cells have been observed in several cancers, including leukaemia, pancreatic and prostate cancer. Such cellular changes may act as physical biomarkers for chemoresistance and cancer recurrence. This study aimed to determine how exposure to paclitaxel affects the intracellular stiffness of human oesophageal cancer of South African origin in vitro. A human oesophageal squamous cell carcinoma cell line WHCO1 was cultured on glass substrates (2D) and in collagen gels (3D) and exposed to paclitaxel for up to 48 hours. Cellular morphology and stiffness were assessed with confocal microscopy, visually aided morpho-phenotyping image recognition, and mitochondrial particle tracking microrheology at 24 and 48 hours. In the 2D environment, the intracellular stiffness was higher for the paclitaxel-treated than for untreated cells at 24 and 48 hours. In the 3D environment, the paclitaxel-treated cells were stiffer than the untreated cells at 24 hours, but no statistically significant differences in stiffness were observed at 48 hours. In 2D, paclitaxel-treated cells were significantly larger at 24 and 48 hours and more circular at 24 but not at 48 hours than the untreated controls. In 3D, there were no significant morphological differences between treated and untreated cells. The shape heterogeneity was lower for paclitaxel-treated than untreated cells in 3D and similar for the treated and untreated groups in 2D. Future studies with patient-derived primary cancer cells and prolonged drug exposure will help identify physical cellular biomarkers to detect chemoresistance onset and assess therapy effectiveness in oesophageal cancer patients.

InsightMechanical changes in cancer cells brought on by chemotherapeutic drug exposure offer possible physical biomarkers to detect chemoresistance and cancer recurrence. This first study on human oesophageal squamous cell carcinoma of South African origin indicated that in vitro paclitaxel treatment induced stiffening and enlarging of the malignant cells in two-dimensional environments at 24 and 48 hours. In physiologically more relevant three-dimensional collagen matrices, however, the paclitaxel treatment led to cellular stiffening at 24 hours but softening after that, without significant changes in cellular size at any time. The in vitro environments influence on experimental outcomes needs to be considered in further studies with prolonged drug exposure and patient-derived primary cancer cells.

sentences: Astrocyte glutamate transport is modulated by motor learning and regulates neuronal correlations and movement encoding by motor cortex neurons While motor cortex is crucial for learning precise and reliable movements, whether and how astrocytes contribute to its plasticity and function during motor learning is unknown. Here we report that primary motor cortex (M1) astrocytes in mice show in vivo plasticity during learning of a lever push task, as revealed by transcriptomic and functional modifications. In particular, we observe changes in expression of glutamate transporter genes and increased coincidence of intracellular calcium events. Astrocyte-specific manipulations of M1 are sufficient to alter motor learning and execution, and neuronal population coding, in the same task. Mice expressing decreased levels of the astrocyte glutamate transporter GLT1 show impaired and variable movement trajectories. Mice with increased astrocyte Gq signaling show decreased performance rates, delayed response times and impaired trajectories, along with abnormally high levels of GLT1. In both groups of mice, M1 neurons have altered inter-neuronal correlations and impaired population representations of task parameters, including response time and movement trajectories. Thus, astrocytes have a specific role in coordinating M1 neuronal activity during motor learning, and control learned movement execution and dexterity through mechanisms that importantly include fine regulation of glutamate transport.

sentences: Keeping time and rhythm by replaying a sensory-motor engram Imagine practicing a piece of music, or a speech, solely within the mind, without any sensory input or motor output. Our ability to implement dynamic internal representations is key for successful behavior, yet how the brain achieves this is not fully understood1-4. Here we trained primates to perceive, and internally maintain, rhythms of different tempos and performed large-scale recordings of neuronal activity across multiple areas spanning the sensory-motor processing hierarchy. Results show that perceiving and maintaining rhythms engage multiple brain areas, including visual, parietal, premotor, prefrontal, and hippocampal regions. Each area displayed oscillatory activity that reflected the temporal and spatial characteristics of an internal metronome which flexibly encoded fast, medium, and slow tempos on a trial-by-trial basis. The presence of widespread metronome-related activity across the brain, in the absence of stimuli and overt actions, is consistent with the idea that time and rhythm are maintained by a mechanism that internally replays the stimuli and actions that define well-timed behavior.

sentences: Mechanisms for Communicating in a Marmoset "Cocktail Party" A fundamental challenge for audition is parsing the voice of a single speaker amid a cacophony of other voices known as the Cocktail Party Problem (CPP). Despite its prevalence, relatively little remains known about how our simian cousins solve the CPP for active, natural communication. Here we employed an innovative, multi-speaker paradigm comprising five computer-generated Virtual Monkeys (VM) whose respective vocal behavior could be systematically varied to construct marmoset cocktail parties and tested the impact of specific acoustic scene manipulations on vocal behavior. Results indicate that marmosets not only employ auditory mechanisms - including attention - for speaker stream segregation, but also selectively change their own vocal behavior in response to the dynamics of the acoustic scene to overcome the challenges of the CPP. These findings suggest notable parallels between human and nonhuman primate audition and highlight the active role that speakers play to optimize communicative efficacy in complex real-world acoustic scenes.

sentences: Can Deep Convolutional Neural Networks Learn Same-Different Relations? Same-different visual reasoning is a basic skill central to abstract combinatorial thought. This fact has lead neural networks researchers to test same-different classification on deep convolutional neural networks (DCNNs), which has resulted in a controversy regarding whether this skill is within the capacity of these models. However, most tests of same-different classification rely on testing on images that come from the same pixel-level distribution as the testing images, yielding the results inconclusive. In this study we tested relational same-different reasoning DCNNs. In a series of simulations we show that DCNNs are capable of visual same-different classification, but only when the test images are similar to the training images at the pixel-level. In contrast, even when there are only subtle differences between the testing and training images, the performance of DCNNs could drop to chance levels. This is true even when DCNNs training regime included a wide distribution of images or when they were trained in a multi-task setup in which training included an additional relational task with test images from the same pixel-level distribution.

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Male genital lobe morphology affects the chance to copulate in Drosophila pachea IntroductionMale genitalia are thought to ensure transfer of sperm through direct physical contact with female during copulation. However, little attention has been given to their pre-copulatory role with respect to sexual selection and sexual conflict. Males of the fruitfly Drosophila pachea have a pair of asymmetric external genital lobes, which are primary sexual structures and stabilize the copulatory complex of female and male genitalia. We wondered if genital lobes in D. pachea may have a role before or at the onset of copulation, before genitalia contacts are made.

ResultsWe tested this hypothesis with a D. pachea stock where males have variable lobe lengths. In 92 mate competition trials with a single female and two males, females preferentially engaged into a first copulation with males that had a longer left lobe and that displayed increased courtship vigor. In 53 additional trials with both males having partially amputated left lobes of different lengths, we observed a weaker and non-significant effect of left lobe length on copulation success. Courtship durations significantly increased with female age and when two males courted the female simultaneously, compared to trials with only one courting male. In addition, lobe length did not affect sperm transfer once copulation was established.

ConclusionLeft lobe length affects the chance of a male to engage into copulation. The morphology of this primary sexual trait may affect reproductive success by mediating courtship signals or by facilitating the establishment of genital contacts at the onset of copulation.

sentences: The impact of multiple species invasion on soil and plant communities increases with invader diversity Despite increasing evidence indicating that invasive species are harming ecological systems and processes, impacts of multiple invasions, and the linkages between these events and changes in vegetation and soil are inadequately documented and remain poorly understood. Addressing multiple invasions would help to highlight high priority invaders and would aid in designing more effective control strategies, contributing to environmental restoration and sustainability. In this work, we tested the impact of three concurring invasive plant species, Amorpha fruticosa, Fraxinus pennsylvanica and Acer negundo, on soil conditions and native plant diversity. The research was conducted in riparian ecosystem and included the following treatments: (1) co-occurrence of the three invasive plant species, (2) occurrence of a single invasive species, and (3) control, i.e., absence of invasive species. Our findings revealed that the impact of invasive plants on soil properties and native plant diversity is magnified by their co-occurrence. Soil in mixed plots (those populated with all three invaders) contained much higher levels of nitrifying bacteria (NB), organic matter (Om), nitrogen (N), and carbon (C) as well as lower carbon to nitrogen ratio (C:N) levels, compared to single species invaded plots and control plots. Mixed plots were also characterized by reduced native plant diversity compared to single species invaded and control plots. Differences in soil conditions and native plant diversity revealed the interactive potential of invasive plants in depleting biodiversity, and thus in affecting ecological and biogeochemical processes. Our results highlight the need to study the impact of multispecies invasion and suggest that sites in riparian areas affected by co-occurring invaders, should be prioritized for ecosystem restoration.

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Generation of excitatory and inhibitory neurons from common progenitors via Notch signaling in the cerebellum Brain neurons arise from relatively few progenitors capable of giving rise to an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis in both the cortex and the cerebellum is that excitatory neurons and inhibitory neurons derive from separate, spatially segregated, progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both excitatory and inhibitory lineages exist and how such a fate decision may be regulated is unknown. Using cerebellar development as a model, we discover that individual embryonic cerebellar progenitors give rise to both inhibitory and excitatory lineages. We find that gradations of Notch activity levels determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate. Daughters with intermediate levels of Notch activity become fate restricted to generate inhibitory neurons, while daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors.

Graphical summary

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sentences: Imaging of the pial arterial vasculature of the human brain in vivo using high-resolution 7T time-of-flight angiography The pial arterial vasculature of the human brain is the only blood supply to the neocortex, but quantitative data on the morphology and topology of these mesoscopic arteries (diameter 50- 300 {micro}m) remains scarce. Because it is commonly assumed that blood flow velocities in these vessels are prohibitively slow, non-invasive time-of-flight MRI angiography (TOF-MRA)--which is well-suited to high 3D imaging resolutions--has not been applied to imaging the pial arteries. Here, we provide a theoretical framework that outlines how TOF-MRA can visualize small pial arteries in vivo, by employing extremely small voxels at the size of individual vessels. We then provide evidence for this theory by imaging the pial arteries at 140-{micro}m isotropic resolution using a 7T MRI scanner and prospective motion correction, and show that pial arteries one voxel-width in diameter can be detected. We conclude that imaging pial arteries is not limited by slow blood flow, but instead by achievable image resolution. This study represents the first targeted, comprehensive account of imaging pial arteries in vivo in the human brain. This ultra-high-resolution angiography will enable the characterization of pial vascular anatomy across the brain to investigate patterns of blood supply and relationships between vascular and functional architecture.

sentences: Population genomics analysis with RAD, reprised: STACKS 2 Restriction enzymes have been one of the primary tools in the population genetics toolkit for fifty years, being coupled with each new generation of technology to provide a more detailed view into the genetics of natural populations. Restriction site-Associated DNA protocols, which joined enzymes with short-read sequencing technology have democratized the field of population genomics, providing a means to assay the underlying alleles in scores of populations. More than ten years on, the technique has been widely applied across the tree of life and served as the basis for many different analysis techniques. Here, we provide a detailed protocol to conduct a RAD analysis from experimental design to de novo analysis - including parameter optimization - as well as reference-based analysis, all in SO_SCPLOWTACKSC_SCPLOW version 2, which is designed to work with paired-end reads to assemble RAD loci up to 1000 nucleotides in length. The protocol focuses on major points of friction in the molecular approaches and downstream analysis, with special attention given to validating experimental analyses. Finally, the protocol provides several points of departure for further analysis.

sentences: Sequential recognition of discrete export signals in flagellar subunits during bacterial Type III secretion Type III Secretion Systems (T3SS) deliver subunits from the bacterial cytosol to nascent cell surface flagella. Early flagellar subunits that form the rod and hook substructures are unchaperoned and contain their own export signals. A gate recognition motif (GRM) docks them at the FlhBc component of the FlhAB-FliPQR export gate, but the gate must then be opened and subunits must be unfolded to pass through the flagellar channel. This induced us to seek further signals on the subunits. Here, we identify a second signal at the extreme N-terminus of flagellar rod and hook subunits and determine that key to the signal is its hydrophobicity. We show that the two export signal elements are recognised separately and sequentially, as the N-terminal signal is recognised by the flagellar export machinery only after subunits have docked at FlhBC via the GRM. The position of the N-terminal hydrophobic signal in the subunit sequence relative to the GRM appeared to be important, as a FlgD deletion variant (FlgDshort), in which the distance between the N-terminal signal and the GRM was shortened,  stalled at the export machinery and was not exported. The attenuation of motility caused by FlgDshort was suppressed by mutations that destabilised the closed conformation of the FlhAB-FliPQR export gate, suggesting that the hydrophobic N-terminal signal might trigger opening of the flagellar export gate.

sentences: Temperature-dependent genetics of thermotolerance between yeast species Many traits of industrial and basic biological interest arose long ago, and manifest now as fixed differences between a focal species and its reproductively isolated relatives. In these systems, extant individuals can hold clues to the mechanisms by which phenotypes evolved in their ancestors. We harnessed yeast thermotolerance as a test case for such molecular-genetic inferences. In viability experiments, we showed that extant Saccharomyces cerevisiae survived at temperatures where cultures of its sister species S. paradoxus died out. Then, focusing on loci that contribute to this difference, we found that the genetic mechanisms of high-temperature growth changed with temperature. We also uncovered an enrichment of low-frequency variants at thermotolerance loci in S. cerevisiae population sequences, suggestive of a history of non-neutral selective forces acting at these genes. We interpret our results in light of a model of gradual acquisition of thermotolerance in the S. cerevisiae lineage by positive selection along a temperature cline. We propose that in an ancestral S. cerevisiae population, alleles conferring defects at a given temperature would have been resolved by adaptive mutations, expanding the range and setting the stage for further temperature advances. Together, our results and interpretation underscore the power of genetic approaches to explore how an ancient trait came to be.

sentences: Drosophila melanogaster  females prioritise dietary sterols for producing high quality eggs Limiting calories or specific nutrients without malnutrition, otherwise known as dietary restriction (DR), has been shown to extend lifespan across a broad range of taxa. Our recent findings in Drosophila melanogaster show that supplementing flies on macronutrient-rich diets with additional cholesterol can extend lifespan to the same extent as DR. Macronutrient-rich diets drive high levels of egg production and in doing so deplete the mothers of somatic sterols that are essential for survival. Thus, DR may be beneficial for lifespan because it reduces egg production which in turn reduces the mothers demand for sterols. If this is true, mothers must be prioritising their available sterols, whether from the diet or from their own bodies, to sustain high quality egg production. To test this, we measured the quality of eggs laid by mothers fed either cholesterol-sufficient or cholesterol-depleted diets. We found that even when the mothers diet was completely devoid of cholesterol, high quality egg production persisted. Furthermore, we show that sterol-supplemented flies with long lives continue to lay high quality eggs that give rise to healthy offspring. Thus, in our assays, long life does not require a fecundity cost.

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Microphaser - a small-scale phasing approach for improved personalized neopeptidome creation MotivationHaplotype phasing approaches have been shown to improve accuracy of the search space of neoantigen prediction by determining if adjacent variants are located on the same chromosomal copy. However, the aneuploid nature of cancer cells as well as the admixture of different clones in tumor bulk sequencing data are challenging the current diploid based phasing algorithms. We present microphaser, a small-scale phasing approach to improve haplotyping variants in cancer samples. Microphaser aims to create a more accurate neopeptidome for downstream neoantigen prediction.

ResultsMicrophaser achieves large concordance with state-of-the-art phasing-aware neoantigen prediction pipeline neoepiscope, with differences in edge cases and an improved filtering step.

AvailabilityMicrophaser is written in the Rust programming language. It is made available via Github (https://github.com/koesterlab/microphaser)and Bioconda. The corresponding prediction pipeline (https://github.com/snakemake-workflows/dna-seq-neoantigen-prediction) has been written within the Snakemake workflow management system and can be deployed as part of the snakemake-workflows project.

sentences: Lamin A/C phosphorylation at serine 22 is a conserved heat shock response to regulate nuclear adaptation during stress The heat shock (HS) response is crucial for cell survival in harmful environments. Nuclear lamin A/C, encoded by LMNA gene, has been shown to contribute towards altered gene expression during heat shock, but the underlying mechanisms are poorly understood. Here we show that reversible lamin A/C phosphorylation at Ser22 upon HS is an evolutionary conserved stress response that is triggered in concert with HSF1 activation in human and mouse cells and can also be observed in D. melanogaster in vivo. Consequently, the phosphorylation increase facilitated nucleoplasmic localization of lamin A/C and nuclear rounding in response to HS. The importance of lamin phosphorylation equilibria in HS was confirmed by lamin A/C knock-out (KO) cells that showed deformed nuclei after HS and were rescued by ectopic expression of wild-type, but not by a phosphomimetic (S22D) lamin A mutant. Furthermore, HS triggered release of lamina-associated protein 2 (Lap2) from its association with lamin A/C and concurrently its downregulation, a response that was perturbed in lamin A/C KO cells and in LMNA mutant patient fibroblasts. The abrogated Lap2 response resulted in impaired cell cycle arrest under HS and compromised survival at the recovery. Taken together, our results suggest that the altered phosphorylation stoichiometry of lamin A/C provides an evolutionary conserved mechanism to regulate lamin structure and serve nuclear adaptation and cell survival during HS.

sentences: Is Palmer's elm leaf goldenrod real? The Angiosperms353 kit provides within-species signal in Solidago ulmifolia s.l. The genus Solidago represents a taxonomically challenging group due to its sheer number of species, putative hybridization, polyploidy, and shallow genetic divergence among species. Here we use a dataset obtained exclusively from herbarium specimens to evaluate the status of Solidago ulmifolia var. palmeri, a morphologically subtle taxon potentially confined to Alabama, Arkansas, Mississippi, and Missouri. A multivariate analysis of both discrete and continuous morphological data revealed no clear distinction between S. ulmifolia var. palmeri and Solidago ulmifolia var. ulmifolia. Solidago ulmifolia var. palmeris status was also assessed with a phylogenomic and SNP clustering analysis of data generated with the "Angiosperms353" probe kit. Neither analysis supported Solidago ulmifolia var. palmeri as a distinct taxon, and we suggest that this name should be discarded. The status of Solidago delicatula (formerly known as Solidago ulmifolia var. microphylla) was also assessed. Both morphological and phylogenic analyses supported the species status of S. delicatula and we suggest maintaining this species at its current rank. These results highlight the utility of the Angiosperms353 probe kit, both with herbarium tissue and at lower taxonomic levels. Indeed, this is the first study to utilize this kit to identify genetic groups within a species.

sentences: A network model of glymphatic flow under different experimentally-motivated parametric scenarios Rapidly growing evidence demonstrates that flow of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) - annular channels surrounding vasculature in the brain - is a critically-important component of neurophysiology. CSF inflow contributes during physiological conditions to clearance of metabolic waste and in pathological situations to edema formation. However, brain-wide imaging methods cannot resolve PVSs, and high-resolution methods cannot access deep tissue or be applied to human subjects, so theoretical models provide essential insight. We model this CSF pathway as a network of hydraulic resistances, built from published parameters. A few parameters have very wide uncertainties, so we focus on the estimated limits of their feasible ranges by analyzing different parametric scenarios. We identify low-resistance PVSs and high-resistance parenchyma (brain tissue) as the scenario that best explains experimental observations. Our results point to the most important parameters that should be measured in future experiments. Extensions of our modeling may help predict stroke severity or lead to neurological disease treatments and drug delivery methods.

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mEos4b photoconversion efficiency depends on laser illumination conditions used in PALM Green-to-red photoconvertible fluorescent proteins (PCFPs) are widely employed as markers in photoactivated localization microscopy (PALM). However, their highly complex photophysical behavior complicates their usage. The fact that only a limited fraction of a PCFP ensemble can form the photoconverted state upon near-UV light illumination, termed photoconversion efficiency (PCE), lowers the achievable spatial resolution in PALM and creates undercounting errors in quantitative counting applications. Here, we show that the PCE of mEos4b is not a fixed property of this PCFP, but strongly depends on illumination conditions. Attempts to reduce long-lived blinking in red mEos4b by application of 488 nm light leads to a reduction of the PCE. Furthermore, the PCE of mEos4b strongly depends on the applied 405-nm power density. A refined photophysical model of mEos4b accounts for the observed effects, involving nonlinear green-state photobleaching upon violet light illumination favored by photon absorption by a putative radical dark state.

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sentences: The ALT pathway generates telomere fusions that can be detected in the blood of cancer patients Telomere fusions (TFs) can trigger the accumulation of diverse genomic rearrangements and the acquisition of oncogenic alterations leading to malignant transformation and resistance to chemotherapy. Despite their relevance in tumour evolution, our understanding of the patterns and consequences of TFs in human cancer remains limited. Here, we have characterized the rates and spectrum of somatic TFs across >30 cancer types using whole-genome sequencing data. TFs are pervasive in human tumours with rates varying markedly across and within cancer types. In addition to end-to-end fusions, we find novel patterns of TFs that we mechanistically link to the activity of the alternative lengthening of telomeres (ALT) pathway. We show that TFs can be detected in the blood of cancer patients, which enables cancer detection with high specificity and sensitivity even for early-stage tumours and cancer types for which early detection remains a high unmet clinical need, such as pancreatic cancer and brain tumours. Overall, we report a novel genomic footprint that enables characterization of the telomere maintenance mechanism of tumours and liquid biopsy analysis, which has implications for early detection, prognosis, and treatment selection.

sentences: Antagonistic modules, SIB1 and LSD1, regulate photosynthesis-associated nuclear genes via GOLDEN2-LIKE transcription factors in Arabidopsis GOLDEN2-LIKE (GLK) transcription factors drive the expression of photosynthesis-associated nuclear genes (PhANGs), indispensable for chloroplast biogenesis. We previously demonstrated that the salicylic acid (SA)-induced SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), a transcription coregulator and positive regulator of cell death, interacts with GLK1 and GLK2 to reinforce their activities. The SIB1-GLK interaction raises the level of light-harvesting antenna proteins in photosystem II, aggravating photoinhibition and singlet oxygen (1O2) burst. 1O2 then contributes to SA-induced cell death via EXECUTER 1 (EX1, 1O2 sensor protein)-mediated retrograde signaling upon reaching a critical level. We now reveal that LESION-SIMULATING DISEASE 1 (LSD1), a transcription coregulator and negative regulator of SA-primed cell death, interacts with GLK1/2 to repress their activities. Consistently, the overexpression of LSD1 represses GLK target genes including PhANGs, whereas the loss of LSD1 increases their expression. Remarkably, LSD1 overexpression inhibits chloroplast biogenesis, resembling the characteristic glk1glk2 double mutant phenotype. The subsequent chromatin immunoprecipitation analysis coupled with quantitative PCR further reveals that LSD1 inhibits the DNA-binding activity of GLK1 towards its target promoters. The SA-induced nuclear-targeted SIB1 appears to counteractively interact with GLK1/2, leading to the activation of EX1-mediated 1O2 signaling. Taken together, we provide a working model that SIB1 and LSD1, mutually exclusive SA-signaling components, antagonistically regulate GLK1/2 to fine-tune the expression of PhANGs, thereby modulating 1O2 homeostasis and related stress responses.

sentences: A novel small molecule that induces cytotoxicity in lung cancer cells inhibits disulfide reductases GSR and TXNRD1 High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD.

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An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex (SKR-1/2-CUL-1-F-box (SCF)), targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin-derivative/mutant AtTIR1 pair (C. elegans AID version 2 (C.e.AIDv2)) that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID targets, addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expands the utility of the AID system and broadens the number of proteins that can be effectively targeted with it.

ARITCLE SUMMARYImplementation of the auxin induced degradation (AID) system has increased the power if the C. elegans model through its ability to rapidly degrade target proteins in the presence of the plant hormone auxin (IAA). The current C.e.AID system is limited in that a substantial level of target degradation occurs in the absence of ligand and full levels of target protein degradation require high levels of auxin inducer. In this manuscript, we modify the AID system to solve these problems.

sentences: Universal Spectrum Explorer: A standalone (web-)application for cross-resource spectrum comparison Here we present the Universal Spectrum Explorer (USE), a web-based tool based on IPSA for cross-resource (peptide) spectrum visualization and comparison (https://www.proteomicsdb.org/use/). Mass spectra under investigation can either be provided manually by the user (table format), or automatically retrieved from online repositories supporting access to spectral data via the universal spectrum identifier (USI), or requested from other resources and services implementing a newly designed REST interface. As a proof of principle, we implemented such an interface in ProteomicsDB thereby allowing the retrieval of spectra acquired within the ProteomeTools project or real-time prediction of tandem mass spectra from the deep learning framework Prosit. Annotated mirror spectrum plots can be exported from the USE as editable scalable high quality vector graphics. The USE was designed and implemented with minimal external dependencies allowing local usage and integration into other websites (https://github.com/kusterlab/universal_spectrum_explorer).

sentences: Multiple lines of evidence indicate survival of the Ivory-billed Woodpecker in Louisiana The history of decline of the Ivory-billed Woodpecker is long, complex, and controversial. The last widely accepted sighting of this species in continental North America was 1944. Reports of Ivory-billed Woodpeckers have continued, yet in 2021 the U.S. Fish and Wildlife Service proposed declaring the species extinct. We draw on 10 years of search effort, and provide trail camera photos and drone videos suggesting the consistent presence of Ivory-billed Woodpeckers at our study site. Data indicate repeated re-use of foraging sites and core habitat. We offer insights into behaviors of the Ivory-billed Woodpecker that contribute to difficulty in finding this species. We discuss results with regard to the value of accumulated evidence, and what repeated observations may indicate for continued survival of this iconic species.

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scDVF: Data-driven Single-cell Transcriptomic Deep Velocity Field Learning with Neural Ordinary Differential Equations Recent advances in single-cell RNA sequencing technology have provided unprecedented opportunities to simultaneously measure the gene expression profile and transcriptional velocity of individual cells, enabling us to sample gene regulatory network dynamics along developmental trajectories. However, traditional methods have faced challenges in modeling gene expression dynamics within individual cells due to sparse, non-linear (e.g., obligate heterodimer transcription factors), and high-dimensional measurements. Here, we present DeepVelo, a neural-network-based ordinary differential equation model that can learn non-linear, high-dimensional single-cell transcriptome dynamics and describe continuous gene expression changes within individual cells across time. We applied DeepVelo to multiple published datasets from different technical platforms and demonstrated its utility to 1) formulate transcriptome dynamics on different timescales, 2) measure the instability of cell states, and 3) identify developmental driver genes upstream of a signaling cascade. Benchmarking against state-of-the-art methods shows that DeepVelo can improve velocity field representation accuracy by at least 50% in out-of-sample cells. Further, perturbation studies revealed that single-cell dynamical systems may exhibit properties similar to those of chaotic systems. In summary, DeepVelo allows for the data-driven discovery of differential equations that delineate single-cell transcriptome dynamics.

TeaserEmbedding neural networks into ordinary differential equations to model gene expression changes within single cells across time.

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Scanning the RBD-ACE2 molecular interactions in Omicron variant The emergence of new SARS-CoV-2 variants poses a threat to the human population where it is difficult to assess the severity of a particular variant of the virus. Spike protein and specifically its receptor binding domain (RBD) which makes direct interaction with the ACE2 receptor of the human has shown prominent amino acid substitutions in most of the Variants of Concern. Here, by using all-atom molecular dynamics simulations we compare the interaction of Wild-type RBD/ACE2 receptor complex with that of the latest Omicron variant of the virus. We observed a very interesting diversification of the charge, dynamics and energetics of the protein complex formed upon mutations. These results would help us in understanding the molecular basis of binding of the Omicron variant with that of SARS-CoV-2 Wild-type.

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sentences: HuR -dependent SOD2 protein synthesis is an early adaptation of ovarian cancer cells in response to anchorage independence During metastasis, cancer cells must adapt to survive a loss of anchorage and evade anoikis. An important pro-survival adaptation is the ability of metastatic tumor cells to increase their antioxidant capacity and restore cellular redox balance. Although much is known about the transcriptional regulation of antioxidant enzymes in response to stress, how cells rapidly adapt to alter antioxidant enzyme levels is less well understood. Using ovarian cancer cells as a model, we demonstrate that an increase in protein expression of the mitochondrial superoxide dismutase SOD2 is a very early event initiated in response to cellular detachment. SOD2 protein synthesis is rapidly induced within 0.5-2 hours of matrix detachment, and polyribosome profiling demonstrates an increase in the number of ribosomes bound to SOD2 mRNA, indicating an increase in SOD2 translation in response to anchorage-independence. Mechanistically, we find that anchorage-independence specifically induces cytosolic accumulation of the RNA binding protein HuR/ELAVL1 and leads to increased HuR binding to SOD2 mRNA. Using HuR siRNA-mediated knock-down, we show that the presence of HuR is necessary for the increase in SOD2 mRNA association with the heavy polyribosome fraction and SOD2 protein synthesis observed in anchorage-independence. Cellular detachment activates the stress-response protein kinase p38 MAPK, which is necessary for HuR-SOD2 mRNA binding and optimal increases in SOD2 protein expression. These findings illustrate a novel post-transcriptional regulatory mechanisms of SOD2, enabling cells to rapidly increase their mitochondrial antioxidant capacity as an acute response to anchorage-independence.

sentences: Individual stochasticity in the life history strategies of animals and plants The life histories of organisms are expressed as rates of development, reproduction, and survival. However, individuals may experience differential outcomes for the same set of rates. Such individual stochasticity generates variance around familiar mean measures of life history traits, such as life expectancy and the reproductive number R0. By writing life cycles as Markov chains, we calculate variance and other indices of variability for longevity, lifetime reproductive output (LRO), age at offspring production, and age at maturity for 83 animal and 332 plant populations from the CO_SCPLOWOMADREC_SCPLOW and CO_SCPLOWOMPADREC_SCPLOW matrix databases. We find that the magnitude within and variability between populations in variance indices in LRO, especially, are surprisingly high. We furthermore use principal components analysis to assess how the inclusion of variance indices affects life history constraints. We find that these indices, to a similar or greater degree than the mean, explain the variation in life history strategies among plants and animals.

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Yclon: Ultrafast clustering of B cell clones from high-throughput immunoglobulin repertoire sequencing data MotivationNext-generation sequencing technologies revolutionize our understanding of immunoglobulin (Ig) profile in different immune states. Clonotyping (grouping Ig sequences into B cell clones) allows the investigation of the diversity of repertoires and how they change upon antigen exposure. Despite its importance, there is no consensus on the best method for clonotyping, and the methods developed for that are computationally intractable for large sequencing datasets. This is the case of Change-O, which compares sequences through hamming distance and uses hierarchical clustering for this task.

ResultsWe propose implementing an approach to identify B cell clones from Ig repertoire data, named Yclon, that makes an alignment-free comparison of the sequences, focusing on reducing the runtime and computer memory usage. Overall, we find that a hierarchical clustering approach grouped Ig sequences into B cell clones similarly to Change-O. However, we observed that Yclon was around 35 times faster and even was able to process larger than 2 million sequences Ig repertoire, which is a critical part of repertoire studies and enables understanding antibody repertoire structure and affinity maturation.

Availability and implementationYClon is written in Python3 and is available on GitHub (https://github.com/jao321/YClon.git)

Contactjoaodgervasio@gmail.com, liza@icb.ufmg.br

sentences: Transcriptional signatures of clonally derived Toxoplasma tachyzoites reveal novel insights into the expression of a family of surface proteins. Toxoplasma gondii has numerous, large, paralogous gene families that are likely critical for supporting its unparalleled host range: nearly any nucleated cell in almost any warm-blooded animal. The SRS (SAG1-related sequence) gene family encodes over 100 proteins, the most abundant of which are thought to be involved in parasite attachment and, based on their stage-specific expression, evading the host immune response. For most SRS proteins, however, little is understood about their function and expression profile. Single-parasite RNA-sequencing previously demonstrated that across an entire population of lab-grown tachyzoites, transcripts for over 70 SRS genes were detected in at least one parasite. In any one parasite, however, transcripts for an average of only 7 SRS genes were detected, two of which, SAG1 and SAG2A, were extremely abundant and detected in virtually all. These data do not address whether this pattern of sporadic SRS gene expression is consistently inherited among the progeny of a given parasite or arises independently of lineage. We hypothesized that if SRS expression signatures are stably inherited by progeny, subclones isolated from a cloned parent would be more alike in their expression signatures than they are to the offspring of another clone. In this report, we compare transcriptomes of clonally derived parasites to determine the degree to which expression of the SRS family is stably inherited in individual parasites. Our data indicate that in RH tachyzoites, SRS genes are variably expressed even between parasite samples subcloned from the same parent within approximately 10 parasite divisions (72 hours). This suggests that the pattern of sporadically expressed SRS genes is highly variable and not driven by inheritance mechanisms, at least under our conditions.

sentences: Soluble guanylate cyclase stimulation mitigates skeletal and cardiac muscle dysfunction in a mdx model of Duchenne muscular dystrophy The impairment of neuronal nitric oxide synthase (nNOS) signaling contributes to disease pathology in the muscle wasting disorder Duchenne muscular dystrophy (DMD). nNOS signal propagation occurs through nitric oxide sensitive soluble guanylate cyclase (sGC), a critical source of cyclic guanosine monophosphate (cGMP) in muscle. Although both nNOS and sGC activity are impaired in DMD patients, little is known about sGC as a therapeutic target. In this study, we tested the hypothesis that stimulating sGC activity with the allosteric agonist BAY41-8543 mitigates striated muscle pathology in the mdx4cv mouse model of DMD. In contrast to DMD patients, mdx mice exhibited greater basal sGC activity than wild type controls with preservation of cGMP levels due partly to upregulation of sGC in some muscles. Stimulating sGC activity in mdx mice with BAY41-8543 substantially reduced skeletal muscle damage, macrophage densities and inflammation and significantly increased resistance to contraction-induced fatigue. BAY41-8543 also enhanced in vivo diaphragm function while reducing breathing irregularities suggesting improved respiratory function. BAY41-8543 attenuated cardiac hypertrophic remodeling, fibrosis and diastolic dysfunction including left atrium enlargement in aged mdx mice. Overall, sGC stimulation significantly mitigated skeletal and cardio-respiratory dysfunction in mdx4cv mice. Importantly, this study provides compelling pre-clinical evidence supporting sGC as a novel target in DMD and the repurposing of FDA-approved sGC stimulators, such as riociguat and veraciguat, as a novel therapeutic approach for DMD.

sentences: Measuring expression heterogeneity of single-cell cytoskeletal protein complexes Multimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in 100s of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing LatA detects a subpopulation (~11%) exhibiting downregulated F-actin, but upregulated microtubule and intermediate filament protein complexes. Thus, some cells upregulate other cytoskeletal complexes to counteract the stress of LatA treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock dysregulates F- and G-actin correlation. The assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

sentences: Emergent Color Categorization in a Neural Network trained for Object Recognition Color is a prime example of categorical perception, yet it is unclear why and how color categories emerge. While prelinguistic infants and animals treat color categorically, several recent modeling endeavors have successfully utilized communicative concepts to predict color categories. Rather than modeling categories directly, we investigate the potential emergence of color categories as a result of acquiring visual skills. Specifically, whether color is represented categorically in a convolutional neural network (CNN) trained to recognize objects in natural images. Systematically training new output layers to the CNN for a color classification task, we find clear borders between new (non-training) colors that are largely invariant to the training colors. Using an evolutionary algorithm that relies on the principle of categorical perception we verify these border locations. These results provide strong evidence that color categorization emerges as a function of basic visual skills and provide a new basis for uncovering how they emerge.

sentences: GENESPACE: syntenic pan-genome annotations for eukaryotes The development of multiple high-quality reference genome sequences in many taxonomic groups has yielded a high-resolution view of the patterns and processes of molecular evolution. Nonetheless, leveraging information across multiple reference haplotypes remains a significant challenge in nearly all eukaryotic systems. These challenges range from studying the evolution of chromosome structure, to finding candidate genes for quantitative trait loci, to testing hypotheses about speciation and adaptation in nature. Here, we address these challenges through the concept of a pan-genome annotation, where conserved gene order is used to restrict gene families and define the expected physical position of all genes that share a common ancestor among multiple genome annotations. By leveraging pan-genome annotations and exploring the underlying syntenic relationships among genomes, we dissect presence-absence and structural variation at four levels of biological organization: among three tetraploid cotton species, across 300 million years of vertebrate sex chromosome evolution, across the diversity of the Poaceae (grass) plant family, and among 26 maize cultivars. The methods to build and visualize syntenic pan-genome annotations in the GENESPACE R package offer a significant addition to existing gene family and synteny programs, especially in polyploid, outbred and other complex genomes.

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Micrococcal nuclease sequencing of porcine sperm suggests a nucleosomal involvement on semen quality and early embryo development BackgroundThe mammalian mature spermatozoon has a unique chromatin structure in which the vast majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The chromatin structure of sperm remains unresolved in most livestock species, including the pig. However, its resolution could provide further light into the identification of the genomic regions related to sperm biology and embryo development and it could also help identifying molecular markers for sperm quality and fertility traits. Here, for the first time in swine, we performed Micrococcal Nuclease coupled with high throughput sequencing on pig sperm and characterized the mono-nucleosomal (MN) and sub-nucleosomal (SN) chromatin fractions.

ResultsWe identified 25,293 and 4,239 peaks in the mono-nucleosomal and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison of nucleosome-associated DNAs in sperm revealed positional conservation of the nucleosome retention between human and pig. Gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and identification of putative transcription factor binding motifs within the mono-nucleosomal peaks showed enrichment for sperm function and embryo development related processes. We found motif enrichment for the transcription factor Znf263, which in humans was suggested to be a key regulator of the genes with paternal preferential expression during early embryo development. Moreover, we found enriched co-occupancy between the RNAs present in pig sperm and the RNA related to sperm quality, and the mono-nucleosomal peaks. We also found preferential co-location between GWAS hits for semen quality in swine and the mono-nucleosomal sites identified in this study.

ConclusionsThese results suggest a clear relationship between nucleosome positioning in sperm and sperm and embryo development.

sentences: Predicting the fitness costs of complex mutations The fitness cost of complex pleiotropic mutations is generally difficult to assess. On the one hand, it is necessary to identify which molecular properties are directly altered by the mutation. On the other, this alteration modifies the activity of many genetic targets with uncertain consequences. Here, we examine the possibility of addressing these challenges by identifying unique predictors of these costs. To this aim, we consider mutations in the RNA polymerase (RNAP) in Escherichia coli as a model of complex mutations. Changes in RNAP modify the global program of transcriptional regulation, with many consequences. Among others is the difficulty to decouple the direct effect of the mutation from the response of the whole system to such mutation. A problem that we solve quantitatively with data of a set of constitutive genes, which better read the global program. We provide a statistical framework that incorporates the direct effects and other molecular variables linked to this program as predictors, which leads to the identification that some genes are more suitable predictors than others. Therefore, we not only identified which molecular properties best anticipate costs in fitness, but we also present the paradoxical result that, despite pleiotropy, specific genes serve as better predictors. These results have connotations for the understanding of the architecture of robustness in biological systems.

sentences: GPI-anchored FGF directs cytoneme-mediated bidirectional signaling to self-regulate tissue-specific dispersion How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and activates target-specific bidirectional FGF-FGFR signaling through cytonemes. FGF-source and recipient ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through CAM-like FGF-FGFR binding. Contact-mediated FGF-FGFR binding induces bidirectional signaling, which, in turn, promotes ASP and source cells to polarize cytonemes toward each other and reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source cell membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion and signaling.

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Identification of novel therapeutic targets for polyglutamine toxicity disorders that target mitochondrial fragmentation Huntingtons disease (HD) is one of at least nine polyglutamine toxicity disorders caused by a trinucleotide CAG repeat expansion, all of which lead to age-onset neurodegeneration. Mitochondrial dynamics and function are disrupted in HD and other polyglutamine toxicity disorders. While multiple studies have found beneficial effects from decreasing mitochondrial fragmentation in HD models by disrupting the mitochondrial fission protein DRP1, disrupting DRP1 can also have detrimental consequences in wild-type animals and HD models. In this work, we examine the effect of decreasing mitochondrial fragmentation in a neuronal C. elegans model of polyglutamine toxicity called Neur-67Q. We find that Neur-67Q worms have deficits in mitochondrial morphology in GABAergic neurons and decreased mitochondrial function. Disruption of drp-1 eliminates differences in mitochondrial morphology and rescues deficits in both movement and longevity in Neur-67Q worms. In testing twenty-four RNA interference (RNAi) clones that decrease mitochondrial fragmentation, we identified eleven clones that increase movement and extend lifespan in Neur-67Q worms. Overall, we show that decreasing mitochondrial fragmentation may be an effective approach to treat polyglutamine toxicity disorders and identify multiple novel genetic targets that circumvent the potential negative side effects of disrupting the primary mitochondrial fission gene drp-1.

Significance StatementPolyglutamine toxicity disorders are caused by a trinucleotide CAG repeat expansion that leads to neurodegeneration. Both mitochondrial dynamics and function are disrupted in these disorders. In this work we use a simple genetic model organism, the worm C. elegans, to define the role of mitochondrial morphology in polyglutamine toxicity disorders. We show that CAG repeat expansion is sufficient to disrupt mitochondrial morphology and that genetic strategies that decrease mitochondrial fragmentation are beneficial in a neuronal model of polyglutamine toxicity. This work identifies multiple novel genes that are protective in worm models of polyglutamine toxicity, which may serve as potential therapeutic targets for Huntingtons disease and other polyglutamine toxicity disorders.

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Single-cell gene regulatory network inference atscale: The Inferelator 3.0 MotivationGene regulatory networks define regulatory relationships between transcription factors and target genes within a biological system, and reconstructing them is essential for understanding cellular growth and function. Methods for inferring and reconstructing networks from genomics data have evolved rapidly over the last decade in response to advances in sequencing technology and machine learning. The scale of data collection has increased dramatically; the largest genome-wide gene expression datasets have grown from thousands of measurements to millions of single cells, and new technologies are on the horizon to increase to tens of millions of cells and above.

ResultsIn this work, we present the Inferelator 3.0, which has been significantly updated to integrate data from distinct cell types to learn context-specific regulatory networks and aggregate them into a shared regulatory network, while retaining the functionality of the previous versions. The Inferelator is able to integrate the largest single-cell datasets and learn cell-type specific gene regulatory networks. Compared to other network inference methods, the Inferelator learns new and informative Saccharomyces cerevisiae networks from single-cell gene expression data, measured by recovery of a known gold standard. We demonstrate its scaling capabilities by learning networks for multiple distinct neuronal and glial cell types in the developing Mus musculus brain at E18 from a large (1.3 million) single-cell gene expression dataset with paired single-cell chromatin accessibility data.

AvailabilityThe inferelator software is available on GitHub (https://github.com/flatironinstitute/inferelator) under the MIT license and has been released as python packages with associated documentation (https://inferelator.readthedocs.io/).

sentences: Late maturation of executive control promotes conceptual development Control processes are critical for the generation of task-appropriate behaviour across cognitive domains, yet children have a long developmental period with reduced executive control. Traditionally, this is viewed as a negative but necessary consequence of the time taken to learn control processes and develop the prefrontal cortex. Here, we exploit a recent computational model of controlled semantic cognition to formally test an alternative (yet perhaps complementary) view that a developmental period without control promotes conceptual knowledge acquisition. Our simulations show that maturational delay and anatomical connectivity conspire to promote conceptual learning. Learning conceptual structure necessitates a connection from control to more peripheral regions rather than the deep multimodal hub. Delayed control speeds conceptual learning without compromising conceptual representations, particularly when control connects to intermediate layers. These results are counterintuitive as delayed control onset can produce overall faster mastery of controlled behaviours. To assess whether delayed semantic control is also observed over development, we conducted a meta-analysis of the classic triadic matching task where participants decide which of two options best matches a third. Matching can be based on taxonomic or thematic relations, and when these conflict, participants must use task context to determine which relation to choose--that is, they must exert semantic control. Children aged 2 have the representations to identify both relations when distractors are unrelated, but when relations conflict, they cannot use context to guide their choices. Context-sensitivity develops later than conceptual structure and shows large increases around 6, a critical time for executive function across domains.

sentences: Abiotic factors modulate interspecies competition mediated by the type VI secretion system effectors in Vibrio cholerae Vibrio cholerae, the etiological pathogen of cholera, relies on its type VI secretion system (T6SS) as an effective weapon to survive in highly competitive communities. The anti-bacterial and anti-eukaryotic functions of T6SS depend on its secreted effectors that target multiple essential cellular processes. However, the mechanisms that account for effector diversity and different effectiveness during interspecies competition remain elusive. Here, we report that environmental cations and temperature play a key role in dictating effector-mediated competition of Vibrio cholerae. We found that V. cholerae could employ its cell-wall-targeting effector TseH to outcompete the otherwise resistant Escherichia coli and the V. cholerae immunity deletion mutant{Delta} tsiH when Ca2+ and Mg2+ were supplemented. The E. coli {Delta}phoQ mutant was more sensitive to TseH-mediated killing during competition, suggesting the metal-sensing PhoPQ two-component system is protective to E. coli from TseH activity. Using transcriptome analysis, we found multiple stress response systems, including acid stress response, oxidative stress response, and osmotic stress response, were activated in E. coli expressing TseH in comparison with E. coli expressing the inactive mutant TseHH64A. The membrane-targeting lipase effector TseL also exhibited reduced killing against E. coli when divalent cations were removed. In addition, competition analysis of E. coli with V. cholerae single-effector active strains reveals a temperature-dependent susceptibility of E. coli to effectors, VasX, VgrG3, and TseL. These findings suggest that abiotic factors, that V. cholerae frequently encounters in natural habitats, play a crucial role in dictating the competitive fitness conferred by the type VI secretion system in complex multispecies communities.

sentences: Nascent peptide-induced translation discontinuation in eukaryotes impacts biased amino acid usage in proteomes Robust translation elongation of any given amino acid sequence is a prerequisite to shape proteomes. Nevertheless, nascent peptides could destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains stochastically can induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in the N-terminal regions could prematurely terminate translation, producing premature products as peptidyl-tRNA species. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we found many endogenous D/E-rich peptidyl-tRNAs in the N-terminal regions in cells lacking a peptidyl-tRNA hydrolase, indicating that the translation of the N-terminal D/E-rich sequences poses an inherent risk. Indeed, a bioinformatics analysis revealed that the N-terminal regions of ORFs avoid D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.

sentences: Semantic and Prosodic Threat Processing in Trait Anxiety. The present study attempts to identify how trait anxiety, measured as worry-level, affects the processing of threatening speech. Two experiments using dichotic listening tasks were implemented; where participants had to identify sentences that convey threat through three different information channels: prosody-only, semantic-only and both semantic and prosody (congruent threat). We expected different ear advantages (left or right) depending on task demands, information type, and worry level. We used a full Bayesian approach for statistical modelling and analysis. Results indicate that when participants made delayed responses (Experiment 1), reaction times barely increased as a function of worry level, but under time pressure (Experiment 2) worry level induced clear decreases in reaction times. We explain these results in terms of multistep models of anxiety and language, concluding that present results mainly indicate effects threat aversion-related over-attention to threat, and that we do not provide enough evidence for supporting the integration of anxiety and language multiphasic models.

sentences: Frequency-dependent hybridization contributes to habitat segregation in monkeyflowers Spatial segregation of closely related species is usually attributed to differences in stress tolerance and competitive ability. For both animals and plants, reproductive interactions between close relatives can impose a fitness cost that is more detrimental to the rarer species. Frequencydependent mating interactions may thus prevent the establishment of immigrants within heterospecific populations, maintaining spatial segregation of species. Despite strong spatial segregation in natural populations, two sympatric California monkeyflowers (Mimulus nudatus and M. guttatus) survive and reproduce in the others habitat when transplanted reciprocally. We hypothesized that a frequency-dependent mating disadvantage maintains spatial segregation of these monkeyflowers during natural immigration. To evaluate this hypothesis, we performed two field experiments. First, we experimentally added immigrants in varying numbers to sites dominated by heterospecifics. Second, we reciprocally transplanted arrays of varying resident and immigrant frequencies. Immigrant seed viability decreased with conspecific rarity for M. guttatus, but not M. nudatus. We observed immigrant minority disadvantage for both species, but driven by different factors- frequency-dependent hybridization for M. guttatus, and competition for resources and/or pollinators for M. nudatus. Overall, our results suggest a major role for reproductive interference in spatial segregation that should be evaluated along with stress tolerance and competitive ability.

sentences: Sniffing Out New Friends: Similarity in Body-Odor Predicts the Quality of Same-Sex None-Romantic Dyadic Interactions Most are familiar with the notion of socially "clicking" with someone, namely sensing an immediate bond that can lead to strong and often long-lasting friendships. The mechanisms underlying such rapid bonding remain unclear. Given that body-odor similarity is a critical cue for social interaction in non-human mammals, we tested the hypothesis that body-odor similarly contributes to bonding in same-sex non-romantic human dyads. We observed that objective ratings obtained with an electronic nose, and subjective ratings obtained from human smellers, converged to suggest that click-friends smell more similar to each other than random dyads. Remarkably, we then found that we could use the electronic nose to predict which strangers would later form better dyadic interactions. Thus, humans may literally sniff-out new friends based on similarities in body-odor.

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Firmicutes and Bacteroidetes explain mass gain variation in an obligate hibernator O_LIBody condition is an important life history challenge that directly impacts individual fitness and is particularly important for hibernating animals, whose maintenance of adequate body fat and mass is essential for survival.
C_LIO_LIIt is well documented that symbiotic microorganisms play a vital role in animal physiology and behaviour. Recent work demonstrates that gut microbes are associated with fat accumulation and obesity; Firmicutes is consistently associated with obesity while Bacteroidetes is associated with leanness both in humans and other animals.
C_LIO_LIThe focus of most microbiome studies has been on human health or involved lab reared animals used as a model system. However, these microbes likely are important for individual fitness in wild populations and provide potential mechanistic insights into the adaptability and survival of wildlife.
C_LIO_LIHere we test whether symbiotic microorganisms within the phyla of Firmicutes and Bacteroidetes are associated with summer mass gain in an exceptionally well-studied wild population of yellow-bellied marmots (Marmota flaviventer) by quantifying microbial abundance over five years of fecal samples (2015 - 2019) collected during their summer active season.
C_LIO_LIResults show that marmots with higher mass gain rates have a greater abundance of Firmicutes. In contrast, higher abundance of Bacteroidetes was associated with lower mass gain rates, but only for marmots living in harsher environments. Similar patterns were found at the family level where Ruminococcaceae, a member of Firmicutes, was associated with higher mass gain rates, and Muribaculaceae, a member of Bacteroidetes, was associated with lower mass gain rates, and similarly in harsher environments.
C_LIO_LIAlthough correlative, these results highlight the importance of symbiotic gut microbiota to mass gain in the wild, a trait associated with survival and fitness in many taxonomic groups.
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sentences: Dynamic task-belief is an integral part of decision-making Natural decisions involve two seemingly separable processes: inferring the relevant task (task-belief) and performing the believed-relevant task. The assumed separability has led to the traditional practice of studying task-switching and perceptual decision-making individually. Here, we used a novel paradigm to manipulate and measure macaque monkeys task-belief, and demonstrated inextricable neuronal links between flexible task-belief and perceptual decision-making. We showed that in animals, but not artificial networks that performed as well or better than the animals, stronger task-belief is associated with better perception. Correspondingly, recordings from neuronal populations in cortical areas 7a and V1 revealed that stronger task-belief is associated with better discriminability of the believed-relevant but not the believed-irrelevant feature. Perception also impacts belief updating: noise fluctuations in V1 help explain how task-belief is updated. Our results demonstrate that complex tasks and multi-area recordings can reveal fundamentally new principles of how biology affects behavior in health and disease.

sentences: Regulation of sexually dimorphic abdominal courtship behaviors in Drosophila by the Tlx/tailless-like nuclear receptor, Dissatisfaction Sexually dimorphic courtship behaviors in Drosophila melanogaster develop from the activity of the sexual differentiation genes, doublesex (dsx) and fruitless (fru), functioning with other regulatory factors that have received little attention. The dissatisfaction gene (dsf) encodes an orphan nuclear receptor homologous to vertebrate Tlx and Drosophila tailless that is critical for the development of several aspects of female- and male-specific sexual behaviors. Here, we report the pattern of dsf expression in the central nervous system and show that the activity of sexually dimorphic abdominal interneurons that co-express dsf and dsx is necessary and sufficient for vaginal plate opening in virgin females, ovipositor extrusion in mated females, and abdominal curling in males during courtship. We find that dsf activity results in different neuroanatomical outcomes in females and males, promoting and suppressing, respectively, female development and function of these neurons depending upon the sexual state of dsx expression. We posit that dsf and dsx interact to specify sex differences in the neural circuitry for dimorphic abdominal behaviors.

sentences: ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1 There is considerable evidence that cross-talk between the Hedgehog pathway and MAPK signaling pathways occurs in several types of cancer, and contributes to the emergence of clinical resistance to Hedgehog pathway inhibitors. Here, we demonstrate that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. We show that ERK2 phosphorylates GLI1 on three evolutionarily-conserved target sites (S102, S116 and S130) located near the high-affinity binding site for the negative regulator SUFU; furthermore, these phosphorylation events cooperate to weaken the affinity of GLI1-SUFU binding by over 25 fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily-conserved SUFU-affinity-modulating region. In cultured mammalian cells, mutant GLI1 variants containing phosphomimetic substitutions of S102, S116 and S130 displayed an increased ability to drive transcription. We conclude that of multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.

sentences: Fatigue Metabolites of Large Yellow Croaker and Construction of Swimming Model A trend in large yellow croaker (Larimichthys crocea) aquaculture is to establish new production sites that are suitable for extreme weather conditions. However, continuous and strong currents can harm fish welfare. To determine the location of the net cage, it is necessary to assess the swimming ability of large yellow croaker. Currently, our research on large yellow croakers is focusing on behavior analysis. This article investigates the effect of swimming large yellow croakers on metabolites in the body by examining the preferred speed of the group and the endurance swimming ability of single-tailed fish. We evaluated the factors that influence the large yellow croakers swimming fatigue by quantifying the content of metabolites and constructed the endurance swimming model using those results. Various results showed large yellow croaker populations tend to grow in low-velocity environments, and this matches their traditional habitat. The samples were taken at different swimming times at a flow rate of 0.35 m/s. According to the results of the metabolite content determination, blood glucose levels is closely related to swimming ability in large yellow croakers. The content of liver glycogen, which regulates blood glucose concentration, decreases in a certain linear relationship. The endurance swimming model of large yellow croaker was constructed according to the changes of liver glycogen content. The goals of this article are to provide a deeper understanding of the physiological characteristics of large yellow croaker swimming, and to provide a reference for choosing fishing and cage sites for large yellow croaker.

sentences: METAMDB: METABOLIC ATOM MAPPING DATABASE AO_SCPLOWBSTRACTC_SCPLOWMetAMDB (https://metamdb.tu-bs.de/) is an open source metabolic atom mapping database, providing atom mappings for around 75000 metabolic reactions. Each atom mapping can be inspected and downloaded either as a RXN file or as a graphic in SVG format. In addition, MetAMDB offers the possibility of automatically creating atom mapping models based on user-specified metabolic networks. These models can be of any size (small to genome scale) and can subsequently be used in standard 13C metabolic flux analysis software.

sentences: EZH2 inhibition enhances TRAIL responses in multiple myeloma but not in quiescent cells Multiple Myeloma is a plasma cell malignancy for which there is currently no cure, despite many novel therapies. TRAIL (Tumour necrosis factor-related apoptosis inducing ligand) is a promising anti-tumour agent although TRAIL-insensitive cells readily emerge when used as a single agent and its effects are limited due to many TRAIL-resistant cells which emerge soon after treatment. EZH2 is a H3K27 histone methyltransferase found to be overexpressed in many cancers including Multiple Myeloma. We tested the hypothesis that epigenetic reprogramming using the EZH2 inhibitor GSK343 would enhance TRAIL sensitivity, overcome TRAIL resistance, and target TRAIL-resistant quiescent cell populations. We show that GSK343 is a potent TRAIL sensitiser in TRAIL-sensitive RPMI 8226, NCI-H 929 and U266, and in TRAIL-resistant OPM-2 and JJN3, the latter showing very potent synergistic induction of apoptosis, primarily via caspase-8 activation but also via caspase-9. GSK343-enhancement of TRAIL responses was further enhanced in a 3D cell culture model of Multiple Myeloma in NCI-H 929 and U266. We show that in TRAIL-resistant sub-populations of Multiple Myeloma cells, GSK343 responses were completely attenuated in RPMI 8226 although synergistic enhancement of apoptosis was observed in NCI-H 929. Furthermore, following isolation of PKH26Hi quiescent cell populations, TRAIL responses and enhancement of TRAIL responses by GSK343 were completely attenuated. These studies show that EZH2 inhibition enhances TRAIL responses both in TRAIL-sensitive and TRAIL-resistant MM cell lines suspension culture and also in 3D cell culture to model the semi-solid Multiple Myeloma lesions in bone. Pre-existing TRAIL resistance was also enhanced by EZH2, and although synergistic enhancement of TRAIL responses by GSK343 was seen in NCI-H 929, responses were completely lost in TRAIL-resistant RPMI 8226 and also in quiescent cells. These studies highlight that although EZH2 inhibitors enhance TRAIL responses, acquired TRAIL resistance, and the presence of quiescent cells may mediate TRAIL-insensitivity in response to GSK343.

sentences: Neural Regression, Representational Similarity, Model Zoology & Neural Taskonomy at Scale in Rodent Visual Cortex How well do deep neural networks fare as models of mouse visual cortex? A majority of research to date suggests results far more mixed than those produced in the modeling of primate visual cortex. Here, we perform a large-scale benchmarking of dozens of deep neural network models in mouse visual cortex with both representational similarity analysis and neural regression. Using the Allen Brain Observatorys 2-photon calcium-imaging dataset of activity in over 6,000 reliable rodent visual cortical neurons recorded in response to natural scenes, we replicate previous findings and resolve previous discrepancies, ultimately demonstrating that modern neural networks can in fact be used to explain activity in the mouse visual cortex to a more reasonable degree than previously suggested. Using our benchmark as an atlas, we offer preliminary answers to overarching questions about levels of analysis (e.g. do models that better predict the representations of individual neurons also predict representational similarity across neural populations?); questions about the properties of models that best predict the visual system overall (e.g. is convolution or category-supervision necessary to better predict neural activity?); and questions about the mapping between biological and artificial representations (e.g. does the information processing hierarchy in deep nets match the anatomical hierarchy of mouse visual cortex?). Along the way, we catalogue a number of models (including vision transformers, MLP-Mixers, normalization free networks, Taskonomy encoders and self-supervised models) outside the traditional circuit of convolutional object recognition. Taken together, our results provide a reference point for future ventures in the deep neural network modeling of mouse visual cortex, hinting at novel combinations of mapping method, architecture, and task to more fully characterize the computational motifs of visual representation in a species so central to neuroscience, but with a perceptual physiology and ecology markedly different from the ones we study in primates.

sentences: Quantitative and temporal measurement of dynamic autophagy rates Autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis. Systematically quantifying flux through this pathway is critical for gaining fundamental insights and effectively modulating this process that is dysregulated during many diseases. Established methods to quantify flux use steady state measurements, which provide limited information about the perturbation and the cellular response. We present a theoretical and experimental framework to measure autophagic steps in the form of rates under non-steady state conditions. We use this approach to measure temporal responses to rapamycin and wortmannin treatments, two commonly used autophagy modulators. We quantified changes in autophagy rates in as little as 10 minutes, which can establish direct mechanisms for autophagy perturbation before feedback begins. We identified concentration-dependent effects of rapamycin on the initial and temporal progression of autophagy rates. We also found variable recovery time from wortmannins inhibition of autophagy, which is further accelerated by rapamycin. In summary, this new approach enables the quantification of autophagy flux with high sensitivity and temporal resolution and facilitates a comprehensive understanding of this process.

sentences: Interplay between intrinsic reprogramming potential and microenvironment controls neuroblastoma cell plasticity and identity Two cell identities, noradrenergic and mesenchymal, have been characterized in neuroblastoma cell lines according to their epigenetic landscapes relying on specific circuitries of transcription factors. Yet, their relationship and relative contribution in patient tumors remain poorly defined. Here, we demonstrate that the knock-out of GATA3, but not of PHOX2A or PHOX2B, in noradrenergic cells induces a mesenchymal phenotype. Our results document spontaneous plasticity in several models between both identities and show that plasticity relies on epigenetic reprogramming. We demonstrate that an in vivo microenvironment provides a powerful pressure towards a noradrenergic identity for these models. Consistently, tumor cells with a mesenchymal identity are not detected in a series of PDX models. Further study of the intra-tumor noradrenergic heterogeneity reveals two distinct cell populations exhibiting features of chromaffin-like or sympathoblast-like cells. This work emphasizes that both external cues of the environment and intrinsic factors control plasticity and cell identity in neuroblastoma.

sentences: A method for stabilising the XX karyotype in female mESC cultures Female mouse embryonic stem cells (mESCs) present differently to male mESCs in several fundamental ways, however complications with their in vitro culture have resulted in an underrepresentation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both of these chromosomes due to absent X chromosome inactivation, sets female mESCs apart from males. To avoid this undesirable state, female mESCs in culture preferentially adopt an XO karyotype, with this adaption leading to loss of their unique properties in favour of a state near indistinguishable from male. If female pluripotency is to be studied effectively in this system, it is critical that high quality cultures of XX mESCs are available. Here we report a method for better maintaining XX female mESCs in culture, which also stabilises the male karyotype, and makes study of female specific pluripotency more feasible.

sentences: Comparison of Neutralizing Antibody Titers Elicited by mRNA and Adenoviral Vector Vaccine against SARS-CoV-2 Variants The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine efficacy. Here, neutralizing antibody titers elicited by mRNA-based and an adenoviral vector-based vaccine against variant pseudotyped viruses were compared. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals were of low neutralizing titer (IC50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest the benefit of a second immunization following Ad26.COV2.S to increase protection against the variants.

sentences: mRNA codon optimization on quantum computers Reverse translation of polypeptide sequences to expressible mRNA constructs is a NP-hard combinatorial optimization problem. Each amino acid in the protein sequence can be represented by as many as six codons, and the process of selecting the combination that maximizes probability of expression is termed codon optimization. This work investigates the potential impact of leveraging quantum computing technology for codon optimization. An adiabatic quantum computer (AQC) is compared to a standard genetic algorithm (GA) programmed with the same objective function. The AQC is found to be competitive in identifying optimal solutions and future generations of AQCs may be able to outperform classical GAs. The utility of gate-based systems is also evaluated using a simulator resulting in the finding that while current generations of devices lack the hardware requirements, in terms of both qubit count and connectivity, to solve realistic problems, future generation devices may be highly efficient.

sentences: Maintenance of Bound or Independent Features in Visual Working Memory is Task-dependent Over the last decade, seemingly conflicting results were obtained regarding the question of whether features of an object are stored separately, or bound together, in visual working memory. Many of these studies are based on an implicit assumption about a default, or fixed, mode of working memory storage. In contrast, here we asked whether the anticipated memory probes used in a given experiment might determine the format in which information is maintained in working memory, consistent with its task-oriented function. In order to test this flexible maintenance hypothesis, we recorded EEG while subjects performed a delayed-match-to-sample task with different requirements and load. In Experiment 1, by contrasting conditions with and without the requirement of maintaining bound features, we found significant differences in EEG signals recorded in central-parietal channels while controlling for differences in visual stimulation. Such difference was confirmed by multivariate pattern analysis (MVPA). Moreover, behavior results, topographical distributions and cross-contrast MVPA decoding jointly suggested that this effect is not the same as task load effect. These results were confirmed in Experiment 2 with an independent group of subjects with the same paradigm, providing reliable evidence that the format of object maintenance in visual working memory could be flexibly shaped by the current task.

sentences: Topologically controlled circuits of human iPSC-derived neurons for electrophysiology recordings Bottom-up neuroscience, which consists of building and studying controlled networks of neurons in vitro, is a promising method to investigate information processing at the neuronal level. However, in vitro studies tend to use cells of animal origin rather than human neurons, leading to conclusions that might not be generalizable to humans and limiting the possibilities for relevant studies on neurological disorders. Here we present a method to build arrays of topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons. The circuits consist of 4 to 50 neurons with mostly unidirectional connections, confined by microfabricated polydimethylsiloxane (PDMS) membranes. Such circuits were characterized using optical imaging and microelectrode arrays (MEAs). Electrophysiology recordings were performed on circuits of human iPSC-derived neurons for at least 4.5 months. We believe that the capacity to build small and controlled circuits of human iPSC-derived neurons holds great promise to better understand the fundamental principles of information processing and storing in the brain.

sentences: Axonal transport of Hrs is activity-dependent and rate limiting for synaptic vesicle protein degradation Turnover of synaptic vesicle (SV) proteins is vital for the maintenance of healthy and functional synapses. SV protein turnover is driven by neuronal activity in an ESCRT (endosomal sorting complex required for transport)-dependent manner. Here, we characterize a critical step in this process: axonal transport of ESCRT-0 component Hrs, necessary for sorting proteins into the ESCRT pathway and recruiting downstream ESCRT machinery to catalyze multivesicular body (MVB) formation. We find that neuronal activity stimulates the formation of presynaptic endosomes and MVBs, as well as the motility of Hrs+ vesicles in axons and their delivery to SV pools. Hrs+ vesicles co-transport ESCRT-0 component STAM1 and comprise a subset of Rab5+ vesicles, likely representing pro-degradative early endosomes. Furthermore, we identify kinesin motor protein KIF13A as essential for the activity-dependent transport of Hrs to SV pools and the degradation of SV membrane proteins. Together, these data demonstrate a novel activity- and KIF13A-dependent mechanism for mobilizing axonal transport of ESCRT machinery to facilitate the degradation of SV membrane proteins.

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E156G and Arg158, Phe-157/del mutation in NTD of spike protein in B.1.617.2 lineage of SARS-CoV-2 leads to immune evasion through antibody escape Emerging variants of SARS-CoV-2 with better immune escape mechanisms and higher transmissibility remains a persistent threat across the globe. B.1.617.2 (Delta) variant was first emerged from Maharashtra, India in December, 2020. This variant is classified to be a major cause and concern of the second wave of COVID-19 in India. In the present study, we explored the genomic and structural basis of this variant through computational analysis, protein modelling and molecular dynamics (MD) simulations approach. B.1.617.2 variant carried E156G and Arg158, Phe-157/del mutations in NTD of spike protein. These mutations in N-terminal domain (NTD) of spike protein of B.1.617.2 variant revealed more rigidity and reduced flexibility compared to spike protein of Wuhan isolate. Further, docking and MD simulation study with 4A8 monoclonal antibody which was reported to bind NTD of spike protein suggested reduced binding of B.1.617.2 spike protein compared to that of spike protein of Wuhan isolate. The results of the present study demonstrate the possible case of immune escape and thereby fitness advantage of the new variant and further warrants demonstration through experimental evidence. Our study identified the probable mechanism through which B.1.617.2 variant is more pathogenically evolved with higher transmissibility as compared to the wild-type.

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High sorbic acid resistance of Penicillium roqueforti is mediated by the SORBUS gene cluster Penicillium roqueforti is a major food-spoilage fungus known for its high resistance to the food preservative sorbic acid. Here, we demonstrate that the minimum inhibitory concentration of undissociated sorbic acid (MICu) ranges between 4.2 and 21.2 mM when 34 P. roqueforti strains were grown on malt extract broth. A genome-wide association study revealed that the six most resistant strains contained the 180 kbp gene cluster SORBUS, which was absent in the other 28 strains. In addition, a SNP analysis revealed five genes outside the SORBUS cluster that may be linked to sorbic acid resistance. A partial SORBUS knock-out (>100 of 180 kbp) in a resistant strain reduced sorbic acid resistance to similar levels as observed in the sensitive strains. Whole genome transcriptome analysis revealed a small set of genes present in both resistant and sensitive P. roqueforti strains that were differentially expressed in the presence of the weak acid. These genes could explain why P. roqueforti is more resistant to sorbic acid when compared to other fungi, even in the absence of the SORBUS cluster. Together, the MICu of 21.2 mM makes P. roqueforti among the most sorbic acid-resistant fungi, if not the most resistant fungus, which is mediated by the SORBUS gene cluster.

Author summaryChemical preservatives, such as sorbic acid, are often used in food to prevent spoilage by fungi, yet some fungi are particularly well-suited to deal with these preservatives. First, we investigated the resistance of 34 Penicillium roqueforti strains to various food preservatives. This revealed that some strains were highly resistant to sorbic acid, while others are more sensitive. Next, we used DNA sequencing to compare the genetic variation between these strains and discovered a specific genetic region (SORBUS) that is unique to the resistant strains. Through comparative analysis with other fungal species the SORBUS region was studied in more detail and with the use of genetic engineering tools we removed this unique region. Finally, the mutant lacking the SORBUS region was confirmed to have lost its sorbic acid resistance. This finding is of particular interest as it suggests that only some, not all, P. roqueforti strains are potent spoilers and that specific genetic markers could help in the identification of resistant strains.

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Obsessive Compulsive Disorder and Response Inhibition: Meta-analysis of the Stop-Signal Task This systematic review and meta-analysis updates evidence pertaining to deficient response inhibition in obsessive-compulsive disorder (OCD) as measured by the stop-signal task (SST). We conducted a meta-analysis of the literature to compare response inhibition in patients with OCD and healthy controls, meta-regressions to determine relative influences of age and sex on response inhibition impairment, and a risk of bias assessment for included studies using the Newcastle-Ottawa Scale (NOS). Stop-signal reaction time (SSRT), which estimates the latency of the stopping process deficit, was significantly longer in OCD samples than in controls, reflecting inferior inhibitory control (Raw mean difference = 23.43ms; p = <0.001; 95% CI = [17.42, 29.45]). We did not observe differences in mean reaction time (MRT) in OCD compared to controls (Raw mean difference = 2.51ms; p = 0.755; 95% CI = [-13.27, 18.30]). Age impacted effect size of SSRT, indicating a greater deficit in older patients than younger ones. We did not observe a significant effect of sex on SSRT or MRT scores.

General Scientific SummaryDifficulty inhibiting responses is an hypothesized deficit in Obsessive-Compulsive Disorder (OCD). The results of this systematic review and meta-analysis of studies using the Stop Signal Task support the notion of deficient response inhibition in OCD and indicate that older individuals with OCD show greater impairments than younger ones.

sentences: Dichotomous thinking and informational waste in neuroimaging Neuroimaging relies on separate statistical inferences at tens of thousands of spatial locations. Such massively univariate analysis typically requires an adjustment for multiple testing in an attempt to maintain the family-wise error rate at a nominal level of 5%. First, we examine three sources of substantial information loss that are associated with the common practice under the massively univariate framework: (a) the hierarchical data structures (spatial units and trials) are not well maintained in the modeling process; (b) the adjustment for multiple testing leads to an artificial step of strict thresholding; (c) information is excessively reduced during both modeling and result reporting. These sources of information loss have far-reaching impacts on result interpretability as well as reproducibility in neuroimaging. Second, to improve inference efficiency, predictive accuracy, and generalizability, we propose a Bayesian multilevel modeling framework that closely characterizes the data hierarchies across spatial units and experimental trials. Rather than analyzing the data in a way that first creates multiplicity and then resorts to a post hoc solution to address them, we suggest directly incorporating the cross-space information into one single model under the Bayesian framework (so there is no multiplicity issue). Third, regardless of the modeling framework one adopts, we make four actionable suggestions to alleviate information waste and to improve reproducibility: 1) abandon strict dichotomization, 2) report full results, 3) quantify effects, and 4) model data hierarchies. We provide examples for all of these points using both demo and real studies, including the recent NARPS investigation.

sentences: A chemical method to sequence 5-formylcytosine on RNA Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present Mal-Seq, a chemical method to sequence 5-formylcytosine (f5C) modifications on RNA based upon selective and efficient malononitrile-mediated labeling of f5C residues to generate adducts that are read as C-to-T mutations upon reverse transcription and PCR amplification. We apply Mal-Seq to characterize the prevalence of f5C at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level f5C modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the f5C epitranscriptome.

sentences: Moore than meets the eye: A modified and regularized indirect Fourier Transform(er) Small angle scattering (SAS) probes the size and shape of particles at low resolution through the analysis of the scattering of X-rays or neutrons passing through a solution of particles. One approach to extracting structural information from SAS data is the indirect Fourier transform (IFT). The IFT approach parameterizes the real space pair distribution function (P (r)) of a particle using a set of orthogonal basis functions, which simultaneously determines the scattering profile (I(q)) using corresponding reciprocal space basis functions. This article presents an extension of an IFT algorithm proposed by Moore which used a trigonometric series to describe the basis functions, where the real space and reciprocal space basis functions are Fourier mates. An equation is presented relating the Moore coefficients to the intensities of the SAS profile at specific positions as well as a series of new equations that describe the size and shape parameters of a particle from this distinct set of intensity values. An analytical real space regularizer is derived to smooth the P (r) curve and ameliorate systematic deviations caused by series termination, commonly used in IFT methods though not described in Moores original approach, which is particularly susceptible to such effects. The algorithm is provided as a script, denss.fit_data.py, as part of the DENSS software package for SAS, which includes both command line and interactive graphical interfaces. Results of the program using experimental data show that it is as accurate, and often more accurate, than existing tools.

sentences: Protein sequence profile prediction using ProtAlbert transformer1 Protein profiles have many applications in bioinformatics. To construct the profile from a protein sequence, the sequence is aligned with database. However, sometimes there are no similar sequences with the query. This paper proposes a method based on pre-trained ProtAlbert transformer to predict the profile for a single protein sequence without alignment. The performance of transformers on natural languages is impressive. Protein sequences can be viewed as a language; therefore, we can benefit from using these models. We analyze the attention heads in different layers of ProtAlbert to show that the transformer can capture five essential protein characteristics of the family from a single protein sequence. These assessments are performed on the CASP13 dataset to find representative heads for each of five protein characteristics. Then, these heads are investigated on one thermophilic and two mesophilic proteins as case studies. The results show the significant attention heads for protein family properties extracted from a single protein sequence. This analysis led us to propose an algorithm called PA_SPP for profile prediction using only a single protein sequence as input. In our algorithm, we apply the masked language modeling method of ProtAlbert. The results display high similarity between the predicted profiles and HSSP profiles.

sentences: Reconstruction and simulation of thalamoreticular microcircuitry Thalamoreticular circuitry is known to play a key role in attention, cognition and the generation of sleep spindles, and is implicated in numerous brain disorders, but the cellular and synaptic mechanisms remain intractable. Therefore, we developed the first detailed computational model of mouse thalamus and thalamic reticular nucleus microcircuitry that captures morphological and biophysical properties of [~]14,000 neurons connected via [~]6M synapses, and recreates biological synaptic and gap junction connectivity. Simulations recapitulate multiple independent network-level experimental findings across different brain states, providing a novel unifying cellular and synaptic account of spontaneous and evoked activity in both wakefulness and sleep. Furthermore, we found that: 1.) inhibitory rebound produces frequency-selective enhancement of thalamic responses during wakefulness, in addition to its role in spindle generation; 2.) thalamic interactions generate the characteristic waxing and waning of spindle oscillations; and 3.) changes in thalamic excitability (e.g. due to neuromodulation) control spindle frequency and occurrence. The model is openly available and provides a new tool to interpret spindle oscillations and test hypotheses of thalamoreticular circuit function and dysfunction across different network states in health and disease.

sentences: Bringing the Pig to Comparative Neuroimaging: A Common Space Approach Contextualizes the Pig and Human Structural Connectome Neuroimagings capability to quickly and rapidly phenotype the cortical organization of the whole brain brings with it the possibility to extend our understanding of cortical organization across the mammalian lineage. However, neuroimaging has thus far generally limited itself to a small number of species, with most animal studies being performed in either rodents or Non-Human Primates. Here we perform a first pass characterization of an animal which has recently seen its stock rise in the neuroscience community with the development of new models of neurological disease; the domestic pig. Characterizing the structural connectome of the pig, we create a white matter atlas, and an anatomical template which we use to build a horizontal translation between the pig and human based on a connectivity blueprint approach. We find that conserved trends of structural connectivity across species enabled spatial prediction of regions of interest between the pig and human, showing the potential horizontal translations have as a tool to assess the translational validity of porcine models of neurological disease. Releasing the anatomical template, white matter atlas, and connectivity blueprints, we hope to ease and promote the acceptance of the pig as an alternative large-animal model by the neuroimaging community.

sentences: Genome-wide characterization of human minisatellite VNTRs: population-specific alleles and gene expression differences Variable Number Tandem Repeats (VNTRs) are tandem repeat (TR) loci that vary in copy number across a population. Using our program, VNTRseek, we analyzed human whole genome sequencing datasets from 2,770 individuals in order to detect minisatellite VNTRs, i.e., those with pattern sizes [≥]7 bp. We detected 35,638 VNTR loci and classified 5,676 as commonly polymorphic (i.e., with non-reference alleles occurring in >5% of the population). Commonly polymorphic VNTR loci were found to be enriched in genomic regions with regulatory function, i.e., transcription start sites and enhancers. Investigation of the commonly polymorphic VNTRs in the context of population ancestry revealed that 1,096 loci contained population-specific alleles and that those could be used to classify individuals into super-populations with near-perfect accuracy. Search for quantitative trait loci (eQTLs), among the VNTRs proximal to genes, indicated that in 187 genes expression differences correlated with VNTR genotype. We validated our predictions in several ways, including experimentally, through the identification of predicted alleles in long reads, and by comparisons showing consistency between sequencing platforms. This study is the most comprehensive analysis of minisatellite VNTRs in the human population to date.

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Transmission of pre-XDR and XDR-TB in the Mumbai Metropolitan Region, India Multidrug-resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis complex (MTBC) strains are a great challenge for tuberculosis (TB) control in India. Still, factors driving the MDR/XDR epidemic in India are not well defined.

To address this, whole genome sequencing (WGS) data from 1 852 MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai were used for phylogenetic strain classification, resistance prediction, and cluster analysis (12 allele distance threshold). Factors associated with pre-XDR/XDR-TB were defined by odds ratios and a multivariate logistic regression model.

Overall, 1 017 MTBC strains were MDR, out of which 57.8 % (n=591) were pre-XDR, and 17.9 % (n=183) were XDR. Lineage 2 (L2) strains represented 41.7 % of the MDR, 77.2 % of the pre-XDR, and 86.3 % of the XDR strains, and were significantly associated with pre-XDR/XDR-TB (P < 0.001). Cluster rates were high among MDR (78 %) and pre-XDR/XDR (85 %) strains with three dominant L2 strain clusters (Cl 1-3) representing half of the pre-XDR and two thirds of the XDR-TB cases. Cl 1 strains accounted for 52.5 % of the XDR MTBC strains. Transmission could be confirmed by identical mutation patterns of particular pre-XDR/XDR strains.

As a conclusion high rates of pre-XDR/XDR strains among MDR-TB patients require rapid changes in treatment and control strategies. Transmission of particular pre-XDR/XDR L2 strains is the main driver of the pre-XDR/XDR-TB epidemic. Accordingly, control of the epidemic in the region requires measures with stopping transmission especially of pre-XDR/XDR L2 strains.

sentences: Inverse Problem Reveals Conditions for Characteristic Retinal Degeneration Patterns in Retinitis Pigmentosa under the Trophic Factor Hypothesis Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy with a prevalence of about 1 in 4000, affecting approximately 1.5 million people worldwide. Patients with RP experience progressive visual field loss as the retina degenerates, destroying light-sensitive photoreceptor cells (rods and cones), with rods affected earlier and more severely than cones. Spatio-temporal patterns of retinal degeneration in human RP have been well characterised; however, the mechanism(s) giving rise to these patterns have not been conclusively determined. One such mechanism, which has received a wealth of experimental support, is described by the trophic factor hypothesis. This hypothesis suggests that rods produce a trophic factor necessary for cone survival; the loss of rods depletes this factor, leading to cone degeneration. In this paper we formulate a partial differential equation mathematical model of RP in one spatial dimension, spanning the region between the retinal centre (fovea) and the retinal edge (ora serrata). Using this model we derive and solve an inverse problem, revealing for the first time experimentally testable conditions under which the trophic factor mechanism will qualitatively recapitulate the spatio-temporal patterns of retinal regeneration observed in human RP.

sentences: Low-dose fluvoxamine modulates endocytic trafficking of SARS-CoV-2 spike protein: a potential mechanism for anti-COVID-19 protection by antidepressants Commonly prescribed antidepressants may be associated with protection against severe COVID-19, with one drug (fluvoxamine) currently undergoing a Phase 3 clinical trial. The mechanism of their action, however, remains unknown. Here, I investigated the effect of fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking may provide a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

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Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids BackgroundAccurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid-throughput analysis for three blood-based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and dried blood.

MethodsOptimal conditions for sample preparation and DIA-MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high-throughput or deep profile analysis with mid-throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of a 5-6-point dilution curve.

ResultUsing our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV<30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV<30% standard. Lower limits of detection and quantitation were determined for proteins and peptides observed in each biofluid and workflow. Combining the analysis of both high-throughput plasma fractions exceeded the number of reliably identified proteins for individual biofluids in the mid-throughput workflows.

ConclusionThe workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.

sentences: Achieving improved accuracy for imputation of ancient DNA Genotype imputation has the potential to increase the amount of information that can be gained from the often limited biological material available in ancient samples. As many widely used tools have been developed with modern data in mind, their design is not necessarily reflective of the requirements in studies of ancient DNA. Here, we investigate if an imputation method based on the full probabilistic Li and Stephens model of haplotype frequencies might be beneficial for the particular challenges posed by ancient data. We present an implementation called prophaser, and compare imputation performance to two alternative pipelines that have been used in the ancient DNA community based on the Beagle software. Considering empirical ancient data downsampled to lower coverages as well as present-day samples with artificially thinned genotypes, we show that the proposed method is advantageous at lower coverages, where it yields improved accuracy and ability to capture rare variation. The software prophaser is optimized for running in a massively parallel manner and achieved reasonable runtimes on the experiments performed when executed on a GPU.

sentences: Cellular and Circuit Organization of the Locus Coeruleus of Adult Mice The locus coeruleus (LC) houses the vast majority of noradrenergic neurons in the brain and regulates many fundamental functions. While efferent projections of the LC have been extensively investigated, little is known about its local circuit organization. Here, we performed large-scale multi-patch recordings of LC noradrenergic neurons to profile their morpho-electric properties while simultaneously examining their interactions. LC neurons were diverse and could be classified into two major morpho-electric types. While chemical transmission was not detected, these neurons electrically connected at a rate comparable to many brain regions. Most connections form between dendrites and were restricted to narrowly spaced pairs or small clusters of neurons of the same type. Furthermore, more than two coupled pairs were often identified from individual recording sets that follow a train-like organizational pattern. The assembly of LC neurons follows a spatial and cell type-specific wiring principle that may be imposed by a unique train-like rule.

sentences: Simultaneously monitoring aquatic and riparian biodiversity using riverine water eDNA Environmental DNA (eDNA) metabarcoding for biodiversity monitoring is a critical technical advance. Both aquatic and terrestrial biodiversity information can be detected in riverine water eDNA. However, it remains unverified whether riverine water eDNA can be used to simultaneously monitor aquatic and terrestrial biodiversity. Our specific objective was to assess the effectiveness of monitoring aquatic and riparian biodiversity using riverine water eDNA. We proposed that the monitoring effectiveness (the proportion of aquatic and terrestrial biodiversity information detected by riverine water eDNA samples) could be approximated by the transportation effectiveness of land-to-river and upstream-to-downstream biodiversity information flow. We conducted a case study in a watershed on the Qinghai-Tibet Plateau and estimated the effectiveness of using riverine water eDNA to monitor aquatic and riparian biodiversity based on comparing the operational taxonomic units (OTUs) and species assemblages of three taxonomic communities detected in riverine water eDNA samples and riparian soil eDNA samples in spring, summer, and autumn. The aquatic and riparian biodiversity of a watershed on the Qinghai-Tibet Plateau could be simultaneously effectively monitored using riverine water eDNA on summer or autumn rainy days. Monitoring bacterial communities was more efficient than monitoring eukaryotic communities. On summer rainy days, 43%-76% of riparian species could be detected in water eDNA samples, 92%-99% of upstream species could be detected in a 1-km downstream eDNA sample, and more than 50% of dead bioinformation (i.e., the bioinformation labeling the biological material without life activity and fertility) could be monitored 4-6 km downstream for eukaryotes and 13-19 km for bacteria. We encourage more studies on the monitoring effectiveness for each taxonomic community in other watersheds with different environmental conditions. We believe that in future ecological research, conservation and management, we could efficiently monitor and assess the aquatic and terrestrial biodiversity by simply using riverine water eDNA samples.

sentences: Large scale in vivo acquisition, segmentation, and 3D reconstruction of cortical vasculature using open-source functional ultrasound imaging platform The brain is composed of a dense and ramified vascular network comprising various sizes of arteries, veins, and capillaries. One way to assess the risk of cerebrovascular pathologies is to use computational models to predict the physiological effects of a reduction of blood supply and correlate these responses with observations of brain damage. Therefore, it is crucial to establish a detailed 3D organization of the brain vasculature, which could be used to develop more accurate in silico models. For this purpose, we have adapted our open-access functional ultrasound imaging platform previously designed for recording brain-wide activity that is now capable of fast and reproducible acquisition, segmentation, and reconstruction of the cortical vasculature. For the first time, it allows us to digitize the cortical vasculature in awake rodents with a [~]100 {micro}m3 spatial resolution. Contrary to most available strategies, our approach can be performed in vivo within minutes. Moreover, it is easy to implement since it neither requires exogenous contrast agents nor long post-processing time. Hence, we performed a cortex-wide reconstruction of the vasculature and its quantitative analysis, including i) classification of descending arteries versus ascending veins in more than 1500 vessels/animal, ii) quick estimation of their length. Importantly, we confirmed the relevance of our approach in a model of cortical stroke, which enables quick visualization of the ischemic lesion. This development contributes to extending the capabilities of ultrasound neuroimaging to understand better cerebrovascular pathologies such as stroke, vascular cognitive impairment, and brain tumors and is highly scalable for the clinic.

sentences: Interferon-gamma mediates skeletal muscle lesions through JAK/STAT pathway activation in inclusion body myositis Dysimmune and Inflammatory Myopathies (DIMs) are acquired idiopathic myopathy associated with immune response dysregulation. Inclusion Body Myositis (IBM), the most common DIMs, is characterized by endomysial infiltrates of cytotoxic T lymphocytes CD8, muscle type II-interferon (IFN{gamma}) signature, and by the lack of response to immunomodulatory therapies. We showed that IBM was pathologically characterized by the presence of chronic degenerative myopathic features including myofiber atrophy, fibrosis, adipose involution, and the altered functions of skeletal muscle stem cells. Here, we demonstrated that protracted systemic exposure to IFN{gamma} delayed muscle regeneration and led to IBM-like muscular degenerative changes in mice. In vitro, IFN{gamma} treatment inhibited the activation, proliferation, migration, differentiation, and fusion of myogenic progenitor cells and promoted their senescence through JAK-STAT-dependent activation. Finally, JAK-STAT inhibitor, ruxolitinib abrogated the deleterious effects of IFN{gamma} on muscle regeneration, suggesting that the JAK-STAT pathway could represent a new therapeutic target for IBM.

sentences: Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug-microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at -80{degrees}C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.

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Sugar Beet Extract Acts as a Natural Bio-Stimulant for Physio-Biochemical analysis of Hordeum Vulgare L. under Induced Salinity Stress Change in climate of the entire globe due to elevated temperature and minimum annual rainfall in barren zone frequently leads to salinity of soil. The current study was aimed to evaluate the importance of sugar beet extract (SBE) as a bio-stimulant to improve the adverse damages of induced salinity stress (40mM) on growth, oosmolytes and antioxidant defense system of barley (Hordeum vulgare L.). Pot experiment was carried in green house under different concentrations of SBE (10%, 20%, 30%, 40%, 50%) pre-soaked seeds of Hordeum vulgare for 5 hours SBE was analyzed for glycine betaine (100mmol/kg), betalains (1.3mg/l), phenolics (1.30g/100ml), flavonoids (0.59mg/ml), carotenoids (0.23ml/100ml), vitamin E (0.002%), vitamin C (8.04g/100ml), sugar (8g/100ml), protein (1.39mg/100ml), and oxalic acid (38mg/100ml) while Ca (13.72mg/l), Mg (7.121 mg/l) and K (11.45mg/l) contents were also determined. We found significant improvement in germination parameters of Hordeum vulgare L. via SB extract on coefficient of velocity of emergence (CVE), mean emergence time (MET), germination energy (GE), timson germination index (TGI), germination rate index (GRI) and time to 50% emergence (E50) under induced salinity stress. However, photosynthetic pigments, e.g., chlorophyll and carotenoids were enhanced using 40% SB extract, soluble sugar, protein, proline, POD, MDA with 50% SB extract while SOD and H2O2 in 20% SBE, respectively. Our findings suggested that SB extract promotes both agronomical and physiological attributes, is a positive way to enhance our economy by increasing crop yields in arid and semi-arid areas along with plant tolerance to under induced salinity stress.

HighlightsThe recent study is of utmost importance owing to the following reasons:[tpltrtarr] Changes in climatic condition results in salinity, droughts, floods, earthquakes, fluctuation in temperature and other environmental hazards.
[tpltrtarr]Salinity stress has myriad of damaging effects on crop growth and productivity and becoming a global issue of major concern especially in the Asian countries including Pakistan. Statistical analysis showed that 6.30 million of lands is affected with salinity.
[tpltrtarr]Barley (Hordeum Vulgare L.) being an important crop of economic and nutritional value has been facing salinity stress condition in Pakistan, resulting in productivity reduction and ultimately paving the way for food scarcity and crippling economy.
[tpltrtarr]The sole purpose of this research article is to assess and ameliorate the physio-biochemical responses via application of sugar beet extract as bio-stimulants in Barley (Hordeum Vulgare L.) grown under induced salinity stress; in order to cope with the dire consequences of salinity stress especially in the southern areas of Pakistan being badly affected by salt stress.
[tpltrtarr]Furthermore, there is a great need for adaptive measures suggested by the present research and other findings made by researchers in pursuit of tackling this alarming global menace.

sentences: Exquisite Sensitivity to Dual BRG1/BRM ATPase Inhibitors Reveals Broad SWI/SNF Dependencies in Acute Myeloid Leukemia Various subunits of mammalian SWI/SNF chromatin remodeling complexes display loss-of-function mutations characteristic of tumor suppressors in different cancers, but an additional role for SWI/SNF supporting cell survival in distinct cancer contexts is emerging. In particular, genetic dependence on the catalytic subunit BRG1/SMARCA4 has been observed in acute myeloid leukemia (AML), yet the feasibility of direct therapeutic targeting of SWI/SNF catalytic activity in leukemia remains unknown. Here, we evaluated the activity of dual BRG1/BRM ATPase inhibitors across a genetically diverse panel of cancer cell lines and observed that hematopoietic cancer cell lines were among the most sensitive compared to other lineages. This result was striking in comparison to data from pooled short hairpin RNA screens, which showed that only a subset of leukemia cell lines display sensitivity to BRG1 knockdown. We demonstrate that combined genetic knockdown of BRG1 and BRM is required to recapitulate the effects of dual inhibitors, suggesting that SWI/SNF dependency in human leukemia extends beyond a predominantly BRG1-driven mechanism. Through gene expression and chromatin accessibility studies, we show that the dual inhibitors act at genomic loci associated with oncogenic transcription factors, and observe a downregulation of leukemic pathway genes including MYC, a well-established target of BRG1 activity in AML. Overall, small molecule inhibition of BRG1/BRM induced common transcriptional responses across leukemia models resulting in a spectrum of cellular phenotypes. Our studies reveal the breadth of SWI/SNF dependency and support targeting SWI/SNF catalytic function as a potential therapeutic strategy in AML.

sentences: Red Knot Stopover Population Size and Migration Ecology at Delaware Bay, USA, 2021 Red Knots (Calidris canutus rufa) stop at Delaware Bay during northward migration to feed on eggs of horseshoe crabs (Limulus polyphemus). In the late 1990s and early 2000s, the number of Red Knots found at Delaware Bay dramatically declined from ~50,000 to ~13,000. Horseshoe crabs have been harvested for use as bait in eel and whelk fisheries since at least 1990, and some avian conservation biologists hypothesized that harvest levels in the 1990s prevented sufficient refueling for successful migration to the breeding grounds, nesting, and survival for the remainder of the annual cycle. Since 2013, the harvest of horseshoe crabs in the Delaware Bay region has been managed using an Adaptive Resource Management (ARM) framework. The objective of the ARM framework is to manage sustainable harvest of Delaware Bay horseshoe crabs while maintaining ecosystem integrity and supporting Red Knot recovery with adequate stopover habitat for Red Knots and other migrating shorebirds. For annual harvest recommendations, the ARM framework requires annual estimates of horseshoe crab population size and the Red Knot stopover population. We conducted a mark-recapture-resight investigation to estimate the passage population of Red Knots at Delaware Bay in 2021. We used a Bayesian analysis of a Jolly-Seber model, which accounts for turnover in the population and the probability of detection during surveys. The 2021 Red Knot mark-resight dataset included a total of 1,591 individual birds that were recorded at least one during mark-resight surveys at Delaware Bay in 2021. The passage population size in 2021 was estimated at 42,271 (95% credible interval: 35,948 - 55,210). Like 2020, the 2021 population estimate is slightly lower than the 2018 and 2019 estimates. The 2021 population size estimate will inform decision making for harvest recommendations in the next management cycle.

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Regulatory perturbations of ribosome allocation reshape the growth proteome with a trade-off in adaptation capacity Bacteria regulate their cellular resource allocation to enable fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655 in glucose minimal medium. Mutants with only 3 of the seven copies of ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied rpoB mutants. Higher growth rates due to increased ribosome content affected resource allocation. Expression profiles of fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. Mutants showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA Polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments. We show how two different regulatory perturbations (rRNA promoters or rpoB mutations) reshape the proteome for growth with a concomitant fitness cost

HighlightsMutants with only 3 ribosomal operons grow faster than wild-type in minimal medium

 {Delta}4 rrn and rpoB mutants share phenotypic traits

Faster growth of mutants is achieved by increased ribosome content

Fast-growing mutants display reduced hedging expression and adaptation trade-offs

Despite similar ribosomal content in rich medium the mutants present growth defects

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A supervised fingerprint-based strategy to connect natural product mass spectrometry fragmentation data to their biosynthetic gene clusters Microbial natural products, in particular secondary or specialized metabolites, are an important source and inspiration for many pharmaceutical and biotechnological products. However, bioactivity-guided methods widely employed in natural product discovery programs do not explore the full biosynthetic potential of microorganisms, and they usually miss metabolites that are produced at low titer. As a complementary method, the use of genome-based mining in natural products research has facilitated the charting of many novel natural products in the form of predicted biosynthetic gene clusters that encode for their production. Linking the biosynthetic potential inferred from genomics to the specialized metabolome measured by metabolomics would accelerate natural product discovery programs. Here, we applied a supervised machine learning approach, the K-Nearest Neighbor (KNN) classifier, for systematically connecting metabolite mass spectrometry data to their biosynthetic gene clusters. This pipeline offers a method for annotating the biosynthetic genes for known, analogous to known and cryptic metabolites that are detected via mass spectrometry. We demonstrate this approach by automated linking of six different natural product mass spectra, and their analogs, to their corresponding biosynthetic genes. Our approach can be applied to bacterial, fungal, algal and plant systems where genomes are paired with corresponding MS/MS spectra. Additionally, an approach that connects known metabolites to their biosynthetic genes potentially allows for bulk production via heterologous expression and it is especially useful for cases where the metabolites are produced at low amounts in the original producer.

SignificanceThe pace of natural products discovery has remained relatively constant over the last two decades. At the same time, there is an urgent need to find new therapeutics to fight antibiotic resistant bacteria, cancer, tropical parasites, pathogenic viruses, and other severe diseases. To spark the enhanced discovery of structurally novel and bioactive natural products, we here introduce a supervised learning algorithm (K-Nearest Neighbor) that can connect known and analogous to known, as well as MS/MS spectra of yet unknowns to their corresponding biosynthetic gene clusters. Our Natural Products Mixed Omics tool provides access to genomic information for bioactivity prediction, class prediction, substrate predictions, and stereochemistry predictions to prioritize relevant metabolite products and facilitate their structural elucidation.

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A lung targeted miR-29 Mimic as a Therapy for Pulmonary Fibrosis microRNAs are non-coding RNAs that negatively regulate gene networks. Previously, we reported a systemically delivered miR-29 mimic MRG-201 that reduced fibrosis in animal models, but at doses prohibiting clinical translation. Here, we generated MRG-229, a next-gen miR-29 mimic with improved chemical stability, conjugated with the internalization moiety BiPPB (PDGFbetaR-specific bicyclic peptide). In TGF-b-treated human lung fibroblasts and precision cut lung slices, MRG-229 decreased COL1A1 and ACTA2 gene expression and reduced collagen production. In bleomycin-treated mice, intravenous or subcutaneous delivery of MRG-229 downregulated profibrotic gene programs at doses more than ten-fold lower than the original compound. In rats and non-human primates, and at clinically relevant doses, MRG-229 was well tolerated, with no adverse findings observed. In human peripheral blood decreased mir-29 concentrations were associated with increased mortality in two cohorts potentially identified as a target population for treatment. Collectively, our results provide support for the development of MRG-229 as a potential therapy in humans with IPF.

One Sentence SummaryOne Sentence Summary: A stabilized, next-generation miR-29 mimic has been developed that demonstrates efficacy at commercially viable doses with a robust safety margin in non-human primates.

sentences: Systemic application of the TRPV4 antagonist GSK2193874 induces tail vasodilation in a mouse model of thermoregulation In humans the skin is a primary thermoregulatory organ, with vasodilation leading to rapid body cooling, whereas in Rodentia the tail performs an analogous function. Many thermodetection mechanisms are likely to be involved including transient receptor potential vanilloid-type 4 (TRPV4), a widely distributed ion channel with both mechanical and thermosensitive properties. Previous studies have shown that TRPV4 can act as a vasodilator by local action in blood vessels, and in this study, we investigated whether TRPV4 activity effects mus muscularis tail vascular tone and thermoregulation. We measured tail blood flow by pressure plethysmography in lightly sedated mus muscularis (CD1 strain) at a range of ambient temperatures, with and without intraperitoneal administration of the blood brain barrier crossing TRPV4 antagonist GSK2193874. We also measured heart rate and blood pressure with and without GSK2193874. As expected for a thermoregulatory organ, we found that tail blood flow increased with temperature. However, unexpectedly we found that the TRPV4 antagonist GSK2193874 increased tail blood flow at all temperatures, and we observed changes in heart rate variability. Since TRPV4 activation stimulates the relaxation of peripheral resistance arteries (vasodilation) that would increase tail blood flow, these data suggest that increases in tail blood flow resulting from the TRPV4 antagonist may arise from a site other than the blood vessels themselves, perhaps in central cardiovascular control centres such as the hypothalamus.

sentences: Interpersonal Comparison of Utility by Measuring Neural Activity Aggregating welfare across individuals is a fundamental problem in our society. There is no rational aggregation procedure without interpersonally comparable utility (Arrows impossibility theorem). However, scientific methods for interpersonal comparison of utility have thus far not been available. Here, we have developed such a method based on brain signals that correlated with changes in expected utility weighted by subjective probability. The signal was larger for participants with lower household incomes than for those with higher household incomes and their ratio coincided with the estimates by "impartial spectators". These findings suggest that our interpersonal comparison method enables scientifically reasonable welfare aggregation.

sentences: Fishing for biodiversity Fisheries are damaging, and seemingly incompatible with the conservation of marine ecosystems. Yet fish are an important source of food, and support the lives of many people in coastal communities. This paper considers an idea that a moderate intensity of fishing, appropriately scaled across species, could help in maintaining biodiversity, rather than reducing it. The scaling comes from an intuition that rates of fishing mortality of species should be kept in line with production rates of the species, a notion known as balanced harvesting. This places species conservation and exploitation on an equal footing in a single equation, showing quantitatively the relative levels of fishing mortality that species of different abundance can support. Using a dynamic model of a marine ecosystem, we give numerical evidence showing for the first time that fishing, if scaled in this way, can protect rarer species, while allowing some exploitation of species with greater production. This works because fishing mortality rates, when scaled by production, are density-dependent. Such fishing, operating adaptively to follow species production rates over time, contains a feedback that would help to protect species from overfishing in the presence of uncertainty about how marine ecosystems work.

sentences: Manifestations of genetic risk for Alzheimer's Disease in the blood: a cross-sectional multi-omic analysis in healthy adults aged 18-90+ Deeply phenotyped cohort data can elucidate differences associated with genetic risk for common complex diseases across an age spectrum. Previous work has identified genetic variants associated with Alzheimers disease (AD) risk from large-scale genome-wide association study meta-analyses. To explore effects of known AD-risk variants, we performed a phenome-wide association study on ~2000 clinical, proteomic, and metabolic blood-based analytes obtained from 2,831 cognitively normal adult clients of a consumer-based scientific wellness company. Results uncovered statistically significant SNP-analyte associations for five genetic variants after correction for multiple testing (for SNPs in or near NYAP1, ABCA7, INPP5D, and APOE). These effects were detectable from early adulthood. Sex modified the effects of four genetic variants, with multiple interrelated immune-modulating effects associated with the PICALM variant. Sex-stratified GWAS results from an independent AD case-control meta-analysis supported sexspecific disease effects of the PICALM variant, highlighting the importance of sex as a biological variable. These analyses support evidence from previous functional genomics studies in the identification of a causal variant within the PILRA gene. Taken together, this study highlights clues to the earliest effects of AD genetic risk variants in individuals where disease symptoms have not (yet) arisen.

sentences: Assessment of Plasmodium falciparum infection and fitness of genetically modified Anopheles gambiae aimed at mosquito population replacement Genetically modified (GM) mosquitoes expressing anti-plasmodial effectors propagating through wild mosquito populations by means of gene drive is a promising tool to support current malaria control strategies. The process of generating GM mosquitoes involves genetic transformation of mosquitoes from a laboratory colony and, often, interbreeding with other GM lines to cross in auxiliary traits. These mosquito colonies and GM lines thus often have different genetic backgrounds and GM lines are invariably highly inbred, which in conjunction with their independent rearing in the laboratory may translate to differences in their susceptibility to malaria parasite infection and life history traits. Here, we show that laboratory Anopheles gambiae colonies and GM lines expressing Cas9 and Cre recombinase vary greatly in their susceptibility to Plasmodium falciparum NF54 infection. Therefore, the choice of mosquitoes to be used as a reference when conducting infection or life history trait assays requires careful consideration. To address these issues, we established an experimental pipeline involving genetic crosses and genotyping of mosquitoes reared in shared containers throughout their lifecycle. We used this protocol to examine whether GM lines expressing the antimicrobial peptide (AMP) Scorpine in the mosquito midgut interfere with parasite infection and mosquito survival. We demonstrate that Scorpine expression in the Peritrophin 1 (Aper1) genomic locus reduces both P. falciparum sporozoite prevalence and mosquito lifespan; both these phenotypes are likely to be associated with the disturbance of the midgut microbiota homeostasis. These data lead us to conclude that the Aper1-Sco GM line could be used in proof-of-concept experiments aimed at mosquito population replacement, although the impact of its reduced fitness on the spread of the transgene through wild populations requires further investigation.

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A tonic nicotinic brake controls spike timing in striatal spiny projection neurons Striatal spiny projection neurons (SPNs) transform convergent excitatory corticostriatal inputs into an inhibitory signal that shapes basal ganglia output. This process is fine-tuned by striatal GABAergic interneurons (GINs), which receive overlapping cortical inputs and mediate rapid corticostriatal feedforward inhibition of SPNs. Adding another level of control, cholinergic interneurons (CINs), which are also vigorously activated by corticostriatal excitation, can disynaptically inhibit SPNs by activating 4{beta}2 nicotinic acetylcholine receptors (nAChRs) on various GINs. Measurements of this disynaptic inhibitory pathway, however, indicate that it is too slow to compete with direct GIN-mediated feed-forward inhibition. Moreover, functional nAChRs are also present on populations of GINs that respond only weakly to phasic activation of CINs, such as parvalbumin-positive fast-spiking interneurons (PV-FSIs), making the overall role of nAChRs in shaping striatal synaptic integration unclear. Using acute striatal slices we show that upon synchronous optogenetic activation of corticostriatal projections blockade of 4{beta}2 nAChRs shortened SPN spike latencies and increased postsynaptic depolarizations. The nAChR-dependent inhibition was mediated by downstream GABA release, and data suggest that the GABA source was not limited to GINs that respond strongly to phasic CIN activation. In particular, the observed decrease in spike latency caused by nAChR blockade was associated with a diminished frequency of spontaneous inhibitory postsynaptic currents in SPNs, a parallel hyperpolarization of PV-FSIs, and was occluded by pharmacologically preventing cortical activation of PV-FSIs. Taken together, we describe a role for tonic (as opposed to phasic) activation of nAChRs in striatal function. We conclude that tonic activation of nAChRs by CINs maintains a GABAergic brake on cortically-driven striatal output by "priming" feedforward inhibition, a process that may shape SPN spike timing, striatal processing and synaptic plasticity.

Impact StatementA novel mechanism describing how tonic activation of nicotinic acetylcholine receptors can modulate the timing of striatal output by priming feed forward inhibition.

sentences: Cell motility driven by long-lived spatial memory Many living cells actively migrate in their environment to perform key biological functions - from unicellular organisms looking for food to single cells such as fibroblasts, leukocytes or cancer cells that can shape, patrol or invade tissues. Cell migration results from complex intracellular processes that enable cell self-propulsion 1,2, and has been shown to also integrate various chemical or physical extracellular signals 3,4,5. While it is established that cells can modify their environment by depositing biochemical signals or mechanically remodeling the extracellular matrix, the impact of such self-induced environmental perturbations on cell trajectories at various scales remains unexplored. Here, we show that cells remember their path: by confining cells on 1D and 2D micropatterned surfaces, we demonstrate that motile cells leave long-lived physicochemical footprints along their way, which determine their future path. On this basis, we argue that cell trajectories belong to the general class of self-interacting random walks, and show that self-interactions can rule large scale exploration by inducing long-lived ageing, subdiffusion and anomalous first-passage statistics. Altogether, our joint experimental and theoretical approach points to a generic coupling between motile cells and their environment, which endows cells with a spatial memory of their path and can dramatically change their space exploration.

sentences: hibayes: An R Package to Fit Individual-Level, Summary-Level and Single-Step Bayesian Regression Models for Genomic Prediction and Genome-Wide Association Studies With the rapid development of sequencing technology, the costs of individual genotyping have been reduced dramatically, leading to genomic prediction and genome-wide association studies being widely promoted and used to predict the unknown phenotypes and to locate causal or candidate genes for animal and plant economic traits and, increasingly, for human diseases. Developing newly advanced statistical models to improve accuracy in predicting and locating for the traits with various genetic architectures has always been a hot topic in those two research domains. The Bayesian regression model (BRM) has played a crucial role in the past decade, and it has been used widely in relevant genetic analyses owing to its flexible model assumptions on the unknown genetic architecture of complex traits. To fully utilize the available data from either a self-designed experimental population or a public database, statistical geneticists have constantly extended the fitting capacity of BRM, and a series of new methodologies have been proposed for different application scenarios. Here we introduce hibayes, which is the only one tool that can implement three types of Bayesian regression models. With the richest methods achieved thus far, it covers almost all the functions involved in genetic analyses for genomic prediction and genome-wide association studies, potentially addressing a wide range of research problems, while retaining an easy-to-use and transparent interface. We believe that hibayes will facilitate the researches conducted by human geneticists, as well as plant and animal breeders. The hibayes package is freely available from CRAN at https://cran.r-project.org/package=hibayes.

sentences: Immunosuppression and Outcomes in Acute Myeloid Leukemia Acute myeloid leukemia (AML) patients rarely have long first remissions (> 5 years) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, short remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing and functional immunologic studies, we characterized 28 Normal Karyotype (NK)-AML patients with >5 year first remissions after chemotherapy (Long First Remissions, LFR) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). Our combined analyses indicated that genetic risk profiling at presentation (as defined by ELN 2017 Criteria) was not sufficient to explain the outcomes of many SFR cases. Single cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T-cells expressed an exhaustion signature and were resistant to activation by T-cell receptor stimulation in the presence of autologous AML cells. T-cell activation could be restored by removing the AML cells, or blocking the inhibitory MHC Class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ T-cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

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Inhibitor of the nuclear transport protein XPO1 enhances the anticancer efficacy of KRAS G12C inhibitors in preclinical models of KRAS G12C mutant cancers The identification of molecules that can bind covalently to KRAS G12C and lock it in an inactive GDP-bound conformation has opened the door to targeting KRAS G12C selectively. These agents have shown promise in preclinical tumor models and clinical trials. FDA has recently granted approval to sotorasib for KRAS G12C mutated non-small cell lung cancer (NSCLC). However, patients receiving these agents as monotherapy may not respond and generally develop drug resistance over time. This necessitates the development of multi-targeted approaches that can potentially sensitize tumors to KRAS inhibitors. We generated KRAS G12C inhibitor-resistant cell lines and observed that they exhibit sensitivity toward selinexor, a selective inhibitor of nuclear export protein exportin1 (XPO1), as a single agent. KRAS G12C inhibitor MRTX1257 in combination with selinexor suppressed the proliferation of KRAS G12C mutant cancer cell lines MiaPaCa-2 and NCI-H2122 in a synergistic manner. Moreover, combined treatment of selinexor with KRAS G12C inhibitors resulted in enhanced spheroid disintegration, reduction in the number and size of colonies formed by G12C mutant cancer cells. A combination of selinexor with KRAS G12C inhibitors potentiated the inhibition of KRAS expression in MiaPaCa-2 cells. NF-kB protein expression was also markedly reduced by selinexor and MRTX1257 combination. In an in vivo KRAS G12C cell-derived xenograft model, oral administration of a combination of selinexor and sotorasib was demonstrated to reduce tumor burden and enhance survival. In conclusion, we have shown that the nuclear transport protein XPO1 inhibitor can enhance the anticancer activity of KRAS G12C inhibitors in preclinical cancer models.

SignificanceIn this study, combining nuclear transport inhibitor selinexor with KRAS G12C inhibitors has resulted in potent antitumor effects in preclinical cancer models. This can be an effective combination therapy for cancer patients that do not respond or develop resistance to KRAS G12C inhibitor treatment.

sentences: Fiber photometry does not reflect spiking activity in the striatum Calcium recording via fiber photometry is commonly used as a proxy for recording population neuronal activity in vivo, yet the biological source of the photometry signal remains unclear. Here, using simultaneous in vivo extracellular electrophysiology and fiber photometry in the striatum, along with endoscopic 1-photon and 2-photon calcium imaging, we determined that the striatal fiber photometry signal reflects primarily non-somatic, and not somatic, changes in calcium.

sentences: Toxoflavin secreted by Pseudomonas alcaliphila inhibits growth of Legionella pneumophila and its host Vermamoeba vermiformis Legionella pneumophila is a natural inhabitant of water systems. From there, it can be transmitted to humans by aerosolization resulting in severe pneumonia. Most large outbreaks are caused by cooling towers contaminated with L. pneumophila. The resident microbiota of the cooling tower is a key determinant for the colonization and growth of L. pneumophila. The genus Pseudomonas correlates negatively with the presence of L. pneumophila, but it is not clear which species is responsible. Therefore, we identified the Pseudomonas species inhabiting 14 cooling towers using a Pseudomonas-specific 16S rRNA amplicon sequencing strategy. Cooling towers free of L. pneumophila contained a high relative abundance of members from the Pseudomonas alcaliphila/oleovorans phylogenetic cluster. In vitro, P. alcaliphila JCM 10630 inhibited the growth of L. pneumophila on agar plates. Analysis of the P. alcaliphila genome revealed the presence of a genes cluster predicted to produce toxoflavin. L. pneumophila growth was inhibited by pure toxoflavin and by extract from P. alcaliphila culture found to contain toxoflavin by LC-ESI-MS. In addition, toxoflavin inhibits growth of Vermameoba vermiformis, a host cell of L. pneumophila. Our study indicates that P. alcaliphila may be important to restrict growth of L. pneumophila in water systems through the production of toxoflavin. A sufficiently high concentration is likely not achieved in the bulk water but might have a local inhibitory effect such as in biofilm.

sentences: The Third Transmembrane Domain of Synaptophysin Regulates Initial Fusion Pore Dynamics during Ca2+ -Triggered Exocytosis in Chromaffin cells Synaptophysin (syp) is a major secretory vesicle protein comprising four transmembrane domains (TMDs) and a large cytoplasmic C-terminus. The C-terminus of syp has been shown to regulate exocytosis, vesicle cycling, and synaptic plasticity, but the roles of its TMDs remain unclear. SNARE TMDs line initial fusion pores, and structural work along with sequence analysis suggest that TMD III of syp may play a similar role. To test this hypothesis, we expressed TMD III tryptophan mutants in chromaffin cells from mice lacking both syp and its homolog synaptogyrin, and used amperometry to evaluate fusion pores. In contrast to SNARE TMDs, tryptophan substitutions in syp TMD III had no effect on the flux through initial fusion pores. However, these mutants increased the fraction of kiss-and-run events and decreased the initial fusion pore lifetime. Thus, syp TMD III does not line the initial fusion pore, but interacts with it to influence its stability and choice of release mode.

sentences: Meta-analysis of virus-induced host gene expression reveals unique signatures of immune dysregulation induced by SARS-CoV-2 The clinical outcome of COVID-19 has an extreme age, genetic and comorbidity bias that is thought to be driven by an impaired immune response to SARS-CoV-2, the causative agent of the disease. The unprecedented impact of COVID-19 on global health has resulted in multiple studies generating extensive gene expression datasets in a relatively short period of time. In order to better understand the immune dysregulation induced by SARS-CoV-2, we carried out a meta-analysis of these transcriptomics data available in the published literature. Datasets included both those available from SARS-CoV-2 infected cell lines in vitro and those from patient samples. We focused our analysis on the identification of viral perturbed host functions as captured by co-expressed gene module analysis. Transcriptomics data from lung biopsies and nasopharyngeal samples, as opposed to those available from other clinical samples and infected cell lines, provided key signatures on the role of the hosts immune response on COVID-19 pathogenesis. For example, severity of infection and patients age are linked to the absence of stimulation of the RIG-I-like receptor signaling pathway, a known critical immediate line of defense against RNA viral infections that triggers type-I interferon responses. In addition, co-expression analysis of age-stratified transcriptional data provided evidence that signatures of key immune response pathways are perturbed in older COVID-19 patients. In particular, dysregulation of antigen-presenting components, down-regulation of cell cycle mechanisms and signatures of hyper-enriched monocytes were strongly correlated with the age of older individuals infected with SARS-CoV-2. Collectively, our meta-analysis highlights the ability of transcriptomics and gene-module analysis of aggregated datasets to aid our improved understanding of the host-specific disease mechanisms underpinning COVID-19.

sentences: A mechanistic model captures the emergence and implications of non-genetic heterogeneity and reversible drug resistance in ER+ breast cancer cells Resistance to anti-estrogen therapy is an unsolved clinical challenge in successfully treating ER+ breast cancer patients. Acquisition of mutations can confer heritable resistance to cancer cells, enabling their clonal selection to establish a drug-resistant population. Recent studies have demonstrated that cells can tolerate drug treatment without any genetic alterations too; however, the mechanisms and dynamics of such non-genetic adaptation remain elusive. Here, we investigate coupled dynamics of epithelial-mesenchymal transition (EMT) in breast cancer cells and emergence of reversible drug resistance. Our mechanism-based model for the underlying regulatory network reveals that these two axes can drive one another, thus conferring bidirectional plasticity. This network can also enable non-genetic heterogeneity in a population of cells by allowing for six co-existing phenotypes: epithelial-sensitive, mesenchymal-resistant, hybrid E/M-sensitive, hybrid E/M-resistant, mesenchymal-sensitive and epithelial-resistant, with the first two ones being most dominant. Next, in a population dynamics framework, we exemplify the implications of phenotypic plasticity (both drug-induced and intrinsic stochastic switching) and/or non-genetic heterogeneity in promoting population survival in a mixture of sensitive and resistant cells, even in the absence of any cell-cell cooperation. Finally, we propose the potential therapeutic use of MET (mesenchymal-epithelial transition) inducers besides canonical anti-estrogen therapy to limit the emergence of reversible drug resistance. Our results offer mechanistic insights into empirical observations on EMT and drug resistance and illustrate how such dynamical insights can be exploited for better therapeutic designs.

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Higher-order effects, continuous species interactions, and trait evolution shape microbial spatial dynamics The assembly and maintenance of microbial diversity in natural communities, despite the abundance of toxin-based antagonistic interactions, presents major challenges for biological understanding. A common framework for investigating such antagonistic interactions involve cyclic dominance games with pairwise interactions. The incorporation of higher-order interactions in such models permits increased levels of microbial diversity, especially in communities where antibiotic producing, sensitive, and resistant strains co-exist. However, most such models involve a small number of discrete species, assume a notion of pure cyclic dominance, and focus on low mutation rate regimes, none of which well represents the highly interlinked, quickly evolving, and continuous nature of microbial phenotypic space. Here, we present an alternative vision of spatial dynamics for microbial communities based on antagonistic interactions--one in which a large number of species interact in continuous phenotypic space, are capable of rapid mutation, and engage in both direct and higher-order interactions mediated by production of and resistance to antibiotics. Focusing on toxin production, vulnerability, and inhibition among species, we observe highly divergent patterns of diversity and spatial community dynamics. We find that species interaction constraints (rather than mobility) best predict spatiotemporal disturbance regimes, whereas community formation time, mobility, and mutation size best explain patterns of diversity. We also report an intriguing relationship among community formation time, spatial disturbance regimes, and diversity dynamics. This relationship, which suggests that both higher-order interactions and rapid evolution are critical for the origin and maintenance of microbial diversity, has broad-ranging links to the maintenance of diversity in other systems.

Significance StatementPersistently diverse microbial communities are one of biologys great puzzles. Using a novel continuous trait space modeling framework that accommodates high mutation rates, elevated species richness, and direct and higher-order antagonistic species interactions, we find that two parameters characterizing mutation size and mobility best explain patterns of microbial diversity. Moreover, community formation time (the duration of the transient phase in community assembly) provides an unexpectedly clear guide to the diversity profiles of the resulting communities. These discoveries showcase how complex, antagonistic interactions mediated by the production of, inhibition of, and vulnerability to toxins (antibiotics) can shape microbial communities, allowing for extraordinarily high levels of diversity and temporal persistence.

sentences: Sensory regulation of absence seizures in a mouse model of Gnb1 encephalopathy Absence seizures, a type of non-convulsive epilepsy manifested by spike-wave discharges (SWD) in the electroencephalogram (EEG), display synchronous reciprocal excitation between the neocortex and thalamus. Recent studies have revealed that inhibitory neurons in the reticular thalamic (RT) nucleus and excitatory thalamocortical (TC) neurons are two key subcortical players in generating SWD. However, the signals that drive SWD-related thalamic activity remain elusive. Here, we show that SWD predominately occurs during wakefulness in several mouse models of absence epilepsy. In more focused studies of Gnb1 mutant mice, we found that sensory input regulates SWD. Using in vivo recording, we demonstrate that TC cells are activated prior to the onset of SWD and then inhibited during SWD. On the contrary, RT cells are slightly inhibited prior to SWD, but are strongly activated during SWD. Furthermore, chemogenetic activation of TC cells leads to the enhancement of SWD in Gnb1 mice. Together, our results indicate that sensory input in the periphery can regulate SWD by activating the thalamocortical pathway.

sentences: FISH-quant v2: a scalable and modular analysis tool for smFISH image analysis Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in-situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling a quantitative understanding of gene expression and its regulatory mechanisms. Recent progress in experimental techniques provides larger data-sets, requiring adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular analysis tool accessible both for non-experts and experts, which we validated and applied on large-scale smFISH image datasets. Our package allows the user to detect isolated and clustered mRNA spots, segment nuclei and cells, quantify RNA localization patterns and visualize these results at the single-cell level.

sentences: Adipose mTORC2 is essential for arborization of sensory neurons in white adipose tissue and whole-body energy homeostasis Adipose tissue, via sympathetic and sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue is poorly characterized. Taking advantage of recent progress in whole-mount three-dimensional imaging of adipose tissue, we identified a neuronal network of calcitonin gene-related protein (CGRP)-positive sensory neurons in white adipose tissue (WAT). Furthermore, we show that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, mediates sensory innervation in WAT. Based on visualization of neuronal networks, mTORC2-deficient WAT displayed reduced arborization of (CGRP)-positive sensory neurons, while sympathetic neurons were unaffected. This selective loss of sensory innervation followed reduced expression of growth-associated protein 43 (GAP43) in CGRP-positive sensory neurons. Finally, we found that loss of sensory innervation in WAT correlated with systemic insulin resistance. Our findings suggest that adipose mTORC2 is necessary for sensory innervation in WAT which likely contributes to WAT-to-CNS communication.

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Larger lesion volume in people with multiple sclerosis is associated with increased transition energies between brain states and decreased entropy of brain activity BackgroundNeuronal loss, demyelination, and inflammation in the brains of people with multiple sclerosis (pwMS) can cause impairment or disability across a range of domains. However, the mapping between severity or location of this brain tissue damage and an individuals impairment remains challenging. Advanced neuroimaging techniques may enable us to better understand the neuropathological or compensatory mechanisms in MS and quantify the link between the brains functional activity and its structural backbone. One recently developed approach uses network control theory to characterize the brains energetic landscape using an individuals structural connectome and brain activity dynamics. While brain activity dynamics have previously been analyzed in MS cohorts, investigating how structural network damage, one of the hallmarks of MS, relates to shifts in activity dynamics has not yet been performed.

AimsWe apply brain activity dynamics analysis and network control theory (NCT) to functional time series and structural connectomes in healthy controls (HC) and pwMS with and without disability and compare the three groups resulting metrics. We also assessed the entropy of the three groups brain activity time series. Our hypotheses were that pwMS with disability would have increased brain state transition energies compared to pwMS without disability and controls, and that greater energies would be related to greater lesion burden and decreased entropy of brain activity.

Materials and MethodsNinety-nine pwMS and 19 HC were included in our study; individuals underwent MRI, including anatomical, diffusion and resting-state functional scans. Expanded Disability Status Scale (EDSS) was used to assess disability; 66 pwMS with EDSS<2 were considered as not having disability. Commonly recurring brain states were identified using k-means clustering of the regional brain activity time series. NCT was used to compute the energy required to transition between each pair of states or to remain in the same state, taking into account an individuals structural connectome. The entropy of brain activity time series was quantified and correlated with overall transition energy and lesion volume. Finally, logistic regression with ridge regularization was used to classify the groups using transition energies and demographics/clinical information and the models feature importance assessed.

ResultsSix brain states were identified, including pairs of states with high and low visual (VIS+/-), ventral attention (VAN)+/- and somatomotor (SOM+/-) network activity. Global entropy was negatively correlated with lesion volume (r=-0.20, p=0.03) as was global transition energy (r=-0.18, p=0.04) in pwMS. Smaller transition energies were associated with pwMS without disability, while larger transition energies were associated with pwMS with disability.

DiscussionWe conjecture that functional compensation of brain activity in pwMS without disability results in decreased transition energies between states compared to controls, but, as this mechanism diminishes, transition energies increase and disability occurs. We also conjecture that larger volumes of MS-related lesions result in greater energetic demands to transition between brain states which then leads to lower overall entropy of brain activity.

sentences: Intermolecular interactions drive protein adaptive and co-adaptive evolution at both species and population levels Proteins are the building blocks for almost all the functions in cells. Understanding the molecular evolution of proteins and the forces that shape protein evolution is essential in understanding the basis of function and evolution. Previous studies have shown that adaptation frequently occurs at the protein surface, such as in genes involved in host-pathogen interactions. However, it remains unclear whether adaptive sites are distributed randomly or at regions associated with particular structural or functional characteristics across the genome, since many proteins lack structural or functional annotations. Here, we seek to tackle this question by combining large-scale bioinformatic prediction, structural analysis, phylogenetic inference, and population genomic analysis of Drosophila protein-coding genes. We found that protein sequence adaptation is more relevant to function-related rather than structure-related properties. Interestingly, intermolecular interactions contribute significantly to protein adaptation. We further showed that intermolecular interactions, such as physical interactions may play a role in the co-adaptation of fast-adaptive proteins. We found that strongly differentiated amino acids across geographic regions in protein-coding genes are mostly adaptive, which may contribute to the long-term adaptive evolution. This strongly indicates that a number of adaptive sites tend to be repeatedly mutated and selected in evolution, in the past, present, and maybe future. Our results highlight the important roles of intermolecular interactions and co-adaptation in the adaptive evolution of proteins both at the species and population levels.

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Transcriptome analysis reveals robust transcriptional reprogramming during early stages of Cicer-Ascochyta interaction Ascochyta blight (AB) caused by a filamentous fungus Ascochyta rabiei is a major threat to global chickpea production. The mechanisms underlying chickpea response to A. rabiei remain elusive. Here, we investigated the comparative transcriptional dynamics of AB-resistant and susceptible chickpea genotypes upon A. rabiei infection to understand the early host defence response. Our findings revealed that AB-resistant plants underwent rapid and extensive transcriptional reprogramming compared to susceptible host. At early stage (24-hpi), mainly cell wall remodeling and secondary metabolite pathways were highly activated, while DEGs related with signaling components viz. protein kinases, transcription factors, and hormonal pathways show remarkable upsurge at 72-hpi, especially in resistant genotype. Notably, our data suggests imperative role of JA, ET, and ABA signaling in providing immunity against A. rabiei. Furthermore, gene co-expression networks and modules corroborated the importance of cell wall remodeling, signal transduction and phytohormone pathways. The hub genes such as MYB14, PRE6 and MADS-SOC1 discovered in these modules might be the master regulators governing chickpea immunity. Overall, we not only provide novel insights for comprehensive understanding of immune signaling components mediating AB resistance/susceptibility at early Cicer-Ascochyta interactions, but also offer a valuable resource for developing AB-resistant chickpea.

HighlightsComparative transcriptomic and co-expression analysis of AB-resistant and susceptible chickpea genotypes reveals high-amplitude transcriptional dynamics in resistant plants, and also identifies TFs, PKs and phytohormone-crosstalk as core regulators for AB-resistance.

sentences: Riboswitch Folds to Holo-Form Like Structure Even in the Absence of Cognate Ligand at High Mg2+ Concentration Riboswitches are non-coding RNA that regulate gene expression by folding into specific three-dimensional structures (holo-form) upon binding by their cognate ligand in the presence of Mg2+. Riboswitch functioning is also hypothesized to be under kinetic control requiring large cognate ligand concentrations. We ask the question under thermodynamic conditions, can the riboswitches populate holo-form like structures in the absence of their cognate ligands only in the presence of Mg2+. We addressed this question using thiamine pyrophosphate (TPP) riboswitch as a model system and computer simulations using a coarse-grained model for RNA. The folding free energy surface (FES) shows that with the initial increase in Mg2+ concentration ([Mg2+]), TPP AD undergoes a barrierless collapse in its dimensions. On further increase in [Mg2+], intermediates separated by barriers appear on the FES, and one of the intermediates has a TPP ligand-binding competent structure. We show that site-specific binding of the Mg2+ aids in the formation of tertiary contacts. For [Mg2+] greater than physiological concentration, AD folds into its holo-form like structure even in the absence of the TPP ligand. The folding kinetics shows that it populates an intermediate due to the misalignment of the two arms in the TPP AD, which acts as a kinetic trap leading to larger folding timescales. The predictions of the intermediate structures from the simulations are amenable for experimental verification.

sentences: Free spermidine evokes superoxide radicals that manifest toxicity Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Besides, a specific mechanism of SpeG function conferring pathogenic fitness to Staphylococcus aureus USA300 strain is unknown. Here, we provide evidence that although spermidine mitigates oxidative stress by lowering hydroxyl radical and hydrogen peroxide levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the E. coli {Delta}speG strain as well as naturally SpeG-deficient S. aureus RN4220 strain. However, wild-type E. coli and S. aureus USA300 with a horizontally-acquired speG gene tolerate applied exogenous spermidine stress. Furthermore, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Superoxide radicals thus generated, further affects redox balance and iron homeostasis. Since iron acquisition and metabolism in the host tissues is a challenging task for S. aureus, the current findings helped us explain that the evolutionary gain of SpeG function by S. aureus USA300 strain allows it to neutralize exogenous spermidine- and spermine-mediated toxicity towards iron metabolism inside the host tissues.

sentences: SLIMP: Supervised learning of metabolite-protein interactions from co-fractionation mass spectrometry data Metabolite-protein interactions affect and shape diverse cellular processes. Yet, despite advances, approaches for identifying metabolite-protein interactions at a genome-wide scale are lacking. Here we present an approach termed SLIMP that predicts metabolite-protein interactions using supervised machine learning on features engineered from metabolic and proteomic profiles from a co-fractionation mass spectrometry-based technique. By applying SLIMP with gold standards, assembled from public databases, along with metabolic and proteomic data sets from multiple conditions and growth stages we predicted over 9,000 and 20,000 metabolite-protein interactions for Saccharomyces cerevisiae and Arabidopsis thaliana, respectively. Extensive comparative analyses corroborated the quality of the predictions from SLIMP with respect to widely-used performance measures (e.g. F1-score exceeding 0.8). SLIMP predicted novel targets of 2, 3 cyclic nucleotides and dipeptides, which we analysed comparatively between the two organisms. Finally, predicted interactions for the dipeptide Tyr-Asp in Arabidopsis and the dipeptide Ser-Leu in yeast were independently validated, opening the possibility for future applications of supervised machine learning approaches in this area of systems biology.

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The Temporal Pattern of Synaptic Activation Determines the Longevity of Structural Plasticity at Single Dendritic Spines Individual synapses are the points at which information is passed between neurons, yet it is unknown how the diverse patterns of activity that are observed in vivo effect plasticity at the level of single inputs. Here, we aimed to determine what are the structural plasticity consequences of naturalistic patterns of activity at single spines, as these reflect changes in synaptic efficacy. Utilizing two- photon fluorescence imaging and glutamate uncaging, we studied structural plasticity of individual CA1 hippocampal dendritic spines using activation patterns sampled from a Poisson distribution, which resemble endogenous firing patterns from their CA3 inputs. We found that while the majority of inputs initially undergo structural changes, the robustness of this plasticity is determined by the timing structure of the Poisson sampled naturalistic stimulation patterns. Further, we found that structural plasticity elicited by these naturalistic patterns is both NMDAR and protein synthesis dependent, consistent with requirements for other forms of plasticity. Lastly, we found that during the delivery of naturalistic activity patterns, spines underwent rapid and dynamic structural growth that predicted the longevity of plasticity, which was not the case during non-naturalistic stimulation protocols. These data suggest that dendritic spines are able to integrate incoming temporal information and accordingly modulate the longevity of plasticity that is induced.

HighlightsO_LINaturalistic stimulation of single dendritic spines of CA1 hippocampal neurons induces long lasting structural plasticity that depends on the temporal distribution of the synaptic events.
C_LIO_LIStructural plasticity induced by naturalistic stimulation patterns requires NMDA receptor activation and new protein-synthesis.
C_LIO_LIRapid spine structural dynamics during naturalistic activity, but not regular patterns, predict the longevity of subsequent structural plasticity.
C_LI

sentences: Intergenerational Effects of Early Life Starvation on Life-History, Consumption, and Transcriptomes of a Holometabolous Insect Intergenerational effects, also known as parental effects in which the offspring phenotype is influenced by the parental phenotype, can occur in response to factors that occur not only in early but also in late parental life. However, little is known about how these parental life stage-specific environments interact with each other and with the offspring environment to influence offspring phenotype, particularly in organisms that realize distinct niches across ontogeny. We examined the effects of parental larval starvation and adult reproductive environment on offspring traits under matching or mismatching offspring larval starvation conditions using the holometabolous, haplo-diploid insect Athalia rosae (turnip sawfly). We show that the parental larval starvation treatment had trait-dependent intergenerational effects on both life-history and consumption traits of offspring larvae, partly in interaction with offspring conditions and sex, while there was no significant effect of parental adult reproductive environment. In addition, while offspring larval starvation led to numerous gene- and pathway-level expression differences, parental larval starvation impacted fewer genes and only the ribosomal pathway. Our findings reveal that parental starvation evokes complex intergenerational effects on offspring life-history traits, consumption patterns as well as gene expression, although the effects are less pronounced than those of offspring starvation.

sentences: The Specious Art of Single-Cell Genomics Dimensionality reduction is standard practice for filtering noise and identifying relevant dimensions in large-scale data analyses. In biology, single-cell expression studies almost always begin with reduction to two or three dimensions to produce  all-in-one visuals of the data that are amenable to the human eye, and these are subsequently used for qualitative and quantitative analysis of cell relationships. However, there is little theoretical support for this practice. We examine the theoretical and practical implications of low-dimensional embedding of single-cell data, and find extensive distortions incurred on the global and local properties of biological patterns relative to the highdimensional, ambient space. In lieu of this, we propose semi-supervised dimensionality reduction to higher dimension, and show that such targeted reduction guided by the metadata associated with single-cell experiments provides useful latent space representations for hypothesis-driven biological discovery.

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A comparison of Remdesivir versus Au22Glutathione18 in COVID-19 golden hamsters: a better therapeutic outcome of gold compound We firstly disclose single compound yields better therapeutic outcome than Remdesivir in COVID-19 hamster treatments as it is armed with direct inhibition viral replication and intrinsic suppression inflammatory cytokines expression. Crystal data reveals that Au (I), released from Au22Glutathione18 (GA), covalently binds thiolate of Cys145 of SARS-CoV-2 Mpro. GA directly decreases SARS-CoV-2 viral replication (EC50: ~0.24 M) and intrinsically down-regulates NF{kappa}B pathway therefore significantly inhibiting expression of inflammatory cytokines in cells. The lung viral load and inflammatory cytokines in GA-treated COVID-19 transgenic mice are found to be significantly lower than that of control mice. When COVID-19 golden hamsters are treated by GA, the lung inflammatory cytokines levels are significantly lower than that of Remdesivir while their lung viral load are decreased to same level. The pathological results show that GA treatment significantly reduce lung inflammatory injuries when compared to that of Remdesivir-treated COVID-19 golden hamsters.

One Sentence SummaryWe found that gold cluster molecule directly inhibits SARS-CoV-2 replication and intrinsically suppresses inflammatory cytokines expression in COVID-19 transgenic mouse and golden hamster model, gold cluster providing a better lung injury protection than Remdesivir in COVID-19 golden hamsters via intranasally dropping administration.

sentences: Global fitting for high-accuracy multi-channel single-molecule localization Multi-channel detection in single-molecule localization microscopy (SMLM) greatly increases information content for various biological applications. Here, we present globLoc, a graphics processing unit (GPU) based global fitting algorithm with flexible PSF modeling and parameter sharing, to extract maximum information from multi-channel single molecule data. We show, both in simulations and experiments, that global fitting can substantially improve the 3D localization precision for biplane and 4Pi SMLM and color assignment for ratiometric multicolor imaging.

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BOXR1030, an anti-GPC3 CAR with exogenous GOT2 expression, shows enhanced T cell metabolism and improved antitumor activity PurposeThe solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models.

Experimental DesignWe evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo.

ResultsExpression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions.

ConclusionsTogether, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose- finding, and preliminary efficacy (NCT05120271).

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Cortical depth-dependent human fMRI of resting-state networks using EPIK Recent laminar-fMRI studies have provided substantial understanding of the evoked cortical responses in multiple sub-systems; in contrast, the laminar component of resting-state networks spread over the whole brain has been less studied due to technical limitations. Animal research strongly suggests that the supragranular layers of the cortex play a critical role in maintaining network communication; however, whether this is true in the human cerebral cortex remains unclear. Here, we used EPIK, which offers unprecedented coverage at sub-millimetre resolution, to investigate cortical broad resting-state dynamics with depth specificity in healthy volunteers. Our results suggest that human default mode network connectivity is primarily supported by intermediate and superficial layers of the cortex and that the preferred cortical depth used for communication can vary from one network to another. We also identified significantly lower ALFF and ReHo laminar slopes in scans acquired during task performance compared to rest, as well as varied laminar connectivity patterns. The evaluation of laminar-fMRI across the brain encompasses unprecedented computational challenges; nonetheless, it enables the investigation of a new dimension of the human neocortex, which may be key in the characterization of neurological disorders from a novel perspective.

Significance statementResting-state networks sustain conscious awareness and diverse cognitive processing in the absence of tasks. In contrast to cortical areas, the cortical depth across different networks has been poorly investigated, mainly due to the small brain coverage enforced in high-resolution imaging methods. Here, using an optimized fMRI sequence, we demonstrate the critical involvement of the supragranular layers of the cerebral cortex in the maintenance of the resting-state dynamics. Given the cytoarchitectonics of the human neocortex, and based on our results, the cortical thickness constitutes an important dimension in the characterization of resting-state oscillations in the healthy brain, and its functional study may facilitate the identification of novel targets in neurological diseases.

Key pointsO_LIEPIK-fMRI of a motor task reproduces previous laminar activation profiles in M1.
C_LIO_LIHuman resting-state network correlations peak at different cortical depths.
C_LIO_LIThe cortical slope of ALFF and ReHo is decreased during task compared to rest.
C_LI

sentences: Inter-chromosomal linkage disequilibrium and linked fitness cost loci influence the evolution of nontarget site herbicide resistance in an agricultural weed The adaptation of weedy plants to herbicide is both a significant problem in agriculture and a model for the study of rapid adaptation under regimes of strong selection. Despite recent advances in our understanding of simple genetic changes that lead to resistance, a significant gap remains in our knowledge of resistance controlled by many loci and the evolutionary factors that influence the maintenance of resistance over time. Here, we perform a multi-level analysis involving whole genome sequencing and assembly, resequencing and gene expression analysis to both uncover putative loci involved in nontarget herbicide resistance and to examine evolutionary forces underlying the maintenance of resistance in natural populations. We found loci involved in herbicide detoxification, stress sensing, and alterations in the shikimate acid pathway to be under selection, and confirmed that detoxification is responsible for glyphosate resistance using a functional assay. Furthermore, we found interchromosomal linkage disequilibrium (ILD), most likely associated with epistatic selection, to influence NTSR loci found on separate chromosomes thus potentially mediating resistance through generations. Additionally, by combining the selection screen, differential expression and LD analysis, we identified fitness cost loci that are strongly linked to resistance alleles, indicating the role of genetic hitchhiking in maintaining the cost. Overall, our work strongly suggests that NTSR glyphosate resistance in I. purpurea is conferred by multiple genes which are maintained through generations via ILD, and that the fitness cost associated with resistance in this species is a by-product of genetic-hitchhiking.

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Re-routing metabolism by the mitochondrial pyruvate carrier inhibitor MSDC-0160 attenuates neurodegeneration in a rat model of Parkinson's disease BackgroundA growing body of evidence supports the idea that mitochondrial dysfunction might represent a key feature of Parkinsons disease (PD). Central regulators of energy production, mitochondria are also involved in several other essential functions such as cell death pathways and neuroinflammation which make them a potential therapeutic target for PD management. Interestingly, recent studies related to PD have reported a neuroprotective effect of targeting mitochondrial pyruvate carrier (MPC) by the insulin sensitizer MSDC-0160. As the sole point of entry of pyruvate into the mitochondrial matrix, MPC plays a crucial role in energetic metabolism which is impacted in PD. This study therefore aimed at providing insights into the mechanisms underlying the neuroprotective effect of MSDC-0160.

MethodsWe investigated behavioral, cellular and metabolic impact of chronic MSDC-0160 treatment in unilateral 6-OHDA PD rats. We evaluated mitochondrial related processes through the expression of pivotal mitochondrial enzymes in dorsal striatal biopsies and the level of metabolites in serum samples using nuclear magnetic resonance spectroscopy (NMR)-based metabolomics.

ResultsMSDC-0160 treatment in unilateral 6-OHDA rats improved motor behavior, decreased dopaminergic denervation and reduced mTOR activity and neuroinflammation. Concomitantly, MSDC-0160 administration strongly modified energy metabolism as revealed by increased ketogenesis, beta oxidation and glutamate oxidation to satisfy energy needs and maintain energy homeostasis.

ConclusionMSDC-0160 exerts its neuroprotective effect through reorganization of multiple pathways connected to energy metabolism.

sentences: Epigenetic cell memory: The gene's inner chromatin modification circuit Epigenetic cell memory allows distinct gene expression patterns to persist in different cell types despite a common genotype. Although different patterns can be maintained by the concerted action of transcription factors (TFs), it was proposed that long-term persistence hinges on chromatin state. Here, we study how the dynamics of chromatin state affect memory, and focus on a biologically motivated circuit motif, among histones and DNA modifications, that mediates the action of TFs on gene expression. Memory arises from time-scale separation among three circuits constituent processes: basal erasure, auto and cross-catalysis, and recruited erasure of modifications. When the two latter processes are sufficiently faster than the former, the circuit exhibits bistability and hysteresis, allowing active and repressed gene states to coexist and persist after TF stimulus removal. The duration of memory is stochastic with a mean value that increases as time-scale separation increases, but more so for the repressed state. This asymmetry stems from the cross-catalysis between repressive histone modifications and DNA methylation and is enhanced by the relatively slower decay rate of the latter. Nevertheless, TF-mediated positive autoregulation can rebalance this asymmetry and even confers robustness of active states to repressive stimuli. More generally, by wiring positively autoregulated chromatin modification circuits under time scale separation, long-term distinct gene expression patterns arise, which are also robust to failure in the regulatory links.

sentences: Functional design of bacterial superoxide:quinone oxidoreductase The superoxide anion - molecular oxygen reduced by a single electron - is produced in large amounts by enzymatic and adventitious reactions and can perform a range of cellular functions, including bacterial warfare and iron uptake, signalling and host immune response in eukaryotes. However, it also serves as precursor for more deleterious species such as the hydroxyl anion or peroxynitrite and therefore, cellular defense mechanisms for superoxide neutralization have evolved. In addition to the soluble proteins superoxide dismutase and superoxide reductase, recently the membrane embedded diheme cytochrome b561(CybB) from E. coli has been proposed to act as a superoxide:quinone oxidoreductase. Here, we confirm superoxide and cellular ubiquinones or menaquinones as natural substrates and show that quinone binding to the enzyme accelerates the reaction with superoxide. The reactivity of the substrates is in accordance with the here determined midpoint potential of the two b hemes (+48 and -23 mV / NHE). Our data suggest that the enzyme can work near the diffusion limit in the forward direction and can also catalyse the reverse reaction efficiently under physiological conditions. The data is discussed in context of described cytochrome b561 proteins and potential physiological roles of CybB.

sentences: A lack of repeatability creates the illusion of a trade-off between basal and plastic cold tolerance. The thermotolerance-plasticity trade-off hypothesis predicts that ectotherms with greater basal thermal tolerance have a lower acclimation capacity. This hypothesis has been tested at both high and low temperatures but the results often conflict. If basal tolerance constrains plasticity (e.g. through shared mechanisms that create physiological constraints), it should be evident at the level of the individual, provided the trait measured is repeatable. Here, we used chill-coma onset temperature and chill-coma recovery time (CCO and CCRT; non-lethal thermal limits) to quantify cold tolerance of Drosophila melanogaster across two trials (pre- and post-acclimation). Cold acclimation improved cold tolerance, as expected, but individual measurements of CCO and CCRT in non-acclimated flies were not (or only slightly) repeatable. Surprisingly, however, there was still a strong correlation between basal tolerance and plasticity in cold-acclimated flies. We argue that this relationship is a statistical artefact (specifically, a manifestation of regression to the mean; RTM) and does not reflect a true trade-off or physiological constraint. Thermal tolerance trade-off patterns in previous studies that used similar methodology are thus likely to be impacted by RTM. Moving forward, controlling and/or correcting for RTM effects is critical to determining whether such a trade-off or physiological constraint truly exists.

sentences: Single and complex spikes relay distinct frequency-dependent circuit information in the hippocampus Hippocampal neurons generate either single spikes or stereotyped bursts of spikes known as complex spikes. Although single and complex spikes co-occur in the same neuron, their contribution to information processing remains unclear. We analyzed hippocampal CA1 neurons in awake mice and in behaving rats, combining cellular membrane voltage imaging with optogenetics and extracellular recordings. We found that network-driven subthreshold membrane rhythms in the theta versus gamma frequencies preferably entrained complex versus single spikes in individual neurons. Optogenetic membrane perturbation revealed a causal link between subthreshold theta and gamma power and the initiation of complex versus single spikes. Further, single and complex spikes exhibited different place field properties and frequency-dependent coding during spatial navigation. Thus, individual hippocampal neurons do not integrate theta and gamma rhythms into a combined spike timing code, but instead, transmit frequency-specific information as distinct output modes of single versus complex spikes during spatial cognition.

sentences: Changes in interactions over ecological time scales influence single cell growth dynamics in a metabolically coupled marine microbial community Microbial communities thrive in almost all habitats on earth. Within these communities, cells interact through the release and uptake of metabolites. These interactions can have synergistic or antagonistic effects on individual community members. The collective metabolic activity of microbial communities leads to changes in their local environment. As the environment changes over time, the nature of the interactions between cells can change. We currently lack understanding of how such dynamic feedbacks affect the growth dynamics of individual microbes and of the community as a whole. Here we study how interactions mediated by the exchange of metabolites through the environment change over time within a simple marine microbial community. We used a microfluidic-based approach that allows us to disentangle the effect cells have on their environment from how they respond to their environment. We found that the interactions between two species - a degrader of chitin and a cross-feeder that consumes metabolic by-products - changes dynamically over time as cells modify their environment. Cells initially interact positively and then start to compete at later stages of growth. Our results demonstrate that interactions between microbes are not static and depend on the state of the environment, emphasizing the importance of disentangling how modifications of the environment affects species interactions. This experimental approach can shed new light on how interspecies interactions scale up to community level processes in natural environments.

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ClusTrast: a short read de novo transcript isoform assembler guided by clustered contigs MotivationTranscriptome assembly from RNA-sequencing data in species without a reliable reference genome has to be performed de novo, but studies have shown that de novo methods often have inadequate reconstruction ability of transcript isoforms. This impedes the study of alternative splicing, in particular for lowly expressed isoforms.

ResultWe present the de novo transcript isoform assembler ClusTrast, which clusters a set of guiding contigs by similarity, aligns short reads to the guiding contigs, and assembles each clustered set of short reads individually. We tested ClusTrast on datasets from six eukaryotic species, and showed that ClusTrast reconstructed more expressed known isoforms than any of the other tested de novo assemblers, at a moderate reduction in precision. An appreciable fraction were reconstructed to at least 95% of their length. We suggest that ClusTrast will be useful for studying alternative splicing in the absence of a reference genome.

Availability and implementationThe code and usage instructions are available at https://github.com/karljohanw/clustrast.

Contactolofem@kth.se

Supplementary informationSupplementary material is available.

sentences: A single de novo substitution in SARS-CoV-2 spike informs enhanced adherence to human ACE2. SARS-CoV-2 initiates colonization of host cells by binding to cell membrane ACE2 receptor. This binding is mediated by the viral spike receptor binding domain (RBD). The COVID-19 pandemic has brought devastating consequences at a clinical, social and economical levels. Therefore, anticipation of potential novel SARS-causing species or SARS-CoV-2 variants with enhanced binding to ACE2 is key in the prevention of future threats to come. We have characterized a de novo single substitution, Q498Y, in SARS-CoV-2 RBD that confers stronger adherence to ACE2. While the SARS-CoV-2 {beta} variant, which includes three simultaneous amino acid replacements, induces a 4-fold stronger affinity, a single Q498Y substitution results in 2.5-fold tighter binding, compared to the Wuhan-Hu-1 SARS-CoV-2 2019 strain. Additionally, we crystallized RBDQ498Y complexed with ACE2 and provide here the structural basis for this enhanced affinity. These studies inform a rationale for prevention of potential SARS-causing viruses to come.

sentences: A comprehensive analysis of gene expression changes in a high replicate and open-source dataset of differentiating hiPSC-derived cardiomyocytes We performed a comprehensive analysis of the transcriptional changes within and across cell populations during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using the single cell RNA-seq combinatorial barcoding method SPLiT-seq, we sequenced >20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation. As expected, differentiated cell populations clustered by time point, with differential expression analysis revealing markers of cardiomyocyte differentiation and maturation changing from D12 to D90. We next performed a complementary cluster-independent sparse regression analysis to identify and rank genes that best assigned cells to differentiation time points. The two highest ranked genes between D12 and D24 (MYH7 and MYH6) resulted in an accuracy of 0.84, and the three highest ranked genes between D24 and D90 (A2M, H19, IGF2) resulted in an accuracy of 0.94, revealing that low dimensional gene features can identify differentiation or maturation stages in differentiating cardiomyocytes. Expression levels of select genes were validated using RNA FISH. Finally, we interrogated differences in differentiation population composition and cardiac gene expression resulting from two differentiation protocols, experimental replicates, and three hiPSC lines in the WTC-11 background to identify sources of variation across these experimental variables.

sentences: Sorting of secretory proteins at the trans-Golgi network by TGN46 Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the transmembrane protein TGN46 as a cargo receptor for the export of secretory proteins in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the TGN46 lumenal domain encodes for its cargo sorting function. Interestingly, this domain forms liquid droplets in vitro by means of liquid-liquid phase separation, a physical mechanism that may assist in TGN46-mediated cargo sorting. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN. These findings could open new therapeutic avenues for diseases that parallel secretory malfunction.

sentences: mRNA-1273 and BNT162b2 mRNA vaccines have reduced neutralizing activity against the SARS-CoV-2 Omicron variant The BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines generate potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the global emergence of SARS-CoV-2 variants with mutations in the spike protein, the principal antigenic target of these vaccines, has raised concerns over the neutralizing activity of vaccine-induced antibody responses. The Omicron variant, which emerged in November 2021, consists of over 30 mutations within the spike protein. Here, we used an authentic live virus neutralization assay to examine the neutralizing activity of the SARS-CoV-2 Omicron variant against mRNA vaccine-induced antibody responses. Following the 2nd dose, we observed a 30-fold reduction in neutralizing activity against the omicron variant. Through six months after the 2nd dose, none of the sera from naive vaccinated subjects showed neutralizing activity against the Omicron variant. In contrast, recovered vaccinated individuals showed a 22-fold reduction with more than half of the subjects retaining neutralizing antibody responses. Following a booster shot (3rd dose), we observed a 14-fold reduction in neutralizing activity against the omicron variant and over 90% of boosted subjects showed neutralizing activity against the omicron variant. These findings show that a 3rd dose is required to provide robust neutralizing antibody responses against the Omicron variant.

sentences: Biochemical characterization of RAD52-mediated D-loop formation using fluorophore-labeled DNA substrates The human RAD52 protein is thought to have multiple roles in the mechanisms of repairing DNA double-strand breaks that are caused by replication errors and reactive oxygen species. One such role is to mediate the formation of a displacement loop (D-loop), which is a critical reaction intermediate in homologous recombinational repair. RAD52 is suggested to promote the formation of D-loops when facilitating DNA synthesis at stalled or collapsed replication forks during mitosis. However, RAD52-mediated D-loop formation remains poorly characterized, and the detailed molecular mechanism of the D-loop formation reaction catalyzed by RAD52 is still unclear. In the present study, we developed a gel-based assay that enables rapid detection of RAD52-mediated D-loop formation. This assay utilizes a fluorophore-labeled, single-stranded DNA substrate. In addition to the rapid detection of D-loops, D-loop extension was observed when DNA polymerase was added to the reaction. This assay can also be used for screening large numbers of compounds that either stimulate or inhibit RAD52-mediated D-loop formation. The D-loop formation assay developed in this study is potentially useful for mechanistic studies of DSB repair involving RAD52-mediated D-loop formation, as well as for screening compounds with potential therapeutic effects.

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Torsions-Driven Root Helical Growth, Waving And Skewing In Arabidopsis Helical growth broadly exists in immobile plants to support their limited movement, and Arabidopsis seedling root exhibiting natural left-handedness helical growth is considered as a simplified model for investigating this interesting behavior. Efforts have been made for understanding the mechanism of root helical growth and consequent root waving and skewing on tilted and impenetrable surface, and several models have been established. Here, previous reports are reviewed and a straightforward torsions-driven mechanism has been emphasized, and additional experiments have been performed to fill up the gaps of this theory in our study.

This study implies that, torsions originating from handedness of both cortical microtubules and cellulose microfibrils play central role in root handed helical growth. Different from torsions directly provided by handed assembled cortical microtubules, torsions originating from right-handed assembled cellulose microfibrils are relaxed by their cross-linking with pectin within cell wall, but only exhibited when their cross-linking is interrupted due to damaged cell wall integrity. To topologically relax these torsions, supercoils of cortical microtubules and/or cellulose microfibrils exhibiting as oblique alignments are formed in root cells, which alter the orientation of root cell files and generate handed helical roots. Working together with gravitropic response, relaxation of torsions originating from helical roots drives roots to elongate with handedness, which therefore produces waved and skewed roots on tilted and impenetrable surface.

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A new method for determining ribosomal DNA copy number shows differences between Saccharomyces cerevisiae populations Ribosomal DNA genes (rDNA) encode the major ribosomal RNAs (rRNA) and in eukaryotic genomes are typically present as one or more arrays of tandem repeats. Species have characteristic rDNA copy numbers, ranging from tens to thousands of copies, with the number thought to be redundant for rRNA production. However, the tandem rDNA repeats are prone to recombination-mediated changes in copy number, resulting in substantial intra-species copy number variation. There is growing evidence that these copy number differences can have phenotypic consequences. However, we lack a comprehensive understanding of what determines rDNA copy number, how it evolves, and what the consequences are, in part because of difficulties in quantifying copy number. Here, we developed a genomic sequence read approach that estimates rDNA copy number from the modal coverage of the rDNA and whole genome to help overcome limitations in quantifying copy number with existing mean coverage-based approaches. We validated our method using strains of the yeast Saccharomyces cerevisiae with previously-determined rDNA copy numbers, and then applied our pipeline to investigate rDNA copy number in a global sample of 788 yeast isolates. We found that wild yeast have a mean copy number of 92, consistent with what is reported for other fungi but much lower than in laboratory strains. We also show that different populations have different rDNA copy numbers. These differences can partially be explained by phylogeny, but other factors such as environment are also likely to contribute to population differences in copy number. Our results demonstrate the utility of the modal coverage method, and highlight the high level of rDNA copy number variation within and between populations.

Author summaryThe ribosomal RNA gene repeats (rDNA) form large tandem repeat arrays in most eukaryote genomes. Their tandem arrangement makes the rDNA prone to copy number variation, and there is increasing evidence that this copy number variation has phenotypic consequences. However, difficulties in measuring rDNA copy number hamper investigation into rDNA copy number dynamics and their significance. Here we developed a novel bioinformatics method for measuring rDNA copy number from whole genome sequence data that is based on the modal sequence read coverage. We established parameters for optimal performance of the method and validated it using yeast strains of known rDNA copy numbers. We then applied the method to a dataset of almost 800 global yeast isolates and demonstrate that yeast populations have different rDNA copy numbers that partially correlate with phylogeny. Our work provides a simple and accurate method for determining rDNA copy number that leverages the growing number of whole genome datasets, and highlights the dynamic nature of rDNA copy number.

sentences: A genetic disorder reveals a hematopoietic stem cell regulatory network co-opted in leukemia The molecular regulation of human hematopoietic stem cell (HSC) maintenance is therapeutically important, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. Here, we have studied a rare genetic disorder due to MECOM haploinsufficiency, characterized by an early-onset absence of HSCs in vivo. By generating a faithful model of this disorder in primary human HSCs and coupling functional studies with integrative single-cell genomic analyses, we uncover a key transcriptional network involving hundreds of genes that is required for HSC maintenance. Through our analyses, we nominate cooperating transcriptional regulators and identify how MECOM prevents the CTCF-dependent genome reorganization that occurs as HSCs differentiate. Strikingly, we show that this transcriptional network is co-opted in high-risk leukemias, thereby enabling these cancers to acquire stem cell properties. Collectively, we illuminate a regulatory network necessary for HSC self-renewal through the study of a rare experiment of nature.

sentences: Chemogenetic activation of Locus Coeruleus Noradrenergic Neurons Modulates the Default Mode Network The default mode network (DMN) of the brain is involved in cognition, emotion regulation, impulsivity, and balancing between internally and externally focused states. DMN dysregulation has been implicated in several neurological and neuropsychiatric disorders. In this study, we used functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and spectral fiber-photometry to investigate the selective neuromodulatory effect of norepinephrine (NE)-releasing noradrenergic neurons in the locus coeruleus (LC) on the DMN in mice. Chemogenetic-induced tonic LC-NE activity decreased cerebral blood volume (CBV) and glucose uptake, and increased synchronous low frequency fMRI activity within the frontal cortices of the DMN. Fiber-photometry results corroborated these findings, showing that LC-NE activation induced NE release, enhanced calcium-weighted neuronal spiking, and reduced CBV in the anterior cingulate cortex. These data suggest that LC-NE alters conventional stimulus-evoked coupling between neuronal activity and CBV in the frontal DMN. We also demonstrated that chemogenetic activation of LC-NE neurons strengthened functional connectivity within the frontal DMN, and this effect was causally mediated by reduced modulatory inputs from retrosplenial and hippocampal regions to the association cortices of the DMN.

sentences: A recombinant selective drug-resistant M. bovis BCG enhances the bactericidal activity of a second-line tuberculosis regimen Drug-resistant tuberculosis (DR-TB) results from infection by Mycobacterium tuberculosis strains resistant to at least rifampin or isoniazid. To improve the treatment outcome in DR-TB, therapeutic vaccines are considered an ideal choice as they can enhance pathogen clearance and minimize disease sequelae. To date, there is no therapeutic vaccine reported to be effective when combined with a chemotherapy regimen against DR-TB. The only available TB vaccine, the M. bovis BCG (BCG) is susceptible to several anti-TB drugs hence not a perfect option for therapeutic vaccination. Herein, we developed a recombinant BCG (RdrBCG) overexpressing Ag85B and Rv2628 with resistance to selected anti-TB drugs. When administered three times adjunct to a second-line anti-TB regimen in a classical murine model of DR-TB, the RdrBCG lowered lung M. tuberculosis colony-forming units by 1 log10. Furthermore, vaccination with the RdrBCG adjunct to TB chemotherapy minimized lung tissue pathology in mice. Most importantly, the RdrBCG maintained the exogenously inserted genes and showed almost the same virulence as its parent BCG Tice strain in severe combined immune-deficient mice. All these suggested that the RdrBCG was stable, safe and effective. Hence, the "recombinant" plus "drug-resistant" BCG strategy could be a useful concept for developing therapeutic vaccines against DR-TB.

sentences: MassGenie: a transformer-based deep learning method for identifying small molecules from their mass spectra The  inverse problem of mass spectrometric molecular identification ( given a mass spectrum, calculate the molecule whence it came) is largely unsolved, and is especially acute in metabolomics where many small molecules remain unidentified. This is largely because the number of experimentally available electrospray mass spectra of small molecules is quite limited. However, the forward problem ( calculate a small molecules likely fragmentation and hence at least some of its mass spectrum from its structure alone) is much more tractable, because the strengths of different chemical bonds are roughly known. This kind of molecular identification problem may be cast as a language translation problem in which the source language is a list of high-resolution mass spectral peaks and the  translation a representation (for instance in SMILES) of the molecule. It is thus suitable for attack using the deep neural networks known as transformers. We here present MassGenie, a method that uses a transformer-based deep neural network, trained on ~6 million chemical structures with augmented SMILES encoding and their paired molecular fragments as generated in silico, explicitly including the protonated molecular ion. This architecture (containing some 400 million elements) is used to predict the structure of a molecule from the various fragments that may be expected to be observed when some of its bonds are broken. Despite being given essentially no detailed nor explicit rules about molecular fragmentation methods, isotope patterns, rearrangements, neutral losses, and the like, MassGenie learns the effective properties of the mass spectral fragment and valency space, and can generate candidate molecular structures that are very close or identical to those of the  true molecules. We also use VAE-Sim, a previously published variational autoencoder, to generate candidate molecules that are  similar to the top hit. In addition to using the  top hits directly, we can produce a rank order of these by  round-tripping candidate molecules and comparing them with the true molecules, where known. As a proof of principle, we confine ourselves to positive electrospray mass spectra from molecules with a molecular mass of 500Da or lower. The transformer method, applied here for the first time to mass spectral interpretation, works extremely effectively both for mass spectra generated in silico and on experimentally obtained mass spectra from pure compounds. The ability to create and to  learn millions of fragmentation patterns in silico, and therefrom generate candidate structures (that do not have to be in existing libraries) directly, thus opens up entirely the field of de novo small molecule structure prediction from experimental mass spectra.

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Modelling Human Gait using a Nonlinear Differential Equation We introduce an innovative method for the investigation of human gait, which is based on the visualisation of the vertical component of the movement of the centre of mass during walking or running, in the space of the coordinates position, velocity, and acceleration of the centre of mass. Collected data has been numerically approximated by the best fitting curve for a non-linear model. The resulting equation for the best fitting plane or curve in this space is a differential equation of second order. The model that we suggest is a Duffing equation with coefficients that depend on the height of a walker or runner and on the angular frequency of the oscillation. Statistics about the distribution of the Duffing stiffness depending on the speed is presented.

1 Author SummaryWe study the human gait modelled by the movement of the centre of mass of the test person. This is an example of a biological process which can be considered as a periodical dynamic system. Roughly, this movement behaves in a similar way to a vibrating mass suspended on a spring, but it is more complex. The vertical component of the movement during walking or running can be visualised as an oscillogram: a graph of the position as a function of time. We suggest a visualisation of the data in 3D space, where the coordinates describe position, velocity, and acceleration. Our new visualisation method allows us to model the movement of a persons centre of mass by a nonlinear differential equation. The resulting curve for an ideal spring-mass movement, without viscosity or external force, is an ellipse in the suggested 3D space. The shape of the data curve shows at which position an additional force was applied, or the movement slowed down. Some deviations are common for all test persons and others are different. In the future we plan to investigate the reasons for these deviations, such as different running techniques or the presence of injuries.

sentences: Hydroxyzine inhibits SARS-CoV-2 Spike protein binding to ACE2 in a qualitative in vitro assay COVID-19 currently represents a major public health problem. Multiple efforts are being performed to control this disease. Vaccinations are already in progress. However, no effective treatments have been found so far. The disease is caused by the SARS-CoV-2 coronavirus that through the Spike protein interacts with its cell surface receptor ACE2 to enter into the host cells. Therefore, compounds able to block this interaction may help to stop disease progression. In this study, we have analyzed the effect of compounds reported to interact and modify the activity of ACE2 on the binding of the Spike protein. Among the compounds tested, we found that hydroxyzine could inhibit the binding of the receptor-binding domain of Spike protein to ACE2 in a qualitative in vitro assay. This finding supports the reported clinical data showing the benefits of hydroxyzine on COVID-19 patients, raising the need for further investigation into its effectiveness in the treatment of COVID-19 given its well-characterized medical properties and affordable cost.

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

sentences: Comparative computational structural genomics highlights divergent evolution of fungal effectors Elucidating the evolution of pathogen effector molecules is critical to understand infection mechanisms of fungal phytopathogens and ensure crop security. However, rapid diversification that diminishes sequence similarities between homologous effectors has largely concealed the roots of effector evolution. We predicted the folds of 26,653 secreted proteins from 21 species with AlphaFold and performed structure-guided comparative analyses on two aspects of effector evolution: uniquely expanded effector families and common folds present across the fungal species. Extreme expansion of nearly lineage-specific effector families was found only in several obligate biotrophs, Blumeria graminis and Puccinia graminis. The highly expanded effector families were the source of conserved sequence motifs, such as the Y/F/WxC motif. We identified additional lineage-specific effector families that include known (a)virulence factors, such as AvrSr35, AvrSr50 and Tin2. These families represented new classes of sequence-unrelated structurally similar effectors. Structural comparisons revealed that the expanded structural folds further diversify through domain duplications and fusion with disordered stretches. Sub-and neo-functionalized structurally similar effectors reconverge on regulation, expanding the functional pools of effectors in the pathogen infection cycle. We also found evidence that many effector families could have originated from ancestral folds that have been conserved across fungi. Collectively, our study highlights diverse mechanisms of effector evolution and supports divergent evolution of effectors that leads to emergence of pathogen virulence from ancestral proteins.

sentences: High-resolution, genome-wide mapping of positive supercoiling in chromosomes Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the "twin-domain" model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

sentences: CELLoGeNe - an Energy Landscape Framework for Logical Networks Controlling Cell Decisions Experimental and computational efforts are constantly made to elucidate mechanisms controlling cell fate decisions during development and cell reprogramming. One powerful method is to consider cell commitment and reprogramming as movements in an energy landscape. Here, we develop CELLoGeNe (Computation of Energy Landscapes of Logical Gene Networks), which maps Boolean implementation of gene regulatory networks (GRNs) into energy landscapes. CELLoGeNe removes inadvertent symmetries in the energy landscapes normally arising from standard Boolean operators. Furthermore, CELLoGeNe provides a tool for visualising multi-dimensional energy landscapes and a platform to stochastically probe and analyse the shapes of the computed landscapes corresponding to the epigenetic landscapes for development and reprogramming. We demonstrate CELLoGeNe on a GRN governing maintenance and self-renewal of pluripotency, identifying attractors experimentally validated. We also apply CELLoGeNe on a network controlling reprogramming from mouse embryonic fibroblast (MEF) to induced pluripotent stem cells (iPSCs) where we identify potential roadblocks as attractors. CELLoGeNe is a general framework that can be applied to various biological systems offering a broad picture of intracellular dynamics otherwise inaccessible with existing methods.

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Contribution of deep soil layers to the transpiration of a temperate deciduous forest: quantification and implications for the modelling of productivity Climate change is imposing drier atmospheric and edaphic conditions on temperate forests. Here, we investigated how deep soil (down to 300 cm) water extraction contributed to the provision of water in the Fontainebleau-Barbeau temperate oak forest over two years, including the 2018 record drought. Deep water provision was key to sustain canopy transpiration during drought, with layers below 150 cm contributing up to 60% of the transpired water in August 2018, despite their very low density of fine roots. We further showed that soil databases used to parameterize ecosystem models largely underestimated the amount of water extractable from the soil by trees, due to a considerable underestimation of the tree rooting depth. The consensus database established for France gave an estimate of 207 mm for the soil water holding capacity (SWHC) at Fontainebleau-Barbeau, when our estimate based on the analysis of soil water content measurements was 1.9 times as high, reaching 390{+/-}17 mm. Running the CASTANEA forest model with the database-derived SWHC yielded a 350 gC m-2 y-1 average underestimation of annual gross primary productivity under current climate, reaching up to 700 gC m-2 y-1 under climate change scenario RCP8.5. It is likely that the strong underestimation of SWHC that we show at our site is not a special case, and concerns a large number of forest sites. Thus, we argue for a generalisation of deep soil water content measurements in forests, in order to improve the estimation of SWHC and the simulation of the forest carbon cycle in the current context of climate change.

HighlightsO_LIForest-atmosphere carbon exchanges remained insensitive to record drought.
C_LIO_LIDeep soil (150-300 cm) provisioned up to 60% of the water transpired by the forest during drought.
C_LIO_LISoil databases were underestimating soil water holding capacity by a factor of two.
C_LIO_LISimulated forest productivity is strongly sensitive to soil water holding capacity parameter.
C_LIO_LIDeep soil water content measurements are urgently needed to correctly estimate the soil water holding capacity.
C_LI

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StrainFLAIR: Strain-level profiling of metagenomic samples using variation graphs Current studies are shifting from the use of single linear references to representation of multiple genomes organised in pangenome graphs or variation graphs. Meanwhile, in metagenomic samples, resolving strain-level abundances is a major step in microbiome studies, as associations between strain variants and phenotype are of great interest for diagnostic and therapeutic purposes.

We developed StrainFLAIR with the aim of showing the feasibility of using variation graphs for indexing highly similar genomic sequences up to the strain level, and for characterizing a set of unknown sequenced genomes by querying this graph.

On simulated data composed of mixtures of strains from the same bacterial species Escherichia coli, results show that StrainFLAIR was able to distinguish and estimate the abundances of close strains, as well as to highlight the presence of a new strain close to a referenced one and to estimate its abundance. On a real dataset composed of a mix of several bacterial species and several strains for the same species, results show that in a more complex configuration StrainFLAIR correctly estimates the abundance of each strain. Hence, results demonstrated how graph representation of multiple close genomes can be used as a reference to characterize a sample at the strain level.

Availabilityhttp://github.com/kevsilva/StrainFLAIR

sentences: Genetic variation among wMel strains of Wolbachia pipientis differentially rescues a bag of marbles partial loss of function mutant in Drosophila melanogaster In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led us to hypothesize that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, we must understand the specificity of the genetic interaction between bam and W. pipientis. Previously, we documented that the W. pipientis variant, wMel, rescued the fertility of the bamBW hypomorphic mutant as a transheterozygote over a bam null. However, bamBW was generated more than 20 years ago in an uncontrolled genetic background and maintained over a balancer chromosome. Consequently, the chromosome carrying bamBW accumulated mutations that have prevented controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation (bamL255F) and a new bam null disruption mutation into the w1118 isogenic background. We assess the fertility of wildtype bam, bamL255F/bamnull hypomorphic, and bamL255F/ bamL255F mutant females, each infected individually with ten W. pipientis wMel variants representing three phylogenetic clades. Overall, we find that all of the W. pipientis variants tested here rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. Additionally, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.

sentences: Genome Size Estimation and Full-length Transcriptome of Sphingonotus tsinlingensis: Genetic Background for the Drought-Adapted Grasshopper Sphingonotus Fieber, 1852 (Orthoptera: Acrididae) is a species-rich grasshopper genus with ~146 species. All species of this genus prefer dry environments, such as: desert, steppe, sand, and stony benchland. This genomic study aimed to understand the evolution and ecology of these grasshopper species. Here, the genome size of Sphingonotus tsinlingensis was estimated using flow cytometry and the first high-quality full-length transcriptome of this species is presented, which may serve as a reference genetic resource for the drought-adapted grasshopper species of Sphingonotus Fieber. The genome size of Sphingonotus tsinlingensis was ~12.8 Gb. Based on the 146.98 Gb Pacbio isoform sequencing data, 221.47 Mb full-length transcripts were assembled. Among these transcripts, 88,693 non-redundant isoforms were identified with an average length of 2,497 bp and an N50 value of 2,726 bp, which was much longer than the formal grasshopper transcriptome assemblies. A total of 48,502 protein coding sequences were determined, and 37,569 were annotated in public gene function databases. A total of 36,488 simple tandem repeats, 12,765 long non-coding RNAs, and 414 transcription factors were also identified. According to gene functions, 70 heat shock proteins and 61 P450 genes that may correspond to drought adaptation of S. tsinlingensis were identified. The genome of Sphingonotus tsinlingensis is an ultra-large and complex genome. Full-length transcriptome sequencing is an ideal strategy for genomic research. This is the first full-length transcriptome of the genus Sphingonotus. The assembly parameters were better than all known grasshopper transcriptomes. This full-length transcriptome may be used to understand its genetic background and the evolution and ecology of grasshoppers.

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Novel Thyroid-Specific Autoantibodies in Patients with Immune-Related Adverse Events Involving the Thyroid Gland AimsProgrammed cell death-1 (PD-1) blockade therapy frequently results in immune-related adverse events involving the thyroid gland (thyroid irAEs). Although clinical features of thyroid irAEs are known, the mechanisms remain unclear. Here, we conducted a pilot study to investigate mechanisms of thyroid irAE development from the perspective of autoantibodies.

MethodsWe performed immunoprecipitation-based assays using sera of 3 patients who developed thyroid irAEs with PD-1 blockade therapy by nivolumab and HEK293T cell lysates, including overexpressed proteins of interest (NKX2-1, PAX8, FOXE1, and HHEX; thyroid-specific transcriptional factors). The pellets were analyzed by western blot to detect the HiBit tag attached to the C-terminus of the proteins.

ResultsRelevant changes to NKX2-1 bands were not seen in all 3 patients, but PAX8 bands were augmented in patient 2 with lung cancer and patient 3 with renal cell carcinoma. In addition, FOXE1 bands were augmented in patient 1 with malignant melanoma and patient 3, and a HHEX band was augmented in patient 3. Thus, we revealed novel thyroid-specific autoantibodies, PAX8Ab, FOXE1Ab, and HHEXAb. Expression patterns of the antigens recognized by these antibodies were not identical to the primary sites, so autoimmune responses in thyroid irAE may originate from the thyroid gland, and not the malignancy. Considering that TPOAb rather than TgAb is often negative in patients with thyroid irAEs, other mechanisms such as cytotoxic T cell and antigenicity of thyroglobulin may be involved.

ConclusionsAlthough the significance of these novel autoantibodies needs further examination, the present study provides new insights for thyroid autoimmunity.

sentences: Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKVs complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoproteins post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.

sentences: Paraoxonase and acylated homoserine lactones in urine from patients with urinary tract infections--relationship to microbial diversity by 16S rRNA gene sequencing Paraoxonase (PON) comprises a trio of mammalian enzymes that have been reported to have a number of roles including the inhibition of bacterial virulence and biofilm formation by microorganisms that quorum sense with acylated homoserine lactones (AHLs). PON have previously been reported to inhibit P. aeruginosa biofilm formation in mammalian airways and skin. An innate immune role for PON in urinary tract infection has not previously been reported. We performed western blots for PON1 in urine from patients with urinary tract infection (UTI), and also tested UTI urine for the presence of AHLs using a cellular reporter system. Urine sample microbiota was assessed through sequencing of the 16S rRNA marker gene. We report here that PON1 was not found in the urine of control subjects, however, in patients with UTI, PON1 was associated with the presence of E. coli in urine. AHLs, but not PON, were found in the bulk urine of those with P. aeruginosa UTI. Microbial consortia of PON positive UTI urine was found to be distinct from PON negative UTI urine; differentially over-represented bacteria in PON positive samples included a number of environmental opportunists. We hypothesize that PON may inhibit the quorum sensing activity of AHLs in UTI, as has previously described in skin and airways.

sentences: Supergenes origin and maintenance in Atlantic cod Supergenes are sets of genes that are inherited as a single marker and encode complex phenotypes through their joint action. They are identified in an increasing number of organisms, yet their origins and evolution remain enigmatic. In Atlantic cod, four megabase-scale supergenes have been identified and linked to migratory lifestyle and environmental adaptations. Here, we investigate the origin and maintenance of these four supergenes through analysis of whole-genome-sequencing data, including a new long-read-based genome assembly for a non-migratory Atlantic cod individual. We corroborate that chromosomal inversions underlie all four supergenes, and show that they originated at different times between 0.40 and 1.66 million years ago. While we found no evidence for a role of introgression in the origin of the four supergenes, we reveal gene flux between supergene haplotypes with derived and ancestral arrangements, occurring both through gene conversion and double crossover. Our results suggest that supergenes can be maintained over long timescales in the same way as hybridizing species, through the purging of introduced genetic variation.

sentences: A population-level invasion by transposable elements triggers genome expansion in a fungal pathogen AO_SCPLOWBSTRACTC_SCPLOWGenome evolution is driven by the activity of transposable elements (TEs). The spread of TEs can have deleterious effects including the destabilization of genome integrity and expansions. However, the precise triggers of genome expansions remain poorly understood because genome size evolution is typically investigated only among deeply divergent lineages. Here, we use a large population genomics dataset of 284 individuals from populations across the globe of Zymoseptoria tritici, a major fungal wheat pathogen. We built a robust map of genome-wide TE insertions and deletions to track a total of 2,456 polymorphic loci within the species. We show that purifying selection substantially depressed TE frequencies in most populations but some rare TEs have recently risen in frequency and likely confer benefits. We found that specific TE families have undergone a substantial genome-wide expansion from the pathogens center of origin to more recently founded populations. The most dramatic increase in TE insertions occurred between a pair of North American populations collected in the same field at an interval of 25 years. We find that both genome-wide counts of TE insertions and genome size have increased with colonization bottlenecks. Hence, the demographic history likely played a major role in shaping genome evolution within the species. We show that both the activation of specific TEs and relaxed purifying selection underpin this incipient expansion of the genome. Our study establishes a model to recapitulate TE-driven genome evolution over deeper evolutionary timescales.

sentences: Harnessing genetic diversity in the USDA pea (Pisum sativum L.) germplasm collection through genomic prediction Phenotypic evaluation and efficient utilization of germplasm collections can be time-intensive, laborious, and expensive. However, with the plummeting costs of next-generation sequencing and the addition of genomic selection to the plant breeders toolbox, we now can more efficiently tap the genetic diversity within large germplasm collections. In this study, we applied and evaluated genomic selections potential to a set of 482 pea accessions - genotyped with 30,600 single nucleotide polymorphic (SNP) markers and phenotyped for seed yield and yield-related components - for enhancing selection of accessions from the USDA Pea Germplasm Collection. Genomic prediction models and several factors affecting predictive ability were evaluated in a series of cross-validation schemes across complex traits. Different genomic prediction models gave similar results, with predictive ability across traits ranging from 0.23 to 0.60, with no model working best across all traits. Increasing the training population size improved the predictive ability of most traits, including seed yield. Predictive abilities increased and reached a plateau with increasing number of markers presumably due to extensive linkage disequilibrium in the pea genome. Accounting for population structure effects did not significantly boost predictive ability, but we observed a slight improvement in seed yield. By applying the best genomic prediction model (e.g., RR-BLUP), we then examined the distribution of genotyped but nonphenotyped accessions and the reliability of genomic estimated breeding values (GEBV). The distribution of GEBV suggested that none of the nonphenotyped accessions were expected to perform outside the range of the phenotyped accessions. Desirable breeding values with higher reliability can be used to identify and screen favorable germplasm accessions. Expanding the training set and incorporating additional orthogonal information (e.g., transcriptomics, proteomics, metabolomics, physiological traits, etc.) into the genomic prediction framework could enhance prediction accuracy.

sentences: Development of a Novel PROTAC using the Nucleic Acid Aptamer as a Targeting Ligand for Tumor Selective Degradation of Nucleolin PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and have poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs.

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Social mobility and biological aging among older adults in the United States Lower socioeconomic status is associated with faster biological aging, the gradual and progressive decline in system integrity that accumulates with advancing age. Efforts to promote upward social mobility may therefore extend healthy lifespan. However, recent studies suggest that upward mobility may also have biological costs related to the stresses of crossing social boundaries. We analyzed blood-chemistry and DNA methylation (DNAm) data from n=9286 participants in the 2016 Health and Retirement Study (HRS) Venous Blood Study to test associations of life-course social mobility with biological aging. We quantified social mobility from childhood to later-life using data on childhood family characteristics, educational attainment, and wealth accumulation. We quantified biological aging using three DNA methylation "clocks" and three blood-chemistry algorithms. We observed substantial social mobility among study participants. Those who achieved upward mobility exhibited less-advanced and slower biological aging. Associations of upward mobility with less-advanced and slower aging were consistent for blood-chemistry and DNAm measures of biological aging and were similar for men and women and for Black and White Americans (Pearson-r effect-sizes ~0.2 for blood-chemistry measures and the DNAm GrimAge clock and DunedinPoAm pace-of-aging measures; effect-sizes were smaller for the DNAm PhenoAge clock). Analysis restricted to educational mobility revealed differential effects by racial identity, suggesting that mediating links between educational mobility and healthy aging may be disrupted by structural racism. In contrast, mobility producing accumulation of wealth appeared to benefit White and Black Americans equally, suggesting economic intervention to reduce wealth inequality may have potential to heal disparities in healthy aging.

Significance StatementUpward social mobility may disrupt effects of early-life disadvantage on aging-related health decline. However, the stresses of crossing social boundaries can have biological costs. To investigate the balance of these forces, we analyzed social mobility from reports of childhood circumstances, education, and later-life wealth in 9,286 older adults in the US Health and Retirement Study. We quantified life-course health impacts of social mobility from blood-chemistry and DNA-methylation analysis of biological aging. We found that educational mobility alone benefited Black Americans less than White Americans, whereas mobility that produced accumulation of wealth into later-life was associated with delayed biological aging across social categories. Black-White disparities in healthy-aging outcomes of educational mobility may reflect inequalities in social gains realized from education.

sentences: CpG enrichment as a vaccine strategy for influenza A virus Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. However, such recoding typically hinders virus growth. A potential solution to this is CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), leading to viral RNA degradation and/or type I interferon stimulation and consequently reduced viral yields. Therefore, in principle, removing the ZAP CpG sensor from a virus propagation system will reverse attenuation of a CpG-enriched virus. Using influenza A viruses (IAVs) engineered for increased CpG content in different genome regions, we show that ZAP mediated virus attenuation is dependent on the viral segment to which CpGs are added. In ZAP-expressing human cells, CpG introduction invariably impaired transcript and protein production, but ZAP-sensitivity did not convey type I interferon activation. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Furthermore, the ZAP-sensitive virus was fully replication competent in MDCK cells and in embryonated hens eggs, both of which are used to propagate live attenuated influenza vaccines. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform for live attenuated vaccine development.

sentences: Establishment and Resilience of Transplanted Gut Microbiota in Aged Mice Fecal microbiota transplantation (FMT), a procedure in which fecal material is transferred from a donor to a recipient, has been increasingly used as a treatment to restore healthy gut microbiota. There is a substantial difference in the composition of gut microbiota between young and aged hosts, but little is known about whether age matching between the FMT donor and recipient affects microbiota restoration and long-term maintenance. In the present investigation, we aimed to study the establishment and resilience of transplanted gut microbiota in aged recipients. We treated naturally aged mice (20 months old) with a broad-spectrum antibiotic cocktail and monitored the restoration of gut microbiota over 8 weeks. The diversity of gut microbiota in aged mice failed to reach the baseline level via spontaneous recovery; in contrast, FMT from either (age-)matched or unmatched donors facilitated the recovery of gut microbiota diversity. The microbiota transplanted from different donors successfully established in the aged recipients and had long-term effects on the gene expression profiles of the host colon. Finally, we evaluated the long-term maintenance of transplanted microbiota via intentional disruption of gut homeostasis. We found that lack of age matching between FMT donors and recipients may decrease the resilience of transplanted gut microbiota against colonic inflammation. The results from our study systematically examining the effects of FMT on the gut homeostasis of aged hosts suggest that the compatibility between donors and recipients should be taken into account when implementing FMT.

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Leveraging big data for classification of children who stutter from fluent peers IntroductionLarge datasets, consisting of hundreds or thousands of subjects, are becoming the new data standard within the neuroimaging community. While big data creates numerous benefits, such as detecting smaller effects, many of these big datasets have focused on non-clinical populations. The heterogeneity of clinical populations makes creating datasets of equal size and quality more challenging. There is a need for methods to connect these robust large datasets with the carefully curated clinical datasets collected over the past decades.

MethodsIn this study, resting-state fMRI data from the Adolescent Brain Cognitive Development study (N=1509) and the Human Connectome Project (N=910) is used to discover generalizable brain features for use in an out-of-sample (N=121) multivariate predictive model to classify young (3-10yrs) children who stutter from fluent peers.

ResultsAccuracy up to 72% classification is achieved using 10-fold cross validation. This study suggests that big data has the potential to yield generalizable biomarkers that are clinically meaningful. Specifically, this is the first study to demonstrate that big data-derived brain features can differentiate children who stutter from their fluent peers and provide novel information on brain networks relevant to stuttering pathophysiology.

DiscussionThe results provide a significant expansion to previous understanding of the neural bases of stuttering. In addition to auditory, somatomotor, and subcortical networks, the big data-based models highlight the importance of considering large scale brain networks supporting error sensitivity, attention, cognitive control, and emotion regulation/self-inspection in the neural bases of stuttering.

sentences: Detection and molecular characterization of a phytoplasma associated with Cucumber (Cucumis sativus) and its first report from India Cucumber (Cucumis sativus) plants exhibiting typical phyllody symptoms were collected from farmers field of Chintamani, Chickballapur districts of Karnataka (India). Disease incidence of phyllody was 2-3%. The etiology of the cucumber phyllody phytoplasma (CuPP) was confirmed by amplifying 16S rRNA gene from symptomatic plants using PCR followed by nested PCR using universal primers pairs P1/P7 and R16F2n/R16R2. After general detection the non-ribosomal SecY and rp (ribosomal protein) genes was amplified using specific primers. The PCR amplified products 16SrRNA (1.2 kb), SecY (1.6 kb) rp gene (1.2 kb) was cloned and sequenced. Sequence and phylogenetic analyses of 16S rRNA, secY and rp genes revealed that the detected phytoplasma is a member of the 16SrI group (Candidatus Phytoplasma asteris). Further on the basis of computer-simulated RFLP (= in silico RFLP) analysis of amplified F2n/R2 region of 16S rRNA gene indicates that, the detected phytoplasma was belongs to the subgroup X (16SrII-X). This is the first report on phytoplasma associated with phyllody disease of cucumber in India.

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In-land long-term culture of colonial ascidians Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.

To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.

Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

HighlightsO_LIFirst artificial seawater based recirculating aquaculture for colonial ascidians
C_LIO_LIOver 3 years of continuous breeding
C_LIO_LISemi-automated setup with minimized maintenance
C_LIO_LIGood biomass production for strain propagation
C_LIO_LI4 different culture conditions for optimized breeding for species of interest
C_LI

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Rapid and Visible RPA-Cas12a fluorescence Assay for Accurate Detection of Zoonotic Dermatophytes BackgroundDermatophytosis is an infectious disease of global significance caused by several fungal species, which affects the hair, nails, or superficial layers of the skin. The most common zoonotic dermatophytes are Microsporum canis, Nannizzia gypsea and Trichophyton mentagrophytes. Woods lamp examination, microscopic identification and fungal culture are the main conventional diagnostic methods used in clinics. Less common methods are dermatophyte PCR and biopsy/histopathology. However, these methods also have limitations for providing both accuracy and timely on-site detection. The recent development of CRISPR-based diagnostic platform provides the possibility of a rapid, accurate, and portable diagnostic tool, which has huge potential for clinical applications.

ObjectivesThe purpose of this study is to establish a molecular method for rapid and accurate diagnosis of clinical dermatophytes, which can accelerate clinical diagnostic testing and help timely treatment.

MethodsIn this paper, we design a Cas12a-based assay combined with recombinase polymerase amplification (RPA) to differentiate three main zoonotic dermatophytes. The limit of detection (LOD) is determined by using standard strains. A total of 25 clinical samples (hair and scurf) are identified to evaluate the sensitivity and specificity of this assay.

ResultsThe RPA-Cas12a method showed high sensitivity and specificity (100% and 100%, respectively). The results could be observed directly by naked-eyes, and all tested samples were consistent with fungal culture and sequencing results.

ConclusionsCompared with other methods, the RPA-Cas12a-fluorescence assay requires less time (30 minutes) and less complicated equipment, and visible changes can be clearly observed, which is suitable for on-site clinical diagnosis.

sentences: SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning SMAD4 regulates gene expression in response to BMP and TGF{beta} signal transduction and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we use an epitope-tagged Smad4 allele for ChIP-seq analysis together with transcriptome analysis of wild-type and mouse forelimb buds lacking Smad4 in the mesenchyme. This analysis identifies the SMAD4 target genes during establishment of the feedback signaling system and establishes that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the initial analysis reveals that the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels reduced in Smad4-deficient limb bud mesenchymal progenitors. The SMAD4 target GRNs includes genes, whose expression in the anterior limb bud is up-regulated by interactions of SMAD4 complexes with enhancers active in the anterior mesenchyme. This reveals a predominant function of SMAD4 in up-regulating target gene expression in the anterior limb bud mesenchyme. Analysis of differentially expressed genes that are shared between Smad4- and Shh-deficient limb buds corroborates the positive role of SMAD4 in transcriptional regulation of anterior genes and reveals a repressive effect on posterior genes that are positively regulated by SHH signaling. This analysis uncovers the overall opposing effects of SMAD4-mediated BMP and SHH signalling on transcriptional regulation during early limb bud development. In summary, this analysis indicates that during early digit patterning and limb bud outgrowth, the anterior/proximal and proximo/distal expression dynamics of co-regulated genes are controlled by distinct and contrasting trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction.

sentences: De-noising Spatial Transcriptomics Data Based on Position and Image Information Spatial transcriptomics (ST) technologies provide gene expression close to or even superior to single-cell resolution while retaining the physical locations of sequencing and often also providing matched pathology images. However, the expression data captured by ST technologies suffer from high noise levels, as a result of the shallow coverage in each sequencing unit. The extra experimental steps for preserving the spatial locations of sequencing could result in even more severe noises, compared to regular single-cell RNA-sequencing (scRNA-seq). Fortunately, such noises could be largely removed by leveraging information from the physical locations of sequencing, and the tissue and cellular organization reflected by corresponding pathology images. In this work, we demonstrated the extensive levels of noise in ST data. We developed a mathematical model, named Sprod, to impute accurate ST gene expression based on latent space and graph learning of matched location and imaging data. We comprehensively validated Sprod and demonstrated its advantages over prior methods for removing drop-outs in scRNA-seq data. We further showed that, after adequate imputation by Sprod, differential expression analyses, pseudotime analyses, and cell-to-cell interaction inferences yield significantly more informative results. Overall, we envision denoising by Sprod to become a key first step to empower ST technologies for biomedical discoveries and innovations.

sentences: TREM2-H157Y Increases Soluble TREM2 Production and Reduces Amyloid Pathology The p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) has been reported to increase Alzheimers disease (AD) risk. This mutation in the extracellular domain of TREM2 localizes at the cleavage site, leading to enhanced shedding. Here, we generated a novel Trem2 H157Y knock-in mouse model to investigate how this H157Y mutation impacts TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology. Consistent with previous in vitro findings, TREM2-H157Y increases the amount of soluble TREM2 (sTREM2) in the cortex and serum of mutant mice compared to the wild type controls. Interestingly, the Trem2 H157Y variant enhances synaptic plasticity without affecting microglial density and morphology. In the presence of amyloid pathology, TREM2-H157Y surprisingly accelerates A{beta} clearance and reduces amyloid burden and microgliosis. Taken together, our findings support a beneficial effect of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD, we speculate TREM2-H157Y might increase AD risk through an amyloid-independent pathway, as such its effects on tauopathy and neurodegeneration merit further investigation.

sentences: The complete sequence of a human genome In 2001, Celera Genomics and the International Human Genome Sequencing Consortium published their initial drafts of the human genome, which revolutionized the field of genomics. While these drafts and the updates that followed effectively covered the euchromatic fraction of the genome, the heterochromatin and many other complex regions were left unfinished or erroneous. Addressing this remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium has finished the first truly complete 3.055 billion base pair (bp) sequence of a human genome, representing the largest improvement to the human reference genome since its initial release. The new T2T-CHM13 reference includes gapless assemblies for all 22 autosomes plus Chromosome X, corrects numerous errors, and introduces nearly 200 million bp of novel sequence containing 2,226 paralogous gene copies, 115 of which are predicted to be protein coding. The newly completed regions include all centromeric satellite arrays and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies for the first time.

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Distributed processing for action control by prelimbic circuits targeting anterior-posterior dorsal striatal subregions. Fronto-striatal circuits have been extensively implicated in the cognitive control of behavioral output for both social and appetitive rewards. The functional diversity of prefrontal cortical populations is strongly dependent on their synaptic targets, with control of motor output strongly mediated by connectivity to the dorsal striatum. Despite evidence for functional diversity along the anterior-posterior axis of the dorsomedial striatum (DMS), it is unclear how distinct fronto- striatal sub-circuits support neural computations essential for action selection. Here we identify prefrontal populations targeting distinct DMS subregions and characterize their functional roles. We first performed neural circuit tracing to reveal segregated prefrontal populations defined by anterior/posterior dorsomedial striatal target. We then probed the functional relevance of these parallel circuits via in vivo calcium imaging and temporally precise causal manipulations during a feedback-based 2-alternative choice task. Single-photon imaging revealed circuit-specific representations of task-relevant information with prelimbic neurons targeting anterior DMS (PL::A- DMS) uniquely encoded choices and responses to negative outcomes, while prelimbic neurons targeting posterior DMS (PL::P-DMS) encoded internal representations of value and positive outcomes contingent on prior choice. Consistent with this distributed coding, optogenetic inhibition of PL::A-DMS circuits strongly impacted choice monitoring and behavioral control in response to negative outcomes while perturbation of PL::P-DMS signals impaired task engagement and strategies following positive outcomes. Di-synaptic retrograde tracing uncovered differences in afferent connectivity that may underlie these pathways functional divergence. Together our data uncover novel PL populations engaged in distributed processing for action control.

SUMMARYO_LIPrelimbic cortex engages A- and P-DMS via distinct circuits
C_LIO_LIPL::A-DMS and PL::P-DMS pathways encode divergent aspects of a simple goal-directed task
C_LIO_LIPL::A-DMS pathways shape responding to negative outcomes via multiple mechanisms
C_LIO_LIPL::P-DMS pathways guide engagement and choices in response to positive outcomes
C_LIO_LIAfferent connectomes of PL neurons defined by A-P DMS target are distinct
C_LI

sentences: Prefrontal cortical connectivity mediates locus coeruleus noradrenergic regulation of inhibitory control in older adults Response inhibition is a core executive function enabling adaptive behaviour in dynamic environments. Human and animal models indicate that inhibitory control and control networks are modulated by noradrenaline, arising from the locus coeruleus. The integrity (i.e., cellular density) of the locus coeruleus noradrenergic system can be estimated from magnetization transfer sensitive magnetic resonance imaging, in view of neuromelanin present in noradrenergic neurons of older adults. Noradrenergic psychopharmacological studies indicate noradrenergic modulation of prefrontal and frontostriatal stopping-circuits in association with behavioural change. Here we test the noradrenergic hypothesis of inhibitory control, in healthy adults. We predicted that locus coeruleus integrity is associated with age-adjusted variance in response inhibition, mediated by changes in connectivity between frontal inhibitory control regions. In a preregistered analysis, we used magnetization transfer MRI images from N=63 healthy adults aged above 50 years who performed a stop-signal task, with atlas-based measurement of locus coeruleus contrast. We confirm that better response inhibition is correlated with locus coeruleus integrity and stronger connectivity between pre-supplementary motor area and right inferior frontal gyrus, but not volumes of the cortical regions. We confirmed a significant role of prefrontal connectivity in mediating the effect of individual differences in the locus coeruleus on behaviour, whereby this effect was moderated by age, over and above adjustment for the mean effects of age. Our results support the hypothesis that in normal populations, as in clinical settings, the locus coeruleus noradrenergic system regulates inhibitory control.

sentences: N-VEGF, the autoregulatory arm of VEGF-A Vascular endothelial growth factor A (VEGF-A) is a secreted protein that stimulates angiogenesis in response to hypoxia. Under hypoxic conditions, a non-canonical long isoform called L-VEGF is concomitantly expressed with VEGF-A. Once translated, L-VEGF it is proteolytically cleaved to generate N-VEGF and VEGF-A. Interestingly, while VEGF-A is secreted and affects the surrounding cells, N-VEGF is mobilized to the nucleus. This suggests that N-VEGF participates in transcriptional response to hypoxia. In this study, we performed a series of complementary experiments to examine the functional role of N-VEGF. Strikingly, we found that the mere expression of N-VEGF followed by its hypoxia-independent mobilization to the nucleus was sufficient to induce key genes associated with angiogenesis, such as Hif1, VEGF-A isoforms, as well as genes associated with cell survival under hypoxia. Complementarily, when N-VEGF was genetically depleted, key hypoxia-induced genes were downregulated and cells were significantly susceptible to hypoxia-mediated apoptosis. This is the first reports of N-VEGF serving as an autoregulatory arm of VEGF-A. Further experiments will be needed to determine the role of N-VEGF in cancer and embryogenesis.

sentences: The Population Genetics of Gene Regulatory Networks The evolution of diverse phenotypes both involves and is constrained by molecular interaction networks. When these networks influence patterns of expression, we refer to them as gene regulatory networks (GRNs). Here, we develop a quasi-species model of GRN evolution. With this model, we prove that-across a broad spectrum of viability and mutation functions-the dynamics converge to a stationary distribution over GRNs. Next, we show from first principles how the frequency of GRNs at equilibrium will be proportional to each GRNs eigenvector centrality in the genotype network. Finally, we determine the structural characteristics of GRNs that are favored in response to a range of selective environments and mutational constraints. Our work connects GRN evolution to quasi-species models, and thus can provide a mechanistic explanation for the topology of GRNs experiencing various evolutionary forces.

sentences: Fluorescence-Activated Droplet Sorting of Polyethylene terephthalate-degrading Enzymes Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. However, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PET-degrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new PET-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of PETases using synthetic biology.

sentences: Genetic analysis of Caenorhabditis elegans pry-1/Axin suppressors identifies genes involved in reproductive structure development, stress response, and aging The Axin family of scaffolding proteins regulates a wide array of developmental and post-developmental processes in eukaryotes. Studies in the nematode, Caenorhabditis elegans, have shown that the Axin homolog, PRY-1, plays essential roles in multiple tissues. To understand the genetic network of pry-1, we focused on a set of genes that are differentially expressed in the pry-1-mutant transcriptome and are linked to reproductive structure development. Eight of the genes (ard-1, rpn-7, cpz-1, his-7, cdk-1, rnr-1, clsp-1, and spp-1), when knocked down by RNA interference, efficiently suppressed the plate-level multivulva phenotype of pry-1 mutants. In every case, other than clsp-1 and spp-1, the ectopic vulval precursor cell (VPC) induction was also inhibited. The suppressor genes are members of known gene families in eukaryotes and perform essential functions. Our genetic interaction experiments revealed that except for clsp-1, the genes participate in one or more pry-1-mediated biological events. While four of them (cpz-1, his-7, cdk-1, and rnr-1) function in VPC induction, stress response, and aging, the other three (spp-1, ard-1, and rpn-7) are specific to one or more of these processes. Further analysis of the genes involved in aging showed that his-7, cdk-1, and rnr-1 also interacted with daf-16/FOXO. The results of genetic epistasis experiments suggested that his-7 functions upstream of daf-16, whereas cdk-1 and rnr-1 act downstream of the pry-1-daf-16 pathway. Altogether, these findings demonstrate the important role of pry-1 suppressors in C. elegans. Given that all of the genes described in this study are conserved, future investigations of their interactions with Axin and their functional specificity promises to uncover the genetic network of Axin under normal and disease states.

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A rare natural lipid induces neuroglobin expression to prevent amyloid oligomers toxicity and retinal neurodegeneration. Most neurodegenerative diseases such as Alzheimers disease are proteinopathies linked to the toxicity of amyloid oligomers. Treatments to delay or cure these diseases are lacking. Using budding yeast, we report that the natural lipid tripentadecanoin induces expression of the nitric oxide oxidoreductase Yhb1 to prevent the formation of protein aggregates during aging and extends replicative lifespan. In mammals, tripentadecanoin induces expression of the Yhb1 orthologue, neuroglobin, to protect neurons against amyloid toxicity. Tripentadecanoin also rescues photoreceptors in a mouse model of retinal degeneration and retinal ganglion cells in a Rhesus monkey model of optic atrophy. Together, we propose that tripentadecanoin affects p-bodies to induce neuroglobin expression and offers a potential treatment for proteinopathies and retinal neurodegeneration.

One Sentence SummaryThe natural lipid tripentadecanoin is cytoprotective against amyloid oligomer toxicity and retinal neurodegeneration by inducing YHBI/neuroglobin expression in yeast and mammals.

sentences: Bacterial-type plant ferroxidases tune local phosphate sensing in root development Fluctuating bioavailability of inorganic phosphate (Pi), often caused by complex Pi-metal interactions, guide root tip growth and root system architecture for maximizing the foraged soil volume. Two interacting genes in Arabidopsis thaliana, PDR2 (P5-type ATPase) and LPR1 (multicopper oxidase), are central to external Pi monitoring by root tips, which is modified by iron (Fe) co-occurrence. Upon Pi deficiency, the PDR2-LPR1 module facilitates cell type-specific Fe accumulation and cell wall modifications in root meristems, inhibiting intercellular communication and thus root growth. LPR1 executes local Pi sensing, whereas PDR2 restricts LPR1 function. We show that native LPR1 displays specific ferroxidase activity and requires a conserved acidic triad motif for high-affinity Fe2+ binding and root growth inhibition under limiting Pi. Our data indicate that substrate availability tunes LPR1 function and implicate PDR2 in maintaining Fe homeostasis. LPR1 represents the prototype of an ancient ferroxidase family, which evolved very early upon bacterial colonization of land. During plant terrestrialization, horizontal gene transfer transmitted LPR1-type ferroxidase from soil bacteria to the common ancestor of Zygnematophyceae algae and embryophytes, a hypothesis supported by homology modeling, phylogenomics, and activity assays of bacterial LPR1-type multicopper oxidases.

sentences: Widespread alterations in microRNA biogenesis in human Huntington's disease putamen Altered microRNA (miRNA) expression is a common feature of Huntingtons disease (HD) and could participate in disease onset and progression. However, little is known about the underlying causes of miRNA disruption in HD. We and others have previously shown that mutant Huntingtin (mHTT) binds to Ago2, a central component of miRNA biogenesis, and disrupts mature miRNA levels. In this study, we sought to determine if miRNA maturation per se was compromised in HD. Towards this end, we characterized t major miRNA biogenesis pathway components and miRNA maturation products (pri-miRNA, pre-miRNA, and mature) in human HD (N=41, Vonsattel grades HD2-4) and healthy control (N=25) subjects. Notably, the striatum (putamen) and cortex (BA39) from the same individuals were analyzed in parallel. We show that Ago2, Drosha, and Dicer were strongly downregulated in human HD at early stages of disease. Using a panel of HD-related miRNAs (miR-10b, miR-196b, miR-132, miR-212, miR-127, miR-128), we uncovered various types of maturation defects in HD brain, the most prominent occurring at the pre-miRNA to mature miRNA maturation step. Consistent with earlier findings, we provide evidence that alterations in autophagy could participate in miRNA maturation defects. Notably, most changes occurred in the striatum, which is more prone to HTT aggregation and neurodegeneration. Likewise, we observed no significant alterations in miRNA biogenesis in human HD cortex and blood, strengthening tissue-specific effects. Overall, these data provide important clues into the underlying mechanisms behind miRNA alterations in HD-susceptible tissues. Further investigations are now required to understand the biological, diagnostic, and therapeutic implications of miRNA/RNAi biogenesis defects in HD and related neurodegenerative disorders.

sentences: Association of stress-related limbic activity and baseline interleukin-6 plasma levels in healthy adults Several studies suggest a link between acute changes in inflammatory parameters due to an endotoxin or (psychological) stressor and the brains stress response. The extent to which basal circulating levels of inflammatory markers are associated with the brains stress response has been hardly investigated so far. In the present study, plasma levels of the cytokine interleukin (IL)-6 were obtained as a baseline measure of peripheral inflammatory processes and linked to neural markers of psychosocial stress using a modified version of the Montreal Imaging Stress Task in a sample of N=65 healthy subjects. Of three a-priori defined regions of interest - the amygdala, anterior insula, and anterior cingulate cortex - baseline IL-6 was significantly and negatively associated with stress-related neural activation in the right amygdala and left anterior insula. Our results suggest that baseline inflammatory physiology might be related to differences in the neural stress response and that this relationship could be inverse to that previously shown by studies on acutely induced inflammation.

sentences: Deficiency of the lysosomal protein CLN5 alters lysosomal function and movement Batten disease is a devastating childhood rare neurodegenerative disease characterized by rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed neutralised lysosomal acidity and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement - a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.

sentences: A single nucleotide polymorphism determines constitutive versus inducible type VI secretion in Vibrio cholerae Vibrio cholerae is a well-studied human pathogen that is also a common inhabitant of marine habitats. In both environments, the bacterium is subject to interbacterial competition. A molecular nanomachine that is often involved in such competitive behavior is the type VI secretion system (T6SS). Interestingly and in contrast to non-pandemic or environmental isolates, the T6SS of the O1 El Tor clade of V. cholerae, which is responsible for the ongoing 7th pandemic, is largely silent under standard conditions. Instead, these strains induce their full T6SS capacity only under specific conditions such as growth on chitinous surfaces (signaled through TfoX and QstR) or when the cells encounter low intracellular c-di-GMP levels (TfoY-driven). In this study, we identified a single nucleotide polymorphism (SNP) within an intergenic region of the major T6SS gene cluster of V. cholerae that determines the T6SS status of the cell. We show that SNP conversion is sufficient to induce T6SS production in numerous pandemic strains, while the converse approach renders non-pandemic/environmental V. cholerae strains T6SS-silent. We further demonstrate that SNP-dependent T6SS production occurs independently of the known T6SS regulators TfoX, QstR, and TfoY. Finally, we identify a putative promoter region adjacent to the identified SNP that is required for all forms of T6SS regulation.

sentences: EphB1 controls proper long-range cortical axon guidance through a cell non-autonomous role in GABAergic cells EphB1 is required for proper guidance of cortical long-range axon projections during brain development, but how EphB1 regulates this process remains unclear. To determine the relevant cell types in which EphB1 functions for proper cortical axon guidance, we generated a new floxed EphB1 mouse. We show here that EphB1 conditional knockout (cKO) in most of the developing cortical excitatory neurons (Emx1-Cre) failed to produce cortical axon guidance defects. However, EphB1 cKO in GABAergic cell populations (Vgat-Cre or dlx1/2-Cre) reproduced fully the cortical axon guidance defects observed in global EphB1-/- mice. Interestingly, in the EphB1 cKOVgat mice, the misguided axon bundles in the dorsal striatum contained comingled striatal GABAergic and cortical glutamatergic axons. Surprisingly, conditional loss of EphB1 in D1 or D2 dopamine receptor-expressing populations did not produce the cortical axon guidance defects, and no D1-or D2-SPN axons were detected within the misguided axon bundles of EphB1 KO mice. In contrast, parvalbumin-positive GABAergic cell axons were observed in the misguided axon bundles in EphB1 KO mice. Finally, a large fraction of the ectopic axons in EphB1 KO mice were in close apposition to the major blood vessels in the ventral telencephalon, suggesting that EphB1 expression in GABAergic cells normally promotes avoidance of these ectopic long-range axons from the developing cerebral vasculature. Together, our data reveal a new role for EphB1 in GABAergic cells to influence proper cortical glutamatergic long-range axon guidance during early brain development.

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Common genetic variants in CHRNA5 alter β-amyloid neuropathology and highlight chandelier cells in human aging and Alzheimer's disease Changes in high-affinity nicotinic acetylcholine receptors are intricately connected to cognitive impairment in Alzheimers Disease (AD). While preclinical work suggests a possible protective and cognitive-enhancing role for the auxiliary nicotinic 5 subunit, mRNA expression of the CHRNA5 gene has not been closely examined in the context of human aging and dementia. Here, we investigate the impact of two common SNPs predicted to have different effects on the nicotinic 5 subunit: the function-compromising rs16969968 and the expression-altering rs1979905. We analyzed data from human prefrontal cortex (922 subjects with matched genotypic and post-mortem RNA sequencing, including 22 subjects with single-nucleus data) of elderly participants in the Religious Orders Study and Memory and Aging Project (ROS/MAP). We tested the impact of SNP haplotypes on prefrontal expression of CHRNA5 as well as that of SNPs in its receptor partners: CHRNA4 and CHRNB2. In the context of cognition and neuropathology, we observed a significant negative association between the high CHRNA5-expressing rs1979905A2 genotype and {beta}-amyloid load in the prefrontal cortex. Our analyses revealed that the greatest abundance of CHRNA5 expression was in chandelier neurons, a sparse but disproportionately-powerful set of interneurons, in addition to layer 5 and 6 pyramidal neurons. In subjects with functionally-unaltered CHRNA5, the proportion of chandelier cells in the prefrontal cortex shows resilience to {beta}-amyloid load, but this resilience is diminished in subjects homozygous for the minor allele of the CHRNA5 missense SNP rs16969968. Taken together, these findings urge further investigation into the role of CHRNA5 and chandelier cells in AD neuroprotection.

Significance statementThe nicotinic 5 subunit is important for attention and cognition, however its relationship to aging and neuropathology is unknown. Our findings suggest that polymorphisms affecting CHRNA5 expression associate with altered brain amyloid pathology. Cell type-specific expression of CHRNA5 in the prefrontal cortex implicates chandelier interneurons as a focus of 5-mediated nicotinic signalling, potentially involved in regulating amyloid load. We find that a missense CHRNA5 polymorphism is correlated with increased sensitivity of this class of interneuron to amyloid pathology. A neuroprotective role for CHRNA5 identifies it as a possible target for therapies to improve neuron survival and excitatory/inhibitory balance in AD pathology.

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Effects of face masks and ventilation on the risk of SARS-CoV-2 respiratory transmission in public toilets: a quantitative microbial risk assessment Public toilets could increase the risk of COVID-19 infection via airborne transmission; however, related research is limited. We aimed to estimate SARS-CoV-2 infection risk through respiratory transmission using a quantitative microbial risk assessment framework by retrieving SARS-CoV-2 concentrations from the swab tests of 251 Thai patients. Three virus-generating scenarios were investigated: an infector breathing, breathing with a cough, and breathing with a sneeze. Infection risk (97.5th percentile) was as high as 10-3 with breathing and increased to 10-1 with a cough or sneeze, thus all higher than the risk benchmark of 5 x 10-5 per event. No significant gender differences for toilet users (receptors) were noted. The highest risk scenario of breathing and a sneeze was further evaluated for risk mitigation measures. Risk mitigation to lower than the benchmark succeeded only when the infector and receptor simultaneously wore an N95 respirator or surgical mask and when the receptor wore an N95 respirator and the infector wore a denim fabric mask. Ventilation up to 20 air changes per hour (ACH), beyond the 12-ACH suggested by the WHO, did not mitigate risk. Virus concentration, volume of expelled droplets, and receptor dwell time were identified as the main contributors to transmission risk.

Highlights- The use of public toilets poses a risk of SARS-CoV-2 respiratory transmission
- Highest risks generated in the order of sneezing, coughing, and breathing
- No gender differences in risk by counteracting dwell times and inhalation rates
- Ventilation did not reduce risk even at 20 ACH, beyond the WHO-recommended value
- N95 and surgical masks offer the most effective risk mitigation to toilet users

Graphical abstract

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sentences: Genetic diversity in chimpanzee transcriptomics does not represent wild populations Chimpanzees (Pan troglodytes) are a genetically diverse species, consisting of 4 highly distinct subspecies. As humans closest living relative they have been a key model organism in the study of human evolution, and comparisons of human and chimpanzee transcriptomes have been widely used to characterise differences in gene expression levels that could underlie the phenotypic differences between the two species. However, the subspecies from which these transcriptomic datasets have been derived is not recorded in metadata available in the public NCBI Sequence Read Archive (SRA). Furthermore, labelling of RNA-seq samples is for the most part inconsistent across studies, and the true number of individuals from whom transcriptomic data is available is difficult to ascertain. Thus we have evaluated genetic diversity at the subspecies and individual level in 486 public RNA-seq samples available in the SRA, spanning the vast majority of public chimpanzee transcriptomic data. Using multiple population genetics approaches we find that nearly all samples (96.6%) have some degree of Western chimpanzee ancestry. At the individual donor level, we identify multiple samples that have been repeatedly analysed across different studies, and identify a total of 135 genetically distinct individuals within our data, a number that falls to 89 when we exclude likely first and second-degree relatives. Altogether, our results show that current transcriptomic data from chimpanzees is capturing low levels of genetic diversity relative to what exists in wild chimpanzee populations. These findings provide important context to current comparative transcriptomics research involving chimpanzees.

sentences: Metagenomic analysis reveals the signature of gut microbiota associated with human chronotypes Patterns of diurnal activity differ substantially between individuals, with early risers and late sleepers being examples of opposite chronotypes. Growing evidence suggests that the late chronotype significantly impacts the risk of developing mood disorders, obesity, diabetes, and other chronic diseases. Despite the vast potential of utilizing chronotype information for precision medicine, those factors that shape chronotypes remain poorly understood. Here, we assessed whether the various chronotypes are associated with different gut microbiome compositions. Using metagenomic sequencing analysis, we established a distinct signature associated with chronotype based on two bacterial genera, Alistipes (elevated in "larks") and Lachnospira (elevated in "owls"). We identified three metabolic pathways associated with the early chronotype, and linked distinct dietary patterns with different chronotypes. Our work demonstrates an association between the gut microbiome and chronotype and may represent the first step towards developing dietary interventions aimed at ameliorating the deleterious health correlates of the late chronotype.

sentences: Calbindin regulates Kv4.1 trafficking and excitability of dentate granule cells via CaMKII-dependent phosphorylation Calbindin, a major Ca2+ buffer in dentate granule cells (GCs), plays a critical role in shaping Ca2+ signals, yet how it regulates neuronal functions remains largely unknown. Here, we found that calbindin knock-out mice (CBKO) exhibited hyperexcitability in dentate GCs and impaired pattern separation, which was concurrent with reduced K+ current due to downregulated surface expression of Kv4.1. Consistently, manipulation of the calbindin expression in HT22 led to changes in CaMKII activation and the level of surface localization of Kv4.1 through phosphorylation at serine 555, confirming the mechanism underlying neuronal hyperexcitability in CBKO. We also discovered that Ca2+ buffering capacity was significantly reduced in the GCs of Tg2576 to the level of CBKO GCs, and this reduction was restored to normal levels by antioxidants, suggesting that calbindin is a target of oxidative stress. Our data suggest that regulation of CaMKII signaling by Ca2+ buffer is crucial for neuronal excitability regulation.

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Brain-wide sensory aberrations in a Parkinson's Disease mouse model revealed by functional MRI Parkinsons disease (PD) is associated with irreparable damage to dopaminergic neurons in brain areas involved in movement. Frequently, a very early symptom of PD involves an impaired sense of smell, while other studies suggest a broader impairment of other sensory modalities. Still, the associated brain-wide mechanisms underlying this phenomenon remain unclear. Here, we harness in vivo functional Magnetic Resonance Imaging (fMRI) at 9.4T to investigate how the olfactory and visual systems are affected in an -Synuclein (Syn) mouse model of PD. We find significant aberrations in fMRI responses along both sensory pathways including decreases in activation extent and in activation amplitude. Deficits in the olfactory system were larger than in the visual pathway, but nevertheless both sensory systems were clearly impacted, suggesting a more overarching mechanism may be involved. To further ensure that the aberrations observed in the fMRI signals were not driven solely by putative vascular deficiency, we quantified c-FOS expression in the olfactory system and found statistically significant decreases in the PD group when compared to age-matched controls. Hence, our findings indicate a neural origin for the observed fMRI aberrations. Our findings thus demonstrate a more global deficiency in sensory systems in the (Syn) mouse model.

One Sentence SummaryParkinsons disease (PD) is associated with an impaired sense of smell and more generally sensory deficits, but the associated brain-wide mechanisms remain unclear; here, we investigate aberrations in BOLD-fMRI responses along sensory pathways in an SYN PD mouse model.

sentences: Concurrent feature-specific reactivation within the hippocampus and neocortex facilitates episodic memory retrieval The hippocampus is a key brain region for the storage and retrieval of episodic memories, but how it performs this function is unresolved. According to the hippocampal indexing theory, the hippocampus stores an event-specific index of the pattern of neocortical activity that occurred during perception. During retrieval, reactivation of the index by a partial cue facilitates the reactivation of the associated neocortical pattern. Therefore, event-specific retrieval requires joint reactivation of the hippocampal index and the associated neocortical networks. To test this theory, we examine the relation between performance on a recognition memory task requiring retrieval of image-specific visual details and feature-specific reactivation within the hippocampus and neocortex. We show that trial-by-trial recognition accuracy correlates with neural reactivation of low-level features (e.g. luminosity and edges) within the posterior hippocampus and early visual cortex for participants with high recognition lure accuracy. As predicted, the two regions interact, such that recognition accuracy correlates with hippocampal reactivation only when reactivation co-occurs within the early visual cortex (and vice-versa). In addition to supporting the hippocampal indexing theory, our findings show large individual differences in the features underlying visual memory and suggest that the anterior and posterior hippocampus represents gist-like and detailed features, respectively.

sentences: Can AlphaFold2 predict protein-peptide complex structures accurately? AO_SCPLOWBSTRACTC_SCPLOWIn this preprint, we investigated whether AlphaFold2, AF2, can predict protein-peptide complex structures only with sequence information. We modeled the structures of 203 protein-peptide complexes from the PepBDB DB and 183 from the PepSet. The structures were modeling with concatenated sequences of receptors and peptides via poly-glycine linker. We found that for more than half of the test cases, AF2 predicted the bound structures of peptides with good accuracy, C-RMSD of a peptide < 3.0 [A]. For about 40% of cases, the peptide structures were modeled with an accuracy of C-RMSD < 2.0 [A]. Our benchmark results clearly show that AF2 has a great potential to be applied to various higher-order structure prediction tasks.

sentences: Hox genes regulate asexual reproductive behavior and tissue segmentation in adult animals Hox genes are highly conserved transcription factors renowned for their roles in the segmental patterning of the embryonic anterior-posterior (A/P) axis1. Emerging evidence for Hox gene expression and function in postnatally derived structures has fueled interest in their additional roles beyond embryogenesis2,3. We report novel functions for Hox genes in A/P adult tissue segmentation and transverse fission behavior underlying asexual reproduction in the planarian flatworm, Schmidtea mediterranea. Silencing of each of the planarian Hox family members identified 5 Hox genes required for asexual reproduction. Among these, silencing of hox3 genes resulted in supernumerary segments, while silencing of post2b eliminated segmentation altogether. The opposing roles of hox3 and post2b in segmentation are paralleled in their respective regulation of fission behavior. Silencing of hox3 increased the frequency of fission behavior initiation, while silencing of post2b eliminated fission behavior entirely. Furthermore, we identified a network of downstream effector genes mediating Hox gene regulation of asexual reproduction, thereby providing insight into their respective mechanisms of action. Our study establishes postembryonic roles for Hox genes in regulating the emergence of tissue segmentation and specific behaviors associated with asexual reproduction in adult animals.

sentences: Parallel inference of hierarchical latent dynamics in two-photon calcium imaging of neuronal populations Dynamic latent variable modelling has provided a powerful tool for understanding how populations of neurons compute. For spiking data, such latent variable modelling can treat the data as a set of point-processes, due to the fact that spiking dynamics occur on a much faster timescale than the computational dynamics being inferred. In contrast, for other experimental techniques, the slow dynamics governing the observed data are similar in timescale to the computational dynamics that researchers want to infer. An example of this is in calcium imaging data, where calcium dynamics can have timescales on the order of hundreds of milliseconds. As such, the successful application of dynamic latent variable modelling to modalities like calcium imaging data will rest on the ability to disentangle the deeper- and shallower-level dynamical systems contributions to the data. To-date, no techniques have been developed to directly achieve this. Here we solve this problem by extending recent advances using sequential variational autoencoders for dynamic latent variable modelling of neural data. Our system VaLPACa (Variational Ladders for Parallel Autoencoding of Calcium imaging data) solves the problem of disentangling deeper- and shallower-level dynamics by incorporating a ladder architecture that can infer a hierarchy of dynamical systems. Using some built-in inductive biases for calcium dynamics, we show that we can disentangle calcium flux from the underlying dynamics of neural computation. First, we demonstrate with synthetic calcium data that we can correctly disentangle an underlying Lorenz attractor from calcium dynamics. Next, we show that we can infer appropriate rotational dynamics in spiking data from macaque motor cortex after it has been converted into calcium fluorescence data via a calcium dynamics model. Finally, we show that our method applied to real calcium imaging data from primary visual cortex in mice allows us to infer latent factors that carry salient sensory information about unexpected stimuli. These results demonstrate that variational ladder autoencoders are a promising approach for inferring hierarchical dynamics in experimental settings where the measured variable has its own slow dynamics, such as calcium imaging data. Our new, open-source tool thereby provides the neuroscience community with the ability to apply dynamic latent variable modelling to a wider array of data modalities.

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Functional organization of frontoparietal cortex in the marmoset investigated with awake resting-state fMRI Frontoparietal networks contribute to complex cognitive functions in humans and macaques such as working memory, attention, task-switching, response suppression, grasping, reaching, and eye movement control. However, little is known about the organization of frontoparietal networks in the New World common marmoset monkey (Callithrix jacchus) which is now widely recognized as a powerful nonhuman primate experimental animal. In this study, we employed hierarchical clustering of interareal BOLD signals to investigate the hypothesis that the organization of the frontoparietal cortex in the marmoset follows the organizational principles of the macaque frontoparietal system. We found that the posterior part of the lateral frontal cortex (premotor regions) was functionally connected to the anterior parietal areas while more anterior frontal regions (frontal eye field (FEF)) were connected to more posterior parietal areas (the area around lateral intraparietal area (LIP)). These overarching patterns of inter-areal organization are consistent with a recent macaque study. These findings demonstrate parallel frontoparietal processing streams in marmosets and support the functional homologies of FEF-LIP and premotor-anterior parietal pathways between marmoset and macaque.

Significant StatementFrontoparietal networks contribute to many cognitive functions in humans and macaques, but little is known about the organization of frontoparietal networks in the New World common marmoset monkey. Here, we investigated the hypothesis that the organization of the frontoparietal cortex in the marmoset follows the organizational principles of the macaque frontoparietal system. These frontoparietal connection showed overarching pattern of interareal organization consistent with Old world monkeys. These findings demonstrate parallel frontoparietal processing streams in marmosets.

sentences: Investigating the causes of stimulus-evoked changes in cone reflectance using a combined adaptive optics SLO-OCT system In vivo functional imaging of human photoreceptors is an emerging field, with compelling potential applications in basic science, translational research, and clinical management of ophthalmic disease. Measurements of light-evoked changes in the photoreceptors has been successfully demonstrated using adaptive optics (AO) coherent flood illumination (CFI), AO scanning light ophthalmoscopy (SLO), AO optical coherence tomography (OCT), and full-field OCT with digital AO (dAO). While the optical principles and data processing of these systems differ greatly, and while these differences manifest in the resulting measurements, we believe that the underlying physiological processes involved in each of those techniques are likely the same. AO-CFI and AO-SLO systems are more widely used than OCT systems. However, those systems produce only two-dimensional images and so, less can be said about the anatomical and physiological origins of the observed signal. OCT signal, on the other hand, provides 3D imaging but at a cost of high volume of data, making it impractical to clinical purposes. In light of this, we employed a combined AO-OCT-SLO system-with point-for-point correspondence between the OCT and SLO images-to measure functional responses simultaneously with both and investigate SLO retinal functional biomarkers based on OCT response. The resulting SLO images reveal reflectance changes in the cones which are consistent with those previously reported using AO-CFI and AO-SLO. The resulting OCT volumes show phase changes in the cone outer segment (OS) consistent with those previously reported by us and others. We recapitulate a model of the cone OS previously proposed to explain AO-CFI reflectance changes, and show how this model can be used to predict the signal in AO-SLO. The limitations of the model is also discussed in this manuscript.

sentences: A universal pocket in FAALs ensures redirection of fatty acid pool away from CoA-based activation Fatty acyl-AMP ligases (FAALs) channelize fatty acids towards biosynthesis of virulent lipids in mycobacteria and other pharmaceutically or ecologically important polyketides and lipopeptides in other microbes. They do so by bypassing the ubiquitous coenzyme A-dependent activation and rely on the acyl carrier protein-tethered 4-phosphopantetheine (holo-ACP). The molecular basis of how FAALs strictly reject chemically identical and abundant acceptors like coenzyme A (CoA) and accept holo-ACP unlike other members of the ANL superfamily remains elusive. We show FAALs have plugged the promiscuous canonical CoA-binding pockets and utilize highly selective alternative binding sites. These alternative pockets can distinguish adenosine 3, 5-bisphosphate-containing CoA from holo-ACP and thus FAALs can distinguish between CoA and holo-ACP. These exclusive features helped identify the omnipresence of FAAL-like proteins and their emergence in plants, fungi, and animals with unconventional domain organisations. The universal distribution of FAALs suggests they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites.

sentences: A minimal model of neural computation with dendritic plateau potentials. AO_SCPLOWBSTRACTC_SCPLOWOver the last two decades, advances in neurobiology have established the essential role of active processes in neural dendrites for almost every aspect of cognition, but how these processes contribute to neural computation remains an open question. We show how two kinds of events within the dendrite, synaptic spikes and localized dendritic plateau potentials, interact on two distinct timescales to give rise to a powerful model of neural computation. In this theoretical model called dendritic plateau computation, a neurons computational function is determined by the compartmentalization of its dendritic tree into functionally independent but mutually coupled segments. We demonstrate the versatility of this mechanism in a simulated navigation experiment, where it allows an individual neuron to reliably detect a specific movement trajectory over hundreds of milliseconds with a high tolerance for timing variability. We conclude by discussing the implications of this model for our understanding of neural computation.

sentences: Evaluation role of miR-124 in neurodegenerative diseases: literature review and in silico analysis Neurodegenerative diseases (ND) are characterized by loss of function and structure of neurons. NDs like Alzheimers disease (AD) and Parkinsons disease (PD) have high burden on the society and patients. Currently microRNAs (miRNAs) approach is growing. miRNAs express in different tissues, especially in the central neuron systems (CNS). miRNAs have a dynamic role in the CNS among this miRNAs, miR-124 significantly express in the CNS. Studies on miR-124 have shown that miR-124 improves ND. In this study, we evaluated the role of miR-124 in the ND by literature review and in silico analysis. We used Pubmed database to find miR-124 function in the Alzheimers disease, Parkinsons disease, Multiple sclerosis, Huntingtons disease and amyotrophic lateral sclerosis. To better understand the role of miR-124 in the neurons, RNA-seq data form miR-124-deleted neuronal cells extracted from GEO database and analyzed in Galaxy platform. According literature review miR-124 attenuates inflammation and apoptosis in the ND by target NF-kb signaling pathway and regulation of BAX/BCL-2. miR-124 targets BACE1 and decreases level of A{beta}. RNA-seq data showed miR-124 downregulation, an increase in chemokine gene like CCL1 and cytokine-cytokine receptor-interaction, as well as MAPK-signaling pathway. Our study shows that miR-124 can be promising therapeutic approaches to ND.

sentences: The Genetics and Evolution of Eye Color in Domestic Pigeons (Columba livia) The avian eye color, generally referred to the color of the iris, results from both pigments and structural coloration. Avian iris colors exhibit striking interspecific and, in some domestic species, intraspecific variations, suggesting unique evolutionary and ecological histories. Here we tackled the genetic basis of the pearl (white) iris color in domestic pigeons (Columba livia), to elucidate the largely unknown genetic mechanism underlying the evolution of avian iris coloration. Using a genome-wide association study (GWAS) in 92 pigeons, we mapped the pearl iris trait to a 9 kb region and a facilitative glucose transporter gene SLC2A11B. A nonsense mutation W49X leading to a premature stop codon in SLC2A11B was identified as the causal variant. Transcriptome analysis suggested that SLC2A11B loss-of-function may downregulate the xanthophore-differentiation gene CSF1R, and a key gene GCH1 involved in biosynthesis of pteridine, whose absence results in pearl iris. Coalescence and phylogenetic analyses indicated the mutation originated about 5,400 years ago coinciding with the onset of pigeon domestication, while positive selection was detected likely associated with artificial breeding. Within Aves, potentially impaired SLC2A11B was found in 10 species from six distinct lineages correlated to their signature brown or blue eyes. Analysis of vertebrate SLC2A11B orthologs revealed relaxed selection in the avian clade, consistent with the scenario that, during and after avian divergence from reptile ancestor, the SLC2A11B-involved development of dermal chromatophores likely degenerated due to feather coverage. Our findings provide new insight into the mechanism of avian iris color variations and the evolution of pigmentation in vertebrates.

sentences: The critical role of natural history museums in advancing eDNA for biodiversity studies: a case study with Amazonian fishes Ichthyological surveys have traditionally been conducted using whole-specimen, capture-based sampling with varied, but conventional fishing gear. Recently, environmental DNA (eDNA) metabarcoding has emerged as a complementary, and possible alternative, approach to whole-specimen methodologies. In the tropics, where much of the diversity remains undescribed, vast reaches continue unexplored, and anthropogenic activities are constant threats; there have been few eDNA attempts for ichthyological inventories. We tested the discriminatory power of eDNA using MiFish primers with existing public reference libraries and compared this with capture-based methods in two distinct ecosystems in the megadiverse Amazon basin. In our study, eDNA provided an accurate snapshot of the fishes at higher taxonomic levels and corroborated its effectiveness to detect specialized fish assemblages. Some flaws in fish metabarcoding studies are routine issues addressed in natural history museums. Thus, by expanding their archives to include eDNA and adopting a series of initiatives linking collection-based research, training and outreach, natural history museums can enable the effective use of eDNA to survey Earths hotspots of biodiversity before taxa go extinct. Our project surveying poorly explored rivers and using DNA vouchered archives to build metabarcoding libraries for Neotropical fishes can serve as a model of this protocol.

sentences: Clathrin mediates both internalization and vesicular release of triggered T cell receptor at the immunological synapse through distinct adaptors Ligation of the T cell receptor (TCR) to peptide-MHC complexes initiates signaling leading to T cell activation. Regulation of T cell responses also requires mechanisms to stop this signaling and to downregulate surface expression of the receptor. T cells achieve this both by TCR internalization and by releasing TCR loaded vesicles directly from the plasma membrane. How these distinct fates are coordinated is unknown. Here we show that clathrin is recruited to TCR microclusters by HRS and STAM2 and that this process is essential for TCR release. Subsequently, EPN1 recruits clathrin to the remaining antigen conjugated TCRs to enable a late wave of TCR endocytosis. With these results we demonstrate two clathrin-dependent mechanisms and show how the clathrin machinery participates in membrane evagination and invagination depending upon the adaptor recruiting it. These sequential mechanisms mediate bi-directional membrane exchange at the immunological synapse, providing a scaffold for critical communication.

sentences: Isolated Grauer's gorilla populations differ in diet and gut microbiome Conservation efforts tend to focus on populations that are genetically differentiated without paying attention to their ecological differentiation. However, isolated populations may be ecologically unique, an important aspect for the design of appropriate conservation measures for endangered species. Here we investigate the interplay between diet and gut microbiome in several geographically isolated and genetically differentiated populations of the critically endangered Grauers gorilla. We find that dietary and gut microbial profiles are population-specific, likely due to geographic isolation and environmental differences. In addition, social groups within each population also differed in diet and, to a lesser extent, in gut microbial composition and diversity. Individuals at low elevation consumed a larger variety of plant taxa than those at high elevation, consistent with the notion that dietary choice is constrained by food availability that changes with elevation. Despite no detectable correlation between the diet and gut microbiome in richness or evenness, dietary and gut microbial composition covaried significantly. As we did not find evidence for an effect of genetic relatedness on the composition of the gut microbiome of Grauers gorillas, this pattern is likely a result of long-term social, ecological, and geographic factors acting on both diet and microbiome. These results reveal that isolated and genetically distinct populations of Grauers gorillas are also ecologically distinct, highlighting the need to dedicate separate conservation efforts for each population.

sentences: Early age-related atrophy of cutaneous lymph nodes precipitates an early functional decline in skin immunity in mice with aging Secondary lymphoid organs (SLO; including the spleen and lymph nodes) are critical both for the maintenance of naive T (TN) lymphocytes and for the initiation and coordination of immune responses. How they age, including the exact timing, extent, physiological relevance, and the nature of age-related changes, remains incompletely understood. We used time-stamping to indelibly mark cohorts of newly generated naive T cells (a.k.a. recent thymic emigrants - RTE) in mice, and followed their presence, phenotype and retention in SLO. We found that SLO involute asynchronously. Skin-draining lymph nodes (LN) atrophied early (6-9 months) in life and deeper tissue-draining LN and the spleen late (18-20 months), as measured by the loss of both TN numbers and the fibroblastic reticular cell (FRC) network. Time-stamped RTE cohorts of all ages entered SLO and successfully completed post-thymic differentiation. However, in older mice, these cells were poorly retained, and those found in SLO exhibited an emigration phenotype (CCR7loS1P1hi). Transfers of adult RTE into recipients of different ages formally demonstrated that the defect segregates with the age of the SLO microenvironment and not with the age of T cells. Finally, upon intradermal immunization, RTE generated in mice as early as 6-7 months of age barely participated in de novo immune responses and failed to produce well-armed effector cells. These results highlight changes in structure and function of superficial secondary lymphoid organs in laboratory mice that are earlier than expected and are consistent with the long-appreciated and pronounced reduction of cutaneous immunity with aging.

sentences: SMaSH: A scalable, general marker gene identification framework for single-cell RNA sequencing and Spatial Transcriptomics Spatial transcriptomics is revolutionising the study of single-cell RNA and tissue-wide cell heterogeneity, but few robust methods connecting spatially resolved cells to so-called marker genes from single-cell RNA sequencing, which generate significant insight gleaned from spatial methods, exist. Here we present SMaSH, a general computational framework for extracting key marker genes from single-cell RNA sequencing data for spatial transcriptomics approaches. SMaSH extracts robust and biologically well-motivated marker genes, which characterise the given data-set better than existing and limited computational approaches for global marker gene calculation.

sentences: Organ- and function-specific anatomical organization and bioelectronic modulation of the vagus nerve Afferent and efferent fibers in the vagus nerve travel inside fascicles and form branches to innervate organs and regulate organ functions. The spatial organization of fibers and fascicles, with respect to the functions they mediate and the organs they innervate, is unknown. Accordingly, it is unknown whether such organization can be leveraged by bioelectronic devices for function- and organ-specific neurostimulation. To characterize the functional microscopic anatomy of the vagus, we developed a pipeline consisting of micro-computed tomography imaging of fascicles and quantitative immunohistochemistry of single fibers. We found that, in swine, the vagus contains clusters of fascicles, organized along 2 overlapping functional axes: one axis specific to sensory vs. motor somatic and visceral functions, with clusters increasingly separating in the cephalad direction, and a second axis specific to innervated organs, including larynx, lungs and heart, with increasing separation in the caudad direction. Large myelinated afferents and efferents, and small unmyelinated efferents occupy large areas of specific clusters of fascicles; small unmyelinated afferents are numerous and occupy small areas across every fascicle. To test whether fiber populations can be selectively modulated, we developed a multi-contact cuff electrode accommodating the nerve fascicular structure. Stimulation targeting separate nerve sections evokes compound action potentials from fibers of distinct functional types and elicits differential organ responses, including laryngeal muscle contraction, cough reflex, and changes in breathing and heart rate. Our results indicate that vagus fibers are anatomically organized according to functions they mediate and organs they innervate and can be differentially modulated by spatially selective nerve stimulation.

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Characteristics of genetically diverse Antarctic sea ice bacteriophages allow adaptation to changing environmental conditions Despite generally appreciated significant roles of microbes in sea ice and polar waters, detailed studies of virus-host systems from such environments have been so far limited by only a few isolates. Here, we investigated infectivity under various conditions, infection cycles, and genetic diversity of Antarctic sea ice bacteriophages: PANV1, PANV2, OANV1, and OANV2. The phages infect common sea ice bacteria belonging to the genera Paraglaciecola or Octadecabacter. Although the phages are marine and cold-active, replicating at 0-5 {degrees}C, they all survived temporal incubations at [≥]30 {degrees}C and remained infectious without any salts or supplemented only with magnesium, suggesting a robust virion assembly maintaining integrity under a wide range of conditions. Host recognition in the cold proved to be effective, and the release of progeny viruses occurred as a result of cell lysis. The analysis of viral genome sequences showed that nearly half of the gene products of each virus are unique, highlighting that sea ice harbors unexplored virus diversity. Based on predicted genes typical for tailed double-stranded DNA phages, we suggest placing the four studied viruses in the class Caudoviricetes. Searching against viral sequences from metagenomic assemblies revealed that related viruses are not restricted to Antarctica, but also found in distant marine environments.

ImportanceVery little is known about sea ice microbes despite the significant role of sea ice in the global oceans as well as microbial input into biogeochemical cycling. Studies on the sea ice viruses have been typically limited to -omics-based approaches and microscopic examinations of sea ice samples. Up to date, only four cultivable viruses have been isolated from Antarctic sea ice. Our study of these unique isolates advances the understanding of the genetic diversity of viruses in sea ice environments, their interactions with host microbes, and possible links to other biomes. Such information contributes to more accurate future sea ice biogeochemical models.

sentences: Cortical layer-specific modulation of neuronal activity after sensory deprivation due to spinal cord injury Cortical areas have the capacity of large-scale reorganization following sensory deprivation. However, it remains unclear whether this phenomenon is a unique process that homogenously affects an entire deprived cortical region or it is suitable to changes depending on neuronal networks across distinct cortical layers. Here, we studied how local circuitries within each layer of the deprived cortex set the basis for neuroplastic changes after immediate sensory deprivation due to thoracic spinal cord injury (SCI) in anaesthetised rats. In vivo electrophysiological recordings from deprived hindlimb somatosensory cortex showed that SCI induces layer-specific changes mediating evoked and spontaneous activity. In supragranular layers 2/3, sensory deprivation increased gamma oscillations and the ability of these neurons to initiate up-states during spontaneous activity, suggesting altered corticocortical network and/or intrinsic properties that may serve to maintain the excitability of the cortical column after deprivation. On the other hand, sensory deprivation enhanced infragranular layers ability to integrate evoked-sensory inputs leading to increased and faster neuronal responses. Delayed evoked-responses onset were also observed in layers 5/6, suggesting alterations in thalamocortical connectivity. Altogether, our data indicate that SCI immediately modifies local circuitries within the deprived cortex allowing supragranular layers to better integrate spontaneous corticocortical information, and thus modifying column excitability, and infragranular layers to better integrate evoked-sensory inputs to preserve subcortical outputs. These layer-specific neuronal changes may guide the long-term alterations in neuronal excitability and plasticity associated to the rearrangements of somatosensory networks and the appearance of central sensory pathologies usually associated with spinal cord injury.

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" Idol with feet of clay ": reliable predictions of forest ecosystem functioning require flawless climate forcings. Climate change affects various aspects of the functioning of ecosystem, especially photosynthesis, respiration and carbon storage. We need accurate modelling approaches (impact models) to simulate the functioning, vitality and provision of ecosystem services of forests in a warmer world. These impact models require climate data as forcings, which are often produced by climate models comparing more or less well with observational climate data. The bias percentage of the climate forcings propagates throughout the modeling chain from the climate model to the impact model.

In this study, we aimed to quantify these bias percentage, addressing three questions: (1) Do the impact model predictions vary when forcing it with different climate models, and how do the predictions under climate model vs. observational climate forcing differ? (2) Does the variability in the impact climate simulations caused by climate forcings fade out at large spatial scale? (3) How the fact of using simulated climatic data affects the process-based model predictions in the case of stressful events?

To answer these questions, we present results obtained over the historical period (e.g. 1970-2010) with the CASTANEA ecophysiological forest model and use the data from three climate models. Our analysis focuses on French forests, studying European beech (Fagus sylvatica), temperate deciduous oaks (Quercus robur and Q. petraea), Scots pine (Pinus sylvestris) and spruce (Picea abies) monospecific stands.

We show that prediction of photosynthesis, respiration and wood growth highly depends on the climate model used, whether debiased or not, and also on species and region considered. Overall, we observed an improvement of prediction after a monthly mean bias or monthly quantile mapping correction for three model considered, but not with the same success. Then we highlighted a large variability in the processes simulated by the impact model under different climate forcings when considering the plot (i.e. scale of a few hectares) scale. This variability fades out at larger scale (e.g. the scale of an ecological region, i.e. 100 km2), owing to an aggregation effect. Moreover, process predictions obtained under different climate forcings are more variable during driest years. These results highlight the necessity to quantify bias and uncertainties in climate forcings before predicting fluxes dynamics with process-based model.

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Soil collected in the Great Smoky Mountains National Park yielded a novel Listeria species, L. swaminathanii, effectively expanding the sensu stricto clade to ten species Soil samples collected in the Great Smoky Mountains National Park yielded a Listeria isolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses confirmed this isolate to be a novel Listeria sensu stricto species with the highest similarity to L. marthii (ANI=93.9%, isDDH=55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit, an automated program for classifying bacterial genomes, further supported delineation as a novel Listeria sensu stricto species, as this tool failed to assign a species identification but identified L. marthii as the closest match. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novel Listeria species described here is phenotypically (i) non-hemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in the Listeria pathogenicity island 1 (LIPI-1), as well as the internalin genes inlA and inlB. While the type strain for the new species is phenotypically catalase-negative (an unusual characteristic for Listeria sensu stricto species), its genome contained an apparently intact catalase gene (kat); hence assessment of this phenotype with future isolates will be important. Rapid species identification systems (Listeria API, VITEK 2, VITEK MS) misidentified this novel species as either L. monocytogenes, L. innocua, or L. marthii. We propose the name L. swaminathanii, and the type strain is FSL L7-0020T (=ATCC TSD-239T).

IMPORTANCEL. swaminathanii is a novel sensu stricto species that originated from a US National Park, and its place of origin is ultimately preventing this species from achieving valid status. The US National Park Service restricts strain accessibility and open access is currently a prerequisite for species validation. Essentially the only criteria that was not met for L. swaminathanii validation is accessibility of the type strain, therefore nomenclature status should not negate the significance of this discovery. As a novel sensu stricto species, L. swaminathanii expands the group of species whose presence is associated with an increased risk of an L. monocytogenes contamination, and therefore could play an important role in public health. While developers of Listeria spp. detection methods historically only included validly publish species in their validation studies, L. swaminathanii is unequivocally a sensu stricto species and should be included as well.

sentences: Botrytis cinerea BcPTP1 is a late infection phase, cysteine rich protein cytotoxic effector Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious crops diseases. To facilitate infection, B. cinerea secretes a large number of effectors that induce plant cell death. In screening secretome data of B. cinerea during infection stage, we identified a phytotoxic protein (BcPTP1) that can also induce immune resistance in plants. BcPTP1 is a small (90 aa), cysteine rich protein without any known domains. Transiently expression of BcPTP1 in leaves caused chlorosis that intensifies with time and eventually lead to cell death. Point mutations in eight of the 10 cysteine residues of BcPTP1 abolished the toxic effect, however residual toxic activity remained after heating the peptide, suggesting contribution of unknown epitopes to protein phytotoxic effect. The transcript level of the bcptp1 gene was low during the first 36 h after inoculation and increased sharply upon transition to the late infection stage, suggesting a role of BcPTP1 in lesion spreading. While statistically insignificant, deletion of the bcptp1 gene led to slightly smaller lesions on bean leaves. Further analyses indicated that BcPTP1 is internalized into plant cells after secreting into the apoplast and its phytotoxic effect is negatively regulated by the receptor-like kinases BAK1 and SOBIR1. Collectively, our findings show that BcPTP1 is a virulence factor that toxifies the host cells and facilitates lesion spreading during the late infection stage.

sentences: A chemical screen based on an interruption of zebrafish gastrulation identifies the HTR2C inhibitor Pizotifen as a suppressor of EMT-mediated metastasis Metastasis is responsible for approximately 90% of cancer-associated mortality but few models exist that allow for rapid and effective screening of anti-metastasis drugs. Current mouse models of metastasis are too expensive and time consuming to use for rapid and high-throughput screening. Therefore, we created a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. We hypothesized that small chemicals that interrupt zebrafish gastrulation might also suppress metastatic progression of cancer cells and developed a phenotype-based chemical screen to test the hypothesis. The screen used epiboly, the first morphogenetic movement in gastrulation, as a marker and enabled 100 chemicals to be tested in five hours. The screen tested 1280 FDA-approved drugs and identified Pizotifen, an antagonist for serotonin receptor 2C (HTR2C) as an epiboly-interrupting drug. Pharmacologic and genetic inhibition of HTR2C suppressed metastatic progression in a mouse model. Blocking HTR2C with Pizotifen restored epithelial properties to metastatic cells through inhibition of Wnt-signaling. In contrast, HTR2C induced epithelial to mesenchymal transition (EMT) through activation of Wnt-signaling and promoted metastatic dissemination of human cancer cells in a zebrafish xenotransplantation model. Taken together, our concept offers a novel platform for discovery of anti-metastasis drugs.

sentences: An integrated in silico-in vitro approach for identification of therapeutic drug targets for osteoarthritis Without the availability of disease-modifying drugs, there is an unmet therapeutic need for osteoarthritic patients. During osteoarthritis, the homeostasis of articular chondrocytes is dysregulated and a phenotypical transition called hypertrophy occurs, leading to cartilage degeneration. Targeting this phenotypic transition has emerged as a potential therapeutic strategy. Chondrocyte phenotype maintenance and switch are controlled by an intricate network of intracellular factors, each influenced by a myriad of feedback mechanisms, making it challenging to intuitively predict treatment outcomes. In this study, we developed a regulatory network model using knowledge-based and data-driven modelling technologies. The in silico high-throughput screening of (pairwise) perturbations operated with that network model highlighted conditions impacting the hypertrophic switch. Several combinations were tested in a murine cell line and primary chondrocytes to validate the predicted conditions potential. Our in silico-in vitro strategy opens a new route for developing osteoarthritis targeting therapies by refining the early stages of drug discovery.

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RNA-silencing induces target gene relocalization toward a specialized nuage domain The authors wish to withdraw this manuscript and apologize for errors in the initial submission. All the original experiments were performed by YY. Unfortunately, JP and members of the Mello lab have not been able to replicate some aspects of the study. JP has failed to independently reproduce the specific results showing RNAi-triggered relocalization of target RNA, and P granule specific accumulation of (the P granule component GLH-1) as reported. The conditions/strains analyzed by JP were as follows: (1) oma-1 FISH on WT worms [control, 6 hr and 12 hr oma-1(RNAi)]. (2) oma-1 FISH on OMA-1:GFP worms [control, 6 hr oma-1(RNAi), or 6 hr gfp(RNAi)]. (3) oma-1 FISH on WT worms [control, 4hr, 6hr, 8 hr, and 10 hr oma-1(RNAi)]. 10-23 gonads were analyzed per experiment. Fixation conditions were essentially as described, with the only known difference being that gonads were not exposed to detergent prior to fixation. Using YYs reagents and protocol the Mello lab has not observed an obvious relocalization of target RNA to P granules (marked by GFP::GLH-1) after 6 hrs oma-1(RNAi); n=92 gonads. CM, JP and DG consider that the published images accurately represent the image stacks provided by YY as representative, raw data, but JP and CM note configurations of FISH signals in germ nuclei and gonad anatomy that they consider unusual. CM, JP and DG have not detected any evidence of image manipulation.

YY states that none of the raw image data were manipulated beyond standard adjustments for brightness and contrast prior to processing images for publication as described. However, YY reports that the images were not representative of the majority of sample gonads, and instead were pre-selected under low magnification for rare examples with asymmetrical, expanded P granules.

Efforts to identify conditions that explain the rare gonads imaged by YY continue in the Mello lab, as do efforts to reproduce independently each of the other reported results; we plan to provide an update in the near future.

sentences: The impact of phosphorylated Pten at threonine 366 on cortical connectivity and behaviour The lipid phosphatase Pten (phosphatase and tensin homologue on chromosome 10) is a key tumour suppressor gene and an important regulator of neuronal signalling. Pten mutations have been identified in patients with autism spectrum disorders, characterized by macrocephaly, impaired social interactions and communication, repetitive behaviour, intellectual disability, and epilepsy. Pten enzymatic activity is regulated by a cluster of phosphorylation sites at the C-terminus of the protein. Here we specifically focussed on the role of Pten T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine (PtenT366A/T366A). We identify that phosphorylation of Pten at T366 controls neuron size and connectivity of brain circuits involved in sensory processing. We show in behavioural tests that PtenT366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in PtenT366A/T366A brains, linked to increases in both dendritic arborization and soma size. In a combinatorial approach of anterograde and retrograde monosynaptic tracing using rabies virus, we characterize differences in connectivity to the primary somatosensory cortex of PtenT366A/T366A brains, with imbalances in long-range cortico-cortical input to neurons. We conclude that phosphorylation of Pten at T366 controls neuron size and connectivity of brain circuits involved in sensory processing and propose that PTEN T366 signalling may account for a subset of autism-related functions of Pten.

sentences: The Origin of Conductive-Pulse Sensing Inside a Nanopore and the Role of Electro-Hydrodynamics Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic reduction of ionic flux during molecular occupancy of a pore, particularly at high salt concentrations. However, conductive pulses are widely reported at low salt conditions wherein electroosmotic flow can be quite significant. Aside from transporting molecules like DNA, we investigated whether electroosmotic flow has other potential impacts on sensing attributes such current enhancements due to the analyte molecule. The overwhelming majority of literature reports counterions as the dominant mechanism of conductive events (a moleculecentric theory for conductive events). Conductive events are not well understood due to the complex interplay between (charged) nanopore walls, DNA grooves, ion mobility, and counterion clouds. Yet, the prevailing consensus of counterions being introduced into the pore by the molecule does not fit well with a growing number of experiments including the fact that proteins can generate conductive events despite having a heterogeneous surface charge. Herein, we demonstrate theory and experiments underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chemical, physical, and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e. a pore-centric theory). We believe these findings will stimulate a useful discussion on the nature of conductive events and their impact on molecular sensing in nanoscale pores.

sentences: Viral and parasitic cell ratios per free-living microbe across ecosystems It is generally assumed that viruses outnumber cells on Earth by at least tenfold1-4. Virus-to-microbe ratios (VMR) are largely based on fluorescently-labelled viral-like particles (VLPs) counts. However, these exclude cell-infecting lytic and lysogenic viruses and potentially include many false positives, e.g. DNA-containing membrane vesicles, genetransfer agents or inert particles. Here, we develop a metagenome-based VMR estimate (mVRM) accounting for all viral stages (virion-lytic-lysogenic) using normalised counts of viral DNA-polymerases and cellular universal single-copy genes as proxies for individuals. To properly estimate mVMR in aquatic ecosystems, we generated metagenomes from co-occurring cellular and viral fractions (>50 kDa-200 m size range) in freshwater, seawater and 6-14-32% salt solar-saltern ponds. Viruses outnumbered cells in non-blooming freshwater and marine plankton by around twofold. However, mVMR in 133 uniformly-analysed metagenomes from diverse ecosystems showed that free-living cells largely exceeded viruses and epibiont DPANN archaea and/or CPR bacteria in compact environments (sediments, soils, microbial mats, host-associated microbiomes). Along the Piggyback-the-winner model5 lines, lysogenic genes significantly correlated with cell, but not necessarily biotope, density. While viruses likely are the most diverse biological entities on Earth6,7, our results suggest that cells are the most abundant and that cellular parasites may exert population-size control in some high-density ecosystems.

sentences: Repurposing statin and L-glutamine: replenishing β-cells in hyperlipidemic T2D mouse model While cases of obesity-induced type 2 diabetes (T2D) are on the upswing, current therapies help only manage the symptoms. Of late, L-glutamine has been implicated in the amelioration of T2D by virtue of its glucagon like peptide-1 (GLP-1) secretagogue property. Alongside, there are mixed reports on adiponectin (insulin sensitizer) potentiating property of statins. We aimed to investigate the effect of pitavastatin (P) and L-glutamine (LG) combination on glycemic control and pancreatic {beta}-cell regeneration in a high-fat diet (HFD)+Streptozotocin (STZ) induced T2D mouse model. C57BL6/J mice treated with HFD+STZ were randomly assigned into four groups: Diabetic control, L-glutamine, Pitavastatin and P+LG. Control group was fed with the chow diet. Significant amelioration in insulin resistance along with plasma glucose, lipid profile, adiponectin levels, and mitochondrial complexes I, II, III activities were observed in P+LG group as compared to HFD+STZ treated group. Phosphoenolpyruvate carboxykinase, glucose 6-phophatase, glycogen phosphorylase, and GLUT2 transcript levels were reduced with increased glycogen synthase transcript levels in liver. Further, the protein levels of Insulin receptor 1-{beta}, pAkt/Akt, and AdipoR1 were restored in the skeletal muscle and a significant increase in islet number as a result of {beta}-cell regeneration and reduced {beta}-cell death were also observed in the combination drug treated group. Thus, L-glutamine and pitavastatin in combination can induce {beta}-cell regeneration and regulate glucose homeostasis to bring about amelioration of HFD+STZ induced T2D.

sentences: Reducing susceptibility distortion related image blurring in diffusion MRI EPI data Diffusion magnetic resonance imaging (MRI) is an increasingly popular technique in basic and clinical neuroscience. One promising application is to combine diffusion MRI with myelin maps from complementary MRI techniques such as multi-parameter mapping (MPM) to produce g-ratio maps that represent the relative myelination of axons and predict their conduction velocity. Statistical Parametric Mapping (SPM) can process both diffusion data and MPMs, making SPM the only widely accessible software that contains all the processing steps required to perform group analyses of g-ratio data in a common space. However, limitations have been identified in its method for reducing susceptibility-related distortion in diffusion data. More generally, susceptibility-related image distortion is often corrected by combining reverse phase-encoded images (blip-up and blip-down) using the arithmetic mean (AM), however, this can lead to blurred images. In this study we sought to (1) improve the susceptibility-related distortion correction for diffusion MRI data in SPM; (2) deploy an alternative approach to the AM to reduce image blurring in diffusion MRI data when combining blip-up and blip-down EPI data after susceptibility-related distortion correction; and (3) assess the benefits of these changes for g-ratio mapping. We found that the new processing pipeline, called consecutive Hyperelastic Susceptibility Artefact Correction (HySCO) improved distortion correction when compared to the standard approach in the ACID toolbox for SPM. Moreover, using a weighted average (WA) method to combine the distortion corrected data from each phase-encoding polarity achieved greater overlap of diffusion and more anatomically faithful structural white matter probability maps derived from minimally distorted multi-parameter maps as compared to the AM. Third, we showed that the consecutive HySCO WA performed better than the AM method when combined with multi-parameter maps to perform g-ratio mapping. These improvements mean that researchers can conveniently access a wide range of diffusion-related analysis methods within one framework because they are now available within the open-source ACID toolbox as part of SPM, which can be easily combined with other SPM toolboxes, such as the hMRI toolbox, to facilitate computation of myelin biomarkers that are necessary for g-ratio mapping.

sentences: COLD ATMOSPHERIC PLASMA DIFFERENTIALLY AFFECTS CELL RENEWAL AND DIFFERENTIATION OF STEM CELLS AND APC-DEFICIENT-DERIVED TUMOR CELLS IN INTESTINAL ORGANOIDS Cold atmospheric plasma (CAP) treatment has been proposed as a potentially innovative therapeutic tool in the biomedical field, notably for cancer due to its proposed toxic selectivity on cancer cells versus healthy cells. In the present study, we addressed the relevance of three-dimensional organoid technology to investigate the biological effects of CAP on normal epithelial stem cells and tumor cells isolated from mouse small intestine. CAP treatment exerted dose-dependent cytotoxicity on normal organoids and induced major transcriptomic changes associated with global response to oxidative stress, fetal-like regeneration reprogramming and apoptosis-mediated cell death. Moreover, we explored the potential selectivity of CAP on tumor-like Apc-deficient versus normal organoids in the same genetic background. Unexpectedly, tumor organoids exhibited higher resistance to CAP treatment, correlating with higher antioxidant activity at baseline as compared to normal organoids. This pilot study suggests that the ex vivo culture system could be a relevant alternative model to further investigate translational medical applications of CAP technology.

sentences: Pupil correlates of decision variables in mice playing a competitive mixed-strategy game In a competitive game involving an animal and an opponent, the outcome is contingent on the choices of both players. To succeed, the animal must continually adapt to competitive pressure, or else risk being exploited and lose out on rewards. In this study, we demonstrate that head-fixed mice can be trained to play the iterative competitive game  matching pennies against a virtual computer opponent. We find that the animals performance is well described by a hybrid computational model that includes Q-learning and choice kernels. Comparing between matching pennies and a non-competitive two-armed bandit task, we show that the tasks encourage animals to operate at different regimes of reinforcement learning. To understand the involvement of neuromodulatory mechanisms, we measure fluctuations in pupil size and use multiple linear regression to relate the trial-by-trial transient pupil responses to decision-related variables. The analysis reveals that pupil responses are modulated by observable variables, including choice and outcome, as well as latent variables for value updating, but not action selection. Collectively, these results establish a paradigm for studying competitive decision-making in head-fixed mice and provide insights into the role of arousal-linked neuromodulation in the decision process.

sentences: Accessory genomic epidemiology of co-circulating Acinetobacter baumannii clones A. baumannii has become one of the most important multidrug resistant nosocomial pathogens all over the world. Nonetheless, very little is known about the diversity of A. baumannii lineages co-existing in hospital settings. Here, using whole-genome sequencing, epidemiological data and antimicrobial susceptibility tests, we uncover the transmission dynamics of extensive and multidrug resistant A. baumannii in a tertiary hospital for a decade. Our core genome phylogeny of almost 300 genomes suggests that there were several introductions of lineages from international clone 2 into the hospital. The molecular dating analysis shows that these introductions happened between 2004 and 2015. Furthermore, using the accessory genome, we show that these lineages were extensively disseminated across many wards in the hospital. Our results demonstrate that accessory genome variation can be a very powerful tool for conducting genomic epidemiology. We anticipate future studies employing the accessory genome as a phylogenomic marker over very short microevolutionary scales.

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Paracrine granules are cytoplasmic RNP granules distinct from stress granules assembling in response to viral infection. To rapidly respond and adapt to stresses, such as viral infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs, together with RNA-binding proteins and signalling components, into cytoplasmic biocondensates called stress granules. Increasing evidence suggests that stress granules contribute to antiviral defense and thus viruses need to evade these response pathways to propagate. In addition, the stress granule pathway is proposed to be dynamic and adaptable to specific stresses. We previously showed that Feline Calicivirus (FCV) impairs SGs assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-granules, suggesting a paracrine response trigged by the infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of paracrine granules. They are different from canonical stress granules and exhibit specific kinetics of assembly-disassembly, protein and RNA composition and are linked to antiviral activity. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells.

Summary statementWe describe a novel type of paracrine induced RNA granules associated with viruses, highlighting how different stresses results in heterogeneous stress granule-like condensates with specific cellular functions.

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Possible Implications of AlphaFold2 for Crystallographic Phasing by Molecular Replacement The AlphaFold2 results in the 14th edition of Critical Assessment of Structure Prediction (CASP14) showed that accurate (low root-mean-square deviation) in silico models of protein structure domains are on the horizon, whether or not the protein is related to known structures through high- coverage sequence similarity. As highly accurate models become available, generated by harnessing the power of correlated mutations and deep learning, one of the aspects of structural biology to be impacted will be methods of phasing in crystallography. We here use the data from CASP14 to explore the prospect for changes in phasing methods, and in particular to explore the prospects for molecular replacement phasing using in silico models.

SynopsisWe discuss the implications of the AlphaFold2 protein structure modelling software for crystallographic phasing strategies.

sentences: Co-evolutionary analysis suggests a role for TLR9 in papillomavirus restriction A.Upon infection, DNA viruses can be sensed by pattern recognition receptors (PRRs) leading to the activation of type I and III interferons, aimed at blocking infection. Therefore, viruses must inhibit these signaling pathways, avoid being detected, or both. Papillomavirus virions are trafficked from early endosomes to the Golgi apparatus and wait for the onset of mitosis to complete nuclear entry. This unique subcellular trafficking strategy avoids detection by cytoplasmic PRRs, a property that may contribute to establishment of infection. However, as the capsid uncoats within acidic endosomal compartments, the viral DNA may be exposed to detection by toll-like receptor (TLR) 9. In this study we characterize two new papillomaviruses from bats and use molecular archeology to demonstrate that their genomes altered their nucleotide composition to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we demonstrate that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for co-evolution between papillomaviruses and their hosts.

sentences: An atlas of lamina-associated chromatin across twelve human cell types revealsan intermediate chromatin subtype Association of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamin-associated domains (LADs) have yet to be mapped genome-wide in a diverse panel of human cell types, which has limited our understanding of the role peripheral chromatin plays in development and disease. To address this gap, we mapped LAMIN B1 (LB1) across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas of peripheral chromatin with publicly available genomic data, as well as gene expression and repressive histone maps generated for this study, revealed that in all twelve cellular contexts lamin-associated chromatin is organized into at least two subtypes defined by differences in LB1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Most genes gain or lose LB1 occupancy consistent with their cell type along developmental trajectories; however, we also identified examples where the enhancer, but not the gene body and promoter, change LAD state. Imaging of fluorescently labeled DNA in single cells validated these transitions and showed intermediate radial positioning of LADs that are gene dense, relatively accessible, and dynamic across development. This atlas represents the largest resource to date for peripheral chromatin organization studies.

sentences: Distinct trans-placental effects of maternal immune activation by TLR3 and TLR7 agonists: implications for schizophrenia risk Exposure to infection in utero predisposes towards psychiatric diseases such as autism, depression and schizophrenia in later life. The mechanisms involved are typically studied by administering mimetics of double-stranded (ds) RNA viral or bacterial infection to pregnant rats or mice. The effect of single-stranded (ss) virus mimetics has been largely ignored, despite evidence linking prenatal ss virus exposure specifically with psychiatric disease. Understanding the effects of gestational ss virus exposure has become even more important with the current SARS-CoV-2 pandemic. In this study, in pregnant mice, we compare directly the effects, on the maternal blood, placenta and the embryonic brain, of maternal administration of ds-virus mimetic poly I:C (to activate toll-like receptor 3, TLR3) and ss-virus mimetic resiquimod (to activate TLR7/8). We find that, 4h after the administration, both poly I:C and resiquimod elevated the levels of IL-6, TNF, and chemokines including CCL2 and CCL5, in maternal plasma. Both agents also increased placental mRNA levels of IL-6 and IL-10, but only resiquimod increased placental TNF mRNA. In foetal brain, poly I:C produced no detectable immune-response-related increases, whereas pronounced increases in cytokine (e.g. Il-6, Tnf) and chemokine (e.g. Ccl2, Ccl5) expression were observed with maternal resiquimod administration. The data show substantial differences between the effect of maternal exposure to a TLR7/8 activator as compared to a TLR3 activator. There are significant implications for future modelling of diseases where maternal ss virus exposure contributes to environmental disease risk in offspring.

sentences: Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans Building strains of filamentous fungi for stable long-term heterologous expression of large biosynthetic pathways is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase-mediated cassette exchange. We built A. nidulans strains harboring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21 kb DNA fragment in a single step. We further evaluated the integration at two loci by analyzing the expression of a fluorescent reporter and the production of a heterologous polyketide metabolite. We compared chromosomal expression at those landing loci to episomal AMA1-based expression, which also shed light on uncharacterized aspects of episomal expression in filamentous fungi. This is the first demonstration of site-specific recombinase-mediated integration in filamentous fungi, setting the foundations for the further development of this tool.

sentences: Evolution of DNA methylation across Ecdysozoa DNA methylation is a crucial, abundant mechanism of gene regulation in vertebrates. It is less prevalent in many other metazoan organisms and completely absent in some key model species, such as D. melanogaster and C. elegans. We report here a comprehensive study of the presence and absence of DNA methyltransferases (DNMTs) in 138 Ecdysozoa, covering Arthropoda, Nematoda, Priapulida, Onychophora, and Tardigrada. Three of these phyla have not been investigated for the presence of DNA methylation before. We observe that the loss of individual DNMTs independently occurred multiple times across ecdysozoan phyla. We computationally predict the presence of DNA methylation based on CpG rates in coding sequences using an implementation of Gaussian Mixture Modelling, MethMod. Integrating both analysis we predict two previously unknown losses of DNA methylation in Ecdysozoa, one within Chelicerata (Mesostigmata) and one in Tardigrada. In the early-branching Ecdysozoa Priapulus caudatus we predict the presence of a full set of DNMTs and the presence of DNA methylation. We are therefore showing a very diverse and independent evolution of DNA methylation in different ecdysozoan phyla spanning a phylogenetic range of more than 700 million years.

sentences: Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.

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CCR4 blockade leads to clinical activity and prolongs survival in a canine model of advanced prostate cancer Targeting regulatory T cell (Treg) infiltration is an emerging strategy for cancer immunotherapy. However, the efficacy of this strategy in advanced prostate cancer remains unclear. Here, we describe the therapeutic efficacy of this strategy in a canine model of advanced prostate cancer. We used dogs with naturally occurring prostate cancer to study the molecular mechanism underlying Treg infiltration into tumor tissues and the effect of anti-Treg treatment. We found that tumor-infiltrating Tregs were associated with poor prognosis in dogs bearing spontaneous prostate cancer. RNA sequencing and protein analyses showed that Treg infiltration was mediated by interaction between the tumor-producing chemokine, CCL17, and the receptor CCR4 expressed on Tregs. Dogs with advanced prostate cancer responded to mogamulizumab, a monoclonal antibody targeting CCR4, with improved survival and low incidence of clinically relevant adverse events. Exploratory analyses showed urinary CCL17 concentration and BRAFV595E mutation to be independently predictive of the response to mogamulizumab. Analysis of a publicly available transcriptomic dataset of human prostate cancer showed that the CCL17/CCR4 axis correlated with the Treg marker, Foxp3. In silico survival analyses showed that high expression of CCL17 was associated with poor prognosis. Immunohistochemistry confirmed that tumor-infiltrating Tregs expressed CCR4 in human patients with prostate cancer. These findings suggest that anti-Treg treatment through the blocking of CCR4 is a promising therapeutic approach for advanced prostate cancer.

One Sentence SummaryTargeting regulatory T cell infiltration by CCR4 blockade induces objective responses and improves survival in a canine model of prostate cancer.

sentences: Efficient ancestry and mutation simulation with msprime 1.0 Stochastic simulation is a key tool in population genetics, since the models involved are often analytically intractable and simulation is usually the only way of obtaining ground-truth data to evaluate inferences. Because of this necessity, a large number of specialised simulation programs have been developed, each filling a particular niche, but with largely overlapping functionality and a substantial duplication of effort. Here, we introduce msprime version 1.0, which efficiently implements ancestry and mutation simulations based on the succinct tree sequence data structure and tskit library. We summarise msprimes many features, and show that its performance is excellent, often many times faster and more memory efficient than specialised alternatives. These high-performance features have been thoroughly tested and validated, and built using a collaborative, open source development model, which reduces duplication of effort and promotes software quality via community engagement.

sentences: Similarities Between Bacterial GAD and Human GAD65:Implications in Gut Mediated Autoimmune Type 1 Diabetes A variety of islet autoantibodies (AAbs) can predict and possibly dictate eventual type 1 diabetes (T1D) diagnosis. Upwards of 75% of those with T1D are positive for AAbs against glutamic acid decarboxylase (GAD65), a producer of gamma-aminobutyric acid (GABA) in human pancreatic beta cells. Interestingly, bacterial populations within the human gut also express GAD65 and produce GABA. Evidence suggests that dysbiosis of the microbiome may correlate with T1D pathogenesis and physiology. Therefore, autoimmune linkages between the gut microbiome and islets susceptible to autoimmune attack need to be further elucidated. Utilizing silico analyses, we show here that 25 GAD sequences from different human gut bacterial sources show sequence and motif similarities to human beta cell GAD65. Our motif analyses determined that a majority of gut GAD sequences contain the pyroxical dependent decarboxylase domain of human GAD65 which is important for its enzymatic activity. Additionally, we showed overlap with known human GAD65 T-cell receptor epitopes which may implicate the immune destruction of beta cells. Thus, we propose a physiological hypothesis in which changes in the gut microbiome in those with T1D result in a release of bacterial GAD, thus causing miseducation of the host immune system. Due to the notable similarities, we found between humans and bacterial GAD, these deputized immune cells may then go on to target human beta cells leading to the development of T1D.

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An Automated Radio-Telemetry System (ARTS) for Monitoring Small Mammals Point 1: The study of animals in nature is essential for developing an ecologically valid understanding of behavior. Small mammals, however, are often fossorial and exceedingly difficult to monitor in the wild. This limits both the taxonomic scope of field observation, and excludes species that are powerful models for the study of behavioral mechanisms.

Point 2: Here, we implement an automated radio telemetry system (ARTS) designed to track small fossorial mammals. Our ARTS uses an isotropic antenna array coupled with broadband receivers. We characterized transmission at our study site and tested the ARTS ability to track 48 prairie voles.

Point 3: We compared position estimates from nonlinear least squares, nonparameteric, and Bayesian trilateration methods and found Bayesian trilateration to have the smallest error. To examine the ability of the system to track biologically significant behavior we used ARTS data to investigate circadian rhythms of freely behaving prairie voles. We used Lomb-Scargle analysis to estimate periodic patterns from irregularly sampled time series of speed. Prairie voles demonstrated ultradian movement at periods of approximately 45 and 90 min, observations on a time scale not possible using data from traditional methods.

Point 4: This ARTS offers a new tool to observe rodent field behavior at time scales and in environments which have not been previously possible, such as investigating social and spatial behaviors on the scale of minutes, hours, and days in natural environments.

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How salience enhances inhibitory control: An analysis of electro-cortical mechanisms Stop-signal tasks (SSTs) combined with human electro-cortical recordings (Event-Related Potentials, ERPs) have revealed mechanisms associated with successful stopping (relative to failed), presumably contributing to inhibitory control. The corresponding ERP signatures have been labeled stop N1 (+/- 100-ms latency), stop N2 (200 ms), and stop P3 (160-250 ms), and argued to reflect more proactive (N1) versus more reactive (N2, P3) mechanisms. However, stop N1 and stop N2, as well as latencies of stop-P3, appear to be quite inconsistent across studies. The present work addressed the possible influence of stop-signal salience, expecting high salience to induce clear stop N1s but reduced stop N2s, and short-latency stop P3s.

Three SST varieties were combined with high-resolution EEG. An imperative visual (go) stimulus was occasionally followed by a subsequent (stop) stimulus that signalled to withhold the just initiated response. Stop-Signal Reaction Times (SSRTs) decreased linearly from visual-low to visual-high-salience to auditory. Auditory Stop N1 was replicated. A C1-like visual evoked potential (latency < 100 ms) was observed only with high salience, but not robustly associated with successful versus failed stops. Using the successful-failed contrast a visual stop-N1 analogue (112-156 ms post-stop-signal) was identified, as was right-frontal stop N2, but neither was sensitive to salience. Stop P3 had shorter latency for high than for low salience, and the extent of the early high-salience stop P3 correlated inversely with SSRT. These results suggest that salience-enhanced inhibitory control as manifest in SSRTs is associated with reactive rather than proactive electrocortical mechanisms.

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An autonomous tail-beating cycle period expressed by a region-specific swimming pattern generator in the Ciona larva Swimming locomotion in aquatic vertebrates, such as fish and tadpoles, is expressed through orchestrated operations of central pattern generators. These parallel neuronal circuits are ubiquitously distributed and mutually coupled along the spinal cord to express undulation patterns accommodated to efferent and afferent inputs. While such sets of schemes have been shown in vertebrates, the evolutionary origin of those mechanisms along the chordate phylogeny remains unclear. Ascidians, representing a sister group of vertebrates, give rise to tadpole larvae that freely swim in seawater. In this study, we tried to locate the swimming pattern generator in larvae of the ascidian Ciona by examining locomotor ability of segmented body fragments. Our experiments demonstrated necessary and sufficient pattern generator activity in a short region ([~]10% of the body length as the longest estimation) including the trunk-tail junction but excluding most of the trunk and tail with major sensory apparatuses therein. Moreover, we found that these "mid-piece" body fragments express periodic tail beating bursts with [~]20-s intervals without any exogenous stimuli. Comparisons among temporal patterns of tail beating bursts expressed by the mid-piece fragments and by whole larvae placed under different sensory conditions suggested that the presence of parts other than the critical mid-piece had effects to shorten swimming burst intervals, especially in the dark, and also to expand the variance in burst durations. We propose that Ciona larvae perform swimming as modified representations of autonomous and periodic pattern generator drives, which operate locally in the region of the trunk-tail junction.

Summary statementMid-piece fragments of tadpole larvae of the ascidian Ciona, lacking most of the anterior trunk and posterior tail, autonomously and periodically express tail beating bursts.

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A single amino acid substitution in PB1 of pandemic H1N1 with A/Ann Arbor/6/60 master donor virus mutations as a novel live-attenuated influenza virus vaccine Influenza A viruses (IAV) remain emerging threats to human public health. Live-attenuated influenza vaccines (LAIV) are one of the most effective prophylactic options to prevent disease caused by influenza infections. However, licensed LAIV remain restricted for use in 2- to 49-year old healthy and non-pregnant people. Therefore, development of LAIV with increased safety, immunogenicity, and protective efficacy is highly desired. The United States (U.S.) licensed LAIV is based on the master donor virus (MDV) A/Ann Arbor/6/60 H2N2 backbone, which was generated by adaptation of the virus to growth at low temperatures. Introducing the genetic signature of the U.S. MDV into the backbone of other IAV strains resulted in varying levels of attenuation. While the U.S. MDV mutations conferred an attenuated phenotype to other IAV strains, the same amino acid changes did not significantly attenuate the pandemic A/California/04/09 H1N1 (pH1N1) strain. To attenuate pH1N1, we replaced the conserved leucine at position 319 with glutamine (L319Q) in PB1 and analyzed the in vitro and in vivo properties of pH1N1 viruses containing either PB1 L319Q alone or in combination with the U.S. MDV mutations using two animal models of influenza infection and transmission, ferrets and guinea pigs. Our results demonstrated that L319Q substitution in the pH1N1 PB1 alone or in combination with the mutations of the U.S. MDV resulted in reduced pathogenicity (ferrets) and transmission (guinea pigs), and an enhanced temperature sensitive phenotype. These results demonstrate the feasibility of generating an attenuated MDV based on the backbone of a contemporary pH1N1 IAV strain.

IMPORTANCEVaccination represents the most effective strategy to reduce the impact of seasonal IAV infections. Although LAIV are superior in inducing protection and sterilizing immunity, they are not recommended for many individuals who are at high risk for severe disease. Thus, development of safer and more effective LAIV are needed. A concern with the current MDV used to generate the U.S. licensed LAIV is that it is based on a virus isolated in 1960. Moreover, mutations that confer the temperature sensitive, cold-adapted, and attenuated phenotype of the U.S. MDV resulted in low level of attenuation in the contemporary pandemic A/California/04/09 H1N1 (pH1N1). Here, we show that introduction of PB1 L319Q substitution, alone or in combination with the U.S. MDV mutations, resulted in pH1N1 attenuation. These findings support the development of a novel LAIV MDV based on a contemporary pH1N1 strain as a medical countermeasure against currently circulating H1N1 IAV.

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Perilacunar bone tissue exhibits sub-micrometer modulus gradation which depends on the recency of osteocyte bone formation Osteocytes are capable of resorbing and replacing bone local to the lacunar-canalicular system (LCS remodeling). However, the impacts of these processes on perilacunar bone quality are not understood. It is well established that aging is associated with reduced whole-bone fracture resistance, reduced osteocyte viability, and truncated LCS geometries, but it remains unclear if aging changes perilacunar bone quality. In this study, we employed atomic force microscopy (AFM) to quantify sub-micrometer gradations from 2D maps surrounding osteocyte lacunae in young (5 mo) and aged (22 mo) female mice. AFM-mapped lacunae were also imaged with confocal laser scanning microscopy to determine which osteocytes had recently deposited bone as determined by the presence of fluorochrome labels. These assays allowed us to quantify gradations in nanoscale mechanical properties of bone-forming/non-bone-forming osteocytes in young and aged mice. This study reports for the first time that there are sub-micrometer gradations in modulus surrounding lacunae and that these gradations are dependent upon recent osteocyte bone formation. Perilacunar bone adjacent to bone-forming osteocytes demonstrated lower peak and bulk modulus values when compared to bone near non-bone-forming osteocytes from the same mouse. Bone-forming osteocytes also showed increased perilacunar modulus variability. Age reduced lacunar size but did not significant effect modulus gradation or variability. In general, lacunar morphology was not a strong predictor of modulus gradation patterns. These findings support the idea that lacunar-canalicular remodeling activity changes the material properties of surrounding bone tissue on a sub-micrometer scale. Therefore, conditions that affect osteocyte health have the potential to impact bone quality.

GRAPHICAL ABSTRACT

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sentences: Two distinct mechanisms underlie dosage sensitivity in Pumilio1-associated diseases Mutations in the RNA-binding protein (RBP) Pumilio1 (PUM1) can cause dramatically different phenotypes. Mild mutations that reduce PUM1 levels by 25% lead to a mild, adult-onset ataxia, whereas more severe mutations that reduce PUM1 levels 40-60% produce an infantile syndrome involving multiple developmental delays and seizures. Why this difference in expression should cause such different phenotypes has been unclear; known PUM1 targets are de-repressed to equal degrees in both diseases. We therefore sought to identify PUM1s protein partners in the murine brain. We identified a number of putative interactors involved in different aspects of RNA metabolism such as silencing, alternative splicing, and polyadenylation. We find that PUM1 haploinsufficiency alters the stability of several interactors and disrupts the regulation of targets of those interactors, whereas mild PUM1 loss only de-represses PUM1-specific targets. We validated these phenomena in patient-derived cell lines and show that normalizing PUM1 levels rescues the levels of interactors and their targets. We therefore propose that dosage sensitivity does not necessarily reflect a linear change in levels but can involve distinct mechanisms. Studying the interactors of RBPs in vivo will be necessary to understand their functions in neurological diseases.

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Factorization-based Imputation of Expression in Single-cell Transcriptomic Analysis (FIESTA) recovers Gene-Cell-State Relationships MotivationSingle cell RNA sequencing (scRNA-seq) is a powerful gene expression profiling technique that is presently revolutionizing the study of complex cellular systems in the biological sciences. Existing single-cell RNA-sequencing methods suffer from sub-optimal target recovery leading to inaccurate measurements including many false negatives. The resulting  zero-inflated data may confound data interpretation and visualization.

ResultsSince cells have coherent phenotypes defined by conserved molecular circuitries (i.e. multiple gene products working together) and since similar cells utilize similar circuits, information about each each expression value or  node in a multi-cell, multi-gene scRNA-Seq data set is expected to also be predictable from other nodes in the data set. Based on this logic, several approaches have been proposed to impute missing values by extracting information from non-zero measurements in a data set. In this study, we applied non-negative matrix factorization approaches to a selection of published scRNASeq data sets to recommend new values where original measurements are likely to be inaccurate and where  zero measurements are predicted to be false negatives. The resulting imputed data model predicts novel cell type markers and expression patterns more closely matching gene expression values from orthogonal measurements and/or predicted literature than the values obtained from other previously published imputation approaches.

Contactbenjamin.spike@hci.utah.edu

Availability and implementationFIESTA is written in R and is available at https://github.com/elnazmirzaei/FIESTA and https://github.com/TheSpikeLab/FIESTA.

sentences: Variance Reducing and Noise Correction in Protein Quantification by Measuring Fluctuations in Fluorescence due to Photobleaching Quantifying protein number using the ratio between the variance and the mean of the protein distribution is a straightforward calibration method in the experimental conditions for microscopy imaging. Recently the model has been expanded to decaying processes with binomial distribution. In this paper, we examine the model proposed, and show how the algorithm can be adapted to the case of variance in the initial number of proteins between cells. We propose improving the algorithm so that the information processing of each frame is done independently from other frames. By doing so, the variance in the process of determining the protein number can be reduced. In addition, we examine the handling of unwanted noises in the measurement, offer a solution for shot noise and background noise, and examine the expected error caused in calculating the decay constant. We also analyze the expected difficulties in conducting a practical experiment, which includes non-exponential decay, and variance in the decay constants of the cells. These methods can be applied to any superposition of n0 discrete decaying processes. However, the evaluation of expected errors in quantification is essential for early planning of the experimental conditions, and for the evaluation of the error.

sentences: Spatiotemporal 3D image registration for mesoscale studies of brain development. Comparison of brain samples representing different developmental stages often necessitates registering the samples to common coordinates. Although the available software tools are successful in registering 3D images of adult brains, registration of perinatal brains remains challenging due to rapid growth-dependent morphological changes and variations in developmental pace between animals. To address these challenges, we propose a multi-step algorithm for the registration of perinatal brains. First, we optimized image preprocessing to increase the algorithms sensitivity to mismatches in registered images. Second, we developed an attention-gated simulated annealing (Monte Carlo) procedure capable of focusing on the differences between perinatal brains. Third, we applied classical multidimensional scaling (CMDS) to align ("synchronize") brain samples in time, accounting for individual development paces. We tested this multi-step algorithm on 28 samples of whole-mounted perinatal mouse brains (P0 - P9) and observed accurate registration results. Our computational pipeline offers a runtime of several minutes per brain on a personal computer and automates brain registration tasks including mapping brain data to atlases, comparison of averaged experimental groups, and monitoring brain development dynamics.

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Living with high potassium: a balance between nutrient acquisition and stress signaling during K-induced salt stress O_LIHigh potassium (K) in the growth medium is more toxic to plants than Na at similar concentrations. However, the molecular mechanisms underlying plant responses to K-induced salt stress are virtually unknown.
C_LIO_LIWe examined Arabidopsis thaliana and its extremophyte relative Schrenkiella parvula, using a comparative multi-omics approach to identify cellular processes affected by excess K and understand which deterministic regulatory pathways are active to avoid tissue damage while sustaining growth.
C_LIO_LIA. thaliana showed limited capacity to curb excess K accumulation and prevent nutrient depletion contrasting to S. parvula which could limit excess K accumulation without restricting nutrient uptake. Facilitated by a targeted transcriptomic response, promoting nitrogen uptake along with other key nutrients and uninterrupted N assimilation into primary metabolites during excess K-stress allowed S. parvula to boost its antioxidant and osmolyte pools concurrently leading to sustained growth. Antithetically, A. thaliana showed transcriptional responses indicative of a poor balance between stress signaling, increased ROS levels, and reduced photosynthesis, subsequently leading to inhibited growth.
C_LIO_LIThe ability to regulate independent nutrient uptake and a coordinated transcriptomic response to avoid non-specific stress signaling are two main deterministic steps towards building stress resilience to excess K+-induced salt stress.
C_LI

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Point-of-care analyte quantification and digital readout via lysate-based cell-free biosensors interfaced with personal glucose monitors Field-deployable diagnostics based on cell-free systems have advanced greatly, but on-site quantification of target analytes remains a challenge. Here we demonstrate that Escherichia coli lysate-based cell-free biosensors coupled to a personal glucose monitor (PGM) can enable on-site analyte quantification, with the potential for straightforward reconfigurability to diverse types of analytes. We show that analyte-responsive regulators of transcription and translation can modulate production of the reporter enzyme {beta}-galactosidase, which in turn converts lactose into glucose for PGM quantification. Because glycolysis is active in the lysate and would readily deplete converted glucose, we decoupled enzyme production and glucose conversion to increase endpoint signal output. This lysate metabolism did, however, allow for one-pot removal of glucose present in complex samples (like human serum) without confounding target quantification. Taken together, we show that integrating lysate-based cell-free biosensors with PGMs enables accessible target detection and quantification at the point of need.

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sentences: Polar and non-polar fractions of deep fried edible oils induce differential cytotoxicity and hemolysis Edible oils are the essential part of diet, however, deep frying process induce oxidative changes in these oils, making them unsuitable for consumption. Deep frying generates various noxious polar and non-polar aldehydes and carbonyls, which may be polar or non-polar in nature. The present study thus evaluated the cytotoxic and hemolytic effects of polar and non-polar fractions of different deep fried edible oils. There observed a significantly elevated level of lipid peroxidation products in the polar fraction of deep fried sunflower (FSO-P) and rice bran oils (FRO-P). The treatment with these fractions induced cytotoxicity in cultured colon epithelial cells, with a higher intensity in FSO-P and FRO-P. Further, an increased TBARS level and catalase activity in RBCs treated with FSO-P and FRO-P led to hemolysis. In comparison, the fried coconut oil (FCO) fractions were less toxic and hemolytic; in addition, the non-polar fraction was more toxic, compared to FCO-P fraction.

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A systems-guided approach to discover the intracellular target of a novel evolution-drug lead A promising approach to prevent antibiotic resistance is design of multivalent antibiotics that constrain bacterial evolvability by blocking multiple escape routes. Previously we discovered that an "evolution drug" lead designed to broadly neutralize bacterial dihydrofolate reductase (DHFR) and its trimethoprim escape variants is multivalent in E. coli cells. In this work we identified the second target of this new lead. We analysed antibiotic induced global metabolic perturbations and deployed metabolic-network analysis and structural similarity search of the putative targets and narrowed down the search to a few enzymes. Further, combining in vivo and in vitro analyses, we identified HPPK (folK), an essential protein in bacterial folate-metabolism, as the non-DHFR target of the lead. We observed that compounds with interaction specificity against WT do not develop multivalency against related proteins. This multivalent-design strategy is effective in blocking the emergence of drug-resistant phenotypes.

HighlightsO_LIAn evolution drug lead designed to block bacterial evolvability manifested behaviours suggesting an intra-cellular multivalent activity.
C_LIO_LIA multi-layered metabolomic and structure guided approach unravelled the second intra-cellular target of the evolution-drug lead, showing folate-pathway inhibitions at two metabolic points.
C_LIO_LIThe on-pathway anti-folate activity is exhibited by inhibiting two essential proteins viz. DHFR and its upstream protein HPPK.
C_LIO_LIMultivalent drug development schemes present a promising anti-evolution strategy to address the problem of "drug-resistance" by inhibiting more than one metabolic protein.
C_LI

sentences: INPP5E regulates CD3ζ enrichment at the immune synapse by phosphoinositide distribution control The immune synapse, a specialized interface formed between T lymphocytes and antigen-presenting cells (APCs) after antigen recognition, is essential for T cell activation and the adaptive immune response. It has been shown that this interface shares similarities with the primary cilium, a sensory organelle in eukaryotic cells, although roles of ciliary proteins on the immune synapse remain elusive. In this study, we find that inositol polyphosphate-5-phosphatase E (INPP5E), a cilium-enriched protein responsible for regulating phosphoinositide localization, accumulated at the immune synapse during antigen-specific conjugation or antibody capping, and formed a complex with CD3{zeta}, ZAP-70, and Lck. Silencing INPP5E in T-cells impaired polarized distribution of CD3{zeta} at the immune synapse, and correlated with a failure of PI(4,5)P2 clearance at the center of the synapse. Moreover, INPP5E silencing decreased proximal TCR signaling, including phosphorylation of CD3{zeta} and ZAP-70, and finally, attenuated IL-2 secretion. Our results suggest that INPP5E is a new player in phosphoinositide manipulation at the synapse, controlling the TCR signaling cascade.

sentences: The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia To study how the 22q11.2 deletion predisposes to psychiatric disease, we generated induced pluripotent stem cells from deletion carriers and controls, as well as utilized CRISPR/Cas9 to introduce the heterozygous deletion into a control cell line. Upon differentiation into neural progenitor cells, we found the deletion acted in trans to alter the abundance of transcripts associated with risk for neurodevelopmental disorders including Autism Spectrum Disorder. In more differentiated excitatory neurons, altered transcripts encoded presynaptic factors and were associated with genetic risk for schizophrenia, including common (per-SNP heritability p ({tau}c)= 4.2 x 10-6) and rare, loss of function variants (p = 1.29x10-12). These findings suggest a potential relationship between cellular states, developmental windows and susceptibility to psychiatric conditions with different ages of onset. To understand how the deletion contributed to these observed changes in gene expression, we developed and applied PPItools, which identifies the minimal protein-protein interaction network that best explains an observed set of gene expression alterations. We found that many of the genes in the 22q11.2 interval interact in presynaptic, proteasome, and JUN/FOS transcriptional pathways that underlie the broader alterations in psychiatric risk gene expression we identified. Our findings suggest that the 22q11.2 deletion impacts genes and pathways that may converge with risk loci implicated by psychiatric genetic studies to influence disease manifestation in each deletion carrier.

sentences: Express detection and discrimination of visual objects by primate superior colliculus neurons Primate superior colliculus (SC) neurons exhibit rich visual feature tuning properties and are implicated in a subcortical network hypothesized to mediate fast threat and/or conspecific detection. However, the mechanisms through which generalized object detection may be mediated by SC neurons remain unclear. Here we explored whether, and how quickly, SC neurons detect and discriminate real-life object stimuli. We presented experimentally-controlled gray-scale images of seven different object categories within the response fields (RFs) of SC neurons, and we also presented a variety of luminance- and spectral-matched image controls. We found that all of our functionally-identified SC neuron types preferentially detected real-life objects even in their very first stimulus-evoked visual bursts (starting within approximately 50 ms from image onset). Intriguingly, even visually-responsive motor-related neurons exhibited such robust early object detection, and they also preferentially discriminated between object categories in their initial visual bursts. We further identified spatial frequency information in visual images as a critical source for early object detection and discrimination by SC neurons. Our results demonstrate rapid and robust SC processing of visual objects, and they underline how the visual processing capabilities of the primate SC support perception and action.

sentences: Directed evolution of angiotensin-converting enzyme 2 (ACE2) variants with enhanced peptidase activity profiles for novel therapeutic applications Angiotensin Converting-Enzyme 2 (ACE2) is currently being investigated for its ability to beneficially modulate the Angiotensin receptor (ATR) therapeutic axis to treat multiple human diseases, but its broad substrate scope and diverse physiological roles limit its potential as a therapeutic agent. In this work we engineer ACE2 variants with enhanced hydrolytic activity and specificity toward Angiotensin-II (Ang-II) that may lead to new therapeutic candidates with improved efficacy and reduced off-target side effects. We established a yeast display-based liquid chromatography screen that enabled use of directed evolution to discover ACE2 variants with improved Ang-II activity and specificity relative to the off-target peptide substrates Apelin-13 and Angiotensin-I (Ang-I). We screened ACE2 active site libraries to reveal three substitution-tolerant positions (M360, T371 and Y510) that can be mutated to enhance ACE2s activity profile and followed up on these hits with focused double mutant libraries to further improve the enzyme. Relative to wildtype ACE2, our top variant (T371L/Y510Ile) displayed a sevenfold increase in Ang-II turnover number (kcat), a sixfold diminished catalytic efficiency (kcat/Km) on Apelin-13, and an overall decreased activity on other ACE2 substrates that were not directly assayed in directed evolution screen. At physiologically relevant substrate concentrations, T371L/Y510Ile hydrolyzes more Ang-II than wildtype ACE2 with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to both established and unexplored ACE2 therapeutic applications and provide a foundation for further ACE2 engineering efforts.

sentences: Establishment of a closed artificial ecosystem to ensure human long-term survival on the moon Bioregenerative life support system (BLSS) is a critical technology maintaining long-term human survival on the Moon or other extraterrestrial bodies. In the current study, we carried out a 370-day integrated high-closure experiment ("Lunar Palace 365" experiment) on the Earth in an upgraded ground-based BLSS experimental facility called "Lunar Palace 1". This experiment was designed to develop techniques to run and adjust system stability under long-term operation and crew shift change conditions. Eight volunteering crew members were divided into two groups with three time phases: Group I stayed in the cabin for the initial 60-day phase; Group II inhabited the cabin instead for a record-breaking duration of 200 days as the second phase; Group I re-entered the cabin, replaced Group II and stayed for the last 105 days. Our results demonstrated the BLSS had excellent stability with a material closure degree of 98.2%. Levels of O2, CO2 and trace harmful gases were well controlled within ranges optimal for crew health and plant production. The system exhibited a strong robustness and could quickly minimize effects of disturbances through self-feedback adjustments. The efficiency of plant production completely met the crews need of plant-based food. The purification efficiency of domestic and sanitary wastewater was up to irrigation standards, and the recovery rate of urine and solid waste achieved 99.7% and 67%, respectively. These results are valuable for further optimization of the BLSS in a lunar base and computer simulations of similar systems.

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Estrogen-related receptor alpha and Rplp1 ribosome protein-dependent translation coordinately regulate starvation response and decrease NASH progression Although general translation declines during fasting, maintaining the translation of a subset or proteins is necessary for metabolic homeostasis and cell viability. Using unbiased proteome analysis of hepatic cells during starvation, we identified a novel pathway in which Esrra-mediated transcription of Rplp1-dependent translation of lysosomal proteins declined during early starvation and recovered after prolonged starvation to restore autophagy-lysosome function. Interestingly, hepatic Esrra-Rplp1-dependent translation rate of lysosomal proteins also was impaired in patients and mice with non-alcoholic steatohepatitis (NASH), and translational response to starvation was dysregulated in mice with NASH. Remarkably, activation of Esrra pharmacologically, genetically, or by alternate day fasting restored protein translation, increased expression of lysosomal proteins, induced autophagy, and reduced lipotoxicity, inflammation, and fibrosis in cell culture and in vivo models of NASH. Thus, hepatic Esrra is essential for ribosome-dependent translation of lysosomal proteins during starvation, and prevention of lipotoxicity and progression in NASH.

Lay summaryFasting for weight loss improves non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH); however, the mechanism is not well understood. Our study shows that a nuclear protein, estrogen related receptor alpha (Esrra), increases ribosome-mediated translation of autophagy and lysosome proteins during chronic starvation to maintain essential metabolic pathways for cell survival. Surprisingly, this translational pathway is impaired during NASH with reduced lysosome-autophagy activity accompanied by increased inflammation and fibrosis gene expression in the liver. Pharmacologic, genetic and dietary activation of Esrra decreases lipid-mediated toxicity in liver cells as well as inflammation and fibrosis in livers from mice with NASH. These findings suggest that the Esrra-ribosome-lysosome pathway is important for liver response to fasting and NASH and thus may be a good therapeutic target for the treatment of NASH.

sentences: Large-scale voltage imaging in the brain using targeted illumination Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold membrane voltage dynamics from single neurons in the mammalian brain. To perform high speed voltage imaging, widefield microscopy remains an essential tool for recording activity from many neurons simultaneously over a large anatomical area. However, the lack of optical sectioning makes widefield microscopy more prone to background signal contamination, and thus far voltage imaging using fully genetically encoded voltage indicators remains limited to simultaneous sampling of a few cells over a restricted field-of-view. We here demonstrate a strategy for large scale voltage imaging using the fully genetically encoded voltage indicator SomArchon and targeted illumination. We implemented a simple, low-cost digital micromirror device based targeted illumination strategy to restrict illumination to the cells of interest, and systematically quantified the improvement of this microscopy design theoretically and experimentally with SomArchon expressing neurons in single layer cell cultures and in the brains of awake mice. We found that targeted illumination, in comparison to widefield illumination, increased SomArchon signal contrast and reduced background cross-contamination in the brain. Such improvement permitted the reduction of illumination intensity, and thus reduced fluorescence photobleaching and prolonged imaging duration. When coupled with a high-speed, large area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in the brain. Thus, the widefield microscopy design with an integrated targeted illumination system described here offers a simple solution for voltage imaging analysis of large neuron populations in behaving animals.

sentences: Interspecific gene flow and vicariance in Northern African rear-edge white oak populations revealed by joint analysis of microsatellites and flanking sequences. Inference of recent population divergence requires fast evolving markers and necessitates to differentiate shared genetic variation caused by ancestral polymorphism and gene flow. Theoretical research shows that the use of compound marker systems integrating linked polymorphisms with different mutational dynamics, such as a microsatellite and its flanking sequences, can improve estimation of population structure and inference of demographic history, especially in the case of complex population dynamics. However, empirical application in natural populations has so far been limited by lack of suitable methods for data collection. A solution comes from the development of sequence-based microsatellite genotyping which we used to study molecular variation at 36 sequenced nuclear microsatellites in seven Quecus canariensis and four Quercus faginea rear-edge populations across Algeria, to decipher their taxonomic relationship, past evolutionary history and recent demographic trajectory. First, we compare the estimation of population genetics parameters and simulation-based inference of demographic history from microsatellite sequence alone, flanking sequence alone or the combination of linked microsatellite and flanking sequence variation. Second, we measure variable importance from random forest approximate Bayesian computation to identify which of these sequence types is most informative. Whereas the analysis of microsatellite variation alone indicates recent interspecific gene flow, additional information gained from combining with nucleotide variation in flanking sequences, by reducing homoplasy, suggests ancient interspecific gene flow followed by drift in isolation instead. The weight of each polymorphism in the inference also demonstrates the value of linked variations with contrasted mutation dynamic to improve estimation of both demographic and mutational parameters.

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Reconstructing mitochondrial genomes from ancient DNA through iterative mapping: an evaluation of software, parameters, and bait reference O_LIWithin evolutionary biology, mitochondrial genomes (mitogenomes) provide useful insights at both population and species level. Several approaches are available to assemble mitogenomes. However, most are not suitable for divergent, extinct species, due to the requirement of a reference mitogenome from a conspecific or close relative, and relatively high-quality DNA.
C_LIO_LIIterative mapping can overcome the lack of a close reference sequence, and has been applied to an array of extinct species. Despite its widespread use, the accuracy of the reconstructed assemblies are yet to be comprehensively assessed. Here, we investigated the influence of mapping software (BWA or MITObim), parameters, and bait reference phylogenetic distance on the accuracy of the reconstructed assembly using two simulated datasets: (i) spotted hyena and various mammalian bait references, and (ii) southern cassowary and various avian bait references. Specifically, we assessed the accuracy of results through pairwise distance (PWD) to the reference conspecific mitogenome, number of incorrectly inserted base pairs (bp), and total length of the reconstructed assembly.
C_LIO_LIWe found large discrepancies in the accuracy of reconstructed assemblies using different mapping software, parameters, and bait references. PWD to the reference conspecific mitogenome, which reflected the level of incorrect base calls, was consistently higher with BWA than MITObim. The same was observed for the number of incorrectly inserted bp. In contrast, the total sequence length was lower. Overall, the most accurate results were obtained with MITObim using mismatch values of 3 or 5, and the phylogenetically closest bait reference sequence. Accuracy could be further improved by combining results from multiple bait references.
C_LIO_LIWe present the first comprehensive investigation of how mapping software, parameters, and bait reference influence mitogenome reconstruction from ancient DNA through iterative mapping. Our study provides information on how mitogenomes are best reconstructed from divergent, short-read data. By obtaining the most accurate reconstruction possible, one can be more confident as to the reliability of downstream analyses, and the evolutionary inferences made from them.
C_LI

sentences: A Novel Mice Model of Catecholaminergic Polymorphic Ventricular Tachycardia Generated by CRISPR/Cas9 Catecholaminergic polymorphic ventricular tachycardia (CPVT) has been considered as one of the most important causes of childrens sudden cardiac death. Mutations in the genes for RyR2 and CASQ2, two mainly subtypes of CPVT, have been identified. However, the mutation in the gene of TECRL was rarely reported, which could be another genetic cause of CPVT. We evaluated myocardial contractility, electrophysiology, calcium handling in Tecrl knockout (Tecrl KO) mice and human induced pluripotent stem cell-derived cardiomyocytes. Immediately after epinephrine plus caffeine injection, Tecrl KO mice showed much more multiple premature ventricular beats and ventricular tachycardia. The Tecrl KO mice demonstrate CPVT phenotypes. Mechanistically, intracellular calcium amplitude was reduced, while time to baseline of 50 was increased in acute isolated cardiomyocytes. RyR2 protein levels decreased significantly upon cycloheximide treatment in TECRL deficiency cardiomyocytes. Overexpression of TECRL and KN93 can partially reverse cardiomyocytes calcium dysfunction, and this is p-CaMKII/CaMKII dependent. Therefore, a new CPVT mouse model was constructed. We propose a previously unrecognized mechanism of TECRL and provide support for the therapeutic targeting of TECRL in treating CPVT.

sentences: Sex Differences in Cerebral Edema after Experimental Traumatic Brain Injury Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. Cerebral edema following TBI is known to play a critical role in injury severity and prognosis. In the current study we used multimodal magnetic resonance imaging (MRI) to assess cerebral edema 24 hours after unilateral contusive TBI in male and female rats. We then directly quantified brain water content in the same subjects ex vivo. We found that both males and females had similarly elevated T2 values after TBI compared with sham controls. Apparent diffusion coefficient (ADC) was more variable than T2 and did not show significant injury effects in males or females. Brain water was elevated in male TBI rats compared with sham controls, but there was no difference between female TBI and sham groups. Notably, MRI biomarkers of edema were more closely correlated with brain water in male rats; female rats did not show any relationship between brain water and T2 or ADC. These observations raise questions about the interpretation of radiological findings traditionally interpreted as edema in female TBI patients. A better understanding of sex differences and similarities in the pathophysiology of post-traumatic edema is needed to help improve patient management and the development of effective treatment strategies for men and women.

sentences: Fear of the new? Geckos hesitate to attack novel prey, feed near objects and enter a novel space Neophobia, the fear of novelty, is an ecologically important response which enables animals to avoid potentially harmful situations. Low levels of neophobia have been linked to elevated dispersal/ migration, invasiveness and living in human modified landscapes albeit only in birds and mammals. In this study, we assessed neophobia in captive Tokay geckos (Gekko gecko). We expected to find low neophobia in our geckos because they are invasive and adopt well to anthropogenic environments. This species is, however, also both predator and prey in the wild which might select for higher neophobia. We tested neophobia in three contexts: attacking novel prey, foraging near novel objects and entering a novel space. We aimed to quantify (1) neophobia in these contexts, (2) individual consistency across trials using different novel stimuli, and (3) correlation of individual responses across the three contexts. We found that geckos hesitate to attack novel prey and prey close to objects (familiar and novel). Geckos hesitated the most when entering novel space and repeatability of behaviour across contexts was low (R = 0.12) indicating that neophobia might not be a single trait. The strength of a neophobic response can indicate how anxious or curious an individual is. This test has great potential to help answer questions about how captivity, enrichment, rearing environment and cognition affect fear responses in different contexts in lizards. By studying reptiles, we can better understand the universality of what is known about the causes leading to difference in neophobia across individuals and species.

sentences: Distinct regionalization patterns of cortical morphology are associated with cognitive performance across different domains Cognitive performance in children is predictive of academic and social outcomes; therefore, understanding neurobiological mechanisms underlying individual differences in cognition during development may be important for improving quality of life. The belief that a single, psychological construct underlies many cognitive processes is pervasive throughout society. However, it is unclear if there is a consistent neural substrate underlying many cognitive processes. Here we show that a distributed configuration of cortical surface area and apparent thickness, when controlling for global imaging measures, is differentially associated with cognitive performance on different types of tasks in a large sample (N=10,145) of 9-11 year old children from the Adolescent Brain and Cognitive DevelopmentSM (ABCD) study. The minimal overlap in these regionalization patterns of association has implications for competing theories about developing intellectual functions. Surprisingly, not controlling for sociodemographic factors increased the similarity between these regionalization patterns. This highlights the importance of understanding the shared variance between sociodemographic factors, cognition and brain structure, particularly with a population-based sample such as ABCD.

sentences: Identifying Uncertainty States during Wayfinding in Indoor Environments: An EEG Classification Study The researchers used a machine-learning classification approach to better understand neurological features associated with periods of wayfinding uncertainty. The participants (n=30) were asked to complete wayfinding tasks of varying difficulty in a virtual reality (VR) hospital environment. Time segments when participants experienced navigational uncertainty were first identified using a combination of objective measurements (frequency of inputs into the VR controller) and behavioral annotations from two independent observers. Uncertainty time-segments during navigation were ranked on a scale from 1 (low) to 5 (high). The machine-learning model, a random forest classifier implemented using scikit-learn in Python, was used to evaluate common spatial patterns of EEG spectral power across the theta, alpha, and beta bands associated with the researcher-identified uncertainty states. The overall predictive power of the resulting model was 0.70 in terms of the area under the Receiver Operating Characteristics curve (ROC-AUC). These findings indicate that EEG data can potentially be used as a metric for identifying navigational uncertainty states, which may provide greater rigor and efficiency in studies of human responses to architectural design variables and wayfinding cues.

sentences: Rare genera differentiate urban green space soil bacterial communities in three cities across the world Vegetation complexity is potentially important for urban green space designs aimed at fostering microbial biodiversity to benefit human health. Exposure to urban microbial biodiversity may influence human health outcomes via immune training and regulation. In this context, improving human exposure to microbiota via biodiversity-centric urban green space designs is an underused opportunity. There is currently little knowledge on the association between vegetation complexity (i.e., diversity and structure) and soil microbiota of urban green spaces. Here, we investigated the association between vegetation complexity and soil bacteria in urban green spaces in Bournemouth, UK; Haikou, China; and the City of Playford, Australia by sequencing the 16S rRNA V4 gene region of soil samples and assessing bacterial diversity. We characterized these green spaces as having  low or  high vegetation complexity and explored whether these two broad categories contained similar bacterial community compositions and diversity around the world. Within cities, we observed significantly different alpha and beta diversities between vegetation complexities; however, these results varied between cities. Rare genera (< 1 % relative abundance individually, on average 35 % relative abundance when pooled) were most likely to be significantly different in sequence abundance between vegetation complexities and therefore explained much of the differences in microbial communities observed. Overall, general associations exist between soil bacterial communities and vegetation complexity, although these are not consistent between cities. Therefore, more in-depth work is required to be done locally to derive practical actions to assist the conservation and restoration of microbial communities in urban areas.

sentences: Tardigrade community microbiomes in North American orchards include putative endosymbionts and plant pathogens The microbiome of tardigrades, a phylum of microscopic animals best known for their ability to survive extreme conditions, is poorly studied worldwide and completely unknown in North America. An improved understanding of tardigrade-associated bacteria is particularly important because tardigrades have been shown to act as vectors of the plant pathogen Xanthomonas campestris in the laboratory. However, the potential role of tardigrades as reservoirs and vectors of phytopathogens has not been investigated further. This study analyzed the microbiota of tardigrades from six apple orchards in central Iowa, USA, and is the first analysis of the microbiota of North American tardigrades. It is also the first ever study of the tardigrade microbiome in an agricultural setting. We utilized 16S rRNA gene amplicon sequencing to characterize the tardigrade community microbiome across four contrasts: location, substrate type (moss or lichen), collection year, and tardigrades versus their substrate. Alpha diversity of the tardigrade community microbiome differed significantly by location and year of collection but not by substrate type. Our work also corroborated earlier findings, demonstrating that tardigrades harbor a distinct microbiota from their environment. We also identified tardigrade-associated taxa that belong to genera known to contain phytopathogens (Pseudomonas, Ralstonia, and the Pantoea/Erwinia complex). Finally, we observed members of the genera Rickettsia and Wolbachia in the tardigrade microbiome; because these are obligate intracellular genera, we consider these taxa to be putative endosymbionts of tardigrades. These results suggest the presence of putative endosymbionts and phytopathogens in the microbiota of wild tardigrades in North America.

sentences: Sub-Cluster Identification through Semi-SupervisedOptimization of Rare-cell Silhouettes (SCISSORS) in Single-Cell Sequencing Single-cell RNA-sequencing (scRNA-seq) has enabled the molecular profiling of thousands to millions of cells simultaneously in biologically heterogenous samples. Currently, common practice in scRNA-seq is to determine cell type labels through unsupervised clustering and the examination of cluster-specific genes. However, even small differences in analysis and parameter choice can greatly alter clustering solutions and thus impose great influence on which cell types are identified. Existing methods largely focus on determining the optimal number of robust clusters, which is not favorable for identifying cells of extremely low abundance due to their subtle contributions towards overall patterns of gene expression. Here we present a carefully designed framework, SCISSORS, which accurately profiles subclusters within major cluster(s) for the identification of rare cell types in scRNA-seq data. SCISSORS employs silhouette scoring for the estimation of heterogeneity of clusters and reveals rare cells in heterogenous clusters by implementing a multi-step, semi-supervised reclustering process. Additionally, SCISSORS provides a method for the identification of marker genes of rare cells, which may be used for further study. SCISSORS is wrapped around the popular Seurat R package and can be easily integrated into existing Seurat pipelines. SCISSORS, including source code and vignettes for two example datasets, is freely available at https://github.com/jrleary/SCISSORS.

sentences: CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say Culex quinquefasciatus Say is a brown, medium sized mosquito distributed widely in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and is responsible for vectoring many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical / chemical) are increasingly ineffective; use of insecticides also imposes some hazards to both human and ecosystem health. Recent advances in genome editing have allowed the development of genetic methods of insect control, which is species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that has been shown to be functional in a range of species. We demonstrate here, the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated exogenous sequence comprising a sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8% similar to rates achieved in other mosquito species. Precise knock-in at the intended locus was confirmed by sequencing. Insertion homozygotes displayed a white eye phenotype in early-mid stage larvae and a recessive lethal phenotype by pupation. This work shows an alternative and efficient method for genetic engineering of C. quinquefasciatus, providing a new tool for researchers interested in developing genetic control tools for this vector.

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Single-cell transcriptomics reveals the identity and regulators of human mast cell progenitors Mast cell accumulation is a hallmark of a number of diseases including allergic asthma and systemic mastocytosis. IgE-mediated crosslinking of the Fc{varepsilon}RI receptors causes mast cell activation and contributes to disease pathogenesis. The mast cell lineage is one of the least studied among the hematopoietic cell lineages and there are still controversies about the identity of the mast cell progenitor, i.e., whether Fc{varepsilon}RI expression appears during the hematopoietic progenitor stage or in maturing mast cells. Here, we used single-cell transcriptomics to reveal a temporal association between the appearance of Fc{varepsilon}RI and the mast cell gene signature in CD34+ hematopoietic progenitors. In agreement with these data, the Fc{varepsilon}RI+ hematopoietic progenitors formed morphologically, phenotypically and functionally mature mast cells in long-term culture assays. Single-cell transcriptomics analysis further revealed the expression patterns of prospective cytokine receptors regulating mast cell progenitor development. Culture assays showed that IL-3 and IL-5 promoted disparate effects on progenitor cell proliferation and survival, respectively, whereas IL-33 caused robust Fc{varepsilon}RI downregulation. Taken together, we have demonstrated that Fc{varepsilon}RI appears during the hematopoietic progenitor stage of mast cell differentiation and that external stimuli may regulate the Fc{varepsilon}RI expression. Thus, the results resolve the controversy regarding the appearance of Fc{varepsilon}RI during mast cell development.

One-sentence summarySingle-cell analysis of human hematopoiesis uncovers the stage at which Fc{varepsilon}RI appears during mast cell differentiation and reveals disparate effects of IL-3, IL-5 and IL-33 on mast cell progenitor proliferation, survival, and suppression of Fc{varepsilon}RI expression.

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High prevalence of Klebsiella pneumoniae in European food products: a multicentric study comparing culture and molecular detection methods Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices, and to characterize food isolates phenotypically and genotypically. Chicken meat (n=160) and salad (n=145) samples were collected in five European countries and screened for KpSC presence using culture-based and ZKIR qPCR methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44{degrees}C/48h) was defined as the most suitable selective culture method for KpSC recovery. High prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples: same food type and same country (15 cases); different food types and same country (1); same food type and two countries (1), suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans.

ImportanceBacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous and K. pneumoniae (Kp) is a leading cause of antibiotic-resistant infections in humans and animals. Despite the urgent public health threat represented by Kp, there is a lack of knowledge on the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing KpSC presence in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low-levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies.

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The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury: a translational in vitro study BackgroundEvidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here we describe the generation of HepG2-derived transmitochondrial cybrids in order to investigate the impact of mtDNA variation upon mitochondrial (dys)function and susceptibility to iDILI against a constant nuclear background. In this study, cybrids were created to contain mitochondrial genotypes of haplogroup H and haplogroup J for comparison.

MethodsBriefly, HepG2 cells were depleted of mtDNA to make rho zero cells before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 distinct transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide and tolcapone, by ATP assays and extracellular flux analysis.

FindingsOverall, baseline mitochondrial function was similar between haplogroups H and J. However, haplogroup specific responses to mitotoxic drugs were observed; haplogroup J was more susceptible to the inhibition of respiratory complexes I and II, and also to the effects of tolcapone, an uncoupler of mitochondrial respiration.

ConclusionsThis study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest, thus providing a practical and reproducible system to investigate the cellular consequences of variation in mitochondrial genome against a constant nuclear background. Additionally the results support that that inter-individual variation in mitochondrial genotype and haplogroup may be a factor in determining sensitivity to mitochondrial toxicants.

FundingThis work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).

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QTL mapping: an innovative method for investigating the genetic determinism of yeast-bacteria interactions in wine The two most commonly used wine microorganisms, Saccharomyces cerevisiae yeast and Oenococcus oeni bacteria, are responsible for completion of alcoholic and malolactic fermentation (MLF), respectively. For successful co-inoculation, S. cerevisiae and O. oeni must be able to complete fermentation, however, this relies on compatibility between yeast and bacterial strains. For the first time, Quantitative Trait Loci (QTL) analysis was used to elucidate whether S. cerevisiae genetic makeup can play a role in the ability of O. oeni to complete MLF. Assessment of 67 progeny of an S. cerevisiae SBxGN cross, co-inoculated with a single O. oeni strain, SB3, revealed a major QTL linked to MLF completion by O. oeni. This QTL encompassed a well-known translocation, XV-t-XVI, that results in increased SSU1 expression and is functionally linked with numerous phenotypes including lag phase duration and sulfite export and production. A reciprocal hemizygosity assay was performed to elucidate the effect of the gene SSU1 in the SBxGN background. Our results instead revealed a strong effect of SSU1 haploinsufficiency on O. oenis ability to complete malolactic fermentation during co-inoculation, and paves the way for the implementation of QTL mapping projects for deciphering the genetic bases of microbial interactions.

Key pointsFor the first time QTL analysis has been used to study yeast-bacteria interactions.

A QTL encompassing a translocation, XV-t-XVI, was linked to MLF outcomes.

S. cerevisiae SSU1 haploinsufficiency positively impacted MLF by O. oeni.

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Development of a novel, pan-variant aerosol intervention for COVID-19 To develop a universal strategy to block SARS-CoV-2 cellular entry and infection represents a central aim for effective COVID-19 therapy. The growing impact of emerging variants of concern increases the urgency for development of effective interventions. Since ACE2 is the critical SARS-CoV-2 receptor and all tested variants bind to ACE2, some even at much increased affinity (see accompanying paper), we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here we show that intranasal administration of APN01 in a mouse model of SARS-CoV-2 infection dramatically reduced weight loss and prevented animal death. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers can now be initiated, with subsequent Phase II testing in individuals with SARS-CoV-2 infection. This strategy could be used to develop a viable and rapidly actionable therapy to prevent and treat COVID-19, against all current and future SARS-CoV-2 variants.

One Sentence SummaryPreclinical development and evaluation of aerosolized soluble recombinant human ACE2 (APN01) administered as a COVID-19 intervention is reported.

sentences: Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies PML nuclear bodies (PML-NBs) are dynamic interchromosomal macromolecular complexes implicated in epigenetic regulation as well as antiviral defense. During herpesvirus infection, PML-NBs induce epigenetic silencing of viral genomes, however, this defense is antagonized by viral regulatory proteins such as IE1 of human cytomegalovirus (HCMV). Here, we show that PML-NBs undergo a drastic rearrangement into highly enlarged PML cages upon infection with IE1-deficient HCMV. Importantly, our results demonstrate that dual signaling by interferon and DNA damage response is required to elicit giant PML-NBs. DNA labeling revealed that invading HCMV genomes are entrapped inside PML-NBs and remain stably associated with PML cages in a transcriptionally repressed state. Intriguingly, by correlative light and transmission electron microscopy (EM), we observed that PML cages also entrap newly assembled viral capsids demonstrating a second defense layer in cells with incomplete first line response. Further characterization by 3D EM showed that hundreds of viral capsids are tightly packed into several layers of fibrous PML. Overall, our data indicate that giant PML-NBs arise via combined interferon and DNA damage signaling which triggers entrapment of both nucleic acids and proteinaceous components. This represents a multilayered defense strategy to act in a cytoprotective manner and to combat viral infections.

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BnaA03.WRKY28, interacting with BnaA09.VQ12, acts as a brake factor of activated BnWRKY33-mediated resistance outburst against Sclerotinia sclerotiorum in Brassica napus Sclerotinia sclerotiorum causes substantial damage to the growth of Brassica napus (rapeseed) and makes a significant loss of crop yield. The plant innate immune system may be the primary solution to defense against S. sclerotiorum for rapeseed. Here, we identify that BnWRKY33, a transcription factor in the innate immune pathway, can be rapidly phosphorylated and activated by the MAPK cascade after rapeseed is infected with S. sclerotiorum. In the MAPK cascade, activated BnaA03.MKK4 phosphorylates and activates BnaA06.MPK3 and BnaC03.MPK3. The activated BnMPK3 acts on the substrate BnWRKY33 to enhance its transcriptional activity and trigger a transcriptional burst of BnWRKY33, which helps plants effectively resist the pathogenic fungi by enhancing the expression of phytoalexin synthesis-related genes. With constant infection, BnaA03.WRKY28 and BnaA09.VQ12 are induced, and BnaA03.WRKY28 physically interacts with BnaA09.VQ12 to form a protein complex. BnaA03.WRKY28 preferentially binds to the promoter of BnWRKY33 with the help of BnaA09.VQ12. Compared with activated BnWRKY33, BnaA03.WRKY28 has a lower transcriptional activity on downstream BnWRKY33, which leads to weaker resistance against S. sclerotiorum for rapeseed in the later stage of infection. Furthermore, the induced BnaA03.WRKY28 may promote axillary bud activity and axillary meristem initiation by regulating the expression of branching-related genes (such as BnBRC1), thus promoting the formation of branches in the leaf axils.

One-sentence summaryUnder constant infection by Sclerotinia sclerotiorum, BnaA03.WRKY28 interacts with BnaA09.VQ12 and takes precedence over phosphorylated BnWRKY33 to bind to the BnWRKY33 promoter, thereby weakening resistance but promoting branching.

sentences: Cryo-EM structure of amyloid fibril formed by α-synuclein familial A53E mutation Synucleinopathies, including Parkinsons disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA) have the same hallmark pathologic feature of misfolded -synuclein protein accumulation in the brain. PD patients who carry -syn hereditary mutations tend to have an earlier onset and more severe clinical symptoms and pathology than sporadic PD patients who carry wild-type (WT) -syn. Therefore, revealing the structural effect of -syn hereditary mutations on the wild-type fibril structure can help us understand synucleinopathies structural basis. Here, we present a 3.38 [A] cryo-electron microscopy structure of -synuclein fibrils containing the hereditary A53E mutation. The A53E fibril is symmetrically composed of two protofilaments, as are many other synucleopathic structures - including WT. Interestingly, the interface between the protofilaments in A53E has significantly less buried surface area than all other documented fibril structures of -syn and its other mutants. The A53E fibril also exhibits slower formation/growth in in vitro fibrillation experiment compared to other mutants. This implies that the structural differences - both in the protofilament and between each protofilament of A53E - change the aggregation mechanism, or in the least, its kinetics of formation. These differences influence the molecular characteristics of each fibril mutant and likely plays a macro-scale role in progressing one clinical pathology over another.

sentences: Spatiotemporal transcriptome at single-cell resolution reveals key radial glial cell population in axolotl telencephalon development and regeneration Brain regeneration requires a precise coordination of complex responses in a time- and region-specific manner. Identifying key cell types and molecules that direct brain regeneration would provide potential targets for the advance of regenerative medicine. However, progress in the field has been hampered largely due to limited regeneration capacity of the mammalian brain and understanding of the regeneration process at both cellular and molecular level. Here, using axolotl brain with extrodinary regeneration ability upon injury, and the SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq), we reconstructed the first architecture of axolotl telencephalon with gene expression profiling at single-cell resolution, and fine cell dynamics maps throughout development and regeneration. Intriguingly, we discovered a marked heterogeneity of radial glial cell (RGC) types with distinct behaviors. Of note, one subtype of RGCs is activated since early regeneration stages and proliferates while other RGCs remain dormant. Such RGC subtype appears to be the major cell population involved in early wound healing response and gradually covers the injured area before presumably transformed into the lost neurons. Altogether, our work systematically decoded the complex cellular and molecular dynamics of axolotl telencephalon in development and regeneration, laying the foundation for studying the regulatory mechanism of brain regeneration in the future.

sentences: Co-expression of recombinant human collagen α1(III) chain with viral prolyl 4-Hydroxylase in P. pastoris GS115 Prolyl 4-hydroxylase (P4H) is essential to maintain the stable triple-helix structure and function of human collagen 1([SHcy]) chain (COL3A1). To obtain hydroxylated human COL3A1, the human COL3A1 and the viral P4H A085R were co-expressed in P. pastoris GS115. The sequence of human COL3A1 without N-terminal and C-terminal was selected for expression. Colony PCR analysis and sequencing after transfection showed that the target gene had inserted successfully. Real-time quantitative PCR (RT-qPCR) indicated that human COL3A1 and P4H were expressed at the mRNA levels. SDS-PAGE and Western blotting analysis of supernatant from the recombinant methylotrophic yest culture showed that recombinant human COL3A1 (rhCOL3A1) was secreted into the culture medium with an apparent molecular mass of approximately 130 kDa. It was noted that the rhCOL3A1 expession quantity was higest at 120 h of induction. Furthermore, mass spectrometry analysis demonstrated that the rhCOL3A1 was expressed successfully. His-tagged rhCOL3A1 protein was purified by Ni-affinity column.

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Keratin dynamics govern the establishment of the maternal-fetal interface. After implantation, the mouse embryo undergoes gastrulation and forms mesoderm and endoderm. Mesoderm participates in embryonic and extra-embryonic tissues including the amnion, yolk sac, chorion and allantois, the umbilical cord precursor.

Extra-embryonic mesoderm is rich in intermediate filaments. Two-photon live imaging of keratin 8-eYFP knock-in embryos allowed recording nucleation and elongation of keratin filaments, which formed apical cables coordinated across multiple cells in amnion, allantois, and blood islands. Embryos lacking all keratins displayed a deflated exocoelomic cavity, a narrow thick amnion, and a short allantois, indicating a hitherto unknown role for keratin filaments in post-implantation extra-embryonic membranes morphogenesis.

Single-cell RNA sequencing of mesoderm cells, microdissected amnion, chorion, and allantois provided an interactive atlas of transcriptomes with germ layer and regional information. Keratin 8high mesenchymal cells in contact with the exocoelom shared a cytoskeleton and adhesion expression profile that might explain the adaptation of extra-embryonic structures to the increasing mechanical pressure.

GRAPHICAL ABSTRACT

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Methodological considerations in analyzing synchronization of resting-state brain networks with the intrinsic electrical rhythm of the stomach: Advantages of weighted phase-locking PurposeTo evaluate the amplitude-weighted phase-locking value (awPLV) as a measure of synchronization of brain resting-state networks (RSNs) with the gastric basal electrical rhythm (BER).

MethodsA recent study combined rsfMRI with concurrent cutaneous electrogastrography (EGG), in a highly-sampled individual who underwent 22 scanning sessions (two 15-minute runs per session) at 3.0 Tesla. After excluding three sessions due to weak EGG signals, 9.5 hours of data remained, from which 18 RSNs were estimated using spatial independent component analysis. Previously, using the phase-locking value (PLV), three of the 18 RSNs were determined to be synchronized with the BER. However, RSN power fluctuations in the gastric frequency band could reduce sensitivity of PLV. Accordingly, the current reanalysis used awPLV to unweight contributions from low power epochs. Mismatched EGG and rsfMRI data (from different days) served as surrogate data; for each RSN, empirical awPLV was compared with chance-level awPLV using a Wilcoxon rank test. P-values were adjusted using with a false discovery rate of 0.05. Additionally, simulations were performed to compare PLV and awPLV error rates under settings with a known ground truth.

ResultsSimulations show high false-negative rates when using PLV, but not awPLV. Reanalysis of the highly-sampled individual data using awPLV indicates that 11 of the 18 RSNs were synchronized with the BER.

ConclusionSimulations indicate that awPLV is a more sensitive measure of stomach/brain synchronization than PLV. Reanalysis results imply communication between the enteric nervous system and brain circuits not typically considered responsive to gastric state or function.

sentences: Histone H2A serine-1 phosphorylation is a chaperone-dependent signal for dimerization with H2B and for enhanced deposition Multiple histone chaperones and histone modifications are involved in the folding, transport, and re-lease of histones onto newly replicated DNA. Little is known about histone H2A-H2B pre-deposition his-tone modifications and their regulation of histone deposition. We previously showed that H2A serine 1 phosphorylation (H2AS1ph) is enriched on the soluble egg histones and on zygotic chromatin in Xenopus embryos. Here, we demonstrate that H2AS1 phosphorylation is required for a timely incorporation of H2A-H2B into the pronuclear chromatin. Our analysis revealed that exogenous H2AS1A-H2B dimers were poorly incorporated into pronuclei in egg extract compared with wildtype and H2AS1E-H2B dimers. Chaperone-mediated deposition using histones purified from pronuclei showed that neither Nap1 nor Nucleoplasmin (Npm2) histone deposition was directly affected by endogenous histone posttranslational modification. We further demonstrate that H2AS1 phosphorylation was dependent on Npm2 and required H2B. Surprisingly, Nap1 was incapable of promoting H2AS1 phosphorylation. These results suggest that serine 1 phosphorylation signals a specific state of H2A-H2B dimer bound by Nucleoplasmin. Neither Npm2 nor Nap1 exhibited preference for binding H2AS1A or H2AS1E mutant histones or dimers with H2B in vitro. We propose that H2AS1 phosphorylation is a pre-deposition modification that signals for the proper dimerization of H2A-H2B, which in turn activates downstream effectors leading to H2A-H2B deposition.

sentences: The joint role of coevolutionary selection and network structure in shaping trait complementarity in mutualisms. Coevolution can sculpt remarkable trait similarity between mutualistic partners. Yet, it remains unclear which network topologies and selection regimes enhance such trait complementarity. To address this, we simulate coevolution in topologically-distinct net-works under a gradient of mutualistic selection strength. We describe three main insights. First, trait matching is jointly influenced by the strength of mutualistic selection and the structural properties of the network where coevolution is unfolding. Second, the strength of mutualistic selection determines the network descriptors better correlated with higher trait matching. When coevolution is weak, network modularity enhances trait matching, but when it is strong, network connectance amplifies trait matching. Third, the structural properties of networks outrank those of modules or species in determining the evolved degree of trait matching. Our findings suggest networks can both enhance or constrain trait complementary, depending on the strength of mutualistic selection.

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Sequence dependencies and mutation rates of localized mutational processes in cancer BackgroundCancer mutations accumulate through replication errors and DNA damage coupled with incomplete repair. Individual mutational processes often show strong sequence and regional preferences. As a result, some sequence contexts mutate at much higher rates than others. Mutational hotspots, with recurrent mutations across cancer samples, represent genomic positions with elevated mutation rates, often caused by highly localized mutational processes.

ResultsWe analyze the mutation rates of all 11-mer genomic sequence contexts using the PCAWG set of 2,583 pan-cancer whole genomes. We further associate individual mutations and contexts to mutational signatures and estimate their relative mutation rates. We show that hotspots generally identify highly mutable sequence contexts. Using these, we show that some mutational signatures are enriched in hotspot sequence contexts, corresponding to well-defined sequence preferences for the underlying localized mutational processes. This includes signature 17b (of unknown etiology) and signatures 62 (POLE), 7a (UV), and 72 (linked to lymphomas). In some cases, the mutation rate increases further when focusing on certain genomic regions, such as signature 62 in poised promoters, where the mutation is increased several thousand folds over the overall data set average.

ConclusionWe summarize our findings in a catalog of localized mutational processes, their sequence preferences, and their estimated mutation rates.

sentences: Human sialomucin CD164 is an essential entry factor for lymphocytic choriomeningitis virus Lymphocytic choriomeningitis virus (LCMV) is a well-studied mammarenavirus that can be fatal in congenital infections. However, our understanding of LCMV and its interactions with human host factors remain incomplete. Here, host determinants affecting LCMV infection were investigated through a genome-wide CRISPR knockout screen in A549 cells, a human lung adenocarcinoma line. We identified and validated a variety of novel host factors that play a functional role in LCMV infection. Among these, knockout of the sialomucin CD164, a heavily glycosylated transmembrane protein, was found to ablate infection with multiple LCMV strains but not other hemorrhagic mammarenaviruses, in several cell types. Further characterization revealed a dependency of LCMV entry on the cysteine-rich domain of CD164, including a N-linked glycosylation site at residue 104 in that region. Given the documented role of LCMV with respect to transplacental human infections, CD164 expression was investigated in human placental tissue and placental cell lines. CD164 was found to be highly expressed in the cytotrophoblast cells, an initial contact site for pathogens within the placenta, and LCMV infection in placental cells was effectively blocked using a monoclonal antibody specific to the cysteine-rich domain of CD164. Together, this study identifies novel factors associated with LCMV infection of human tissues, and highlights the importance of CD164, a sialomucin that has previously not been associated with viral infection.

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Common variation in EDN1 regulatory regions highlights the role of PPARγ as a key regulator of Endothelin in vitro Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the commonest targeted by current treatments.

We sequenced the EDN1 untranslated regions of a small subset of PAH patients, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors predicted for these regions (PPAR{gamma}, KLF4, and VDR) to assess Endothelin-1 expression in cell culture and validate the binding sites.

First, we detected a SNP in the 5UTR (rs397751713) and another in the 3regulatory region (rs2859338) that increased luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPAR{gamma} could be binding in the rs397751713 and VDR in rs2859338. By using siRNAs and luciferase, we determined that PPAR{gamma} binds differentially in rs397751713. PPAR{gamma} and VDR Knock-Down, increased EDN1 mRNA levels and Endothelin-1 production in PAECs, while PPAR{gamma} and KLF4 Knock-Down increased the Endothelin-1 production in HeLa.

In conclusion, common variants in EDN1 regulatory regions could alter Endothelin-1 levels. We were able to validate that PPAR{gamma} binds in rs397751713 and is key to regulate Endothelin-1. Also, KLF4 and VDR regulate Endothelin-1 production in a cell-dependent manner, but for VDR this interaction does not happen by binding directly to the regions we studied.

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Knowledge Graph-based Recommendation Framework Identifies Novel Drivers of Resistance in EGFR mutant Non-small Cell Lung Cancer Resistance to EGFR inhibitors (EGFRi) presents a major obstacle in treating non-small cell lung cancer (NSCLC). One of the most exciting new ways to find potential resistance markers involves running functional genetic screens, such as CRISPR, followed by manual triage of significantly enriched genes. This triage process to identify  high value hits resulting from the CRISPR screen involves significant manual curation that requires specialized knowledge and can take even experts several months to comprehensively complete.

To find key drivers of resistance faster we built a hybrid recommendation system on top of a heterogeneous biomedical knowledge graph integrating preclinical, clinical, and literature evidence. Genes were ranked based on trade-offs between diverse types of evidence linking them to potential mechanisms of EGFRi resistance. This unbiased approach identified 36 resistance markers from >3,000 genes, reducing hit identification time from months to minutes. In addition to reproducing known resistance markers, our method identified novel resistance mechanisms that we prospectively validated.

sentences: Evidence of long-term purging of mutation load in killer whale genomes Genomes of high latitude killer whales harbour signatures of post-glacial founding and expansion. Here, we investigate whether reduced efficacy of selection increased mutation load in founder populations, or whether recessive deleterious mutations exposed to selection in homozygous genotypes were purged. Comparing the accumulation of synonymous and non-synonymous mutations across pairs of globally sampled genomes reveals that the most significant outliers are high latitude North Atlantic genomes, which have accumulated significantly fewer non-synonymous mutations than all other populations. Comparisons with the genome of a 7.5-Kyr-old North Atlantic killer whale, inferred to be closely related to the population directly ancestral to present-day Icelandic and Norwegian populations, calibrates the timing of the action of selection on non-synonymous mutations predominantly to the mid-late Holocene. Non-synonymous mutations purged in modern Norwegian killer whale genomes are found as globally shared standing variation in heterozygote genotypes more often than expected, suggesting overdominance. Taken together, our findings are consistent with purging of recessive non-synonymous mutations exposed to selection in founder-associated homozygous genotypes.

sentences: Septins are essential for protrusion and detachment in collective border cell migration Collective cell migration is prevalent throughout development and common in metastatic tumors, yet this process is not fully understood. In this study, we explore the role of septins (Sep) in collective cell migration, using the Drosophila border cell model. We show that Sep2 and Pnut are expressed in migrating border cells and Sep1, 2, 4, and Peanut (Pnut) are required for migration. Pnut stability depends on the expression of Sep1 and Sep2 in epithelial follicle cells and migratory border cells. We show that knockdown of septins prevents normal protrusion and detachment behaviors. High resolution Airyscan imaging reveals Pnut localization in rings at the base of protrusions. While septins function independently of Cdc42, they colocalize dynamically with nonmuscle myosin II. We suggest that septin polymers may stabilize growing protrusions until sufficient myosin is recruited to retract them.

sentences: Biophysical Characterisation of Human LincRNA-p21 Sense and Antisense Alu Inverted Repeats Human Long Intergenic Noncoding RNA-p21 (LincRNA-p21) is a regulatory noncoding RNA that plays an important role in promoting apoptosis. LincRNA-p21 is also critical in down-regulating many p53 target genes through its interaction with a p53 repressive complex. The interaction between LincRNA-p21 and the repressive complex is likely dependent on the RNA tertiary structure. Previous studies have determined the two-dimensional secondary structures of the sense and antisense human LincRNA-p21 AluSx1 IRs using SHAPE. However, there were no insights into its three-dimensional structure. Therefore, we in vitro transcribed the sense and antisense regions of LincRNA-p21 AluSx1 Inverted Repeats (IRs) and performed analytical ultracentrifugation, size exclusion chromatography, light scattering, and small angle X-ray scattering (SAXS) studies. Based on these studies, we determined low-resolution, three-dimensional structures of sense and antisense LincRNA-p21. By adapting previously known two-dimensional information, we calculated their sense and antisense high-resolution models and determined that they agree with the low-resolution structures determined using SAXS. Thus, our integrated approach provides insights into the structure of LincRNA-p21 Alu IRs. Our study also offers a viable pipeline for combining the secondary structure information with biophysical and computational studies to obtain high-resolution atomistic models for long noncoding RNAs.

sentences: Mapping the Central and Peripheral Projections of Lung Innervating Sensory Neurons While best known as the gas exchange organ, the lung is also critical for sensing and responding to the aerosol environment in part through interaction with the nervous system. The rich diversity of lung innervating neurons remains poorly understood. Here, we interrogated the cell body location, projection pattern and targets of lung-innervating sensory neurons. Retrograde tracing from the lung labeled neurons primarily in the vagal ganglia, in a spatially distributed population expressing markers including Vglut2, Trpv1, Tac1, Calb1 or Piezo2. Centrally, they project to the nucleus of the solitary tract in the brainstem. Peripherally, they project along the branching airways and terminate on airway smooth muscles, vasculature including lymphatics, and selected alveoli. Notably, a discrete population of Calb1+ neurons preferentially innervate pulmonary neuroendocrine cells, a demonstrated airway sensor population. This comprehensive illustration of the properties of lung innervating sensory neurons serves as a foundation for understanding their function in lung.

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Competition within low-density bacterial populations as an unexpected factor regulating carbon decomposition in bulk soil Bacterial decomposition of organic matter in soils is generally believed to be mainly controlled by the access bacteria have to their substrate. The influence of bacterial traits on this control has, however, received little attention. Here, we develop a bioreactive transport model to screen the interactive impacts of dispersion and bacterial traits on mineralization. We compare the model results with two sets of previously performed cm-scale soil-core experiments in which the mineralization of the pesticide 2,4-D was measured under well-controlled initial distributions and transport conditions. Bacterial dispersion away from the initial substrate location induced a significant increase in 2,4-D mineralization, revealing the existence of a regulation of mineralization by the bacterial decomposer density, in addition to the dilution of substrate. This regulation of degradation by density becomes dominant for bacteria with an efficient uptake of substrate at low substrate concentrations (a common feature of oligotrophs). The model output suggests that the distance between bacteria adapted to oligotrophic environments is a stronger regulator of degradation than the distance between these bacteria and the substrate initial location. Such oligotrophs, commonly found in soils, compete with each other for substrate even at remarkably low population densities. The ratio-dependent Contois growth model, which includes a density regulation in the expression of the uptake efficiency, provide a more versatile representation than the substrate-dependent Monod model in these conditions. In view of their strong interactions, bioreactive and transport processes cannot be handled independently but should be integrated, in particular when reactive processes of interest are carried out by oligotrophs.

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- Decomposition can be reduced by competition between bacteria even at low densities
- Bacterial density regulation counterbalances substrate accessibility regulation
- Regulation of decomposition by bacterial density is more acute for oligotrophs

sentences: IRG1 controls host responses to restrict Mycobacterium tuberculosis infection Mycobacterium tuberculosis (Mtb), the pathogen causing human tuberculosis, has evolved multiple strategies to successfully prevent clearance by immune cells and to establish dissemination and long-term survival in the host. The modulation of host immunity to maximize pathogen elimination while minimizing inflammation-mediated tissue damage may provide another tool to fight drug-resistant Mtb strains. Metabolic reprogramming of phagocytes can dramatically influence the intracellular colonization by Mtb and the key players involved in this process remain a matter of debate. Here, we demonstrate that aconitate decarboxylase 1 (Acod1; also known as immune-responsive gene 1, IRG1), which converts cis-aconitate into the metabolite itaconate, is a major player in controlling the acute phase of Mtb infection. Exposure of IRG1-deficient mice to a virulent Mtb strain (H37Rv) was lethal, while M. bovis BCG and the H37Ra attenuated Mtb strain induced neither lethality nor severe lung immunopathology. Lungs of IRG1-deficient mice infected by Mtb H37Rv displayed large areas of necrotizing granulomatous inflammation and neutrophil infiltration, accompanied by reduced levels of B and T lymphocytes and increased levels of alveolar and interstitial macrophage populations, compared to their wild type counterparts. Next, we show that IRG1, beyond its recruitment to Mtb-containing vacuoles, restricts Mtb replication and lipid droplets accumulation in phagocytes, hallmarks of a tight interplay between the bacillus and the host. Altogether, IRG1 confines the host response to create a favourable phagocytic environment for Mtb controlled intracellular replication.

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Gradual development of non-adjacent dependency learning during early childhood In order to become proficient native speakers, children have to learn the morpho-syntactic relations between distant elements in a sentence, so-called non-adjacent dependencies (NADs). Previous research suggests that NAD learning in children comprises different developmental stages, where until 2 years of age children are able to learn NADs associatively under passive listening conditions, while starting around the age of 3 to 4 years children fail to learn NADs during passive listening. To test whether the transition between these developmental stages occurs gradually, we tested childrens NAD learning in a foreign language using event-related potentials (ERPs). We found ERP evidence of NAD learning across the ages of 1, 2 and 3 years. The amplitude of the ERP effect indexing NAD learning, however, decreased with age. These findings might indicate a gradual transition in childrens ability to learn NADs associatively. Cognitively, this transition might be driven by childrens increasing knowledge of their native language, hindering NAD learning in novel contexts. Neuroanatomically, maturation of the prefrontal cortex might play a crucial role, promoting top-down learning, affecting bottom-up, associative learning. In sum, our study suggests that NAD learning under passive listening conditions undergoes a gradual transition between different developmental stages during early childhood.

Research HighlightsO_LITransition between developmental stages of non-adjacent dependency (NAD) learning
C_LIO_LIChildren between 1 and 3 years of age showed learning of NADs in a foreign language
C_LIO_LIBrain responses revealed associative NAD learning, triggered by passive listening
C_LIO_LIGradual decrease of the strength of associative non-adjacent dependency learning
C_LI

sentences: Stiffness of fluid and gel phase lipid nanovesicles: weighting the contributions of membrane bending modulus and luminal pressurization The mechanical properties of biogenic membranous compartments are thought to be relevant in numerous biological processes; however, their quantitative measurement remains challenging for most of the already available Force Spectroscopy (FS)-based techniques. In particular, the debate on the mechanics of lipid nanovesicles and on the interpretation of their mechanical response to an applied force is still open. This is mostly due to the current lack of a unified model being able to describe the mechanical response of gel and fluid phase lipid vesicles and to disentangle the contributions of membrane rigidity and luminal pressure. In this framework, we herein propose a simple model in which the contributions of membrane rigidity and luminal pressure to the overall vesicle stiffness are described as a series of springs; this approach allows estimating the two contributions for both gel and fluid phase liposomes. Atomic Force Microscopy-based FS (AFM-FS), performed on both vesicles and Supported Lipid Bilayers (SLBs), is exploited for obtaining all the parameters involved in the model. Moreover, the use of coarse-grained full-scale molecular dynamics simulations allowed for better understanding the differences in the mechanical responses of gel and fluid phase bilayers and supported the experimental findings. Results suggest that the pressure contribution is similar among all the probed vesicle types; however, it plays a dominant role in the mechanical response of lipid nanovesicles presenting a fluid phase membrane, while its contribution becomes comparable to the one of membrane rigidity in nanovesicles with a gel phase lipid membrane. The herein presented results offer a simple way to quantify two of the most important parameters in vesicular nanomechanics, and as such represent a first step towards a currently unavailable, unified model for the mechanical response of gel and fluid phase lipid nanovesicles.

sentences: A test statistic to quantify treelikeness in phylogenetics Most phylogenetic analyses assume that the evolutionary history of an alignment (either that of a single locus, or of multiple concatenated loci) can be described by a single bifurcating tree, the so-called the treelikeness assumption. Treelikeness can be violated by biological events such as recombination, introgression, or incomplete lineage sorting, and by systematic errors in phylogenetic analyses. The incorrect assumption of treelikeness may then mislead phylogenetic inferences. To quantify and test for treelikeness in alignments, we develop a test statistic which we call the tree proportion. This statistic quantifies the proportion of the edge weights in a phylogenetic network that are represented in a bifurcating phylogenetic tree of the same alignment. We extend this statistic to a statistical test of treelikeness using a parametric bootstrap. We use extensive simulations to compare tree proportion to a range of related approaches. We show that tree proportion successfully identifies non-treelikeness in a wide range of simulation scenarios, and discuss its strengths and weaknesses compared to other approaches. The power of the tree-proportion test to reject non-treelike alignments can be lower than some other approaches, but these approaches tend to be limited in their scope and/or the ease with which they can be interpreted. Our recommendation is to test treelikeness of sequence alignments with both tree proportion and mosaic methods such as 3Seq. The scripts necessary to replicate this study are available at https://github.com/caitlinch/treelikeness

sentences: A partially nested cortical hierarchy of neural states underlies event segmentation in the human brain A fundamental aspect of human experience is that it is segmented into discrete events. This may be underpinned by transitions between distinct neural states. Using an innovative data-driven state segmentation method, we investigate how neural states are organized across the cortical hierarchy and where in the cortex neural state boundaries and perceived event boundaries overlap. Our results show that neural state boundaries are organized in a temporal cortical hierarchy, with short states in primary sensory regions, and long states in lateral and medial prefrontal cortex. State boundaries are shared within and between groups of brain regions that resemble well-known functional networks. Perceived event boundaries overlap with neural state boundaries across large parts of the cortical hierarchy, particularly when those state boundaries demarcate a strong transition or are shared between brain regions. Taken together, these findings suggest that a partially nested cortical hierarchy of neural states forms the basis of event segmentation.

sentences: Dual RNA modulation of protein mobility and stability within phase-separated condensates One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a novel sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1 and TDP-43 proteins along with that of their corresponding prion-like domains and RNA-recognition motifs, from absence to moderately high RNA concentration. By characterising the phase diagram, key molecular interactions, surface tension and transport properties of the condensates, we report a dual RNA-induced behavior: On the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whilst at high concentration it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity, even at high concentration, but barely modify protein self-diffusion, which mainly depends on RNA concentration and on its own effect on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.

sentences: Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. By directly probing tension we found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

sentences: Chemical interplay and complementary adaptative strategies toggle bacterial antagonism and co-existence Bacterial communities are in a continuous adaptive and evolutionary race for survival. A myriad of molecules that kill, defend, or mediate communication between bacterial cells of different lineages shape the final structure of the microbial community. In this work we expand our knowledge on the chemical interplay and specific mutations that modulate the transition from antagonism to co-existence between two plant-beneficial bacteria, Pseudomonas chlororaphis PCL1606 and Bacillus amyloliquefaciens FZB42. We reveal that the bacteriostatic activity of bacillaene produced by Bacillus relies on an interaction with the protein elongation factor FusA and how mutations in this protein lead to tolerance to bacillaene and other protein translation inhibitors. Additionally, we describe how the unspecific tolerance to antimicrobials associated with mutations in the glycerol kinase GlpK is provoked mainly by a decrease of Bacillus cell membrane permeability among other pleiotropic cellular responses. We conclude that nutrient specialization and mutations in basic biological functions are bacterial evolutive and adaptive strategies that lead to the coexistence of two primary competitive bacterial species rather than their mutual eradication.

sentences: Genomic evidence for a chemical link between redox conditions and microbial community composition Environmental influences on community structure are often assessed through multivariate analyses in order to relate microbial abundances to separately measured physicochemical variables. However, genes and proteins are themselves chemical entities; in combination with genome databases, differences in microbial abundances directly encode for chemical variability. We predicted that the carbon oxidation state of inferred community proteomes, obtained by combining taxonomic abundances from published 16S rRNA gene sequencing datasets with predicted microbial proteomes from the NCBI Reference Sequence (RefSeq) database, would reflect environmental oxidation-reduction conditions in various natural and engineered settings including shale gas wells. Our analysis confirms the geobiochemical predictions for environmental redox gradients within and between hydrothermal systems and stratified lakes and marine environments. Where they are present, a common set of taxonomic groups (Gamma- and Deltaproteobacteria and Clostridia) act as drivers of the community-level differences in oxidation state, whereas Flavobacteria most often oppose the overall changes. The geobiochemical signal is largest for the steep redox gradients associated with hydrothermal systems and between surface water and produced fluids from shale gas wells, demonstrating the ability to determine the magnitude of redox effects on microbial communities from 16S sequencing alone.

sentences: Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

sentences: Exploring the lncRNA localization landscape within the retinal pigment epithelium under normal and stress conditions Long noncoding RNAs (lncRNAs) are emerging as a class of genes whose importance has yet to be fully realized. It is becoming clear that the primary function of lncRNAs is to regulate gene expression, and they do so through a variety of mechanisms that are critically tied to their subcellular localization. Although most lncRNAs are poorly understood, mapping lncRNA subcellular localization can provide a foundation for understanding these mechanisms. Here, we present an initial step toward uncovering the localization landscape of lncRNAs in the human retinal pigmented epithelium (RPE) using high throughput RNA-Sequencing (RNA-Seq). To do this, we differentiated human induced pluripotent stem cells (iPSCs) into RPE, isolated RNA from nuclear and cytoplasmic fractions, and performed RNA-Seq on both. Furthermore, we investigated lncRNA localization changes that occur in response to oxidative stress. We discovered that, under normal conditions, most lncRNAs (76%) are seen in both the nucleus and the cytoplasm to a similar degree, but of the transcripts that are highly enriched in one compartment, more are nuclear (18.6%) than cytoplasmic (5.6%). Interestingly, under oxidative stress conditions, we observed an increase in lncRNA localization in both nuclear (23.5%) and cytoplasmic (9.7%) fractions. In addition, we found that nuclear localization was partially attributable to the presence of previously described nuclear retention motifs, while adenosine to inosine (A-to-I) RNA editing appeared to play a very minimal role. Our findings regarding lncRNA localization in the RPE provide two avenues for future research: 1) how lncRNAs function in the RPE, and 2) how one environmental factor, in isolation, may play a role in retinal disease pathogenesis.

sentences: Alkane-priming of Beauveria bassiana strains to improve biocontrol of the redbanded stink bug Piezodorus guildinii and the bronze bug Thaumastocoris peregrinus Insect Epicuticle hydrocarbons (CHC) are known to be important determinants in the susceptibility degree of insects to fungal entomopathogens. Five Beauveria bassiana isolates were phenotypically analyzed regarding their response to CHC nutrition and their pathogenicity and virulence towards high fungal-susceptible Thaumastocoris peregrinus and low fungal-susceptible Piezodorus guildinii, which are important hemipteran pests in eucalyptus and soybean plantations, respectively. Two of these isolates, resulting the most (ILBB308) and the least (ILBB299) virulent to P. guildinii, were also evaluated at gene expression level after growth on n-pentadecane. B. bassiana most virulent isolate ILBB308 showed the lowest growth on most evaluated CHC media. However, this isolate distinctively induced most of the analyzed genes involved in CHC assimilation, cuticle degradation and stress tolerance. Virulence towards low susceptibility P. guildinii was enhanced in both hypervirulent ILB308 and hypovirulent ILBB299 isolates after growth on n-pentadecane as the sole carbon source, whereas virulence enhancement towards high susceptibility T. peregrinus was not observed in alkane-grown fungi. Virulence enhancement towards P. guildinii could be mostly explained by a priming effect produced by CHC on the induction of some genes related to hydrocarbon assimilation in ILB 205 and ILB 308, such as hydrophobin (Bbhyd2) and cytochrome P450 genes (BbCyp52g11 and BbCyp52x1), and partially by the induction of genes related to cuticle degradation (Bbchit and Bbcdep1) and stress tolerance (Bbsod1) observed only in ILB308.

sentences: BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2 SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.

sentences: Genomic variation in the tea leafhopper reveals the basis of adaptive evolution The tea green leafhopper (TGL), Empoasca onukii, is of biological and economic interest. Despite numerous studies, the mechanisms underlying its adaptation and evolution remain enigmatic. Here, we used previously untapped genome and population genetics approaches to examine how this pest so rapidly has adapted to different environmental variables and thus has expanded geographically. We complete a chromosome-level assembly and annotation of the E. onukii genome, showing notable expansions of gene families associated with adaptation to chemoreception and detoxification. Genomic signals indicating balancing selection highlight metabolic pathways involved in adaptation to a wide range of tea varieties grown across ecologically diverse regions. Patterns of genetic variation among 54 E. onukii samples unveil the population structure and evolutionary history across different tea-growing regions in China. Our results demonstrate that the genomic change in key pathways, including those linked to metabolism, circadian rhythms and immune system function, may underlie the successful spread and adaptation of E. onukii. This work highlights the genetic and molecular bases underlying the evolutionary success of a species with broad economic impact, and provides insight into insect adaptation to host plants, which will ultimately facilitate more sustainable pest management.

sentences: Blood flow modeling under LVAD physiology. From global circulation to local hemodynamics This document presents the modeling strategy to address the in-silico study of different LVAD patho-physiological scenarios. The proposed approach employs a closed-loop lumped-parameter compartmental representation of the global circulation in the cardiovascular system (CVS). The CVS is coupled to a HeartMate 3 LVAD, and different cardiovascular conditions are proposed by modification of model parameters. Once the simulation for these conditions are performed, the cardiac function is analyzed in detail, and the global circulation model delivers flow rate waveforms which are employed as boundary conditions in a 3D hemodynamic simulation. This local circulation model is built using a patient-specific geometry of the aortic arch, containing 7 inlet/outlet boundaries, namely: LVAD cannula, aortic root, left and right subclavian arteries, left and right common carotid arteries and thoracic aorta. This model is exploited to investigate the impact of global cardiovascular conditions in the local hemodynamic features, particularly the wall shear stress (WSS) in different spatial regions.

sentences: Swift Formation of Optimal Single Spheroids towards In-Vitro 3-Dimensional Tumour Models Monolayer cell culture, while useful for basic in vitro studies, are not physiologically relevant. Spheroids, on the other hand provide a more complex 3-dimensional (3D) structure which more resemble the in vivo tumour growth thereby allowing results obtained with those on proliferation, cell death, differentiation, metabolism, and various anti-tumour therapies to be more predictive of in vivo outcomes. However, the cost associated with their generation often involve expensive, plate, media, and growth supplements, which have limited their use for high throughput experiments. The protocol herein presents a novel and rapid generation for single spheroids of various cancer cell lines, U87 MG; SEBTA-027; SF188, brain cancer cells, DU-145, TRAMP-C1, prostate cancer cells, in 96-round bottom well plates. Cells are washed with anti-adherent solution, and the homogeneous compact spheroid morphology was evidenced as early as 24 hours after 10 minutes centrifugation for the seeded cells. By using confocal microscopy, the proliferating cells were traced in the rim and the dead cells were found inside the core region of the spheroid. The H&E stain of spheroid slices and the western blotting were utilised to investigate the tightness of the cell packaging by adhesion proteins. Carnosine was used as an example of treatment for U87 single spheroids. The protocol allows the rapid generation of spheroids, which will help towards reducing the number of tests performed on animals.

sentences: The limits of evolutionary convergence in sympatry: reproductive interference and historical constraints leading to local diversity in warning traits Mutualistic interactions between defended species represent a striking case of evolutionary convergence in sympatry, driven by the increased protection against predators brought by mimicry in warning traits. However, such convergence is often limited: sympatric defended species frequently display different or imperfectly similar warning traits. The phylogenetic distance between sympatric species may indeed prevent evolution towards the exact same signal. Moreover, warning traits are also involved in mate recognition, so that trait convergence might result in heterospecific courtship and mating. Here, we develop a mathematical model to investigate the strength and direction of evolution of warning trait in defended species with different ancestral traits. Specifically, we determine the effect of phenotypic distances between ancestral trait states of sympatric defended species and of costs of heterospecific sexual interactions on imperfect mimicry and trait divergence. Our analytical results confirm that reproductive interference and historical constraints limit the convergence of warning traits, leading to either complete divergence, or imperfect mimicry. Our model reveals that imperfect mimicry evolves only when ancestral trait values differ between species, due to historical constraints and highlights the importance of female and predator discriminations in the evolution of such imperfect mimicry. Our study thus provides new predictions on how reproductive interference interacts with historical constraints and may promote the emergence of novel warning traits, enhancing mimetic diversity.

sentences: Rabies anterograde monosynaptic tracing reveals organization of spinal sensory circuits Somatosensory neurons detect vital information about the environment and internal status of the body, such as temperature, touch, itch and proprioception. The circuit mechanisms controlling the coding of somatosensory information and the generation of appropriate behavioral responses are not clear yet. In order to address this issue, it is important to define the precise connectivity patterns between primary sensory afferents dedicated to the detection of different stimuli and recipient neurons in the central nervous system. In this study we used a rabies tracing approach for mapping spinal circuits receiving sensory input from distinct, genetically defined, modalities. We analyzed the anatomical organization of spinal circuits involved in coding of thermal and mechanical stimuli and showed that somatosensory information from distinct modalities is relayed to partially overlapping ensembles of interneurons displaying stereotyped laminar organization, thus highlighting the importance of positional features and population coding for the processing and integration of somatosensory information.

sentences: Sequence and functional characterization of a public HIV-specific antibody clonotype Public antibody clonotypes shared among multiple individuals have been identified for several pathogens. However, little is known about the limits of what constitutes a public clonotype. Here, we characterize the sequence and functional properties of antibodies from a public HIV-specific clonotype comprising sequences from 3 individuals. Our results showed that antigen specificity for the public antibodies was modulated by the VH, but not VL, germline gene. Non-native pairing of public heavy and light chains from different donors resulted in antibodies with consistent antigen specificity, suggesting functional complementation of sequences within the public antibody clonotype. The strength of antigen recognition appeared to be dependent on the specific antibody light chain used, but not on other sequence features such as germline or native-antibody sequence identity. Understanding the determinants of antibody clonotype "publicness" can provide insights into the fundamental rules of host-pathogen interactions at the population level, with implications for clonotype-specific vaccine development.

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Gene exchange networks define species-like units in marine prokaryotes Post-submission note. Since the original submission of this manuscript to bioRxiv, we discovered that some of our results may be impacted by the limitations of some of the comparative genomics tools used in this study. We are working on a revised version of the manuscript.

Although horizontal gene transfer is recognized as a major evolutionary process in Bacteria and Archaea, its general patterns remain elusive, due to difficulties tracking genes at relevant resolution and scale within complex microbiomes. To circumvent these challenges, we analyzed a randomized sample of >12,000 genomes of individual cells of Bacteria and Archaea in the tropical and subtropical ocean - a well-mixed, global environment. We found that marine microorganisms form gene exchange networks (GENs) within which transfers of both flexible and core genes are frequent, including the rRNA operon that is commonly used as a conservative taxonomic marker. The data revealed efficient gene exchange among genomes with <28% nucleotide difference, indicating that GENs are much broader lineages than the nominal microbial species, which are currently delineated at 4-6% nucleotide difference. The 42 largest GENs accounted for 90% of cells in the tropical ocean microbiome. Frequent gene exchange within GENs helps explain how marine microorganisms maintain millions of rare genes and adapt to a dynamic environment despite extreme genome streamlining of their individual cells. Our study suggests that sharing of pangenomes through horizontal gene transfer is a defining feature of fundamental evolutionary units in marine planktonic microorganisms and, potentially, other microbiomes.

sentences: Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells Brain development is regulated by conserved transcriptional programs across species, but little is known about divergent mechanisms that create species-specific characteristics. Among brain regions, the cerebellum is now recognized to contribute to human cognitive evolution having a broad range of non-motor cognitive functions in addition to motor control. Emerging studies highlight the complexity of human cerebellar histogenesis, compared with non-human primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. Here we report a rapid and simple protocol for the directed derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSC), and strategies to decrease culture variability; a common limitation in hPSC studies. Upon transplantation into juvenile mice, early postmitotic hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification (TRAP)-seq, the ATOH1 lineage most closely resembled human cerebellar tissue in the second trimester. Unexpectedly, TRAP-seq identified a heterochronic shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, within granule cell progenitors (GCPs) in the human external granule layer. This molecular divergence may provide the mechanism by which the GCP pool persists into year two post birth in humans, but only lasts for two weeks in mice. Our approach provides a scalable in vitro model of the human ATOH1 lineage that yields cerebellar granule cells within 48 days as well as a strategy for identifying uniquely human cellular and molecular characteristics.

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Restricted X chromosome introgression and support for Haldane's rule in hybridizing damselflies Contemporary hybrid zones act as natural laboratories for the investigation of species boundaries and allow to shed light on the little understood roles of sex chromosomes in species divergence. Sex chromosomes are considered to function as a hotspot of genetic divergence between species; indicated by less genomic introgression compared to autosomes during hybridisation. Moreover, they are thought to contribute to Haldanes rule which states that hybrids of the heterogametic sex are more likely to be inviable or sterile. To test these hypotheses, we used contemporary hybrid zones of Ischnura elegans, a damselfly species that has been expanding its range into the northern and western regions of Spain, leading to chronic hybridization with its sister species Ischnura graellsii. We analysed genome-wide SNPs in the Spanish I. elegans and I. graellsii hybrid zone and found (i) that the X chromosome shows less genomic introgression compared to autosomes and (ii) that males are underrepresented among admixed individuals as predicted by Haldanes rule. This is the first study in Odonata that suggests a role of the X chromosome in reproductive isolation.

Moreover, our data adds to the few studies on species with X0 sex determination system and contradicts the hypothesis that the absence of a Y chromosome causes exceptions to Haldanes rule.

sentences: Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation Langerhans cells are specialized antigen-presenting cells localized within the epidermis and mucosal epithelium. Upon contact with Langerhans cells, pathogens are captured by the C- type lectin langerin and internalized into a structurally unique vesicle known as a Birbeck granule. Although the immunological role of Langerhans cells and Birbeck granules have been extensively studied, the mechanism by which the characteristic zippered membrane structure of Birbeck granules is formed remains elusive. In this study, we observed isolated Birbeck granules using cryo-electron tomography and reconstructed the 3D structure of the repeating unit of the honeycomb lattice of langerin at 6.4 [A] resolution. We found that the interaction between the two langerin trimers was mediated by docking the flexible loop at residues 258-263 into the secondary carbohydrate-binding cleft. Mutations within the loop inhibited Birbeck granule formation and the internalization of HIV pseudovirus. These findings suggest a molecular mechanism for membrane zippering during Birbeck granule biogenesis and provide insight into the role of langerin in the defense against viral infection.

sentences: Sex and estrous cycle affect experience-dependent plasticity in mouse primary visual cortex Sex hormones can affect cellular physiology and modulate synaptic plasticity, but it is not always clear whether or how sex-dependent differences identified in vitro express themselves as functional dimorphisms in the brain. Historically, most experimental neuroscience has been conducted using only male animals and the literature is largely mute about whether including female mice in will introduce variability due to inherent sex differences or endogenous estrous cycles. Though this is beginning to change following an NIH directive that sex should be included as a factor in vertebrate research, the lack of information raises practical issues around how to design experimental controls and apply existing knowledge to more heterogeneous populations. Various lines of research suggest that visual processing can be affected by sex and estrous cycle stage. For these reasons, we performed a series of in vivo electrophysiological experiments to characterize baseline visual function and experience-dependent plasticity in the primary visual cortex (V1) of male and female mice. We find that sex and estrous stage have no statistically significant effect on baseline acuity measurements, but that both sex and estrous stage have can modulate two mechanistically distinct forms of experience dependent cortical plasticity. We also demonstrate that resulting variability can be largely controlled with appropriate normalizations. These findings suggest that V1 plasticity can be used for mechanistic studies focusing on how sex hormones effect experience dependent plasticity in the mammalian cortex.

sentences: Mechanism of R-loop formation and conformational activation of Cas9 Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage1-3. The programmable activity of Cas9 has been widely utilized for genome editing applications4-6, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-EM structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2/3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2/3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

sentences: Cryo-EM structure of native human thyroglobulin The thyroglobulin (Tg) protein is essential to thyroid hormone synthesis, playing a vital role in the regulation of metabolism, development and growth. Its structure is conserved among vertebrates. Tg is delivered through the secretory pathway of the thyroid follicular unit to the central colloid depository, where it is iodinated at specific tyrosine sites to form mono- or diiodotyrosine, which combine to produce triiodothyronine (T3) and thyroxine (T4), respectively. Synthesis of these hormones depends on the precise 3D structure of Tg, which has remained unknown despite decades of research. Here, we present the cryo-electron microscopy structure of human thyroglobulin (hTg) to a global resolution of 3.2 [A]. The structure provides detailed information on the location of the hTg hormonogenic sites and reveals the position as well as the role of many of its glycosylation sites. Our results offer structural insight into thyroid hormonogenesis and provide a fundamental understanding of clinically relevant hTg mutations, which can improve treatment of thyroid diseases.

sentences: Evidence of Absence Regression: A Binomial N-Mixture Model for Estimating Fatalities at Wind Power Facilities Estimating bird and bat fatalities caused by wind-turbine facilities is challenging when carcass counts are rare and produce counts that are either exactly zero or very near zero. The rarity of found carcasses is exacerbated when live members of a particular species are rare and when carcasses degrade quickly, are removed by scavengers, or are not detected by observers. With few observed carcass counts, common statistical methods like logistic, Poisson, or negative binomial regression are unreliable (statistically biased) and often fail to provide answers (i.e., fail to converge). Here, we propose a binomial N-mixture model that estimates fatality rates as well as the total number of carcass counts when these rates are expanded. Our model extends the  evidence of absence model (Huso et al., 2015; Dalthorp, Huso, and Dail, 2017) by relating carcass deposition rates to study covariates and by incorporating terms that naturally scale counts from facilities of different sizes. Our model, which we call Evidence of Absence Regression (EoAR), can estimate the total number of birds or bats killed at a single wind energy facility or a fleet of wind energy facilities based on covariate values. Furthermore, with accurate prior distributions the models results are extremely robust to sparse data and unobserved combinations of covariate values. In this paper, we describe the model, show its low bias and high precision via computer simulation, and apply it to bat carcass counts observed at 21 wind energy facilities in Iowa.

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Single cell trajectory analysis of human pluripotent stem cells differentiating towards lung and hepatocyte progenitors Understanding the development of human respiratory tissues is crucial for modeling and treating lung disorders. The molecular details for the specification of lung progenitors from human pluripotent stem cells (hPSCs) are unclear. Here, we use single cell RNA-sequencing with high temporal resolution along an optimized differentiation protocol to determine the transcriptional hierarchy of lung specification from human hPSCs and map out the underlying single cell trajectories. We show that Sonic hedgehog, TGF-{beta} and Notch activation are required in an ISL1/NKX2-1 trajectory that gives rise to lung progenitors during the foregut endoderm stage. Induction of HHEX marks an alternative trajectory at the early definitive endoderm stage, which precedes the lung trajectory and generates a major hepatoblast population. Moreover, neither KDR+ nor mesendoderm progenitors are apparent intermediate states of lung and hepatic lineages. Our hierarchical multistep model predicts mechanisms leading to lung organogenesis, and creates a basis for studying early human lung development, as well as hPSC based disease and drug research.

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sentences: Protective mucosal immunity against SARS-CoV-2 after heterologous systemic RNA-mucosal adenoviral vector immunization Several effective SARS-CoV-2 vaccines are currently in use, but in the light of waning immunity and the emergence of novel variants, effective boost modalities are needed in order to maintain or even increase immunity. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic DNA or mRNA priming result in strong systemic and mucosal immunity in mice. In contrast to two intramuscular injections with an mRNA vaccine, the mucosal boost with adenoviral vectors induced high levels of IgA and tissue-resident memory T cells in the respiratory tract. Mucosal neutralization of virus variants of concern was also enhanced by the intranasal boosts. Importantly, priming with mRNA provoked a more comprehensive T cell response consisting of circulating and tissue-resident memory T cells after the boost, while a DNA priming induced mostly mucosal T cells. Concomitantly, the intranasal boost strategies provided protection against symptomatic disease. Therefore, a mucosal booster immunization after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses.

sentences: PRC2-mediated repression is essential to maintain identity and function of differentiated dopaminergic and serotonergic neurons How neurons in the CNS can maintain cellular identity over an entire lifespan remains largely unknown. Here we show that long-term maintenance of identity in differentiated dopaminergic and serotonergic neurons is critically reliant on the Polycomb repressive complex 2 (PRC2). Deletion of the obligate PRC2-component, Eed, in these neurons, resulted in global loss of H3K27me3, followed by a gradual activation of genes harbouring both H3K27me3 and H3K9me3 modifications. Notably, H3K9me3 was also lost at these PRC2-targets prior to gene activation. Neuronal survival was not compromised, instead there was a reduction in subtype specific gene expression as well as a progressive impairment of dopaminergic or serotonergic neuronal function leading to behavioural deficits characteristic of Parkinsons disease (PD) or mood disorders, respectively. Single cell analysis revealed an unexpected subtype specific vulnerability to loss of PRC2-repression in dopamine neurons of the substantia nigra, the neurons primarily affected in PD. Taken together, our study reveals that a PRC2-dependent non-permissive chromatin state is essential to maintain subtype identity and function of dopaminergic and serotonergic neurons.

sentences: Receptor binding domain of SARS-CoV-2 is a functional αv-integrin agonist Among the novel mutations distinguishing SARS-CoV-2 from similar respiratory coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2 (ACE2)-binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. In the present study, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared to RGD-containing, integrin-binding fragments of fibronectin. S1-RBD supported adhesion of both fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells. Cell adhesion to S1-RBD was cation- and RGD-dependent, and was inhibited by blocking antibodies against v and {beta}3, but not 5 or {beta}1, integrins. Similarly, direct binding of S1-RBD to recombinant human v{beta}3 and v{beta}6 integrins, but not 5{beta}1 integrins, was observed by surface plasmon resonance. Adhesion to S1-RBD initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin, Akt activation, and supported cell proliferation. Together, these data demonstrate that the RGD sequence within S1-RBD can function as an v-selective integrin agonist. This study provides evidence that cell surface v-containing integrins can respond functionally to spike protein and raise the possibility that S1-mediated dysregulation of ECM dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.

sentences: Transcriptional Diversity of Medium Spiny Neurons in the Primate Striatum The striatum is the interface between dopamine reward signals and cortico-basal ganglia circuits that mediate diverse behavioral functions. Medium spiny neurons (MSNs) constitute the vast majority of striatal neurons and are traditionally classified as direct- or indirect-pathway neurons. However, that traditional model does not explain the anatomical and functional diversity of MSNs. Here, we defined molecularly distinct MSN types in the primate striatum, including (1) dorsal striatum MSN types associated with striosome and matrix compartments, (2) ventral striatum types associated with the nucleus accumbens shell and olfactory tubercle, and (3) an MSN-like type restricted to -opioid receptor rich islands in the ventral striatum. These results lay the foundation for achieving cell type-specific transgenesis in the primate striatum and provide a blueprint for investigating circuit-specific processing.

sentences: Broad variation in rates of polyploidy and dysploidy across flowering plants is correlated with lineage diversification Changes in chromosome number are considered an important driver of diversification in angiosperms. Single chromosome number changes caused by dysploidy may produce strong reproductive barriers leading to speciation. Polyploidy, or whole genome duplication, yields new species that are often reproductively isolated from progenitors and may exhibit novel morphology or ecology that may further facilitate diversification. Here, we examined the rates of polyploidy, dysploidy, and diversification across the angiosperms. Our analyses of nearly 30,000 taxa representing 46 orders and 147 families found that rates of polyploidy and dysploidy differed by two to three orders of magnitude. The rates of polyploidy and dysploidy were positively correlated with diversification rates, but relative importance analyses indicated that variation in polyploidy was better correlated with diversification rates than dysploidy. Our results provide an overview of angiosperm chromosomal evolution and a roadmap for future research on the complex relationships among polyploidy, dysploidy, and diversification.

sentences: First observation of Aedes albopictus in the Tshuapa province (Boende) of the Democratic Republic of the Congo In May-June 2021, we detected Aedes albopictus adults near the central hospital in Boende, the capital city of the Tshuapa province of the Democratic Republic of the Congo (DRC). We identified the mosquitoes using morphological and molecular techniques (COI barcoding). This is the first report of this species in the DRC outside of Kinshasa and Kongo Central. Given the central location of Boende in the Congo Basin, our finding suggests that the vector might also have spread to other cities which are located upstream of the Congo River and its major tributaries. Because Aedes albopictus is an important vector for human arbovirus transmission, we highlight the need to investigate its distribution range and to update disease risk maps in Central Africa.

sentences: Greater Neural Pattern Dissimilarity and Higher Meaningfulness in the Retrieval Stage are Enhanced by Spaced Learning The question of whether repeated studies bring more variability or less to our brain is a critical problem before scientist continue their further study about memory. In the past ten years, a series of neural pattern representation studies have found that under the condition of spaced learning, the neural pattern similarity(NPS) between two encoding stages increases, and the researchers claimed that these results support the idea that repeated studies bring more similarity along with repetition in our brain, which is conflicted with the encoding variability theory(Feng et al., 2019; Y. Lu et al., 2015; Xue et al., 2010, 2011a). However, we doubt this viewpoint because we think the difference between encoding processing cannot be used to represent the difference between memory states. In current experiments, we used a new experimental paradigm with a longer lag and elaboration learning task to test the encoding variability theory. By comparing the difference between neural pattern dissimilarity(NPDS1)(spaced learning - one-time learning) and NPDS2(massed learning - one-time learning) in the final test (retrieval) stage, we get the result that the NPDS1 was significantly greater than NPDS2 in the parietal lobe of 400ms and the right frontal lobe of 600ms, which is more fitting to the encoding variability mechanism. However, we believe that there is no contradiction between these two experimental evidences. On the contrary, we think they reflect different aspects of the process of spaced learning. We propose that the deficient processing in encoding stage exactly lead to less encoding variability in our memory. More importantly, this result gives us reason to double the paper published ten years ago in Science, which claimed repetition brings greater neural pattern similarity.

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3D-imaging inspired improvements to hearing model in parasitoid fly Ormia ochracea This manuscript was compiled on October 5, 2021 Although most binaural organisms localize sound sources using neurological structures to amplify the sounds they hear, some animals use mechanically coupled hearing organs instead. One of these animals, the parasitoid fly Ormia ochracea, has astoundingly accurate sound localization abilities and can locate objects in the azimuthal plane with a precision of 2{degrees}, equal to that of humans. This is accomplished despite an intertympanal distance of only 0.5 mm, which is less than 1/100th of the wavelength of the sound emitted by the crickets that it parasitizes. In 1995, Miles et al. developed a model of hearing mechanics in O. ochracea, which works well for incoming sound angles of less than {+/-}30{degrees}, but suffers from reduced accuracy (up to 60% error) at higher angles. Even with this limitation, it has served as the basis for multiple bio-inspired microphone designs for decades. Here, we present critical improvements to the classic O. ochracea hearing model based on information from 3D reconstructions of O. ochraceas tympana. The 3D images reveal that the tympanal organ has curved lateral faces in addition to the flat front-facing prosternal membranes represented in the Miles model. To mimic these faces, we incorporated spatially-varying spring and damper coefficients that respond asymmetrically to incident sound waves, making a new quasi-two-dimensional (q2D) model. The q2D model has high accuracy (average errors of less than 10%) for the entire range of incoming sound angles. This improved biomechanical hearing model can inform the development of new technologies and may help to play a key role in developing improved hearing aids.

Significance StatementThe ability to identify the location of sound sources is critical to organismal survival and for technologies that minimize unwanted background noise, such as directional microphones for hearing aids. Because of its exceptional auditory system, the parasitoid fly Ormia ochracea has served as an important model for binaural hearing and a source of bioinspiration for building tiny directional microphones with outsized sound localization abilities. Here, we performed 3D imaging of the flys tympanal organs and used the morphological information to improve the current model for hearing in O. ochracea. This model greatly expands the range of biological accuracy from {+/-}30{degrees} to all incoming sound angles, providing a new avenue for studies of binaural hearing and further inspiration for fly-inspired technologies.

sentences: Persistent postnatal migration of interneurons into the human entorhinal cortex The entorhinal cortex (EC) is a highly-interconnected hub for multisensory integration and memory processing1-3, containing diverse neuronal subtypes4,5 including subpopulations that are uniquely spatially-tuned6,7. Although many spatial and memory functions develop in infancy, it is considered that neurogenesis and neuronal migration to the EC occurs prenatally. Here we show that the postnatal human temporal lobe contains a prominent stream with large chains of young migrating neurons and many individual neurons breaking away directed into the EC. The EC stream forms between the second and third trimesters of prenatal development when the lateral ventricle walls in the temporal lobe collapse, displacing the subventricular zone (SVZ) and dividing radial glia. At birth, the EC stream follows a path of radial glial [fi]bers in the site of the collapsed ventricle. Migratory chains persist up to 11 months postnatally; however, many individually migrating young neurons can still be detected in the EC at 2 years of age and a few isolated cells at 3 years of age. Within the EC at birth, immature neurons are a mixed population expressing markers of the medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE), but postnatally rapidly become primarily CGE-derived. Using single-nuclei RNAseq we identified these lineages and found that the MGE-derived neurons matured at earlier postnatal ages compared to those derived from the CGE. The CGE interneurons arriving and maturing the latest included subtypes expressing calretinin (CR), reelin (RELN), and vasoactive intestinal protein (VIP) many of which settle in layer II of the entorhinal cortex. This study reveals that the human EC is still being constructed during the first years of life revealing the largest known postnatal stream of migratory neurons in humans. The protracted postnatal arrival of a diverse population of interneurons could contribute to plasticity8,9 and proper excitation-inhibition balance10,11 within these highly connected brain circuits.

sentences: pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

sentences: A panoramic perspective on human phosphosites Protein phosphorylation is the most common post-translational reversible modification of proteins and is key in the regulation of many cellular processes. Due to this importance, phosphorylation is extensively studied, resulting in the availability of a large amount of mass spectrometry based phospho-proteomics data. Here, we leverage the information in these large-scale phospho-proteomics datasets, as contained in Scop3P, to analyze and characterize proteome-wide protein phosphorylation sites (P-sites). First, we set out to differentiate correctly observed P-sites from false positive sites using five complementary site properties. We then describe the context of these P-sites in terms of protein structure, solvent accessibility, structural transitions and disorder, and biophysical properties. We also investigate the relative prevalence of disease-linked mutations on and around P-sites. Moreover, we also assess structural dynamics of P-sites in their phosphorylated and unphosphorylated state. Our study shows that the residues that gets phosphorylated are more flexible than their equivalent non-phosphorylated residues. Our structural and biophysical analyses of P-sites in solvent inaccessible (buried) regions of proteins show that these sites are primarily found in multi-site phospho-proteins, where highly dynamic structural transitions can occur upon binding with another protein. Finally, our analysis of the biophysical properties of P-site mutations shows that P-site mutations that occur in structurally rigid regions are more often involved in disease.

sentences: Decreasing Mitochondrial RNA Polymerase Activity Reverses Biased Inheritance of Hypersuppressive mtDNA Faithful inheritance of mitochondrial DNA (mtDNA) is crucial for cellular respiration/oxidative phosphorylation and mitochondrial membrane potential. However, how mtDNA is transmitted to progeny is not fully understood. We utilized hypersuppressive mtDNA, a class of respiratory deficient Saccharomyces cerevisiae mtDNA that is preferentially inherited over wild-type mtDNA (rho+), to uncover the factors governing mtDNA inheritance. We found that regions of rho+ mtDNA persisted after hypersuppressive takeover indicating that hypersuppressive preferential inheritance may partially be due to active destruction of rho+ mtDNA. From a multicopy suppression screen, we found that overexpression of putative mitochondrial RNA exonuclease PET127 reduced hypersuppressive biased inheritance. This suppression required PET127 binding to the mitochondrial RNA polymerase RPO41 but not PET127 exonuclease activity. A temperature-sensitive allele of RPO41 improved rho+ mtDNA inheritance relative to hypersuppressive mtDNA at semi-permissive temperatures revealing a previously unknown role for rho+ transcription in promoting hypersuppressive mtDNA inheritance.

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Permeability profiling of all 13 Arabidopsis PIP aquaporins using a high throughput yeast approach Plant aquaporins have many more functions than just transporting water. Within the diversity of plant aquaporins are isoforms capable of transporting signaling molecules, nutrients, metalloids and gases. It is established that aquaporin substrate discrimination depends on combinations of factors such as solute size, pore size and polarity, and post-translational protein modifications. But our understanding of the relationships between variation in aquaporin structures and the implications for permeability is limited. High-throughput yeast-based assays were developed to assess diverse substrate permeabilities to water, H2O2, boric acid, urea and Na+. All 13 plasma membrane intrinsic proteins (PIPs) from Arabidopsis (AtPIPs) were permeable to both water and H2O2, although their effectiveness varied, and none were permeable to urea. AtPIP2 isoforms were more permeable to water than AtPIP1s, while AtPIP1s were more efficient at transporting H2O2 with AtPIP1;3 and AtPIP1;4 being the most permeable. Among the AtPIP2s, AtPIP2;2 and AtPIP2;7 were also permeable to boric acid and Na+. Linking AtPIP substrate profiles with phylogenetics and gene expression data enabled us to align substrate preferences with known biological roles of AtPIPs and importantly guide towards unidentified roles hidden by functional redundancy at key developmental stages and within tissue types. This analysis positions us to more strategically test in planta physiological roles of AtPIPs in order to unravel their complex contributions to the transport of important substrates, and secondly, to resolve links between aquaporin protein structure, substrate discrimination, and transport efficiency.

One sentence summaryYeast based high throughput assays were developed to assess the permeability of each Arabidopsis PIP aquaporin isoform to water, H2O2, boric acid, urea and sodium.

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Inhibiting microglia proliferation after spinal cord injury improves recovery in mice and nonhuman primates No curative treatment is available for any deficits induced by spinal cord injury (SCI). Following injury, microglia undergo highly diverse activation processes, including proliferation, and play a critical role on functional recovery.

In a translational objective, we investigated whether a transient pharmacological reduction of microglia proliferation after injury is beneficial for functional recovery after SCI in mice and nonhuman primates. The colony stimulating factor-1 receptor (CSF1R) regulates proliferation, differentiation, and survival of microglia, we thus used an oral administration of GW2580, a CSF1R inhibitor.

First, transient post-injury GW2580 administration in mice improves motor function recovery, promotes tissues preservation and/or reorganization (identified by coherent anti-stokes Raman scattering microscopy), and modulates glial reactivity.

Second, post-injury GW2580-treatment in nonhuman primates reduces microglia proliferation, improves functional motor function recovery, and promotes tissue protection. Notably, three months after lesion microglia reactivity returned to baseline value.

Finally, to initiate the investigation on molecular mechanisms induced by a transient post-SCI GW2580-treatment, we used microglia-specific transcriptomic analysis in mice. Notably, we detected a downregulation in the expression of inflammatory-associated genes and we identified genes that were up-regulated by SCI and further downregulated by the treatment.

Thus, a transient oral GW2580 treatment post-injury may provide a promising therapeutic strategy for SCI patients and may also be extended to other central nervous system disorders displaying microglia activation.

sentences: The Interplay of Electrostatics and Chemical Positioning in the Evolution of Antibiotic Resistance in TEM β-Lactamases The interplay of enzyme active site electrostatics and chemical positioning are important for understanding the origin(s) of enzyme catalysis and the design of novel catalysts. We reconstruct the evolutionary trajectory of TEM-1 {beta}-lactamase to TEM-52 towards extended-spectrum activity to better understand the emergence of antibiotic resistance and to provide insights into the structure-function paradigm and non-covalent interactions involved in catalysis. Utilizing a detailed kinetic analysis and the vibrational Stark effect, we quantify the changes in rates and electric fields in the Michaelis and acyl-enzyme complexes for penicillin G and cefotaxime to ascertain the evolutionary role of electric fields to modulate function. These data are combined with MD simulations to interpret and quantify the substrate-dependent structural changes during evolution. We observe that this evolutionary trajectory utilizes a large preorganized electric field and substrate-dependent chemical positioning to facilitate catalysis. This governs the evolvability, substrate promiscuity, and protein fitness landscape in TEM {beta}-lactamase antibiotic resistance.

sentences: Polycomb suppresses a female gene regulatory network to ensure testicular differentiation Gonadal sex determination is controlled by the support cells of testes and ovaries. In testes, the epigenetic mechanism that maintains cellular memory to suppress female sexual differentiation remains unknown. Here, we show that Polycomb suppresses a female gene regulatory network in Sertoli cells, the specific support cells for postnatal testes. Through genetic ablation, we removed Polycomb repressive complex 1 (PRC1) from embryonic Sertoli cells after sex determination. PRC1-depleted postnatal Sertoli cells exhibited defective proliferation and cell death, leading to the degeneration of adult testes. In adult Sertoli cells, PRC1 suppressed the specific, critical genes required for granulosa cells, the support cells of ovaries, thereby inactivating the female gene regulatory network. The underlying chromatin of female genes was coated with Polycomb-mediated repressive modifications: PRC1-mediated H2AK119ub and PRC2-mediated H3K27me3. Taken together, we identify a critical mechanism centered on Polycomb that maintains the male fate in adult testes.

sentences: Differentially accessible Cdc4 phospho-degrons regulate Ctf19CCAN kinetochore subunit stability in mitosis Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. How effective, yet strictly centromere-dependent kinetochore assembly is coupled to cell cycle progression is incompletely understood. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-Phase and targets the protein for degradation. Binding of the Mtw1 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature-sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic to cells, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits protects against ectopic kinetochore assembly and contributes to mitotic checkpoint silencing. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.

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B cell-specific XIST complex enforces X-inactivation and restrains atypical B cells The long noncoding RNA (lncRNA) XIST establishes X chromosome inactivation (XCI) in female cells in early development and thereafter is thought to be largely dispensable. Here we show XIST is continually required in adult human B cells to silence a subset of X-linked immune genes such as TLR7. XIST-dependent genes lack promoter DNA methylation and require continual XIST-dependent histone deacetylation. XIST RNA-directed proteomics and CRISPRi screen reveal distinctive somatic cell-specific XIST complexes, and identify TRIM28 that mediates Pol II pausing at promoters of X-linked genes in B cells. XIST dysregylation, reflected by escape of XIST-dependent genes, occurs in CD11c+ atypical memory B cells across single-cell transcriptome data in patients with female-biased autoimmunity and COVID-19 infection. XIST inactivation with TLR7 agonism suffices to promote isotype-switched atypical B cells. These results suggest cell-type-specific diversification of lncRNA-protein complexes increase lncRNA functionalities, and expand roles for XIST in sex-differences in biology and medicine.

HIGHLIGHTSO_LIXIST prevents escape of genes with DNA hypomethylated promoters in B cells.
C_LIO_LIXIST maintains X-inactivation through continuous deacetylation of H3K27ac.
C_LIO_LIXIST ChIRP-MS and allelic CRISPRi screen reveal a B cell-specific XIST cofactor TRIM28.
C_LIO_LIXIST loss and TLR7 stimulation promotes CD11c+ atypical B cell formation.
C_LI

sentences: Adaptive coding of stimulus information in human fronto-parietal cortex during visual classification The neural mechanisms of how frontal and parietal brain regions support flexible adaptation of behavior remain poorly understood. Here, we used functional magnetic resonance imaging (fMRI) and model-based representational similarity analysis (RSA) to investigate frontoparietal representations of stimulus information during visual classification under varying task demands. Based on prior research, we predicted that increasing perceptual task difficulty should lead to more categorical coding of stimulus information, and that exemplar-level stimulus coding would be restricted to posterior, sensory brain regions. Counter to our expectations, however, we found frontoparietal regions encoded exemplar-level stimulus information. Interestingly, the anterior intraparietal sulcus (aIPS) encoded stimuli equally well regardless of perceptual difficulty, and these representations were directly related to choice behavior (proportion of guessing). Overall, these findings reveal unexpected exemplar-level stimulus coding in frontoparietal cortex, and highlight the role of aIPS in supporting adaptive behavior.

sentences: Hydrogen properties and charges in a sub-1.2 A resolution cryo-EM structure revealed by a cold field emission beam The cold field emission (CFE) beam produces the less-attenuated contrast transfer function of electron microscopy, thereby enhancing high-resolution signals and this particularly benefits higher-resolution single particle cryogenic electron microscopy. Here, we present a sub-1.2 [A] resolution structure of a standard protein sample, apoferritin. Image data were collected with the CFE beam in a high-throughput scheme while minimizing beam tilt deviations from the coma-free axis. A difference map reveals positive densities for most hydrogen atoms in the core region of the protein complex including those in water molecules, while negative densities around acidic amino-acid side chains likely represent negative charges. The position of the hydrogen densities depends on parent bonded-atom type, which is validated by an estimated level of coordinate errors.

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Sinorhizobium meliloti functions required for resistance to antimicrobial NCR peptides and bacteroid differentiation Legumes of the Medicago genus form symbiosis with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of the intracellular symbionts. Members of the Nodule-specific Cysteine Rich peptide (NCRs) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts, including an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes, lipopolysaccharide modifications mediated by lpsB and lpxXL as well as rpoH1, encoding a stress sigma factor. Mutants of these genes show in vitro a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions including peptide transporters, the bacterial envelope structures and stress response regulators to resist the aggressive assault of NCR peptides in the nodule cells.

ImportanceThe nitrogen fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plant needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane composition and the stress response regulator RpoH1.

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KIMGENS: A novel method to estimate kinship in organisms with mixed haploid diploid genetic systems robust to population structure MotivationKinship estimation is necessary for evaluating violations of assumptions or testing certain hypotheses in many population genomic studies. However, kinship estimators are usually designed for diploid systems and cannot be used in populations with mixed haploid diploid genetic systems. The only estimators for different ploidies require datasets free of population structure, limiting their usage.

ResultsWe present KIMGENS, an estimator for kinship estimation among individuals of various ploidies, that is robust to population structure. This estimator is based on the popular KING-robust estimator but uses diploid relatives of the individuals of interest as references of heterozygosity and extends its use to haploid-diploid and haploid pairs of individuals. We demonstrate that KIMGENS estimates kinship more accurately than previously developed estimators in simulated panmictic, structured and admixed populations, but has lower accuracy when the individual of interest is inbred. KIMGENS also outperforms other estimators in a honeybee dataset. Therefore, KIMGENS is a valuable addition to a population geneticists toolbox.

Availability and ImplementationKIMGENS and its association simulation tool are implemented and available open-source at https://github.com/YenWenWang/HapDipKinship.

ContactYen-Wen Wang Email: ywang883@wisc.edu

sentences: Caveats to deep learning approaches to RNA secondary structure prediction Machine learning (ML) and in particular deep learning techniques have gained popularity for predicting structures from biopolymer sequences. An interesting case is the prediction of RNA secondary structures, where well established biophysics based methods exist. These methods even yield exact solutions under certain simplifying assumptions. Nevertheless, the accuracy of these classical methods is limited and has seen little improvement over the last decade. This makes it an attractive target for machine learning and consequently several deep learning models have been proposed in recent years. In this contribution we discuss limitations of current approaches, in particular due to biases in the training data. Furthermore, we propose to study capabilities and limitations of ML models by first applying them on synthetic data that can not only be generated in arbitrary amounts, but are also guaranteed to be free of biases. We apply this idea by testing several ML models of varying complexity. Finally, we show that the best models are capable of capturing many, but not all, properties of RNA secondary structures. Most severely, the number of predicted base pairs scales quadratically with sequence length, even though a secondary structure can only accommodate a linear number of pairs.

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Contrasting recruitment of skin-associated adipose depots during cold challenge of mouse and human Mammalian skin impacts metabolic efficiency system-wide, controlling the rate of heat loss and consequent heat production. Here we compare the unique fat depots associated with mouse and human skin, to determine whether they have corresponding function and regulation. For human, we assay a skin-associated fat (SAF) body-wide depot to distinguish it from the subcutaneous fat pads characteristic of abdomen and upper limbs. We show that the thickness of SAF is not related to general adiposity; it is much thicker (1.6-fold) in women than men, and highly subject-specific. We used molecular and cellular assays of {beta}-adrenergic induced lipolysis and found that dermal white adipose tissue (dWAT) in mice is resistant to lipolysis; in contrast, the body-wide human SAF depot becomes lipolytic, generating heat in response to {beta}-adrenergic stimulation. In mice challenged to make more heat to maintain body temperature (either environmentally or genetically), there is a compensatory increase in thickness of dWAT: A corresponding {beta}-adrenergic stimulation of human skin adipose (in vivo or in explant) depletes adipocyte lipid content. We summarize the regulation of skin-associated adipocytes by age, sex, and adiposity, for both species. We conclude that the body-wide dWAT depot of mice shows unique regulation that enables it to be deployed for heat preservation; combined with the actively lipolytic subcutaneous mammary fat pads they enable thermal defense. The adipose tissue that covers human subjects produces heat directly, providing an alternative to the brown adipose tissues.

KEY POINTS SUMMARYO_LISeveral distinct strategies produce and conserve heat to maintain body temperature of mammals, each associated with unique physiologies, with consequence for wellness and disease susceptibility
C_LIO_LIHighly regulated properties of skin offset the total requirement for heat production
C_LIO_LIWe hypothesize that the adipose component of skin is primarily responsible for modulating heat flux; here we evaluate the relative regulation of adipose depots in mouse and human, to test their recruitment to heat production and conservation
C_LIO_LIWe found that insulating mouse dermal white adipose tissue accumulates in response to environmentally- and genetically-induced cool stress; this layer is one of two adipose depots closely apposed to mouse skin, where the subcutaneous mammary gland fat pads are actively recruited to heat production
C_LIO_LIIn contrast, the body-wide adipose depot associated with human skin produces heat directly, potentially creating an alternative to the centrally regulated brown adipose tissue
C_LI

sentences: Development of a flexible split prime editor using truncated reverse transcriptase Prime Editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9 - which affects editing efficiency - is limited due to the large size of PE. Furthermore, the potential effect of overexpressing RT in mammalian cells is largely unknown. Here, we developed a compact PE with complete deletion of the RNase H domain of reverse transcriptase (RT), which showed comparable editing to full-length PE. Using compact PE, we tested the effect of 4 different Cas9 split sites and found that the Glu 573 split site supports robust editing (up to 93% of full-length PE). The compact PE, but not PE2, abolished its binding to eRF1 and showed minimal effect on stop codon readthrough, which therefore might reduce the effects on protein biosynthesis. This study identifies a safe and efficient compact PE2 that enables flexible split-PE design to facilitate efficient delivery in vivo and advance the utility of prime editing.

sentences: Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting eleven essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e., dnaB, ftsZ, and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that the growth inhibitory capacity of 9mer PNAs is generally as effective as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.

sentences: A single-cell resolved cell-cell communication model explains lineage commitment in hematopoiesis Cells do not function in isolation. Arguably, every cell fate decision occurs in response to environmental signals. In many cases cell-cell communication alters the dynamics of a cells internal gene regulatory network to initiate cell fate transitions, yet models rarely take this into account. Here we develop a multiscale perspective to study the granulocyte-monocyte vs. megakaryocyte-erythrocyte fate decisions. This transition is dictated by the GATA1-PU.1 network, a classical example of a bistable cell fate system. We show that, for a wide range of cell communication topologies, even subtle changes in signaling can have pronounced effects on cell fate decisions. We go on to show how cell-cell coupling through signaling can spontaneously break the symmetry of a homogenous cell population. Noise, both intrinsic and extrinsic, shapes the decision landscape profoundly, and affects the transcriptional dynamics underlying this important hematopoietic cell fate decision-making system.

sentences: Meta-analysis of Microbiome Association Networks Reveal Patterns of Dysbiosis in Diseased Microbiomes The human gut microbiome is composed of a diverse and dynamic population of microbial species which play key roles in modulating host health and physiology. While individual microbial species have been found to be associated with certain disease states, increasing evidence suggests that higher-order microbial interactions may have an equal or greater contribution to host fitness. To better understand microbial community dynamics, we utilize complex networks to study interactions through a meta-analysis of microbial association networks between healthy and disease gut microbiomes. Taking advantage of the large number of metagenomes derived from healthy individuals and patients with various diseases, together with recent advances in network inference that can deal with sparse compositional data, we inferred microbial association networks based on co-occurrence of gut microbial species and made the networks publicly available as a resource (github repository named GutNet). Through our meta-analysis of inferred networks, we were able to identify network-associated features that help stratify between healthy and disease states such as the differentiation of various bacterial phyla and enrichment of Proteobacteria interactions in diseased networks. Additionally, our findings show that the contributions of taxa in microbial associations are disproportionate to their abundances and that rarer taxa of microbial species play an integral part in shaping dynamics of microbial community interactions. Overall, this meta-analysis revealed valuable insights into microbial community dynamics between healthy and disease phenotypes.

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PvC3H29 interacts with and inhibits DNA binding of PvNAPs to finetune leaf senescence in switchgrass (Panicum virgatum) Finetuning the progression of leaf senescence is important for plants fitness in nature, while  staygreen with delayed leaf senescence has been considered as a valuable agronomic trait in crop genetic improvement. In this study, a switchgrass CCCH-type Zinc finger gene, PvC3H29, was characterized as a suppressor of leaf senescence that over-expressing or suppressing the gene led to delayed or accelerated leaf senescence, respectively. Transcriptomic analysis marked that chlorophyll catabolic pathway genes were involved in the PvC3H29-regulated leaf senescence. PvC3H29 was a nucleus-localized protein with no transcriptional activity. By Y2H screening, we identified its interacting proteins, including a pair of paralogous transcription factors, PvNAP1&2. Over-expressing the PvNAPs led to precocious leaf senescence at least partially by directly targeting and transactivating chlorophyll catabolic genes to promote chlorophyll degradation. PvC3H29, through protein-protein interaction, repressed the DNA-binding efficiency of PvNAPs and alleviated its transactivating effect on downstream genes, thereby functioned as a  brake in the progression of leaf senescence. Moreover, over-expressing PvC3H29 resulted in up to 47% higher biomass yield and improved biomass feedstock quality, reiterating the importance of leaf senescence regulation in the genetic improvement of switchgrass and other feedstock crops.

One-sentence summaryPvC3H29 interacts with transcription factors, PvNAP1&2, to inhibit their transactivation on chlorophyll catabolism and leaf senescence in switchgrass.

sentences: Exogenous application of dsRNA for the control of viruses in cucurbits The recurrent emergence of virus diseases in intensive horticultural crops requires alternative control strategies. Topical application of dsRNA molecules homologous to pathogens has been proposed as a tool for plant virus control. These dsRNAs induce the silencing mechanism (RNAi) that degrades homologous dsRNAs. Cucumber green mottle mosaic virus (CGMMV) represents a serious threat to cucurbit crops. Since genetic resistance to the virus is not yet available in commercial varieties, we aimed to control this virus by RNAi. For this purpose, we obtained constructions both for expressing dsRNA in bacteria to treat cucumber plants by topical application and for agroinoculation in experiments done in the growth chamber. Besides, greenhouse tests were performed in spring and in summer when plants were challenged with the virus and differences in several parameters were investigated, including severity of symptoms, dry weight, total height, virus accumulation and virus-derived small interfering RNAs (vsiRNAs). Spraying of plants with dsRNA reduced significatively CGMMV symptoms in the plants in growth chamber tests. Agroinfiltration experiments done under identical conditions were also effective in limiting the progress of CGMMV disease. In the greenhouse assay performed in spring, symptoms were significatively reduced in dsRNA-sprayed plants and the development of the plants improved with respect to non-treated plants. Virus titters and vsiRNAs were clearly reduced in dsRNA-treated plants. The effect of protection of the dsRNA was less evident in the greenhouse assay carried out in summer. Besides, we investigated the mobility of long (ds)RNA derived from spraying or agroinfiltrated dsRNA and found that it could be detected in local, close distal and far distal points from the site of application. VsiRNAs were also detected in local and distal points and the differences in accumulation were compared. In parallel, we investigated the capacity of dsRNAs derived from genes of tomato leaf curl New Delhi virus (ToLCNDV), another economically important virus in cucurbits, to limit the disease in zucchini, both by agroinfiltration or direct spraying, but found no protection effect. In view of the results, topical application of dsRNAs is postulated as a promising strategy for CGMMV control in cucumber.

sentences: Consequences of human Tau aggregation in the hippocampal formation of ageing mice in vivo Intracellular aggregation of hyperphosphorylated Tau (pTau) in the brain is associated with cognitive and motor impairments, and ultimately neurodegeneration. We investigate how human pTau affects cells and network activity in the hippocampal formation of THY-Tau22 tauopathy model mice in vivo. We find that pTau preferentially accumulates in deep-layer pyramidal neurons, leading to neurodegeneration, and we establish that pTau spreads to oligodendrocytes. During goal-directed virtual navigation in aged transgenic mice, we detect fewer high-firing pyramidal cells, with the remaining cells retaining their coupling to theta oscillations. Analysis of network oscillations and firing patterns of pyramidal and GABAergic neurons recorded in head-fixed and freely-moving mice suggests preserved neuronal coordination. In spatial memory tests, transgenic mice have reduced short-term familiarity but spatial working and reference memory are surprisingly normal. We hypothesize that unimpaired subcortical network mechanisms implementing cortical neuronal coordination compensate for the widespread pTau aggregation, loss of high-firing cells and neurodegeneration.

sentences: Dietary methionine restriction impairs anti-tumor immunity through gut microbiota Dietary methionine restriction has been reported to repress cancer growth and improve therapeutic responses in several pre-clinical settings. However, how this dietary intervention impacts cancer progression in the context of the intact immune system is unknown. Here we report that methionine restriction exacerbates cancer growth and influences the outcomes of anti-tumor immunotherapy through gut microbiota and immune suppression in immunocompetent settings. Methionine restriction reduces T cell activation, increases tumor growth, and impairs response to anti-tumor immunotherapy. Mechanistically, methionine restriction alters composition of gut microbiota and reduces microbial production of hydrogen sulfide. Fecal transplantation from methionine-restricted tumor-free animals is sufficient to repress T cell activation and enhance tumor growth in tumor-bearing recipient mice. Conversely, dietary supplementation of a hydrogen sulfide donor or methionine stimulates anti-tumor immunity and suppresses tumor progression. Our findings reveal a vital role of gut microbiota in mediating methionine restriction-induced suppression of anti-tumor immunity and suggest that any possible anti-cancer benefits of methionine restriction require careful considerations of both the microbiota and the immune system.

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Prediction of allosterically acting mutations using MD simulations and Rosetta Computationally modeling how mutations affect protein binding not only helps uncover the biophysics of protein interfaces, but also enables the redesign and optimization of protein-protein interactions. Traditional high-throughput methods for estimating binding free energies are currently limited to mutations directly at the interface due to difficulties in accurately modeling how more distant allosterically acting mutations propagate their effects through the protein structure. However, the modeling and design of such allosteric mutations is of substantial interest as it allows for greater control and flexibility in protein design applications. We have developed a method that combines high-throughput Rosetta-based side-chain optimization with conformational sampling using classical molecular dynamics simulations, finding significant improvements in our ability to accurately predict allosterically acting mutational perturbations to protein binding. Our approach uses an analytical framework grounded in rigorous alchemical free energy calculations while enabling exploration of a vastly larger sequence space. When comparing to experimental data, we find that our method can predict allosteric perturbations with a level of accuracy similar to that of traditional methods in predicting the effects of non-allosteric interface mutations. This work represents a new and generalizable approach to optimize protein free energy landscapes for desired biological functions.

Author SummaryProtein-protein interactions are vital to almost all biological processes, and therefore the ability to accurately and efficiently predict how mutations alter protein binding has far-reaching applications in protein analysis and design. Current approaches to predict such mutational free energy changes are limited to mutations directly at the interaction interface. Much research has underlined the prevalence of allosteric protein regulation in biological processes, indicating the importance of understanding and predicting the effects of mutations which act over long distances. In this work we develop a novel method based on theoretically rigorous alchemical free energy calculations and the Rosetta macromolecular modeling suite which can predict the effects of allosteric mutations with levels of accuracy rivaling state of the art interface-specific methods. We hope that our method will serve as a novel framework for high-throughput allosteric mutational analysis and therefore benefit future protein design efforts.

sentences: Single Cell, Single Nucleus and Spatial RNA Sequencing of the Human Liver Identifies Hepatic Stellate Cell and Cholangiocyte Heterogeneity The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation related cell perturbation can limit the ability to fully capture the human livers parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at single-cell resolution, revealed the presence of rare subtypes of hepatic stellate cells previously only seen in disease, and detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and NK cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell-types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte and stellate cell populations by an independent spatial transcriptomics dataset and immunohistochemistry. Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver.

sentences: Conserved Circuits for Direction Selectivity in the Primate Retina The detection of motion direction is a fundamental visual function and a classic model for neural computation1,2. In the non-primate mammalian retina, direction selectivity arises in starburst amacrine cell (SAC) dendrites, which provide selective inhibition to ON and ON-OFF direction selective retinal ganglion cells (dsRGCs)3,4. While SACs are present in primates5, their connectivity is unknown and the existence of primate dsRGCs remains an open question. Here we present a connectomic reconstruction of the primate ON SAC circuit from a serial electron microscopy volume of macaque central retina. We show that the structural basis for the SACs ability to compute and confer directional selectivity on post-synaptic RGCs6 is conserved in primates and that SACs selectively target a single ganglion cell type, a candidate homolog to the mammalian ON-sustained dsRGCs that project to the accessory optic system and contribute to gaze-stabilizing reflexes7,8. These results indicate that the capacity to compute motion direction is present in the retina, far earlier in the primate visual system than classically thought, and they shed light on the distinguishing features of primate motion processing by revealing the extent to which ancestral motion circuits are conserved.

sentences: Novel Feline Papillomavirus Isolate P20 Assembled from Metagenomic Data Isolated from Human Skin of a House Cat Owner A feline papillomavirus was assembled from metagenomic sequencing data collected from the human skin of a house cat owner. This circular papillomavirus strain P20 is 8069 bp in length, has a GC content of 54.38%, and displays genome organization typical of feline papillomaviruses with six annotated protein coding regions. The genome exhibits approximately 75% sequence similarity to other feline papillomavirus genomes.

sentences: Exploring Extreme Signaling Failures in Intracellular Molecular Networks Developing novel methods for the analysis of intracellular signaling networks is essential for understanding interconnected biological processes that underlie complex human disorders. A fundamental goal of this research is to quantify the vulnerability of a signaling network to the dysfunction of one or multiple molecules, when the dysfunction is defined as an incorrect response to the input signals. In this study, we propose an efficient algorithm to identify the extreme signaling failures that can induce the most detrimental impact on the physiological function of a molecular network. The algorithm basically finds the molecules, or groups of molecules, with the maximum vulnerability, i.e., the highest probability of causing the network failure, when they are dysfunctional. We propose another algorithm that efficiently accounts for signaling feedbacks in this analysis. The algorithms are tested on two experimentally verified ERBB and T cell signaling networks. Surprisingly, results reveal that as the number of concurrently dysfunctional molecules increases, the maximum vulnerability values quickly reach to a plateau following an initial increase. This suggests the specificity of vulnerable molecule (s) involved, as a specific number of faulty molecules cause the most detrimental damage to the function of the network. Increasing a random number of simultaneously faulty molecules does not further deteriorate the function of the network. Such a group of specific molecules whose dysfunction causes the extreme signaling failures can better elucidate the molecular mechanisms underlying the pathogenesis of complex trait disorders, and can offer new insights for the development of novel therapeutics.

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Dynamics of global gene expression and chromatin accessibility of the peripheral nervous system in animal models of persistent pain Efforts to understand genetic variability involved in an individuals susceptibility to chronic pain support a role for upstream regulation by epigenetic mechanisms. To examine the transcriptomic and epigenetic basis of chronic pain that resides in the peripheral nervous system, we used RNA-seq and ATAC-seq of the rat dorsal root ganglion (DRG) to identify novel molecular pathways associated with pain hypersensitivity in two well-studied persistent pain models induced by Chronic Constriction Injury (CCI) of the sciatic nerve and intra-plantar injection of Complete Freunds Adjuvant (CFA) in rats. Our RNA-seq studies identify a variety of biological process related to synapse organization, membrane potential, transmembrane transport, and ion binding. Interestingly, genes that encode transcriptional regulators were disproportionately downregulated in both models. Our ATAC-seq data provide a comprehensive map of chromatin accessibility changes in the DRG. A total of 1123 regions showed changes in chromatin accessibility in one or both models when compared to the naive and 31 shared differentially accessible regions (DAR)s. Functional annotation of the DARs identified disparate molecular functions enriched for each pain model which suggests that chromatin structure may be altered differently following sciatic nerve injury and hind paw inflammation. Motif analysis identified 17 DNA sequences known to bind transcription factors in the CCI DARs and 33 in the CFA DARs. Two motifs were significantly enriched in both models. Our improved understanding of the changes in chromatin accessibility that occur in chronic pain states may identify regulatory genomic elements that play essential roles in modulating gene expression in the DRG.

SummaryShared transcriptomic and epigenetic changes in two animal models improves our understanding of how chromatin structural changes alter DRG gene expression under persistent pain conditions.

sentences: Ectopic expression of lipid homeostasis genes in rare cells of the teleost pituitary gland Directing both organismal homeostasis and physiological adaptation, the pituitary is a key endocrine gland in all vertebrates. It communicates the needs of the organism to different organs by secreting hormones into the bloodstream. Here, we have used the model fish medaka to investigate the developmental dynamics in the pituitary using a comprehensive RNA-seq time series. By linking developmental expression trends to single-cell RNA-seq profiles, we show how the transcriptional activities of cell types change during sexual maturation. One of the most prominent changes is the decline of the non-endocrine folliculo-stellate cell populations, and especially of rare cells expressing genes encoding secreted lipid transport proteins. As these genes are typically associated with the liver, this reveals the existence of unexpected connections between endocrine communication, lipid homeostasis, and sexual maturation.

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High-throughput screening of aqueous two-phase systems for polyphenol extraction from A. nodosum: a green chemistry approach Brown macroalgae are an attractive third-generation feedstock of natural products, in order to design green chemistry-compliant processes and reduce the use of organic solvents in bioactive product extraction, aqueous two-phase systems (ATPS) was applied. This research aimed to develop a high-throughput screening (HTS) to recover polyphenols from Ascophyllum nodosum using ATPS. In total, 384 different 2-phase systems were assessed using an automated liquid-handling system to evaluate polyphenol recovery using a model system of phloroglucinol to establish an optimal 2-phase system for polyphenol partitioning. Various ratios of PEG:potassium phosphate solutions were explored to evaluate partitioning of polyphenols via a scale-down approach. Scale-down selected system showed a recovery of phloroglucinol of 62.9{+/-}12.0%, this system was used for scale-up trials. Scale-up studies confirmed that the HTS method was able to recover polyphenols with a 54.8{+/-}14.2% in the phloroglucinol model system. When the optimised ATPS system was tested with a polyphenol extract, 93.62{+/-}8.24% recovery was observed. When ATPS was applied to a fucoidan and alginate biorefinery residue, 88.40{+/-}4.59% polyphenol was recovered. These findings confirm that ATPS is a valuable addition to the bioprocess toolkit for sustainable extraction of natural products from macroalgae in a multiproduct biorefinery approach.

Practical applicationSelection of the best concentrations of phase-forming components and recovery conditions for the application of aqueous two-phase systems in an industrial setup has been proved to be laborious and cumbersome. This paper presents an automated platform to rapidly assess several ATPS to recover polyphenols from brown macroalgae and a subsequent confirmation with the scale-up of the potential candidates and contrasted with two case studies. This methodology allows a quick screening for the best aqueous two-phase system and can be expanded to recover high-value products from other types of macroalgae or microalgae.

sentences: Semi-supervised identification of SARS-CoV-2 molecular targets SARS-CoV-2 genomic sequencing efforts have scaled dramatically to address the current global pandemic and aid public health. In this work, we analyzed a corpus of 66,000 SARS-CoV-2 genome sequences. We developed a novel semi-supervised pipeline for automated gene, protein, and functional domain annotation of SARS-CoV-2 genomes that differentiates itself by not relying on use of a single reference genome and by overcoming atypical genome traits. Using this method, we identified the comprehensive set of known proteins with 98.5% set membership accuracy and 99.1% accuracy in length prediction compared to proteome references including Replicase polyprotein 1ab (with its transcriptional slippage site). Compared to other published tools such as Prokka (base) and VAPiD, we yielded an 6.4- and 1.8-fold increase in protein annotations. Our method generated 13,000,000 molecular target sequences-- some conserved across time and geography while others represent emerging variants. We observed 3,362 non-redundant sequences per protein on average within this corpus and describe key D614G and N501Y variants spatiotemporally. For spike glycoprotein domains, we achieved greater than 97.9% sequence identity to references and characterized Receptor Binding Domain variants. Here, we comprehensively present the molecular targets to refine biomedical interventions for SARS-CoV-2 with a scalable high-accuracy method to analyze newly sequenced infections.

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LSMMD-MA: Scaling multimodal data integration for single-cell genomics data analysis MotivationModality matching in single-cell omics data analysis--i.e., matching cells across data sets collected using different types of genomic assays--has become an important problem, because unifying perspectives across different technologies holds the promise of yielding biological and clinical discoveries. However, single-cell dataset sizes can now reach hundreds of thousands to millions of cells, which remains out of reach for most multi-modal computational methods.

ResultsWe propose LSMMD-MA, a large-scale Python implementation of the MMD-MA method for multimodal data integration. In LSMMD-MA we reformulate the MMD-MA optimization problem using linear algebra and solve it with KeOps, a CUDA framework for symbolic matrix computation in Python. We show that LSMMD-MA scales to a million cells in each modality, two orders of magnitude greater than existing implementations.

AvailabilityLSMMD-MA is freely available at https://github.com/google-research/large_scale_mmdma

Contactlpapaxanthos@google.com

sentences: Interrogation of cancer gene dependencies reveals novel paralog interactions of autosome and sexchromosome encoded genes Genetic networks are characterized by extensive buffering. During tumour evolution, disruption of these functional redundancies can create de novo vulnerabilities that are specific to cancer cells. In this regard, paralog genes are of particular interest, as the loss of one paralog gene can render tumour cells dependent on a remaining paralog. To systematically identify cancer-relevant paralog dependencies, we searched for candidate dependencies using CRISPR screens and publicly available loss-of-function datasets. Our analysis revealed >2,000 potential candidate dependencies, several of which were subsequently experimentally validated. We provide evidence that DNAJC15-DNAJC19, FAM50A-FAM50B and RPP25-RPP25L are novel cancer relevant paralog dependencies. Importantly, our analysis also revealed unexpected redundancies between sex chromosome genes. We show that chrX- and chrY- encoded paralogs, as exemplified by ZFX-ZFY, DDX3X-DDX3Y and EIF1AX-EIF1AY, are functionally linked so that tumour cell lines from male patients with Y-chromosome loss become exquisitely dependent on the chrX-encoded gene. We therefore propose genetic redundancies between chrX- and chrY- encoded paralogs as a general therapeutic strategy for human tumours that have lost the Y-chromosome.

sentences: Normalization benchmark of ATAC-seq datasets shows the importance of accounting for GC-content effects Modern assays have enabled high-throughput studies of epigenetic regulation of gene expression using DNA sequencing. In particular, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) allows the study of chromatin configuration for an entire genome. Despite the gain in popularity of the assay, there have been limited studies investigating the analytical challenges related to ATAC-seq data, and most studies leverage tools developed for bulk transcriptome sequencing (RNA-seq). Here, we show that GC-content effects are omnipresent in ATAC-seq datasets. Since the GC-content effects are sample-specific, they can bias downstream analyses such as clustering and differential accessibility analysis. We introduce a normalization method based on smooth-quantile normalization within GC-content bins, and evaluate it together with eleven different normalization procedures on eight public ATAC-seq datasets. Our work clearly shows that accounting for GC-content effects in the normalization is crucial for common downstream ATAC-seq data analyses, leading to improved accuracy and interpretability of the results. Using two case studies, we show that exploratory data analysis is essential to guide the choice of an appropriate normalization method for a given dataset.

sentences: Targeting diverse operational regimes in recurrent spiking networks Neural computations emerge from recurrent neural circuits that comprise hundreds to a few thousand neurons. Continuous progress in connectomics, electrophysiology, and calcium imaging require tractable spiking network models that can consistently incorporate new information about the network structure and reproduce the recorded neural activity features. However, it is challenging to predict which spiking network connectivity configurations and neural properties can generate fundamental operational states and specific experimentally reported nonlinear cortical computations. Theoretical descriptions for the computational state of cortical spiking circuits are diverse, including the balanced state where excitatory and inhibitory inputs balance almost perfectly or the inhibition stabilized state (ISN) where the excitatory part of the circuit is unstable. It remains an open question whether these states can co-exist with experimentally reported nonlinear computations and whether they can be recovered in biologically realistic implementations of spiking networks. Here, we show how to identify spiking network connectivity patterns underlying diverse nonlinear computations such as XOR, bistability, inhibitory stabilization, supersaturation, and persistent activity. We established a mapping between the stabilized supralinear network (SSN) and spiking activity which allowed us to pinpoint the location in parameter space where these activity regimes occur. Notably, we found that biologically-sized spiking networks can have irregular asynchronous activity that does not require strong excitation-inhibition balance or large feedforward input and we showed that the dynamic firing rate trajectories in spiking networks can be precisely targeted without error-driven training algorithms.

sentences: Electronic nicotine delivery systems exhibit reduced bronchial epithelial cells toxicity compared to cigarette: The Replica Project Cigarette smoking is the leading cause of preventable deaths worldwide. Electronic Nicotine Delivery Systems (ENDS) may reduce health risks associated with chronic exposure to smoke and their potential benefits have been the matter of intense scientific debate. Here we replicated three key published studies from the Tobacco Industry on cytotoxic and inflammatory effects of cigarette smoke and ENDS aerosol in an independent multicentric study. We aimed to establish the reliability of results and the robustness of conclusions by replicating the authors experimental protocols and further validating them with different techniques. We exposed human bronchial epithelial cell (NCI-H292) to cigarette smoke and to aerosol from ENDS. All the exposure were conducted at air-liquid interface to assess cytotoxicity effects of smoke and aerosol. Moreover, we aimed to assess different inflammatory mediators release (IL-6, IL-8 and MMP-1) from cells exposed to whole smoke and to smoke without particulate matter (vapor phase). We were able to replicate the results obtained in the original studies on cytotoxicity confirming that almost 80% of the cytotoxic effect of smoke is due to the vapor phase of smoke. Moreover, our results substantiated the reduced cytotoxic effects of ENDS aerosol in respect to cigarette smoke. However, our data are significantly different from the original ones in terms of inflammatory and remodeling activity triggered by smoke. Taken all together, the data obtained independently in different laboratories clearly demonstrate the reduced toxicity of ENDS products compared to cigarettes and thus providing a valuable tool to the harm reduction strategies in smokers.

sentences: Machine Learning Differentiates Enzymatic and Non-Enzymatic Metals in Proteins Metalloenzymes are 40% of all enzymes and can perform all seven classes of enzyme reactions. Because of the physicochemical similarities between the active sites of metalloenzymes and inactive metal binding sites, it is challenging to differentiate between them. Yet distinguishing these two classes is critical for the identification of both native and designed enzymes. Because of similarities between these two types of metal binding sites, finding physicochemical features that distinguish active and inactive metal sites can indicate aspects that are critical to enzyme function. In this work, we develop the largest structural dataset of enzymatic and non-enzymatic metalloprotein sites to date. We then use a decision-tree ensemble machine learning model to classify metals bound to proteins as enzymatic or non-enzymatic with 92.2% precision and 90.1% recall. Our model scores electrostatic and pocket lining features as more important than pocket volume, despite the fact that volume is the most quantitatively different feature between enzyme and non-enzymatic sites. Finally, we find our model has overall better performance in a side-to-side comparison against other methods that differentiate enzymatic from non-enzymatic sequences. We anticipate that our models ability to correctly identify which metal sites are responsible for enzymatic activity could enable identification of new enzymatic mechanisms and de novo enzyme design.

sentences: Newly developed ad hoc molecular assays show how eDNA can witness and anticipate the monk seal recolonization of central Mediterranean The monk seal Monachus monachus is the most endangered pinniped worldwide and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic decline in the last few decades. Data on its status are scattered due to both its rarity and evasiveness, and records are biased towards occasional, mostly coastal, encounters. Nowadays molecular techniques allow us to detect and quantify minute amounts of DNA traces released in the environment (eDNA) by any organism. We present three qPCR-assays targeting the monk seal mitogenome. The assays were soundly tested on an extensive and diversified sample set (n=73), including positive controls from Madeira breeding population collected during the peak of abundance, and two opportunistic Mediterranean eDNA-sample collections (offshore/coastal) from on-going projects. Monk seal DNA was detected in 47.2% and 66.7% of the samples collected in the Tyrrhenian from a ferry platform (2018-2019) and in the Pelagie archipelago -Strait of Sicily- (2020) respectively, anticipating (up to 2 year) visual observations occurred subsequently in proximity of the sampled areas. In the Tyrrhenian, detection occurrence increased between 2018 and 2019. Monk seal DNA recoveries were commoner in night-time ferry-samples, suggesting nocturnal predatory activity in pelagic waters. The proposed technique provides a non-invasive and yet highly-sensitive tool for defining the monk seal actual distribution and home range, its recovery rate and pinpoint coastal/offshore localities where prioritizing conservation, research, citizen science and education initiatives.

sentences: Structure of a Plant-Specific Partitivirus Capsid Reveals a Novel Coat Protein Architecture with a Hypervariable Protrusion Persistent plant viruses may be the most common viruses in wild plants. A growing body of evidence for mutualism between such viruses and their hosts, suggests that they play an important role in ecology and agriculture. Here we present the structure of a plant-specific partitivirus capsid at 2.9 [A] resolution by Cryo-EM. Structural features, including the T=1 arrangement of 60 coat protein dimers, are shared with fungal partitiviruses and the picobirnavirus lineage of dsRNA viruses. However, the topology of the capsid is markedly different with protrusions emanating from, and partly comprising, the binding interface of coat protein dimers. We show that a disordered region at the apex of the protrusion is not required for capsid assembly and represents a hypervariable site characteristic of the plant-specific partitiviruses. These results suggest a structural basis for the acquisition of additional functions by partitivirus coat proteins that enables mutualistic relationships with diverse plant hosts.

sentences: Azithromycin possesses biofilm-inhibitory activity and potentiates non-bactericidal colistin methanesulfonate against Klebsiella pneumonia Novel antibiotic combinations may act synergistically to inhibit the growth of multidrug-resistant bacterial pathogens but predicting which will be successful is difficult, and standard antimicrobial susceptibility testing may not identify important physiological differences between planktonic free-swimming and biofilm-protected surface-attached sessile cells. Using a nominally macrolide-resistant model Klebsiella pneumoniae strain (ATCC 10031) we demonstrate the effectiveness of several macrolides in inhibiting biofilm growth in multi-well plates, and the ability of azithromycin (AZM) to improve the effectiveness of the antibacterial last-agent-of-choice for K. pneumoniae infections, colistin methanesulfonate (CMS), against biofilms. This synergistic action was also seen in biofilm tests of several K. pneumoniae hospital isolates and could also be identified in polymyxin B disc-diffusion assays on azithromycin plates. Our work highlights the complexity of antimicrobial-resistance in bacterial pathogens and the need to test antibiotics with biofilm models where potential synergies might provide new therapeutic opportunities not seen in liquid culture or colony-based assays.

sentences: Rational Exploration of Fold Atlas for Human Solute Carrier Proteins Solute carrier (SLC) superfamily is the largest group responsible for transmembrane transport of substances in human cells. It includes more than 400 members which are organized into 65 families according to their physiological function and sequence similarity. Different families of solute transporters can adopt the same or different folds that determine the working mechanism and reflect the evolutionary relationship between SLC members. Analysis of structural data in literatures shows that there are 13 different folds in the solute carrier superfamily covering 40 families and 343 members. To further study their working mechanism, we systematically explored the SLC superfamily to look for more folds. Based on our results, at least three new folds were found for SLC superfamily, and one was experimentally verified in the SLC44 family. Our work has laid a foundation and provided important insights for the systematic and comprehensive study on the structure and function of solute carriers.

sentences: A Phosphorus-Limitation Induced, Functionally Conserved DUF506 Protein is a Repressor of Root Hair Elongation in Arabidopsis thaliana Root hairs (RHs) function in nutrient and water acquisition, root metabolite exudation, soil anchorage and plant-microbe interactions. Longer or more abundant RHs are potential breeding traits for developing crops that are more resource-use efficient and can improve soil health. RH elongation is controlled by both environmental and endogenous factors. While many genes are known to promote RH elongation, relatively little is known about genes and mechanisms that constrain RH growth. Here we demonstrate that a DOMAIN OF UNKNOWN FUNCTION 506 (DUF506) protein, AT3G25240, negatively regulates Arabidopsis thaliana RH growth. The AT3G25240 gene is strongly and specifically induced during P-limitation. Mutants of this gene, which we call REPRESSOR OF EXCESSIVE ROOT HAIR ELONGATION 1 (RXR1), have much longer RHs, while over-expression of the gene results in much shorter RHs. RXR1 physically interacts with a Rab-GTPase (RXR2), and an rxr2 mutant phenocopies the rxr1 mutant. Overexpression of a Brachypodium distachyon RXR1 homolog resulted in repression of RH elongation in Brachypodium. Taken together, our results reveal a DUF506-GTPase module with a prominent role in repression of RH elongation that is conserved in monocots and dicots.

sentences: Temporal order of mutations influences cancer initiation dynamics Cancer is a set of genetic diseases that are driven by mutations. It was recently discovered that the temporal order of genetic mutations affects the cancer evolution and even the nature of the decease itself. The mechanistic origin of these observations, however, remain not well understood. Here we present a theoretical model for cancer initiation dynamics that allows us to quantify the impact of the temporal order of mutations. In our approach, the cancer initiation process is viewed as a set of stochastic transitions between discrete states defined by the different numbers of mutated cells. Using a first-passage analysis, probabilities and times before the cancer initiation are explicitly evaluated for two alternative sequences of two mutations. It is found that the probability of cancer initiation is determined only by the first mutation, while the dynamics is specified by both mutations. In addition, it is shown that the acquisition of a mutation with higher fitness before mutation with lower fitness increases the probability of the tumor formation but delays the cancer initiation. Theoretical results are explained using effective free-energy landscapes.

sentences: Recurrent micropollutant exposure leads to divergent degradation abilities and community succession in a freshwater microbiome The increasing-volume release of micropollutants into natural surface waters has raised great concern due to their environmental accumulation. Persisting micropollutants can impact multiple generations of organisms, but their microbially-mediated degradation and their influence on community assembly remain understudied. Here, freshwater microbes were treated with several common micropollutants, alone or in combination, and then transferred every 5 days to fresh medium containing the same micropollutants to mimic the repeated exposure of microbes. Metabarcoding of 16S rRNA gene makers was chosen to study the succession of bacterial assemblages following micropollutant exposure. The removal rates of micropollutants were then measured to assess degradation capacity of the associated communities. The degradation of micropollutants did not accelerate over time but altered the microbial community composition. Community assembly was dominated by stochastic processes during early exposure, via random community changes and emergence of seedbanks, and deterministic processes later in the exposure, via advanced community succession. Early exposure stages were characterized by the presence of sensitive microorganisms such as Actinobacteria and Planctomycetes, which were then replaced by more tolerant bacteria such as Bacteroidetes and Gammaproteobacteria. Our findings have important implication for ecological feedback between microbe-micropollutants under anthropogenic climate change scenarios.

sentences: Sex-specific effects of chronic paternal stress on offspring development are partially mediated via mothers. Paternal stress exposure is known to impact the development of stress-related behaviors in offspring. Previous work has highlighted the importance of sperm mediated factors, such as RNAs, in transmitting the effects of parental stress. However, a key unanswered question is whether mothers behavior could drive or modulate the transmission of paternal stress effects on offspring development. Here we investigate how chronic variable stress in Balb/C mice influences the sex-specific development of anxiety- and depression-like neural and behavioral development in offspring. Moreover, we examined how stressed fathers influenced mate maternal investment towards their offspring and how this may modulate the transmission of paternal stress effects on offspring. We show that paternal stress leads to sex-specific effects on offspring behavior. Males that are chronically stressed sire female offspring that show increased anxiety and depression-like behaviors. However, male offspring of stressed fathers show reductions in anxiety- and depression-behaviors and are generally more exploratory. Moreover, we show that females mated with stressed males gain less weight during pregnancy and provide less care towards their offspring which additionally influenced offspring development. These data indicate that paternal stress can influence offspring development directly and indirectly via changes in mothers, with implications for divergent development between male and female offspring.

sentences: Genetic architecture of a mutation's expressivity Genetic background often influences the phenotypic consequences of mutations, resulting in variable expressivity. How standing genetic variants collectively cause this phenomenon is not fully understood. Here, we comprehensively identify loci in a budding yeast cross that impact the growth of individuals carrying a spontaneous missense mutation in the nuclear-encoded mitochondrial ribosomal gene MRP20. Initial results suggested that a single large effect locus influences the mutations expressivity, with one allele causing inviability in mutants. However, further experiments revealed this simplicity was an illusion. In fact, many additional loci shape the mutations expressivity, collectively leading to a wide spectrum of mutational responses. These results exemplify how complex combinations of alleles can produce a diversity of qualitative and quantitative responses to the same mutation.

sentences: CoRe: A robustly benchmarked R package for the identification of core fitness genes in genome-wide pooled CRISPR knock-out screens CRISPR-Cas9 genome-wide screens are being increasingly performed, allowing systematic explorations of cancer dependencies at unprecedented accuracy and scale. Identifying from these screens the genes that are essential for cell survival invariantly across tissues, conditions, and genomic-contexts (core-fitness genes), is of paramount importance to assess the safety profile of candidate therapeutic targets and for elucidating mechanisms involved in tissue-specific genetic diseases. We present CoRe: An R package implementing novel methods for identifying core-fitness genes from joint analyses of multiple CRISPR-Cas9 screens. We demonstrate that CoRe outperforms state-of-the-art tools, yielding more reliable sets of core-fitness genes than existing and widely used reference sets.

sentences: NEDD4 Regulates Ubiquitination and Stability of the Cell adhesion Molecule IGPR-1 via Lysosomal Pathway The cell adhesion molecule immunoglobulin and proline-rich receptor-1 (IGPR-1) regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy. However, the molecular mechanisms governing IGPR-1 cell surface expression levels remains unknown. In the present study, we used an in vitro ubiquitination assay and identified ubiquitin E3 ligase NEDD4 and the ubiquitin conjugating enzyme UbcH6 involved in the ubiquitination of IGPR-1. In vitro GST-pulldown and in vivo co-immunoprecipitation assays demonstrated that NEDD4 binds to IGPR-1. Over-expression of wild-type NEDD4 downregulated IGPR-1 and deletion of WW domains (1-4) of NEDD4 revoked its effects on IGPR-1. Similarly, knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Furthermore, deletion of 57 amino acids encompassing polyproline rich (PPR) motif on the C-terminus of IGPR-1 nullified the binding of NEDD4 with IGPR-1. Moreover, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulated lysosomal degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine and ammonium chloride increased IGPR-1 levels in the HEK-293 cells ectopically expressing IGPR-1 and in multiple human skin melanoma cell lines. Hence, these findings suggest that ubiquitin E3 ligase NEDD4 is a key regulator of IGPR-1 with a significant implication in the therapeutic targeting of IGPR-1.

sentences: How many replicates to accurately estimate fish biodiversity using environmental DNA on coral reefs? Quantifying the diversity of species in rich tropical marine environments remains challenging. Environmental DNA (eDNA) metabarcoding is a promising tool to face this challenge through the filtering, amplification, and sequencing of DNA traces from water samples. However, the reliability of biodiversity detection from eDNA samples can be low in marine environments because eDNA density is low and certainly patchy in this vast, heterogenous and dynamic environment. So, the number of sampling replicates and filtered volume necessary to obtain accurate estimates of biodiversity in rich tropical marine environments using eDNA metabarcoding is still unknown. Here, we used a paired sampling design of 30L per replicate on 68 reef transects from 8 sites in three tropical regions and identified fish Molecular Taxonomic Units (MOTUs) using a 12S marker. We quantified local biodiversity variation as MOTU richness, compositional turnover and compositional nestedness between replicated pairs of seawater samples. We report strong turnover of MOTUs between replicated pairs of samples undertaken in the same location, time, and conditions. Paired samples contained non-overlapping assemblages rather than subsets of one-another. As a result, localised diversity accumulation curves showed that even 6 replicates (180L) in the same location underestimated local diversity (for an area <1km). However, sampling of regional diversity using [~]25 replicates in variable locations (often covering 10s of km) achieved saturation of biodiversity accumulation curves. Our results demonstrate high variability of diversity estimates perhaps arising from heterogeneous and local distribution of eDNA distribution in seawater or highly skewed frequencies of eDNA traces. This high compositional variability has consequences for using eDNA to monitor temporal and spatial biodiversity changes of local assemblages. Future biomonitoring efforts could be strongly undermined by a high level of false-negative detections under low replication protocols. We reveal the need to increase replicates or increase sampled water volume to better inform management of marine biodiversity using eDNA.

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Early Computational Detection of Potential High Risk SARS-CoV-2 Variants The ongoing COVID-19 pandemic is leading to the discovery of hundreds of novel SARS-CoV-2 variants on a daily basis. While most variants do not impact the course of the pandemic, some variants pose significantly increased risk when the acquired mutations allow better evasion of antibody neutralisation in previously infected or vaccinated subjects, or increased transmissibility. Early detection of such high risk variants (HRVs) is paramount for the proper management of the pandemic. However, experimental assays to determine immune evasion and transmissibility characteristics of new variants are resource-intensive and time-consuming, potentially leading to delayed appropriate responses by decision makers. Here we present a novel in silico approach combining Spike protein structure modelling and large protein transformer language models on Spike protein sequences, to accurately rank SARS-CoV-2 variants for immune escape and fitness potential. We validate our immune escape and fitness metrics with in vitro pVNT and binding assays. These metrics can be combined into an automated Early Warning System (EWS) capable of evaluating new variants in minutes and risk monitoring variant lineages in near real-time. The EWS flagged 12 out of 13 variants, designated by the World Health Organisation (WHO, Alpha-Omicron) as potentially dangerous, on average two months ahead of them being designated as such, demonstrating its ability to help increase preparedness against future variants. Omicron was flagged by the EWS on the day its sequence was made available, with immune evasion and binding metrics subsequently confirmed through our in vitro experiments.

One-Sentence SummaryA COVID-19 Early Warning System combining structural modelling with AI to detect and monitor high risk SARS-CoV-2 variants, identifying >90% of WHO designated variants on average two months in advance.

sentences: Evaluation of lamin A/C mechanotransduction under different surface topography in LMNA related muscular dystrophy. Most of the single point mutations of the LMNA gene are associated with distinct muscular dystrophies, marked by heterogenous phenotypes but primarily the loss and symmetric weakness of skeletal muscle tissue. The molecular mechanism and phenotype-genotype relationships in these muscular dystrophies are poorly understood. An effort has been here to delineating the adaptation of mechanical inputs into biological response by mutant cells of lamin A associated muscular dystrophy. In this study we implement engineered smooth and pattern surfaces of particular young modulus to mimic muscle physiological range. Using fluorescence and atomic force microscopy we present distinct architecture of the actin filament along with abnormally distorted cell and nuclear shape in mutants, which showed a tendency to deviate from wild type cells. Topographic features of pattern surface antagonizes the binding of the cell with it. Correspondingly, from the analysis of genome wide expression data in wild type and mutant cells, we report differential expression of the gene products of the structural components of cell adhesion as well as LINC (linkers of nucleoskeleton and cytoskeleton) protein complexes. This study also reveals mis expressed downstream signaling processes in mutant cells, which could potentially lead to onset of the disease upon the application of engineered materials to substitute the role of conventional cues in instilling cellular behaviors in muscular dystrophies. Collectively, these data support the notion that lamin A is essential for proper cellular mechanotransduction from extracellular environment to the genome and impairment of the muscle cell differentiation in the pathogenic mechanism for lamin A associated muscular dystrophy.

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Dynamical Origin for Winner-Take-All Competition in A Biological Network of The Hippocampal Dentate Gyrus We consider a biological network of the hippocampal dentate gyrus (DG). The DG is a pre-processor for pattern separation which facilitates pattern storage and retrieval in the CA3 area of the hippocampus. The main encoding cells in the DG are the granule cells (GCs) which receive the input from the entorhinal cortex (EC) and send their output to the CA3. We note that the activation degree of GCs is so low (~ 5%). This sparsity has been thought to enhance the pattern separation. We investigate the dynamical origin for winner-take-all (WTA) competition which leads to sparse activation of the GCs. The whole GCs are grouped into lamellar clusters. In each GC cluster, there is one inhibitory (I) basket cell (BC) along with excitatory (E) GCs. There are three kinds of external inputs into the GCs; the direct excitatory EC input, the indirect inhibitory EC input, mediated by the HIPP (hilar perforant path-associated) cells, and the excitatory input from the hilar mossy cells (MCs). The firing activities of the GCs are determined via competition between the external E and I inputs. The E-I conductance ratio [Formula] (given by the time average of the external E to I conductances) may represents well the degree of such external E-I input competition. It is thus found that GCs become active when their [Formula] is larger than a threshold [Formula], and then the mean firing rates of the active GCs are strongly correlated with [Formula]. In each GC cluster, the feedback inhibition of the BC may select the winner GCs. GCs with larger [Formula] than the threshold [Formula] survive, and they become winners; all the other GCs with smaller [Formula] become silent. In this way, WTA competition occurs via competition between the firing activity of the GCs and the feedback inhibition from the BC in each GC cluster. In this case, the hilar MCs are also found to play an essential role of enhancing the WTA competition in each GC cluster by exciting both the GCs and the BC.

PACS numbers: 87.19.lj, 87.19.lm, 87.19.lv

sentences: Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica Genome-wide functional genetic screens have been successful in discovering genotype-phenotype relationships and in engineering new phenotypes. While broadly applied in mammalian cell lines and in E. coli, use in non-conventional microorganisms has been limited, in part, due to the inability to accurately design high activity CRISPR guides in such species. Here, we develop an experimental-computational approach to sgRNA design that is specific to an organism of choice, in this case the oleaginous yeast Yarrowia lipolytica. A negative selection screen in the absence of non-homologous end-joining, the dominant DNA repair mechanism, was used to generate single guide RNA (sgRNA) activity profiles for both SpCas9 and LbCas12a. This genome-wide data served as input to a deep learning algorithm, DeepGuide, that is able to accurately predict guide activity. DeepGuide uses unsupervised learning to obtain a compressed representation of the genome, followed by supervised learning to map sgRNA sequence, genomic context, and epigenetic features with guide activity. Experimental validation, both genome-wide and with a subset of selected genes, confirms DeepGuides ability to accurately predict high activity sgRNAs. DeepGuide provides an organism specific predictor of CRISPR guide activity that could be broadly applied to fungal species, prokaryotes, and other non-conventional organisms.

sentences: Envelope analysis of the human alpha rhythm reveals EEG Gaussianity The origin of the human alpha rhythm has been a matter of debate since Lord Adrian attributed it to synchronous neural populations in the occipital cortex. Although some authors have pointed out the Gaussian characteristics of the alpha rhythm, their results have been repeatedly disregarded in favor of Adrians interpretation; even though the first EEG Gaussianity reports can be traced back to the origins of the field. Here we revisit this problem using the envelope analysis -- a method that relies on the fact that the coefficient of variation of the envelope (CVE) for continuous-time zero-mean Gaussian noise (as well as for any filtered sub-band) is equal to [Formula], thus making the CVE a fingerprint for Gaussianity. As a consequence, any significant deviation from [Formula] is linked to synchronous neural dynamics. We analyzed occipital EEG and iEEG data from massive public databases. Our results showed the human alpha rhythm can be characterized either as a synchronous or as a Gaussian signal based on the value of its CVE. Furthermore, Fourier analysis showed the canonical spectral peak at {approx} 10[Hz] is present in both the synchronous and Gaussian cases, thus demonstrating this same peak can be produced by different underlying neural dynamics. This study confirms the original interpretation of Adrian regarding the origin of the alpha rhythm but also opens the door for the study of Gaussianity in brain dynamics. These results suggest a broader interpretation for event-related synchronization/desynchronization (ERS/ERD) may be needed. Envelope analysis constitutes a novel complement to Fourier-based methods for neural signal analysis relating amplitude modulation patterns (CVE) to signal energy.

sentences: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites. The precise control of phagosome maturation is critical for innate and adaptive immunity, determining whether phagocytosed material is destroyed or used to present antigens. We observed previously that non-fusogenic contacts between the endoplasmic reticulum (ER) and phagosomes, called membrane contact sites (MCS), are tethered by the calcium regulator STIM1 and fine-tune phagosomal maturation. The secretory pathway SNARE protein Sec22b has been implicated in controlling phagocytosis, phagosome maturation and antigen presentation, though its effects are controversial, and its mechanism of action poorly understood. Recently, Sec22b was shown to tether MCS at the plasma membrane without mediating membrane fusion. Here, we show that Sec22b localizes to and regulates the frequency of ER-phagosome contacts independently of STIM proteins. Sec22b knockdown and overexpression of a an MCS-disrupting mutant Sec22b-P33 induced only mild or no effect on global and local calcium signalling. However, Sec22b knockdown altered phagosomal phospholipids including PI(3)P, PI(4)P and PS, but not PI(4,5)P2. Increased PI(4)P in shSec22b cells was rescued by re-expression of Sec22b or the artificial MCS tether MAPPER but not the P33 mutant. Moreover, Sec22b co-precipitated and was co-recruited to phagosomes with the PS/PI(4)P lipid exchange protein ORP8. Expression of wild-type, but not mutant ORP8, also rescued phagosomal PI(4)P. Concordantly, Sec22b, MAPPER and ORP8 but not P33 or the ORP8 mutant decreased phagolysosome fusion in shSec22b cells. These results clarify a novel mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.

sentences: Local light signalling at the leaf tip drives remote differential petiole growth through auxin-gibberellin dynamics Although plants are immobile, many of their organs are flexible to move in response to environmental cues. In dense vegetation plants detect neighbours through far-red light perception with their leaf tip. They respond remotely, with asymmetrical growth between the abaxial and adaxial sides of the leafstalk, the petiole. This results in upward movement that brings the leaf blades into better lit zones of the canopy. The plant hormone auxin is required for this response, but it is not understood how non-differential leaf tip-derived auxin can remotely regulate movement. Here we show that remote light signalling promotes auxin accumulation in the abaxial petiole by reinforcing an intrinsic auxin transport directionality. In the petiole, auxin elicits a response of both auxin as well as a second growth promoter; gibberellin. We show that this dual regulation is necessary for hyponastic leaf movement in response to light. Our results reveal how plants can spatially relay information about neighbour proximity from their sensory leaf tips to the petiole base, thus driving adaptive growth.

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The virulome of Streptomyces scabiei in response to cello-oligosaccharides elicitors 2.The development of spots or lesions symptomatic of the common scab disease on root and tuber crops is caused by few pathogenic Streptomyces with Streptomyces scabiei 87-22 as the model species. Thaxtomin phytotoxins are the primary virulence determinants, mainly acting by impairing cellulose synthesis, and their production in S. scabiei is in turn boosted by the cello-oligosaccharides released from host plants. In this work we aimed to determine which molecules and which biosynthetic gene clusters (BGCs) of the specialized metabolism of S. scabiei 87-22 show a production and/or transcriptional response to cello-oligosaccharides. Comparative metabolomic and transcriptomic analyses revealed that molecules of the virulome of S. scabiei induced by cellobiose and cellotriose include i) thaxtomins and concanamycins phytotoxins (and to a lesser extent N-coronafacoyl-L-isoleucine), ii) desferrioxamines, scabichelin and turgichelin siderophores in order to acquire iron essential for housekeeping functions, iii) ectoine for protection against osmotic shock once inside the host, and iv) bottromycins and concanamycins antimicrobials possibly to prevent other microorganisms from colonizing the same niche. Importantly, both cell-oligosaccharides reduced the production of the spore germination inhibitors germicidins and the plant growth regulators rotihibins. The metabolomic study also revealed that cellotriose is in general a more potent elicitor of the virulome compared to cellobiose. This result supports an earlier hypothesis that suggested that the trisaccharide would be the real virulence-triggering factor released from the plant cell wall through the action of thaxtomins. Interestingly, except for thaxtomins, none of these BGCs expression seems to be under direct control of the cellulose utilization repressor CebR suggesting the existence of another master regulator sensing the internalization of cello-oligosaccharides. Finally, we found nine additional cryptic and orphan BGCs that have their expression awakened by cello-oligosaccharides, demonstrating that other and yet to be discovered metabolites are part of the virulome of S. scabiei.

3. Impact statementUnveiling the environmental triggers that signal proper conditions for host colonization and what is the composition of the arsenal of metabolites specialized for this task (the virulome) is key to understand host-pathogen interactions. In this work, focused on the induction of the common scab disease caused by Streptomyces species, we provided further knowledge to both aspects i.e., i) highlighting the capability of cellotriose to trigger the entire virulome and not only the production of thaxtomin phytotoxins, and ii) identifying the set of metabolites that specifically respond to cello-oligosaccharides emanating from the plant under attack. Importantly, we also revealed that the expression of nine cryptic/orphan biosynthetic gene clusters (BGCs) involved in the production of unknown compounds was drastically activated upon cello-oligosaccharides import suggesting that a significant part of the virulome of S. scabiei remains to be discovered. Finally, we unexpectedly found that the expression control of most of the known and cryptic BGCs does not depend on the cello-oligosaccharide utilization repressor CebR which suggests the existence of another and yet unknown master regulator of the virulence in S. scabiei.

4. Significance as a BioResource to the communityNot Applicable

5. OutcomeNot Applicable

6. Data summary[A section describing all supporting external data including the DOI(s) and/or accession numbers(s), and the associated URL.]

The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. RNAseq data were publicly deposited, and our experimental and analytical pipeline were described on the GEO database repository (Accession number: GSE181490)

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Assembly-free rapid differential gene expression analysis in non-model organisms using DNA-protein alignment BackgroundRNA-seq is being increasingly adopted for gene expression studies in a panoply of non-model organisms, with applications spanning the fields of agriculture, aquaculture, ecology, and environment. For organisms that lack a well-annotated reference genome or transcriptome, a conventional RNA-seq data analysis workflow requires constructing a de-novo transcriptome assembly and annotating it against a high-confidence protein database. The assembly serves as a reference for read mapping, and the annotation is necessary for functional analysis of genes found to be differentially expressed. However, assembly is computationally expensive. It is also prone to errors that impact expression analysis, especially since sequencing depth is typically much lower for expression studies than for transcript discovery.

ResultsWe propose a shortcut, in which we obtain counts for differential expression analysis by directly aligning RNA-seq reads to the high-confidence proteome that would have been otherwise used for annotation. By avoiding assembly, we drastically cut down computational costs - the running time on a typical dataset improves from the order of tens of hours to under half an hour, and the memory requirement is reduced from the order of tens of Gbytes to tens of Mbytes. We show through experiments on simulated and real data that our pipeline not only reduces computational costs, but has higher sensitivity and precision than a typical assembly-based pipeline. A Snakemake implementation of our workflow is available at: https://bitbucket.org/project_samar/samar

ConclusionsThe flip side of RNA-seq becoming accessible to even modestly resourced labs has been that the time, labor, and infrastructure cost of bioinformatics analysis has become a bottleneck. Assembly is one such resource-hungry process, and we show here that it can be avoided for quick and easy, yet more sensitive and precise, differential gene expression analysis in non-model organisms.

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Synaptic density and neuronal metabolic function measured by PET in the unilateral 6-OHDA rat model of Parkinson's disease Parkinsons disease (PD) is caused by progressive neurodegeneration and characterised by motor dysfunction. Neurodegeneration of dopaminergic neurons also causes aberrations within the cortico-striato-thalamo-cortical (CSTC) circuit, which has been hypothesised to lead to non-motor symptoms such as depression. Individuals with PD have both lower synaptic density and changes in neuronal metabolic function in the basal ganglia, as measured using [11C]UCB-J and [18F]FDG positron emission tomography (PET), respectively. However, the two radioligands have not been directly compared in the same PD subject or in neurodegeneration animal models. Here, we investigate [11C]UCB-J binding and [18F]FDG uptake in the CSTC circuit following a unilateral dopaminergic lesion in rats and compare it to sham lesioned rats.

Rats received either a unilateral injection of 6-hydroxydopamine (6-OHDA) or saline in the medial forebrain bundle and rostral substantia nigra (n=4/group). After three weeks, all rats underwent two PET scans using [18F]FDG, followed by [11C]UCB-J on a separate day. [18F]FDG uptake and [11C]UCB-J binding were both lower in the ipsilateral striatal regions compared to the contralateral regions. Using [11C]UCB-J, we could detect an 8.7% decrease in the ipsilateral ventral midbrain, compared to a 2.9% decrease in ventral midbrain using [18F]FDG. Differential changes between hemispheres for [11C]UCB-J and [18F]FDG outcomes were also evident in the CSTC circuits cortical regions, especially in the orbitofrontal cortex and medial prefrontal cortex where higher synaptic density yet lower neuronal metabolic function was observed, following lesioning.

In conclusion, [11C]UCB-J and [18F]FDG PET can detect divergent changes following a dopaminergic lesion in rats, especially in cortical regions that are not directly affected by the neurotoxin. These results suggest that combined [11C]UCB-J and [18F]FDG scans could yield a better picture of the heterogeneous cerebral changes in neurodegenerative disorders.

sentences: Disrupted PDZ-domain binding motif of the dopamine transporter uniquely alters nanoscale distribution, dopamine homeostasis and reward motivation The dopamine transporter (DAT) is part of a presynaptic multi-protein network involving interactions with scaffold proteins via its C-terminal PDZ-domain binding sequence. In a mouse model expressing DAT with mutated PDZ binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here we show by application of direct Stochastic Reconstruction Microscopy (dSTORM) not only that the striatal level of transporter is reduced in DAT-AAA mice, but also that the nanoscale distribution of the transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal dopamine (DA) adaptations and changes in DA-related behaviors different from those seen in other genetic DAT mouse models. DA levels in striatum are reduced to [~]45% of wild type (WT), accompanied by elevated DA turnover. Nonetheless, Fast-Scan Cyclic Voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared to WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels while immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed-ratio (FR1) and progressive-ratio (PR) schedule of reinforcement, but a reduction compared to WT when using cocaine as reinforcer. Summarized, our data demonstrate how disruption of PDZ-domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics.

sentences: Development, Structure, and Mechanism of Synthetic Antibodies that Target Claudin and Clostridium perfringens Enterotoxin Complexes Strains of the Gram-positive bacterium Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflict millions of humans and domesticated animals annually by causing prevalent gastrointestinal illnesses. CpEs C-terminal domain (cCpE) binds cell surface receptors then its N-terminal domain restructures to form a membrane-penetrating {beta}-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are the receptors for CpE, and also control the architecture and function of cell/cell contacts called tight junctions that create barriers to intercellular transport of solutes. CpE binding disables claudin and tight junction assembly and induces cytotoxicity via {beta}-pore formation, disrupting gut homeostasis. Here, we aimed to develop probes of claudin/CpE assembly using a phage display library encoding synthetic antigen-binding fragments (sFabs) and discovered two that bound complexes between human claudin-4 and cCpE. We established each sFabs unique modes of molecular recognition, their binding affinities and kinetics, and determined structures for each sFab bound to ~35 kDa claudin-4/cCpE in three-protein comprised complexes using cryogenic electron microscopy (cryoEM). The structures reveal a recognition epitope common to both sFabs but also that each sFab distinctly conforms to bind their antigen, which explain their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in further binding changes. Together, these findings validate the structures and uncover the mechanism of targeting claudin-4/cCpE complexes by these sFabs. Based on these structural insights we generate a model for CpEs cytotoxic claudin-bound {beta}-pore that predicted that these two sFabs would not prevent CpE cytotoxicity, which we verify in vivo with a cell-based assay. This work demonstrates the development and targeting mechanisms of sFabs against claudin/cCpE that enable rapid structural elucidation of these small membrane protein complexes using a cryoEM workflow. It further provides a structure-based framework and therapeutic strategies for utilizing these sFabs as molecular templates to target claudin/CpE assemblies, obstruct CpE cytotoxicity, and treat CpE-linked gastrointestinal diseases that cause substantial economic and quality of life losses throughout the world.

sentences: Identification of anthracnose (Colletotrichum lentis) race 1 resistance loci in lentil by integrating linkage mapping and a genome-wide association study Anthracnose, caused by Colletotrichum lentis, is a devastating disease of lentil in Western Canada. Growing resistant lentil cultivars is the most cost-effective and environmentally friendly approach to prevent seed yield losses that can exceed 70%. To identify loci conferring resistance to anthracnose race 1 in lentil, biparental quantitative trait loci (QTL) mapping of two recombinant inbred line (RIL) populations was integrated with a genome-wide association study (GWAS) using 200 diverse lentil accessions from a lentil diversity panel (LDP). A major-effect QTL (qAnt1.Lc-3) conferring resistance to race 1 was mapped to lentil chromosome 3 and co-located on the lentil physical map for both RIL populations. Clusters of candidate nucleotide binding-leucine-rich repeats (NB-LRR) and other defense-related genes were uncovered within the QTL region. A GWAS detected 14 significant SNP markers associated with race 1 resistance on chromosomes 3, 4, 5, and 6. The most significant GWAS SNPs on chromosome 3 supported qAnt1.Lc-3 and delineated a region of 1.6 Mb containing candidate resistance genes. The identified SNP markers can be directly applied in marker-assisted selection to accelerate the introgression of race 1 resistance in lentil breeding.

sentences: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.

sentences: Reliable cell retention of mammalian suspension cells in microfluidic cultivation chambers We present a new microfluidic trapping concept to retain randomly moving suspension cells inside a cultivation chamber. In comparison to previously published complex multilayer structures, we achieve cell retention by a thin PDMS barrier, which can be easily integrated into various PDMS-based cultivation devices. Cell loss during cultivation is effectively prevented while diffusive media supply is still ensured.

sentences: How does temperature affect the dynamics of SARS-CoV-2 M proteins? Insights from Molecular Dynamics Simulations Enveloped viruses, in general, have several transmembrane proteins and glycoproteins, which assist the virus in entry and attachment onto the host cells. These proteins also play a significant role in determining the shape and size of the newly formed virus particles. The lipid membrane and the embedded proteins affect each other in non-trivial ways during the course of the viral life cycle. Unravelling the nature of the protein-protein and protein-lipid interactions, under various environmental and physiological conditions, could therefore prove to be crucial in development of therapeutics. Here, we study the M protein of SARS-CoV-2 to understand the effect of temperature on the properties of the protein-membrane system. The membrane embedded dimeric M proteins were studied using atomistic and coarse-grained molecular dynamics simulations at temperatures ranging between 10 and 50 {degrees}C. While temperature induced fluctuations should be monotonic, we observe a steady rise in the protein dynamics up to 40 {degrees}C, beyond which it surprisingly reverts back to the low temperature behaviour. Detailed investigation reveals disordering of the membrane lipids in the presence of the protein, which induces additional curvature around the transmembrane region. Coarse-grained simulations indicate temperature dependent aggregation of M protein dimers. Our study clearly indicates that the dynamics of membrane lipids and integral M protein of SARS-CoV-2 enables it to better associate and aggregate only at a certain temperature range (i.e., ~30 to 40 {degrees}C). This can have important implications in the protein aggregation and subsequent viral budding/fission processes.

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New from old: discovery of the novel antibiotic actinomycin L in Streptomyces sp. MBT27 Streptomycetes are major producers of bioactive natural products, including the majority of the antibiotics. While much if the low-hanging fruit has been discovered, it is predited that less than 5% of the chemical space has been mined. Here, we describe the novel actinomycins L1 and L2, which are produced by Streptomyces sp. MBT27. The molecules were discovered via metabolic analysis combined with molecular networking of cultures grown with different combinations of carbon sources. Actinomycins L1 and L2 are diastereoisomers, and the structure of actinomycin L2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via a unique spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X2, prior to the condensation of the actinomycin halves. Feeding anthranilamide to cultures of Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27 but only produces actinomycin X2, resulted in the production of actinomycin L. This shows that actinomycin L results from joining two distinct metabolic pathways, namely those for actinomycin X2 and for anthranilamide. Actinomycins L1 and L2 showed significant antimicrobial activity against Gram- positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families.

IMPORTANCEActinomycin was the first antibiotic discovered in an actinobacterium by Selman Waksman and colleagues, as early as 1940. This period essentially marks the start of the  golden era of antibiotic discovery. Over time, emerging antimicrobial resistance (AMR) and the declining success rate of antibiotic discovery resulted in the current antibiotic crisis. We surprisingly discovered that under some growth conditions, Streptomyces sp. MBT27 can produce actinomycins that are significantly different from those that have been published so far. The impact of this work is not only that we have discovered a novel molecule with very interesting chemical modifications in one of the oldest antibiotics ever described, but also that this requires the combined action of primary and secondary metabolic pathways, namely the biosynthesis of anthranilamide and of actinomycin X2, respectively. The implication of the discovery is that even the most well-studied families of natural products may still have surprises in store for us.

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Cell Adhesion-Dependent Biphasic Axon Outgrowth Elucidated by Femtosecond Laser Impulse Axon outgrowth is promoted by the mechanical coupling between the dynamic actin cytoskeleton and adhesive substrates via clutch and adhesion molecules in the axonal growth cone. In this study, we utilized a femtosecond laser-induced impulse to break the coupling between an axonal growth cone and an adhesive substrate, enabling us to evaluate the strength of the binding between proteins in the growth cone and a laminin substrate, and also determine the contribution of adhesion strength to axon outgrowth. We found that the adhesion strength of axonal L1 cell adhesion molecule (L1CAM)-laminin binding increased with the density of the laminin substrate. In addition, fluorescent speckle microscopy revealed that the retrograde flow of actin filaments in the axonal growth cone was dependent on the laminin density such that the flow speed reduced with increasing L1CAM-laminin binding. However, axon outgrowth did not increase monotonically with increased L1CAM-laminin binding but rather exhibited biphasic behavior, in which the outgrowth was suppressed by excessive L1CAM-laminin binding. Our quantitative evaluations of the adhesion strength suggest that the biphasic outgrowth is regulated by the balance between traction force and adhesion strength as a result of changes in the number of L1CAM-laminin interactions. These results imply that adhesion modulation is key to the regulation of axon guidance.

Significance StatementThere is a lack of a method to evaluate an adhesion strength of axonal growth cone. We evaluated the adhesion strength of axonal growth cones to a substrate by utilizing the force applied from a femtosecond laser impulse. This study shows that the adhesion strength between the growth cone and substrate is strengthened by L1CAM-laminin binding. Axon outgrowth did not increase monotonically with increased L1CAM-laminin binding but rather exhibited biphasic behavior, indicating that existence of suitable adhesion strength for axonal growth. Our findings suggest that the balance between growth cone adhesion strength and the traction force transmitted via cytoskeletal flow is a key factor in axon guidance.

sentences: Integrated single-cell profiling dissects cell-state-specific enhancer landscapes of human tumor-infiltrating T cells. Despite extensive studies on the chromatin landscape of exhausted T cells, the transcriptional wiring underlying the heterogeneous functional and dysfunctional states of human tumor-infiltrating lymphocytes (TILs) is incompletely understood. Here, we identify tissue-specific and general gene-regulatory landscapes in the wide breadth of CD8+ TIL functional states covering four cancer entities using single-cell chromatin profiling. We map enhancer-promoter interactions in human TILs by integrating single-cell chromatin accessibility with single-cell RNA-seq data from tumor entity-matching samples, and prioritize key elements by super-enhancer analysis. Our results reveal a human core chromatin trajectory to TIL dysfunction and identify involved key enhancers, transcriptional regulators, and deregulated target genes in this process. Finally, we validate enhancer regulation at immunotherapeutically relevant loci by targeting non-coding regulatory elements with potent CRISPR activators and repressors. In summary, our study provides a framework for understanding and manipulating cell-state-specific gene-regulatory cues from human tumor infiltrating lymphocytes.

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Roadkill islands: carnivore extinction shifts seasonal use of roadside carrion by generalist avian scavenger O_LIGlobal road networks facilitate habitat modification and are integral to human expansion. Many animals, particularly scavengers, use roads as they provide a reliable source of food, such as carrion left after vehicle collisions. Tasmania is often cited as the  roadkill capital of Australia, with the isolated offshore islands in the Bass Strait experiencing similar, if not higher, levels of roadkill. However, native mammalian predators on the islands are extirpated, meaning the remaining scavengers are likely to experience lower interference competition.
C_LIO_LIIn this study, we use a naturally occurring experiment to examine how the loss of mammalian carnivores within a community impacts roadside foraging behaviour by avian scavengers.
C_LIO_LIWe monitored the locations of roadkill and forest ravens (Corvus tasmanicus), an abundant scavenger species, on eight road transects across the Tasmanian mainland (high scavenging competition) and the Bass Strait islands (low scavenging competition). We represented raven observations as one-dimensional point patterns, using hierarchical Bayesian models to investigate the dependence of raven spatial intensity on habitat, season, distance to roadkill and route location.
C_LIO_LIWe found that roadkill carcasses were a strong predictor of raven presence along road networks. The effect of roadkill was amplified on roads on the Bass Strait islands, where roadside carrion was a predictor of raven presence across the entire year. In contrast, ravens were more often associated with roadkill on Tasmanian mainland roads in the autumn, when other resources were low. This suggests that in the absence of competing mammalian scavengers, ravens choose to feed on roadside carrion throughout the year, even in seasons when other resources are available. This low interference competition could be disproportionately benefiting forest ravens, leading to augmented raven populations and changes to the vertebrate community structure.
C_LIO_LIOur study provides evidence that scavengers modify their behaviour in response to reduced scavenger species diversity, potentially triggering trophic shifts and highlighting the importance of conserving or reintroducing carnivores within ecosystems.
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Individually Unique Dynamics of Cortical Connectivity Reflect the Ongoing Intensity of Chronic Pain BackgroundChronic pain diseases are characterised by an ongoing and fluctuating endogenous pain, yet it remains to be elucidated how this is reflected by the dynamics of ongoing functional cortical connections. The present study addresses this disparity by taking the individual perspective of pain patients into account, which is the varying intensity of endogenous pain.

MethodsTo this end, we investigated the cortical encoding of 20 chronic back pain patients and 20 chronic migraineurs in four repeated fMRI sessions. During the recording, the patients were asked to continuously rate their pain intensity. A brain parcellation approach subdivided the whole brain into 408 regions. A 10 s sliding-window connectivity analysis computed the pair-wise and time-varying connectivity between all brain regions across the entire recording period. Linear mixed effects models were fitted for each pair of brain regions to explore the relationship between cortical connectivity and the observed trajectory of the patients fluctuating endogenous pain.

ResultsTwo pain processing entities were taken into account: pain intensity (high, middle, low pain) and the direction of pain intensity changes (rising vs. falling pain). Overall, we found that periods of high and increasing pain were predominantly related to low cortical connectivity. For chronic back pain this applies to the pain intensity-related connectivity for limbic and cingulate areas, and for the precuneus. The change of pain intensity was subserved by connections in left parietal opercular regions, right insular regions, as well as large parts of the parietal, cingular and motor cortices. The change of pain intensity direction in chronic migraine was reflected by decreasing connectivity between the anterior insular cortex and orbitofrontal areas, as well as between the PCC and frontal and ACC regions.

ConclusionsInterestingly, the group results were not mirrored by the individual patterns of pain-related connectivity, which is suggested to deny the idea of a common neuronal core problem for chronic pain diseases. In a similar vein, our findings are supported by the experience of clinicians, who encounter patients with a unique composition of characteristics: personality traits, various combinations of symptoms, and a wide range of individual responses to treatment. The diversity of the individual cortical signatures of chronic pain encoding results adds to the understanding of chronic pain as a complex and multifaceted disease. The present findings support recent developments for more personalised medicine.

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The Androgen Receptor: A Therapeutic Target in Desmoplastic Small Round Cell Sarcoma Desmoplastic small round cell tumor (DSRCT) is an aggressive, usually incurable sarcoma subtype that predominantly occurs in post-pubertal young males. Recent evidence suggests that the androgen receptor (AR) can promote tumor progression in DSRCTs. However, the mechanism of AR-induced oncogenic stimulation remains undetermined. Herein, we demonstrate that enzalutamide and AR-directed antisense oligonucleotides (AR-ASO) block 5-dihydrotestosterone (DHT)-induced DSRCT cell proliferation and reduce xenograft tumor burden. Gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to elucidate how AR signaling regulates cellular epigenetic programs. Remarkably, ChIP-seq revealed novel DSRCT-specific AR DNA binding sites adjacent to key oncogenic regulators, including WT1 (the C-terminal partner of the pathognomonic fusion protein) and FOXF1. Additionally, AR occupied enhancer sites that regulate the Wnt pathway, neural differentiation, and embryonic organ development, implicating AR in dysfunctional cell lineage commitment. Our findings have immediate clinical implications given the widespread availability of FDA-approved androgen-targeted agents used for prostate cancer.

ONE SENTENCE SUMMARYWe demonstrate that DSRCT, an aggressive pediatric cancer, is an AR-driven malignancy capable of responding to androgen deprivation therapy.

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Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium ObjectiveMethotrexate (MTX) is an anchor drug for rheumatoid arthritis (RA) treatment; however, the exact mechanisms by which MTX improves RA activity are still debatable. This study aimed to understand the roles of molecules whose expression is affected by MTX in RA patients and find novel therapeutic targets.

MethodsCD4+ T cells from 28 treatment naive RA patients before and 3 months after the initiation of MTX treatment were subjected to DNA microarray analyses. The expression of Semaphorin 3G (Sema3G), as one of the differentially-expressed genes, and its receptor, Neuropilin-2 (Nrp2), was evaluated in RA synovium and collagen-induced arthritis (CIA) synovium. CIA and collagen antibody-induced arthritis (CAIA) were induced in Sema3G-deficient (Sema3G-/-) mice and control mice, and the clinical score, histological score, and serum cytokines were assessed. The migration and proliferation of Sema3G-stimulated bone marrow-derived macrophages (BMMs) were analyzed in vitro. The effect of local Sema3G administration during CAIA on the clinical score and the quantity of infiltrating macrophages was evaluated.

ResultsThe expression of Sema3G in CD4+ T cells was downregulated by MTX treatment in RA patients. Sema3G was expressed in RA but not osteoarthritis synovium, and its receptor Nrp2 was mainly expressed on activated macrophages. Sema3G deficiency ameliorated CIA and CAIA. Sema3G stimulation enhanced the migration and proliferation of BMMs. The local administration of Sema3G deteriorated CAIA and increased infiltrating macrophages.

ConclusionsUpregulation of Sema3G in RA synovium is a novel mechanism to deteriorate joint inflammation through the accumulation of macrophages.

Key messagesSemaphorin 3G is expressed in the inflamed synovium in human and mice.

The receptor of Semaphorin 3G is mainly expressed on M1 macrophages.

Semaphorin 3G deteriorates inflammatory arthritis through macrophage proliferation and migration.

sentences: Rv0233 is not essential for the survival of Mycobacterium tuberculosis in stress conditions Mycobacterium tuberculosis is an important global pathogen. We were interested in understanding the role of Rv0233, a proposed subunit of the class IB ribonucleotide reductase. We constructed an in-frame, unmarked deletion strain of M. tuberculosis and characterized its growth and survival under replicating and non-replicating conditions. We confirmed previous studies that Rv0233 is not essential for aerobic growth or survival in the presence of nitrite. We demonstrate that the deletion of Rv0233 does not affect susceptibility to frontline tuberculosis drugs or hydrogen peroxide. The deletion strain survived equally well under nutrient starvation or in hypoxia and was not attenuated for growth in macrophages.

sentences: Transcription activation is enhanced by multivalent interactions independent of liquid-liquid phase separation Transcription factors (TFs) consist of a DNA binding and an activation domain (AD) that are considered to be independent and exchangeable modules. However, recent studies conclude that also the physico-chemical properties of the AD can control TF assembly at chromatin by driving a phase separation into transcriptional condensates. Here, we dissected transcription activation by comparing different synthetic TFs at a reporter gene array with real-time single-cell fluorescence microscopy readouts. In these experiments, binding site occupancy, residence time and co-activator recruitment in relation to multivalent TF interactions were compared. While phase separation propensity and activation strength of the AD were correlated, the actual formation of liquid-like TF droplets had a neutral or inhibitory effect on transcription activation. Rather, we conclude that multivalent AD mediated interactions increase the transcription activation capacity of a TF by stabilizing chromatin binding and mediating the recruitment of co-activators independent of phase separation.

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A unicellular walker controlled by a microtubule-based finite state machine Cells are complex biochemical systems whose behavior emerges from interactions among myriad molecular components. Computation is often invoked as a general framework for navigating this cellular complexity. However, the manner in which cells might embody computational processes such that theories of computation, including finite state machine models, could be productively applied, remains to be seen. Here we demonstrate finite state machine-like processing embodied in cells using the walking behavior of Euplotes eurystomus, a ciliate that walks across surfaces using fourteen motile appendages (cirri). We found that cellular walking entails highly regulated transitions between a discrete set of gait states. The set of observed transitions decomposes into a small group of high-probability, temporally irreversible transitions forming a cycle and a large group of low-probability time-symmetric transitions, thus revealing stereotypy in sequential patterns of state transitions. Taken together, these findings implicate a finite state machine-like process. Cirri are connected by microtubule bundles, and we found that the dynamics of cirri involved in different state transitions are associated with the structure of the microtubule bundle system. Perturbative experiments revealed that the fibers mediate gait coordination, suggesting a mechanical basis of gait control.

One sentence summaryThe ciliate Euplotes walks across surfaces with an unusual gait, which involves modular subcellular structure and combines elements of stereotypy and variability according to a computational process actively coordinated by a microtubule fiber system.

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Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen Most human genetic variation is classified as VUS - variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a new method termed "Raft-Seq". We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator MFN2, even when the cellular phenotype is subtle. We also show that our platform achieves its efficacy using multiple cellular features, which can be configured on-the-fly. Raft-Seq enables a new way to perform pooled screening on sets of mutations in biologically relevant cells, with the ability to physically capture any cell with a perturbed phenotype and expand it clonally, directly from the primary screen.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/434746v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1d1a431org.highwire.dtl.DTLVardef@72aba5org.highwire.dtl.DTLVardef@62feb1org.highwire.dtl.DTLVardef@13f2b2d_HPS_FORMAT_FIGEXP  M_FIG Graphical Abstract

Here, we address the need to evaluate the impact of a multitude of genetic variants. This manuscript depicts the methods of using machine learning on a biologically relevant phenotype to predict specific point mutations, followed by physically capturing those mutated cells.

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sentences: A closed-loop stimulation approach with real-time estimation of the instantaneous phase of neural oscillations by a Kalman filter We propose a novel method to estimate the instantaneous oscillatory phase to implement a real-time system for closed-loop sensory stimulation in electroencephalography (EEG) experiments. The method uses Kalman filter-based prediction to estimate current and future EEG signals. We tested the performance of our method in a real-time situation. We demonstrate that the performance of our method shows higher accuracy in predicting the EEG phase than the conventional autoregressive model-based method. A Kalman filter allows us to easily estimate the instantaneous phase of EEG oscillations based on the automatically estimated autoregressive model implemented in a real-time signal processing machine. The proposed method has a potential for versatile applications targeting the modulation of EEG phase dynamics and the plasticity of brain networks in relation to perceptual or cognitive functions.

sentences: Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2 Activation of G protein-coupled receptors (GPCRs) is an allosteric process. It involves conformational coupling between the orthosteric ligand binding site and the G protein binding site. Factors that bind at sites other than the orthosteric ligand binding site and alter this allosteric activation process are allosteric modulators and are important class of therapeutics. For many receptors, how modulation of signaling is represented at the structural level is unclear. Here, we developed FRET sensors to quantify receptor modulation at each of the three structural domains of metabotropic glutamate receptor 2 (mGluR2). We identified the conformational fingerprint for several allosteric modulators in live cells. This approach enabled us to derive a receptor-centric representation of allosteric modulation and to correlate structural modulation to the standard signaling modulation metrics. Single-molecule FRET analysis revealed that a NAM increases the occupancy of one of the intermediate states while a PAM increases the occupancy of the active state. Moreover, we found that the effect of allosteric modulators on the receptor dynamics is complex and depend on the orthosteric ligand. Collectively, our findings provide a structural mechanism of allosteric modulation in mGluR2 and suggest possible strategies for design of future modulators.

sentences: Mmp-13 deletion in cells of the mesenchymal lineage increases bone mass, decreases endocortical osteoclast number, and attenuates the cortical bone loss caused by estrogen deficiency in mice The protective effect of estrogens against cortical bone loss is mediated via direct actions on mesenchymal lineage cells, but functional evidence for the precise molecular mechanism(s) and the mediators of these effects has only recently began to emerge. We report that the matrix metalloproteinase 13 (MMP-13) is the highest up-regulated gene in calvaria or bone marrow cells from mice lacking the estrogen receptor (ER) alpha in osteoprogenitors. We, therefore, generated mice with conditional Mmp-13 deletion in Prrx1 expressing cells (Mmp-13{Delta}Prrx1) and compared the effect of estrogen deficiency on their bone phenotype to that of control littermates (Mmp-13f/f). Femur and tibia length was decreased in sham-operated Mmp-13{Delta}Prrx1 mice as compared to Mmp-13f/f. Cortical thickness and trabecular bone volume in the femur and tibia were increased and osteoclast number at the endocortical surfaces was decreased in the sham-operated female Mmp-13{Delta}Prrx1 mice; whereas bone formation rate was unaffected. Ovariectomy (OVX) caused a decrease of cortical thickness in the femur and tibia of Mmp-13f/f control mice. This effect was attenuated in the Mmp-13{Delta}Prrx1 mice; but the decrease of trabecular bone caused by OVX was not affected. These results reveal that mesenchymal cell-derived MMP-13 regulates osteoclast number, bone resorption, and bone mass. We have recently reported that the loss of cortical, but not trabecular bone, caused by OVX is also attenuated in Cxcl12{Delta}Prrx1 mice. Together with the present report, this functional genetic evidence provides proof of principle that increased production of mesenchymal cell-derived factors, such as CXCL12 and MMP-13, are important mediators of the adverse effect of estrogen deficiency on cortical, but not trabecular, bone. Therefore, the mechanisms responsible for the protective effect of estrogens on these two major bone compartments are different.

sentences: Suicidal chemotaxis in bacteria Bacteria commonly live in communities on surfaces where steep gradients of antibiotics and other chemical compounds routinely occur. While many species of bacteria can move on surfaces, we know surprisingly little about how such antibiotic gradients affect cell motility. Here we study the behaviour of the opportunistic pathogen Pseudomonas aeruginosa in stable spatial gradients of a range of antibiotics by tracking thousands of cells in microfluidic devices as they form biofilms. Unexpectedly, these experiments reveal that individual bacteria use pili-based ( twitching) motility to actively navigate towards regions with higher antibiotic concentrations. Our analyses suggest that this biased migration is driven, at least in part, by a direct response to the antibiotics. Migrating cells can reach antibiotic concentrations hundreds of times higher than their minimum inhibitory concentration in a few hours and remain highly motile. However, isolating these cells - using fluid-walled microfluidic devices that can be reconfigured in situ - suggests that these bacteria are terminal and not able to reproduce. In spite of moving towards their death, we show that migrating cells are capable of entering a suicidal program to release bacteriocins that are used to kill other bacteria. Our work suggests that bacteria respond to antibiotics as if they come from a competing colony growing in the neighbourhood, inducing them to invade and attack. As a result, clinical antibiotics have the potential to serve as a bait that lures bacteria to their death.

sentences: Rapid Estimation of SNP Heritability using Predictive Process approximation in Large scale Cohort Studies With the advent of high throughput genetic data, there have been attempts to estimate heritability from genome-wide SNP data on a cohort of distantly related individuals using linear mixed model (LMM). Fitting such an LMM in a large scale cohort study, however, is tremendously challenging due to its high dimensional linear algebraic operations. In this paper, we propose a new method named PredLMM approximating the aforementioned LMM motivated by the concepts of genetic coalescence and gaussian predictive process. PredLMM has substantially better computational complexity than most of the existing LMM based methods and thus, provides a fast alternative for estimating heritability in large scale cohort studies. Theoretically, we show that under a model of genetic coalescence, the limiting form of our approximation is the celebrated predictive process approximation of large gaussian process likelihoods that has well-established accuracy standards. We illustrate our approach with extensive simulation studies and use it to estimate the heritability of multiple quantitative traits from the UK Biobank cohort.

sentences: Predicting the Toxicity of Druggable Proteins to Human Tissues Assessing in vivo tissue toxicity of therapeutic targets remains a major challenge in drug development and drug safety research. We developed TissueTox, an algorithm that learns from multi-omic features of a target protein and predicts toxicity in human body systems and tissues. Predicted TissueTox scores accurately differentiate drugs that failed clinical trials from those that succeeded, and, importantly, can be used to identify the tissues where toxic events occurred.

sentences: AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in Castrate-Resistant Prostate Cancer Expression of the AR splice variant, AR-V7, in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin- /{beta} pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions.

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Sialic acid ligands of CD28 block co-stimulation of T cells Effector T cells comprise the cellular arm of the adaptive immune system and are essential for mounting immune responses against pathogens and cancer. To reach effector status, co-stimulation through CD28 is required. Here, we report that sialic acid-containing glycans on the surface of both T cells and APCs are alternative ligands of CD28 that compete with binding to its well-documented activatory ligand CD80 on the APC, resulting in attenuated co-stimulation. Removal of sialic acids enhances T cell activation and enhances the activity of effector T cells made hypofunctional via chronic viral infection through a mechanism that is synergistic with antibody blockade of the inhibitory PD-1 axis. These results reveal a previously unrecognized role for sialic acids in attenuation of CD28 mediated co-stimulation of T cells.

One Sentence SummarySialic acids attenuate the second signal required for T cell activation.

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Refining conformational ensembles of flexible proteins against small-angle X-ray scattering data Intrinsically disordered proteins and flexible regions in multi-domain proteins display substantial conformational heterogeneity. Characterizing the conformational ensembles of these proteins in solution typically requires combining one or more biophysical techniques with computational modelling or simulations. Experimental data can either be used to assess the accuracy of a computational model or to refine the computational model to get a better agreement with the experimental data. In both cases, one generally needs a so-called forward model, i.e. an algorithm to calculate experimental observables from individual conformations or ensembles. In many cases, this involve one or more parameters that need to be set, and it is not always trivial to determine the optimal values or to understand the impact on the choice of parameters. For example, in the case of small-angle X-ray scattering (SAXS) experiments, many forward models include parameters that describe the contribution of the hydration layer and displaced solvent to the background-subtracted experimental data. Often, one also needs to fit a scale factor and a constant background for the SAXS data, but across the entire ensemble. Here, we present a protocol to dissect the effect of free-parameters on the calculated SAXS intensities, and to identify a reliable set of values. We have implemented this procedure in our Bayesian/Maximum Entropy framework for ensemble refinement, and demonstrate the results on four intrinsically disordered proteins and a three-domain protein connected by flexible linkers. Our results show that the resulting ensembles can depend on the parameters used for solvent effects, and suggests that these should be chosen carefully. We also find a set of parameters that work robustly across all proteins.

SIGNIFICANCEThe flexibility of a protein is often key to its biological function, yet understanding and characterizing its conformational heterogeneity is difficult. We here describe a robust protocol for combining small-angle X-ray scattering experiments with computational modelling to obtain a conformational ensemble. In particular, we focus on the contribution of protein hydration to the experiments and how this is included in modelling the data. Our resulting algorithm and software should make modelling intrinsically disordered proteins and multi-domain proteins more robust, thus aiding in understanding the relationship between protein dynamics and biological function.

sentences: Custom long non-coding RNA capture enhances detection sensitivity in different human sample types Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.

sentences: An early-morning flowering trait in rice helps retain grain yield under heat stress field conditions at flowering stage Early-morning flowering (EMF) trait is supposed to be effective in retaining grain yield due to mitigation of heat-induced spikelet sterility at flowering in rice. This study evaluated (i) phenotypic differences between a near-isogenic line carrying a QTL for EMF trait, designated as IR64+qEMF3, and a recurrent parent, IR64, under wide variation in climates and (ii) whether an EMF trait can retain grain yield under heat stress at flowering. IR64+qEMF3 had significant earlier flower opening time (FOT) in diverse environmental conditions including temperate, subtropical, and tropical regions. Under normal temperatures at flowering, IR64+qEMF3 had similar grain yield to IR64 with some significant changes in agronomic traits and yield components. Field trials in heat-vulnerable regions of central Myanmar for seven crop seasons showed that higher percentage of filled grains contributed to the significantly higher grain yield in IR64+qEMF3 among yield components when plants were exposed to daily maximum air temperatures around 36.5 {degrees}C or higher. Lower spikelet sterility in IR64+qEMF3 was attributed to the earlier FOT during cooler early morning hours. This is the first field study that clearly demonstrates the advantage of the EMF trait for retaining grain yield by stabilizing percentage of fertile grains under heat stress at flowering.

sentences: A second view on the evolution of flight in stick and leaf insects (Phasmatodea) The re-evolution of complex characters is generally considered impossible, yet, studies of recent years have provided several examples of phenotypic reversals shown to violate Dollos law. Along these lines, the regain of wings in stick and leaf insects (Phasmatodea) was hypothesised to have occurred several times independently after an ancestral loss, a scenario controversially discussed among evolutionary biologists. Here, we revisit the recovery of wings by reconstructing a phylogeny based on a comprehensive taxon sample of over 500 representative phasmatodean species to infer the evolutionary history of wings. We additionally explored the presence of ocelli, the photoreceptive organs used for flight stabilisation in winged insects, which might provide further information for interpreting flight evolution. Our findings support an ancestral loss of wings and that the ancestors of most major lineages were wingless. While the evolution of ocelli was estimated to be dependent on the presence of (fully-developed) wings, ocelli are nevertheless absent in the majority of all examined winged species and only appear in the members of few subordinate clades, albeit winged and volant taxa are found in every lineage. The disjunct distribution of ocelli substantiates the hypothesis on trait reacquisition and that wings were regained in Phasmatodea.

sentences: Expression Based Species Deconvolution and Realignment Removes Misalignent Error in Multi-species Single Cell Data Although single-cell RNA sequencing of xenograft samples has been widely used, no comprehensive bioinformatics pipeline is available for human and mouse mixed single-cell analyses. Considering the numerous homologous genes across the human and mouse genomes, misalignment errors should be evaluated, and a new algorithm is required. We assessed the extents and effects of misalignment errors when using human and mouse combined reference data and developed a new bioinformatics pipeline with expression-based species deconvolution to minimize errors. We also evaluated false-positive signals for a species presumed to originate from ambient RNA of the other species and applied a computational method to remove them. Misaligned reads account for an average of 0.5% of total reads, but such reads were concentrated to few genes that were greatly affected. Human and mouse mixed single-cell data, analyzed using our pipeline, clustered well with unmixed data and showed higher k-nearest-neighbor batch effect test and Local Inverse Simpsons Index scores than those derived from Cell Ranger. We also applied our pipeline to multispecies multisample single-cell library containing breast cancer xenograft tissue and successfully identified all samples using genomic array and expression. Moreover, diverse cell types in the tumor microenvironment were well captured. We present our bioinformatics pipeline for mixed human and mouse single-cell data, which can also be applied to pooled libraries to obtain cost-effective single-cell data. We also address major consideration points when analyzing multispecies single-cell data for other applications.

sentences: Structure-based discovery of small molecule inhibitors of the autocatalytic proliferation of alpha-synuclein aggregates The presence of amyloid fibrils of -synuclein is closely associated with Parkinsons disease and related synucleinopathies. It is still very challenging, however, to systematically discover small molecules that prevent the formation of these aberrant aggregates. Here, we describe a structure-based approach to identify small molecules that specifically inhibit the surface-catalyzed secondary nucleation step in the aggregation of -synuclein by binding to the surface of the amyloid fibrils. The resulting small molecules are screened using a combination of kinetic and thermodynamic assays for their ability to bind -synuclein fibrils and prevent the further generation of toxic oligomers. This study demonstrates that the combination of structure-based and kinetic-based drug discovery methods can lead to the identification of small molecules that selectively inhibit the autocatalytic proliferation of -synuclein aggregates.

sentences: An optimized live bacterial delivery platform for the production and delivery of therapeutic nucleic acids and proteins There is an unmet need for delivery platforms that realize the full potential of next-generation therapeutic and vaccine technologies, especially those that require intracellular delivery of nucleic acids. The in vivo usefulness of the current state-of-the-art delivery systems is limited by numerous intrinsic weaknesses, including lack of targeting specificity, inefficient entry and endosomal escape into target cells, undesirable immune activation, off-target effects, a small therapeutic window, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we present our characterization of a delivery platform based on the use of engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli strain SVC1) for intracellular cargo delivery. The SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to escape the endosome upon intracellularization, and to have minimal immunogenicity. Here we report findings on key features of this system. First, we demonstrated that bacterial delivery of a short hairpin RNA (shRNA) can target and silence a gene in an in vitro mammalian respiratory cell model. Next, we used an in vivo mouse model to demonstrate that SVC1 bacteria are invasive to epithelial cells of various tissues and organs (eye, nose, mouth, stomach, vagina, skeletal muscle, and lungs) via local administration. We also showed that repeat dosing of SVC1 bacteria to the lungs is minimally immunogenic and that it does not have adverse effects on tissue homeostasis. Finally, to validate the potential of SVC1 bacteria in therapeutic applications, we demonstrated that bacterial delivery of influenza-targeting shRNAs to the respiratory tissues can mitigate viral replication in a mouse model of influenza infection. Our ongoing work is focused on further refining this platform for efficient delivery of nucleic acids, gene editing machinery, and therapeutic proteins, and we expect that this platform technology will enable a wide range of advanced therapeutic approaches.

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Modular biosynthesis of plant hemicellulose and its impact on yeast cells BackgroundThe carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past five years, exciting possibilities have emerged in the engineering of polysaccharide-based biomaterials. Despite impressive advances on bacterial cellulose-based hydrogels, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes.

ResultsHere, we assembled cellulose synthase-like A (CSLA) enzymes using an optimized Pichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan and glucomannan synthases, we engineered chimeric CSLA proteins that made {beta}-1,4-linked mannan in quantities surpassing those of the native enzymes while minimizing the burden on yeast growth. Prolonged expression of a glucomannan synthase from Amorphophallus konjac was toxic to yeast cells: reducing biomass accumulation and ultimately leading to compromised cell viability. However, an engineered glucomannan synthase as well as CSLA pure mannan synthases and a CSLC glucan synthase did not inhibit growth. Interestingly, Pichia cell size could be increased or decreased depending on the composition of the CSLA protein sequence. HM yield and glucose incorporation could be further increased by co-expressing chimeric CSLA proteins with a MANNAN-SYNTHESIS-RELATED (MSR) co-factor from Arabidopsis thaliana.

ConclusionThe results provide novel routes for the engineering of polysaccharide-based biomaterials that are needed for a sustainable bioeconomy. The characterization of chimeric cellulose synthase-like enzymes in yeast offers an exciting avenue to produce plant polysaccharides in a tunable manner. Furthermore, cells modified with non-toxic plant polysaccharides such as {beta}-mannan offer a modular chassis to produce and encapsulate sensitive cargo such as therapeutic proteins.

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Integrated Omics Analysis Reveals Sirtuin Signaling is Central to Hepatic Response to a High Fructose Diet BackgroundDietary high fructose (HFr) is a known metabolic disruptor contributing to development of obesity and diabetes in Western societies. Initial molecular changes from exposure to HFr on liver metabolism may be essential to understand the perturbations leading to insulin resistance and abnormalities in lipid and carbohydrate metabolism. We studied vervet monkeys (Clorocebus aethiops sabaeus) fed a HFr (n=5) or chow diet (n=5) for 6 weeks, and obtained clinical measures of liver function, blood insulin, cholesterol and triglycerides. In addition, we performed untargeted global transcriptomics, proteomics, and metabolomics analyses on liver biopsies to determine the molecular impact of a HFr diet on coordinated pathways and networks that differed by diet.

ResultsWe show that integration of omics data sets improved statistical significance for some pathways and networks, and decreased significance for others, suggesting that multiple omics datasets enhance confidence in relevant pathway and network identification. Specifically, we found that sirtuin signaling and a peroxisome proliferator activated receptor alpha (PPARA) regulatory network were significantly altered in hepatic response to HFr. Integration of metabolomics and miRNAs data further strengthened our findings.

ConclusionsOur integrated analysis of three types of omics data with pathway and regulatory network analysis demonstrates the usefulness of this approach for discovery of molecular networks central to a biological response. In addition, metabolites aspartic acid and docosahexaenoic acid (DHA), protein ATG3, and genes ATG7, HMGCS2 link sirtuin signaling and the PPARA network suggesting molecular mechanisms for altered hepatic gluconeogenesis from consumption of a HFr diet.

sentences: Revisiting regulatory decoherence and phenotypic integration: accounting for temporal bias in co-expression analyses Environment can alter the degree of phenotypic variation and covariation, potentially influencing evolutionary trajectories. However, environment-driven changes in phenotypic variation remain understudied. In an effort to exploit the abundance of RNASequencing data now available, an increasing number of ecological studies rely on population-level correlation to characterize the plastic response of the entire transcriptome and to identify environmentally responsive molecular pathways. These studies are fundamentally interested in identifying groups of genes that respond in concert to environmental shifts. We show that population-level differential co-expression exhibits biases when capturing changes of regulatory activity and strength in rice plants responding to elevated temperature. One possible cause of this bias is regulatory saturation, the observation that detectable co-variance between a regulator and its target may be low as their transcript abundances are induced. This phenomenon appears to be particularly acute for rapid-onset environmental stressors. However, our results suggest that temporal correlations may be a reliable means to detect transient regulatory activity following rapid onset environmental perturbations such as temperature stress. Such temporal bias is likely to confound the studies of phenotypic integration, where high-order organismal traits are hypothesized to be more integrated with strong correlation under stressful conditions, while recent transcriptome studies exhibited weaker coexpression between genes under stressful conditions. Collectively, our results point to the need to account for the nuances of molecular interactions and the possibly confounding effects that these can introduce into conventional approaches to study transcriptome datasets.

sentences: Cell type specific labeling and partial connectomes of dopaminergic circuits reveal non-synaptic communication and large-scale axonal remodeling after exposure to cocaine Dopaminergic (DA) neurons exert profound influences on behavior including addiction. However, how DA axons communicate with target neurons and how those communications change, for example, with drug exposure, remains poorly understood. We combine recent advances in cell type specific labeling for electron microscopy with large volume three-dimensional serial electron microscopy -  connectomics- to detail DA connections in the Nucleus Accumbens (NAc) across multiple animals and after exposure to cocaine. We find that DA axonal varicosities are of four general types: 38% are empty, 25% contain few small ([~]50 nm diameter) vesicles, 19% contain few large ([~]133 nm diameter) vesicles, and 18% have mixed small and large vesicles, suggesting that DA axons may use multiple types of neurotransmitters. Individual DA axons were significantly more likely to contain multiple varicosities of the same type relative to chance, suggesting a new method of DA axon classification. Across all categories, we find only rare examples (<2%, 6/410) of varicosities making specific synapses with any neighboring neuron with the few examples being made exclusively on the shafts and soma of resident NAc neurons. Instead, we find much more frequently (15%) that DA varicosities form spinule-like structures: physical membrane interdigitations with nearby dendrites or excitatory and inhibitory axons. Days after a brief exposure to cocaine, DA axons were extensively branched relative to controls but with similar densities of varicosities and spinules. Additionally, cocaine exposure results in the formation of blind-ended "bulbs" in DA axons, filled with mitochondria, and reminiscent of axonal retraction in the developing and damaged brain. Every bulb was surrounded by elaborated glia further suggestive of active remodeling. Finally, mitochondrial lengths increased by [~]2.2 times relative to control throughout DA axons and NAc spiny dendrites after cocaine exposure but not in DA soma or DA dendrites. We conclude that DA axonal transmission is unlikely to be mediated via classical synapses in the NAc and that the major locus of anatomical plasticity of DA circuits after exposure to cocaine are large scale axonal rearrangements with correlated changes in mitochondria.

sentences: Biochemistry and nanomechanical properties of human colon cells upon simvastatin, lovastatin and mevastatin supplementations - Raman imaging and AFM studies One of the most important areas of medical science is oncology, which is responsible for both the diagnostics and treatment of cancer diseases. Over the years, there has been an intensive development of cancer diagnostics and treatment. This paper shows the comparison of normal (CCD-18Co) and cancerous (CaCo-2) cell lines of the human gastrointestinal tract on the basis of nanomechanical and biochemical properties in order to obtain information on the cancer biomarkers useful in oncological diagnostics. The research techniques used were: Atomic Force Microscopy and Raman spectroscopy and imaging. In addition, the studies included also the effect of the statins compounds: mevastatin, lovastatin and simvastatin and their influence on nanomechanical and biochemical changes of properties of cells tracking using AFM and Raman imaging techniques. The cytotoxicity of mevastatin and lovastatin was determined by using XTT tests.

sentences: Combining evolutionary and assay-labelled data for protein fitness prediction Predictive modelling of protein properties has become increasingly important to the field of machine-learning guided protein engineering. In one of the two existing approaches, evolutionarily-related sequences to a query protein drive the modelling process, without any property measurements from the laboratory. In the other, a set of protein variants of interest are assayed, and then a supervised regression model is estimated with the assay-labelled data. Although a handful of recent methods have shown promise in combining the evolutionary and supervised approaches, this hybrid problem has not been examined in depth, leaving it unclear how practitioners should proceed, and how method developers should build on existing work. Herein, we present a systematic assessment of methods for protein fitness prediction when evolutionary and assay-labelled data are available. We find that a simple baseline approach we introduce is competitive with and often outperforms more sophisticated methods. Moreover, our simple baseline is plug-and-play with a wide variety of established methods, and does not add any substantial computational burden. Our analysis highlights the importance of systematic evaluations and sufficient baselines.

sentences: MG132 induces progerin clearance and improves disease phenotypes in fibroblasts of patients affected with Hutchinson-Gilford Progeria-like syndromes Progeroid Syndromes (PS), including Hutchinson-Gilford Progeria Syndrome (HGPS, OMIM #176670), are premature and accelerated aging that clinically resemble some aspects of advancing physiological aging. Most classical HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type Lamins. This mutation activates a cryptic splice site leading to the deletion of 50 amino acids at its carboxy-terminal domain, resulting in a truncated and permanently farnesylated Prelamin A called Prelamin A {Delta}50 or Progerin that accumulates in HGPS cell nuclei and is a hallmark of the disease. Some patients with PS carry other LMNA mutations affecting exon 11 splicing, leading to defects in nuclear A-type Lamins and are named "HGPS-like" patients. They also produce Progerin and/or other truncated Prelamin A isoforms ({Delta}35 and {Delta}90). We recently found that MG132, a proteasome inhibitor, induced progerin clearance in classical HGPS through autophagy activation and splicing regulation. Here, we show that MG132 induces aberrant prelamin A clearance and improves cellular phenotypes in HGPS-like patient cells. These results provide preclinical proof of principle for the use of a promising class of molecules toward a potential therapy for children with HGPS-like, who may therefore be eligible for inclusion in a therapeutic trial based on this approach, together with classical HGPS patients.

sentences: AtPQT11, a P450 enzyme, detoxifies paraquat via N-demethylation Paraquat is one of the most widely used nonselective herbicides in agriculture. Due to its wide use, paraquat resistant weeds have emerged and is becoming a potential threat to agriculture. The molecular mechanisms of paraquat resistance in weeds remain largely unknown. Physiological studies indicated that the impaired translocation of paraquat and enhanced antioxidation could improve paraquat resistance in plants. However, the detoxification of paraquat via active metabolism by plants has not been reported to date. Here we report that an activated expression of At1g01600 encoding the P450 protein CYP86A4 confers paraquat resistance as revealed by the gain-of-function mutant paraquat tolerance 11D (pqt11D), in which a T-DNA with four 35S enhancers was inserted at 1646 bp upstream the ATG of At1g01600. The paraquat resistance can be recapitulated in Arabidopsis wild type by overexpressing AtPQT11 (At1g01600), while its knockout mutant is hypersensitive to paraquat. Moreover, AtPQT11 also confers paraquat resistance in E. coli when overexpressed. We further demonstrate that AtPQT11 has P450 enzyme activity that converts paraquat to N-demethyl paraquat nontoxic to Arabidopsis, therefore detoxifying paraquat in plants. Taken together, our results unequivocally demonstrate that AtPQT11/ CYP86A4 detoxifies paraquat via active metabolism, thus revealing a novel molecular mechanism of paraquat resistance in plants and providing a means potentially enabling crops to resist paraquat.

sentences: The Euler Characteristic and Topological Phase Transitions in Complex Systems In this work, we use methods and concepts of applied algebraic topology to comprehensively explore the recent idea of topological phase transitions (TPT) in complex systems. TPTs are characterized by the emergence of nontrivial homology groups as a function of a threshold parameter. Under certain conditions, one can identify TPTs via the zeros of the Euler characteristic or by singularities of the Euler entropy. Recent works provide strong evidence that TPTs can be interpreted as a complex networks intrinsic fingerprint. This work illustrates this possibility by investigating some classic network and empirical protein interaction networks under a topological perspective. We first investigate TPT in protein-protein interaction networks (PPIN) using methods of topological data analysis for two variants of the Duplication-Divergence model, namely, the totally asymmetric model and the heterodimerization model. We compare our theoretical and computational results to experimental data freely available for gene co-expression networks (GCN) of Saccharomyces cerevisiae, also known as bakers yeast, as well as of the nematode Caenorhabditis elegans. Supporting our theoretical expectations, we can detect topological phase transitions in both networks obtained according to different similarity measures. Later, we perform numerical simulations of TPTs in four classical network models: the Erd[o]s-Renyi model, the Watts-Strogatz model, the Random Geometric model, and the Barabasi-Albert. Finally, we discuss some perspectives and insights on the topic. Given the universality and wide use of those models across disciplines, our work indicates that TPT permeates a wide range of theoretical and empirical networks.

sentences: Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and antiviral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost minimal weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo, K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection of the lung and cardiovascular system, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice.

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Prevalence Of Multi Drug Resistant Bacteria On Environmental And Medical Device Surfaces Of Korea Nepal Friendship Hospital The microbial monitoring of environmental and medical-devices surface is used to evaluate efficacy of routine cleaning and disinfection practises and to detect the presence of specific Nosocomial Pathogens. The prevalence of Multidrug Resistance organisms in hospital premises projects serious problems in transmitting to susceptible hosts which is difficult to treat. A cross sectional descriptive research was conducted from December 2016 to June 2017 at the pathology laboratory of Korea Nepal Friendship Hospital (KNFH). A total 140 samples were considered, encompassing the medical devices of the hospital (100), housekeeping surfaces (15) and air (25). Susceptibility test for bacterial isolates was done by disk diffusion assay. Of the total 140 samples taken and analysed, 100% showed growth positivity. In most of the swabs taken, Coagulase Negative Staphylococci was dominant, followed by Staphylococcus aureus, Streptococcus spp. Micrococcus spp., E coli, Pseudomonas spp., Bacillus spp., Acinetobacter spp., Klebsiella spp., Fungi, and least Proteus spp. The dry surfaces were dominantly contaminated by gram positive bacteria whereas moistened surfaces like wash basin were contaminated by gram negative as well as gram positive bacteria. Total 277 strains were exposed to various class of antibiotics, among the gram positive environmental isolates, Coagulase Negative Staphylococci 16 (34.78%) had highest MDR prevalence followed by Staphylococcus aureus 8 (29.62%), Streptococcus spp. 4 (12.90%), Micrococcus spp. 4 (9.30%) and no MDR was shown by any Bacillus spp isolates. Whereas, in case of gram negative, Klebsiella spp. 6 (35.29%) had highest MDR prevalence

followed by Acinetobacter spp. 6 (31.57%), E. coli 8 (27.58%), Pseudomonas spp. 4 (18.18%), and lastly Proteus spp. with no MDR at all. The thick dirt covering the cotton swabs and heavy microbial load on them has displayed not only disinfecting practice but also cleaning practice is missing. Heavy contamination shows possible NIs breakout, its important to have routine microbial assessment with standard protocol and find ways to decrease its load.

sentences: Traces of semantization - from episodic to semantic memory in a spiking cortical network model Episodic memory is the recollection of past personal experiences associated with particular times and places. This kind of memory is commonly subject to loss of contextual information or "semantization", which gradually decouples the encoded memory items from their associated contexts while transforming them into semantic or gist-like representations. Novel extensions to the classical Remember/Know behavioral paradigm attribute the loss of episodicity to multiple exposures of an item in different contexts. Despite recent advancements explaining semantization at a behavioral level, the underlying neural mechanisms remain poorly understood. In this study, we suggest and evaluate a novel hypothesis proposing that Bayesian-Hebbian synaptic plasticity mechanisms might cause semantization of episodic memory. We implement a cortical spiking neural network model with a Bayesian-Hebbian learning rule called Bayesian Confidence Propagation Neural Network (BCPNN), which captures the semantization phenomenon and offers a mechanistic explanation for it. Encoding items across multiple contexts leads to item-context decoupling akin to semantization. We compare BCPNN plasticity with the more commonly used spike-timing dependent plasticity (STDP) learning rule in the same episodic memory task. Unlike BCPNN, STDP does not explain the decontextualization process. We also examine how selective plasticity modulation of isolated salient events may enhance preferential retention and resistance to semantization. Our model reproduces important features of episodicity on behavioral timescales under various biological constraints whilst also offering a novel neural and synaptic explanation for semantization, thereby casting new light on the interplay between episodic and semantic memory processes.

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Differential stimulation of pluripotent stem cell-derived human microglia leads to exosomal proteomic changes affecting neurons Microglial exosomes are an emerging communication pathway, implicated in fulfilling homeostatic microglial functions and transmitting neurodegenerative signals. Gene variants of the triggering receptor expressed myeloid cells-2 (TREM2) are associated with an increased risk of developing dementia. We investigated the influence of the TREM2 Alzheimers disease risk variant, R47Hhet, on the microglial exosomal proteome consisting of 3019 proteins secreted from human iPS-derived microglia (iPS-Mg). Exosomal protein content changed according to how iPS-Mg were stimulated. Thus lipopolysaccharide (LPS) induced microglial exosomes to contain more inflammatory signals, whilst stimulation with the TREM2 ligand phosphatidylserine (PS+) increased metabolic signals within the microglial exosomes. We tested the effect of these exosomes on neurons and found that the exosomal protein changes were functionally relevant and influenced downstream functions in both neurons and microglia. Exosomes from R47Hhet iPS-Mg contained disease-associated microglial (DAM) signature proteins, and were less able to promote outgrowth of neuronal processes and increase mitochondrial metabolism in neurons compared with exosomes from TREM2 common variant iPS-Mg. Taken together these data highlight the importance of microglial exosomes in fulfilling microglial functions. Additionally, variations in the exosomal proteome influenced by the R47Hhet TREM2 variant may underlie the increased risk of Alzheimers disease associated with this variant.

Graphical Abstract

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Ubiquitin ligases and a processive proteasome facilitate protein clearance during the oocyte-to-embryo transition in Caenorhabditis elegans The ubiquitin-mediated degradation of oocyte translational regulatory proteins is a conserved feature of the oocyte-to-embryo transition (OET). In the nematode Caenorhabditis elegans, multiple translational regulatory proteins, including the TRIM-NHL RNA-binding protein LIN-41/Trim71 and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11, are degraded during the OET. Degradation of each protein requires activation of the M-phase cyclin-dependent kinase CDK-1, is largely complete by the end of the first meiotic division and does not require the anaphase promoting complex (APC). However, only LIN-41 degradation requires the F-box protein SEL-10/FBW7/Cdc4p, the substrate recognition subunit of an SCF-type E3 ubiquitin ligase. This finding suggests that PUF-3 and PUF-11, which localize to LIN-41-containing ribonucleoprotein particles (RNPs), are independently degraded through the action of other factors and that the oocyte RNPs are disassembled in a concerted fashion during the OET. We develop and test the hypothesis that PUF-3 and PUF-11 are targeted for degradation by the proteasome-associated HECT-type ubiquitin ligase ETC-1/UBE3C/Hu15, which is broadly expressed in C. elegans. We find that several GFP-tagged fusion proteins that are degraded during the OET, including fusions with PUF-3, PUF-11, LIN-41, IFY-1/Securin and CYB-1/Cyclin B, are incompletely degraded when ETC-1 function is compromised. However, it is the fused GFP moiety that appears to be the critical determinant of this proteolysis defect. These findings are consistent with a conserved role for ETC-1 in promoting proteasome processivity and suggest that proteasomal processivity is an important element of the OET during which many key oocyte regulatory proteins are rapidly targeted for degradation.

Article SummaryThe ubiquitin-mediated degradation of translational regulatory RNA-binding proteins is a conserved feature of the oocyte-to-embryo transition (OET). C. elegans LIN-41 is a master regulator of oogenesis and is found in a large translational regulatory ribonucleoprotein (RNP) complex with more than 1000 maternal transcripts and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11. We show that the concerted action of ubiquitin-mediated protein degradation and proteasome processivity rapidly disassemble LIN-41-containing RNPs during the OET thereby relieving repression of many maternal transcripts.

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Cooperation enhances structural stability in mutualistic systems Dynamical systems on graphs allow to describe multiple phenomena from different areas of Science. In particular, many complex systems in Ecology are studied by this approach. In this paper we analize the mathematical framework for the study of the structural stability of each stationary point, feasible or not, introducing a generalization for this concept, defined as Global Structural Stability. This approach would fit with the proper mathematical concept of structural stability, in which we find a full description of the complex dynamics on the phase space due to nonlinear dynamics. This fact can be analyzed as an informational field grounded in a global attractor whose structure can be completely characterized. These attractors are stable under perturbation and suppose the minimal structurally stable sets. We also study in detail, mathematically and computationally, the zones characterizing different levels of biodiversity in bipartite graphs describing mutualistic antagonistic systems of population dynamics. In particular, we investigate the dependence of the region of maximal biodiversity of a system on its connectivity matrix. On the other hand, as the network topology does not completely determine the robustness of the dynamics of a complex network, we study the correlation between structural stability and several graph measures. A systematic study on synthetic and biological graphs is presented, including 10 mutualistic networks of plants and seed-dispersal and 1000 random synthetic networks. We compare the role of centrality measures and modularity, concluding the importance of just cooperation strength among nodes when describing areas of maximal biodiversity. Indeed, we show that cooperation parameters are the central role for biodiversity while other measures act as secondary supporting functions.

Author summaryWe introduce the concept of Global Structural Stability as a proper mathematical concept to fully understand biodiversity in some ecological systems. Our concept retakes the definitions in the classical works of R. Thom [1] and Andronov-Pontryagin [2]. Moreover, there exists a close relation between the structure of a complex network, described as a graph, and its associated dynamics. Mutualistic networks introduce cooperation links between two groups of species, as plant and pollinators or seed-dispersal. The understanding of organizational aspects leading to maximizing biodiversity is one of the more important research areas in Theoretical and Applied Ecology. In this work we introduce a systematic study on different graph measures in order to identify optimal organization for maximal biodiversity (defined as structural stability). Our results conclude that, for mutualistic systems, the strength in cooperation parameters are the core fact, i.e., cooperation is the real fact optimizing biodiversity among other possible structural configurations.

sentences: Restriction of RecG translocation by DNA mispairing The RecG DNA helicase plays a crucial role in stalled replication fork rescue as the guardian of the bacterial genome. We have recently demonstrated that single-strand DNA binding protein (SSB) promotes binding of RecG to the stalled replication fork by remodeling RecG, enabling the helicase to translocate ahead of the fork. We also hypothesized that mispairing of DNA could limit such translocation of RecG, which plays the role of roadblocks for the fork movement. Here, we used atomic force microscopy (AFM) to directly test this hypothesis and investigate how sensitive RecG translocation is to different types of mispairing. We found that a C-C mismatch at a distance of 30 bp away from the fork position prevents translocation of RecG over this mispairing. A G-bulge placed at the same distance also has a similar roadblock efficiency. However, a C-C mismatch 10 bp away from the fork does not prevent RecG translocation, as 10 bp from fork is within the distance of footprint of RecG on fork DNA. Our findings suggest that retardation of RecG translocation ahead of the replication fork can be a mechanism for the base pairing control for DNA replication machinery.

sentences: Integrative analysis allows a global and precise identification of functional miRNA target genes in mESCs MicroRNA (miRNA) loaded Argonaute (AGO) complexes regulate gene expression via direct base pairing with their mRNA targets. Current prediction approaches identified that between 20 to 60% of mammalian transcriptomes are regulated by miRNAs, but it remains largely unknown which fraction of these interactions are functional in a specific cellular context. Here, we integrated transcriptome data from a set of miRNA-depleted mouse embryonic stem cell (mESC) lines with published miRNA interaction predictions and AGO-binding profiles. This integrative approach, combined with molecular validation data, identified that only 6% of expressed genes are functionally and directly regulated by miRNAs in mESCs. In addition, analyses of the stem cell-specific miR-290-295 cluster target genes identified TFAP4 as an important transcription factor for early development. The extensive datasets developed in this study will support the development of improved predictive models for miRNA-mRNA functional interactions.

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Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 Spike protein BackgroundSARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NF{kappa}B and ENaC, and expression of high levels of cytokines and chemokines. Post-infection inflammation may contribute to "Long COVID", and there are also other long term consequences of acute severe COVID-19, which double or triple the chances of dying from any cause within a year. Enhanced signaling by NF{kappa}B and ENaC also marks the airway of patients suffering from cystic fibrosis, a lethal proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 Spike protein.

MethodsThis hypothesis was tested using the hTERT-transformed BCi-NS1.1 basal stem cell, previously derived from small airway epithelia, which were differentiated into a model of small airway epithelia on an air-liquid-interface (ALI). Cyclic AMP-activated CFTR chloride channel activity was measured using an Ussing Chamber. Cell surface-CFTR was labeled with the impermeant biotin method.

ResultsExposure of differentiated airway epithelia to SARS-CoV-2 Spike protein resulted in loss of CFTR protein expression. As hypothesized, TNF/NF{kappa}B signaling was activated, based on increased protein expression of TRADD, the first intracellular adaptor for the TNF/TNFR1 complex, TNFR1, the TNF receptor, phosphorylated I{kappa}B, and the chemokine IL8. ENaC activity was also activated, based on specific changes in molecular weights for  and {gamma} ENaC. Exposure of the epithelia to viral Spike protein suppressed cAMP-activated CFTR chloride channel activity. However, addition of 30 nM concentrations of cardiac glycoside drugs ouabain, digitoxin and digoxin, prevented loss of channel activity. ACE2 and CFTR were found to co-immunoprecipitate (co-IP) in both basal cells and epithelia, suggesting that the mechanism for Spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, Spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane, suggesting that failure of CFTR recovery from endosomal recycling might be a mechanism for Spike-dependent loss of CFTR.

ConclusionBased on experiments with this model of small airway epithelia, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by SARS-CoV-2 Spike protein, thus inducing a CFTR-null, cystic fibrosis-like clinical phenotype. Descriptions of COVID-19 in CF carriers with only one copy of wildtype CFTR suggest that this model-based conclusion might be consistent with patient-based experience.

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Linked-Read Sequencing of Eight Falcons Reveals a Unique Genomic Architecture in Flux Falcons are diverse birds of cultural and economic importance. They have undergone major lineage-specific chromosomal rearrangements, resulting in greatly-reduced chromosome counts relative to other birds. Here, we use 10X Genomics linked reads to provide new high-contiguity genomes for two gyrfalcons, a saker falcon, a lanner falcon, three subspecies of peregrine falcons, and the common kestrel. Assisted by a transcriptome sequenced from 22 gyrfalcon tissues, we annotate these genomes for a variety of genomic features, estimate historical demography, and then investigate genomic equilibrium in the context of falcon-specific chromosomal rearrangements. We find that falcon genomes are not in AT-GC equilibrium with a bias in mutations towards higher AT content; this bias is predominantly driven by--but not dependent on--hypermutability of CpG sites. Small indels and large structural variants were also biased towards insertions rather than deletions. Patterns of disequilibrium were linked to chromosomal rearrangements: falcons have lost GC content in regions that have fused to larger chromosomes from microchromosomes and gained GC content in regions of macrochromosomes that have translocated to microchromosomes. Inserted bases have accumulated on regions ancestrally belonging to microchromosomes, consistent with insertion-biased gene conversion. We also find an excess of interspersed repeats on regions of microchromosomes that have fused to macrochromosomes. Our results reveal that falcon genomes are in a state of flux. They further suggest that many of the key differences between microchromosomes and macrochromosomes are driven by differences in chromosome size, and indicate a clear role for recombination and biased-gene-conversion in determining genomic equilibrium.

SignificanceFalcons are a particularly diverse and widespread genus of birds of particular cultural and economic importance. Falcons have also undergone recent large-scale chromosomal rearrangements to arrive at atypically low chromosome counts relative to other birds. We produced eight new high-quality falcon genomes to support general research on falcons, and we analyze these genomes to assess how chromosome loss has influenced specific aspects of genomic architecture.

sentences: Live-cell imaging and physical modeling reveal control of chromosome folding dynamics by cohesin and CTCF Physical proximity between genomic sequences in mammalian chromosomes controls key biological processes such as transcriptional regulation and DNA repair. Yet it is currently unknown if chromosomal contacts are rare and stable or instead frequent and dynamic, and how they depend on the loop extrusion activity of cohesin or barriers such as CTCF. By imaging chromosomal locations at high spatial and temporal resolution over several hours in living cells, we show that sequences within topological associating domains (TADs) frequently come into physical proximity during the course of a cell cycle and remain close to each other only for a few minutes. Such contacts become nonetheless substantially longer and more frequent in the presence of convergent CTCF sites, resulting in a suppression of variability in chromosome folding in single cells across time. Supported by physical models of chromosome dynamics, our data additionally suggests that individual CTCF-anchored loops last around 10 minutes. The estimates of chromosomal contact dynamics in our study provide a novel quantitative framework to link chromosome structure to function and show that cohesin and CTCF stabilize otherwise highly dynamic chromosome structures to facilitate selected subsets of chromosomal interactions.

sentences: Edge strand of Escherichia coli BepA interacts with immature LptD on the β-barrel assembly machine to direct it to on- and off-pathways The outer membrane (OM) of gram-negative bacteria functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of LptD, a {beta}-barrel protein component of the lipopolysaccharide translocon. BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions probably on the {beta}-barrel assembly machinery (BAM) complex. However, how BepA recognizes and directs an immature LptD to different pathways remains unclear. Here, we explored the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge-strand located adjacent to the active site with LptD was crucial not only for proteolysis but also, unexpectedly, for assembly promotion of LptD. Site-directed crosslinking analyses indicated that the unstructured N-terminal half of the {beta}-barrel-forming domain of an immature LptD contacts with the BepA edge-strand. Furthermore, the C-terminal region of the {beta}-barrel-forming domain of the BepA-bound LptD intermediate interacted with a "seam" strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the functional mechanism of BepA.

sentences: Extreme Heterogeneity in Genomic Differentiation between Phenotypically Divergent Songbirds: A Test of Mitonuclear Co-introgression Comparisons of genomic variation among closely related species often show more differentiation in mitochondrial DNA (mtDNA) and sex chromosomes than in autosomes, a pattern expected due to the differing effective population sizes and evolutionary dynamics of these genomic components. Yet, introgression can cause species pairs to deviate dramatically from general differentiation trends. The yellowhammer (Emberiza citrinella) and the pine bunting (E. leucocephalos) are hybridizing avian sister species that differ greatly in appearance and moderately in nuclear DNA, but that show no mtDNA differentiation. This mitonuclear discordance is best explained by adaptive mtDNA introgression--a process that can select for co-introgression at nuclear genes with mitochondrial functions (mitonuclear genes). To better understand the extent of mitonuclear discordance and characterize nuclear differentiation patterns in this system, we investigated genome-wide differentiation between allopatric yellowhammers and pine buntings and compared it to what was seen previously in mtDNA. We found significant nuclear differentiation that was highly heterogeneous across the genome, with a particularly wide differentiation peak on the sex chromosome Z. We further tested for preferential introgression of mitonuclear genes and found statistical support for this process in yellowhammers. A role for mitonuclear coevolution in this system is supported by a stronger signal of co-introgression in genes coding for subunits of the mitoribosome and electron transport chain complexes. Altogether, our study emphasizes the extreme variation seen in differentiation across genomic components and study systems as well as highlights the ramifications of this variation in species evolution.

sentences: NEIL1 and NEIL2 DNA glycosylases regulate anxiety and learning in a cooperative manner Oxidative DNA damage in the brain has been implicated in neurodegeneration and cognitive decline. DNA glycosylases initiate base excision repair (BER), the main pathway for oxidative DNA base lesion repair. NEIL1 and NEIL3 DNA glycosylases alter cognition in mice, the role of NEIL2 remains unclear. Here, we investigate the impact of NEIL2 and its potential overlap with NEIL1 on behavior in single and double knock-out mouse models. Neil1-/-Neil2-/- mice displayed hyperactivity, reduced anxiety and improved learning. Hippocampal oxidative DNA base lesion levels were comparable between genotypes, no mutator phenotype was found. Impaired canonical repair was thus not the cause of altered behavior. Electrophysiology indicated reduced stratum oriens afferents in the hippocampal CA1 region in Neil1-/-Neil2-/-. Within CA1, NEIL1 and NEIL2 jointly regulated transcription in genes relevant for synaptic function. Thus, we postulate a cooperative function of NEIL1 and NEIL2 in genome regulation beyond canonical BER modulating memory formation and anxiety.

sentences: Sexual maturation in Atlantic salmon male parr may be triggered both in early spring and late summer under standard farming conditions Male Atlantic salmon (Salmo salar) display different sexual strategies, maturing either as parr during the freshwater phase (as sneaky spawners), or as post smolts following one or several years at sea. First sexual maturation (puberty) occurs at different times depending on environmental and genetic factors. To improve our knowledge on the timing (age and season) of first sexual maturation in Atlantic salmon male parr, we investigated pubertal activation in second generation farmed salmon from the Norwegian river Figgjo, reared under natural conditions of photoperiod and water temperature. Histological analysis, in combination with morphometric measurements, plasma androgen levels and pituitary gonadotropin gene expression analysis revealed that, as previously reported, some male parr initiated early sexual maturation in spring at one year of age. Interestingly, some male parr were observed to initiate sexual maturation already in autumn, six months after hatching (under-yearlings), much earlier than reported in previous studies. One-year old maturing males showed a low induction in gonadotropin levels, while under-yearling maturing males displayed a significant increase in fshb transcripts as compared to immature fish. Plasma testosterone, detectable also in immature males, increased constantly during testes development, while 11-ketotestosterone, undetectable in immature and early maturing males, increased during more advanced stages of maturation. A mild feminization of the testes (ovotestes) was detected in a subset of samples. This study brings new knowledge on the little investigated field of sexually maturing under-yearlings in Atlantic salmon. This is also the first study comparing the physiology of under-yearling vs one-year old maturing male parr, thus bringing new insights to the remarkable plasticity of Atlantic salmon puberty.

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Uncovering the hologenomic basis of an extraordinary plant invasion While invasive species are a key driver of the global biodiversity crisis, the drivers of invasiveness remain debated. To investigate the genomic basis of invasiveness in plants, we use the invasive weed Ambrosia artemisiifolia, introduced to Europe in the late 19th century, resequencing 655 ragweed genomes, including 308 herbarium specimens collected up to 190 years ago. In introduced European populations, we report selection signatures in defense genes and lower prevalence of particular plant pathogens in the invasive range. Together with temporal changes in population structure associated with introgression from closely related Ambrosia species, escape from microbial enemies likely favoured the plants remarkable success as an invasive species.

One-Sentence SummaryThe invasive success of European ragweed was facilitated by release from enemy microbes and inter-species hybridization.

sentences: Gene losses in the common vampire bat illuminate molecular adaptations to blood feeding Feeding exclusively on blood, vampire bats represent the only obligate sanguivorous lineage among mammals. To uncover genomic changes associated with adaptations to this unique dietary specialization, we generated a new haplotype-resolved reference-quality genome of the common vampire bat (Desmodus rotundus) and screened 26 bat species for genes that were specifically lost in the vampire bat lineage. We discovered previously-unknown gene losses that relate to metabolic and physiological changes, such as reduced insulin secretion (FFAR1, SLC30A8), limited glycogen stores (PPP1R3E), and a distinct gastric physiology (CTSE). Other gene losses likely reflect the biased nutrient composition (ERN2, CTRL) and distinct pathogen diversity of blood (RNASE7). Interestingly, the loss of REP15 likely helped vampire bats to adapt to high dietary iron levels by enhancing iron excretion and the loss of the 24S-hydroxycholesterol metabolizing enzyme CYP39A1 could contribute to their exceptional cognitive abilities. Finally, losses of key cone phototransduction genes (PDE6H, PDE6C) suggest that these strictly-nocturnal bats completely lack cone-based vision. These findings enhance our understanding of vampire bat biology and the genomic underpinnings of adaptations to sanguivory.

sentences: clampFISH 2.0 enables rapid, scalable amplified RNA detection in situ RNA labeling in situ has enormous potential to visualize transcripts and quantify their levels in single cells, but it remains challenging to produce high levels of signal while also enabling multiplexed detection of multiple RNA species simultaneously. Here, we describe clampFISH 2.0, a method that uses an inverted padlock design to efficiently detect and exponentially amplify signals from many RNA species at once, while also reducing time and cost compared to the prior clampFISH method. We leverage the increased throughput afforded by multiplexed signal amplification and sequential detection by demonstrating the ability to detect 10 different RNA species in over 1 million cells. We also show that clampFISH 2.0 works in tissue sections. We expect the advantages offered by clampFISH 2.0 will enable many applications in spatial transcriptomics.

sentences: Reduced variability of bursting activity during working memory Working memories have long been thought to be maintained by persistent spiking. However, mounting evidence from multiple-electrode recording (and single-trial analyses) shows that the underlying spiking is better characterized by intermittent bursts of activity. A counterargument suggested this intermittent activity is at odds with observations that spike-time variability reduces during task performance. However, this counterargument rests on assumptions, such as randomness in the timing of the bursts, that may not be correct. Thus, we analyzed spiking and LFPs from the prefrontal cortex (PFC) of monkeys to determine if task-related reductions in variability can co-exist with intermittent spiking. We found that it does because both spiking and associated gamma bursts were task-modulated, not random. In fact, the task-related reduction in spike variability could be explained by a related reduction in gamma burst variability. Our results provide further support for the intermittent activity models of working memory as well as novel mechanistic insights into how spike variability is reduced during cognitive tasks.

sentences: Anti-GD2 antibody disrupts GD2:Siglec-7 interactions and synergizes with CD47 blockade to mediate tumor eradication The disialoganglioside GD2 is consistently overexpressed in neuroblastoma and osteosarcoma, and is variably expressed in other sarcomas, gliomas, neuroendocrine tumors, and epithelial cancers. Anti-GD2 antibodies have improved the survival rates of patients with neuroblastoma only when administered as part of intense chemotherapy-based cytotoxic regimens, which are associated with debilitating late effects including hearing loss, growth retardation, and secondary leukemias. Despite broad expression of GD2 on osteosarcoma, anti-GD2 antibody has not mediated significant antitumor activity in that disease or any other GD2+ cancers. CD47 is a checkpoint molecule overexpressed on tumor cells that inhibits macrophage activity, and CD47 blockade has demonstrated promising clinical activity in early human trials. We investigated whether anti-CD47 antibody could enhance the efficacy of anti-GD2 antibody in neuroblastoma and other GD2+ malignancies. We demonstrate substantial synergy of these two agents, resulting in the recruitment of tumor associated macrophages (TAMs) to mediate robust and durable anti-tumor responses. The responses are driven by GD2-specific factors that reorient the balance of macrophage activity towards phagocytosis of tumor cells, including disruption of a newly described GD2:Siglec-7 axis. These results demonstrate the unique synergy of combining anti-GD2 with anti-CD47, which has the potential to significantly enhance outcomes for children with neuroblastoma and osteosarcoma and will soon be investigated in a first-in-human clinical trial.

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Environmental and taxonomic drivers of bacterial extracellular vesicle production in marine ecosystems Extracellular vesicles are small ([~]50-250 nm diameter) membrane-bound structures released by cells into their surrounding environment. Vesicles are abundant in the global oceans and likely play a number of ecological roles in these microbially dominated ecosystems, yet we know nothing about what influences their production and distributions. Here we examine how vesicle production varies among different strains of cultivated marine microbes and explore the degree to which this is influenced by some key environmental variables. We show that vesicle production rates - the number of vesicles produced per cell per generation - vary across an order of magnitude in cultures of marine Proteobacteria, Cyanobacteria, and Bacteroidetes. Vesicle production rates further differ among strains of the cyanobacterium Prochlorococcus, and vary across temperature and light gradients. These data suggest that both community composition and local environmental conditions modulate the production and standing stock of vesicles in the oceans. Examining samples from the oligotrophic North Pacific Gyre, we show depth-dependent changes in the abundance of vesicle-like particles in the upper water column in a manner broadly consistent with culture observations: highest vesicle abundances are found near the surface, where light irradiances and temperatures are greatest, and then decrease with depth. This work represents the beginnings of a quantitative framework for describing extracellular vesicle dynamics in the oceans - essential as we begin to incorporate vesicles into our ecological and biogeochemical understanding of marine ecosystems.

ImportanceBacteria secrete extracellular vesicles containing a wide variety of cellular compounds, including lipids, proteins, nucleic acids, and small molecules, into their surrounding environment. These structures are found in diverse microbial habitats, including the oceans, where their distributions vary throughout the water column. Differences in vesicle abundances likely affect their functional impacts within microbial ecosystems, but the factors influencing vesicle distributions in the environment remain poorly understood. Using quantitative analysis of marine microbial cultures, we show that bacterial vesicle production in the oceans is shaped by a combination of biotic and abiotic factors. Our data indicate that different marine taxa release vesicles at rates varying across an order of magnitude, and that vesicle production can change dynamically as a function of environmental conditions. Taken together with direct measurements of vesicle concentrations in the oceans, these culture-based measurements further provide a window into estimating vesicle loss rates. These findings represent a step forward in our understanding of marine vesicle distributions and provide a basis for quantitatively exploring vesicle dynamics in natural ecosystems.

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Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda BackgroundBovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health.

Materials and methodsA cross-sectional study employing a systematic random sampling of cattle (n=300) with the collection of retropharyngeal lymph nodes and tonsils (n=300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence.

FindingsOf the 300 samples, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. M. bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistance (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle.

ConclusionsThis study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is prevalent in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.

Authors summaryTuberculosis in cattle (bTB) causes financial losses to livestock owners and is a disease transmissible to humans especially those with an occupational risk through exposure to infected animals and animal products. This study aimed to identify the prevalence of bTB and characterize the mycobacterial species from cattle slaughtered in the six abattoirs in Rwanda. Four M. bovis, as well as one rifampicin-resistant (RR) M. tuberculosis, were identified from slaughtered cattle and, thus, the apparent bTB prevalence was 1.7% (5/300). Likely, the RR M. tuberculosis isolate was mostly likely of human origin and transmitted to cattle during close human-cattle contact. It is therefore essential to control bTB in cattle and reinforce the protection of farmworkers and abattoir workers who are always exposed to infected animals.

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Constitutive depletion of brain serotonin differentially affects rats' social and cognitive abilities BackgroundCentral serotonin is an essential neuromodulator for mental disorders. It appears a promising transdiagnostic marker of distinct psychiatric disorders and a common modulator of some of their key behavioral symptoms. We aimed to identify the behavioral markers of serotonergic function in rats and compare them to human deficits.

MethodsWe applied a comprehensive profiling approach in adult male Tph2-/- rats constitutively lacking central serotonin. Under classical and ethological testing conditions, we tested each individuals cognitive, social and non-social abilities and characterized the group organization (i.e. social network, hierarchy). Using unsupervised machine learning, we identified the functions most dependent on central serotonin.

ResultsIn classical procedures, Tph2-/- rats presented an unexpected normal cognitive profile. Under the complex and experimenter-free conditions of their home-cage, the same Tph2-/- rats presented drastic changes in their daily life. Brain serotonin depletion induced compulsive aggression and sexual behavior, hyperactive and hypervigilant stereotyped behavior, reduced self-care and body weight, and exacerbated corticosterone levels. Group-housed Tph2-/- rats showed strong social disorganization with disrupted social networks and hierarchical structure, which may arise from communication deficits and cognitive blunting.

ConclusionsSerotonin depletion induced a profile reminiscent of the symptomatology of impulse control and anxiety disorders. Serotonin was necessary for behavioral adaptation to dynamic social environments. In classical testing conditions, our animal model challenged the concept of an essential role of serotonin in decision-making, flexibility, and impulsivity, although developmental compensations may have occurred. These contrasting findings highlight the need to generalize the evaluation of animal models multidimensional functions within the complexity of the social living environment.

sentences: Transport among protocells via tunneling nanotubes We employ model protocell networks for evaluation of molecular transport through lipid nanotubes as potential means of communication among primitive cells on the early Earth. Network formation is initiated by deposition of multilamellar lipid reservoirs onto a silicon oxide surface in an aqueous environment. These reservoirs autonomously develop into surface-adhered protocells interconnected via lipid nanotubes, and encapsulate solutes from the ambient buffer in the process. We prepare networks in the presence of DNA and RNA and observe encapsulation of these molecules, and their diffusive transport between the lipid compartments via the interconnecting nanotubes. By means of an analytical model we determine key physical parameters affecting the transport, such as nanotube diameter and compartment size. We conclude that nanotube-mediated transport in self-organized nanotube-vesicle networks could have been a possible pathway of chemical communication between primitive, self-assembled protocells under early earth conditions, circumventing the necessity for crossing the membrane barrier. We suggest this transport within a closed protocell network as a feasible means of RNA and DNA exchange under primitive prebiotic conditions, possibly facilitating early replication.

sentences: Targeted isolation of panels of diverse human broadly neutralizing antibodies against SARS-like viruses The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1-7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10-13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14-18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

sentences: Exponential magnetophoretic gradient for the direct isolation of basophils from whole blood in a microfluidic system Despite their rarity in peripheral blood, basophils play important roles in allergic disorders and other diseases including sepsis and COVID-19. Existing basophil isolation methods require many manual steps and suffer from significant variability in purity and recovery. We report an integrated basophil isolation device (i-BID) in microfluidics for negative immunomagnetic selection of basophils directly from 100 L of whole blood within 10 minutes. We use a simulation-driven pipeline to design a magnetic separation module to apply an exponentially increasing magnetic force to capture magnetically tagged non-basophils flowing through a microtubing sandwiched between magnetic flux concentrators sweeping across a Halbach array. The exponential profile captures non-basophils effectively while preventing their excessive initial buildup causing clogging. The i-BID isolates basophils with a mean purity of 93.9%{+/-}3.6% and recovery of 95.6%{+/-}3.4% without causing basophil degradation or unintentional activation. Our i-BID has the potential to enable basophil-based point-of-care diagnostics such as rapid allergy assessment.

sentences: Synthetic lethality-based prediction of anti-SARS-CoV-2 targets Novel strategies are needed to identify drug targets and treatments for the COVID-19 pandemic. The altered gene expression of virus-infected host cells provides an opportunity to specifically inhibit viral propagation via targeting the synthetic lethal (SL) partners of such altered host genes. Pursuing this antiviral strategy, here we comprehensively analyzed multiple in vitro and in vivo bulk and single-cell RNA-sequencing datasets of SARS-CoV-2 infection to predict clinically relevant candidate antiviral targets that are SL with altered host genes. The predicted SL-based targets are highly enriched for infected cell inhibiting genes reported in four SARS-CoV-2 CRISPR-Cas9 genome-wide genetic screens. Integrating our predictions with the results of these screens, we further selected a focused subset of 26 genes that we experimentally tested in a targeted siRNA screen using human Caco-2 cells. Notably, as predicted, knocking down these targets reduced viral replication and cell viability only under the infected condition without harming non-infected cells. Our results are made publicly available, to facilitate their in vivo testing and further validation.

sentences: SARS-CoV-2 B.1.351 (beta) variant shows enhanced infectivity in K18-hACE2 transgenic mice and expanded tropism to wildtype mice compared to B.1 variant SARS-CoV-2 variants display enhanced transmissibility and/or immune evasion and can be generated in humans or animals, like minks, thus generating new reservoirs. The continuous surveillance of animal susceptibility to new variants is necessary to predict pandemic evolution. In this study we demonstrate that, compared to the B.1 SARS-CoV-2 variant, K18-hACE2 transgenic mice challenged with the B.1.351 variant displayed a faster progression of infection. Furthermore, we also report that B.1.351 can establish infection in wildtype mice, while B.1 cannot. B.1.351-challenged wildtype mice showed a milder infection than transgenic mice, confirmed by detectable viral loads in oropharyngeal swabs and tissues, lung pathology, immunohistochemistry and serology. In silico models supported these findings by demonstrating that the Spike mutations in B.1.351 resulted in increased affinity for both human and murine ACE2 receptors. Overall, this study highlights the plasticity of SARS-CoV-2 animal susceptibility landscape, which may contribute to viral persistence and expansion.

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From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection rates approaching 40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2, WTD fawns shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD. Infectious virus was recovered from respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field.

Author summaryThe high susceptibility of white-tailed deer (WTD) to SARS-CoV-2, their ability to transmit the virus to other deer, and the recent findings suggesting widespread SARS-CoV-2 infection in wild WTD populations in the U.S. underscore the need for a better understanding of the infection and transmission dynamics of SARS-CoV-2 in this potential reservoir species. Here we investigated the transmission dynamics of SARS-CoV-2 over time and defined the major sites of virus replication during the acute phase of infection. Additionally, we assessed the evolution of the virus as it replicated and transmitted between animals. The work provides important information on the infection dynamics of SARS-CoV-2 in WTD, an animal species that - if confirmed as a new reservoir of infection - may provide many opportunities for exposure and potential zoonotic transmission of the virus back to humans.

sentences: Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling Pulmonary arterial hypertension (PAH) is a fatal disease characterized by pathological pulmonary artery remodeling. Endothelial cells (EC) injury including DNA damage is critically involved in the vascular remodeling in PAH, and persistent injury leads to cellular senescence in ECs. Here, we show that EC senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling. EC-specific progeroid mice that we recently generated showed exacerbated pulmonary hypertension after chronic hypoxia exposure, accompanied by the enhanced pulmonary arterial smooth muscle cells (PASMCs) proliferation in the distal pulmonary arteries. Mechanistically, we identified that senescent ECs highly expressed Notch ligands, and thus activated Notch signaling in PASMCs, leading to enhanced PASMCs proliferation and migration capacities. Consistently, pharmacological inhibition of Notch signaling attenuated the effects of senescent ECs on SMCs functions in vitro, and on the pulmonary hypertension in EC-specific progeroid mice in vivo. These data establish EC senescence as a crucial disease-modifying facor in PAH.

sentences: Phased Mutations and Complex Rearrangements in Human Prostate Cancer Cell Lines through Linked-Read Whole Genome Sequencing A limited number of cell lines have fueled the majority of preclinical Prostate cancer (PCa) research. Despite tremendous effort in characterizing their molecular profiles, comprehensive whole genome sequencing with allelic phasing of somatic genome alterations has not been undertaken to date. Here, we utilized whole genome Linked-read sequencing to obtain haplotype information from the seven most commonly used PCa cell lines (PC3, LNCaP, DU145, CWR22Rv1, VCaP, LAPC4, MDA-PCa-2b), four castrate resistant (CR) subclones (LNCaP_Abl, LNCaP_C42b, VCaP-CR, LAPC4-CR), and an immortalized prostate epithelial line RWPE-1. Phasing of mutations allowed derivation of "Gene-level Haplotype" to assess whether a gene harbored heterozygous mutations in one or both alleles, providing a comprehensive catalogue of mono or bi-allelically inactivated genes. Phased structural variant analysis allowed identification of complex rearrangement chains consistent with chromothripsis and chromoplexy, with breakpoints occurred across a single allele, providing further evidence that complex SVs occurred in a concerted event, rather than through accumulation of multiple independent rearrangements. Additionally, comparison of parental and CR subclones revealed previously known and novel genomic alterations associated with the CR clones. This study therefore comprehensively characterized phased genomic alterations in the commonly used PCa cell lines and provided a useful resource for future cancer research.

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Fluorescent protein expression as a proxy of bacterial fitness in a high throughput assay Bacterial growth is classically assessed by measuring the increase in optical density of pure cultures in shaken liquid media. Measuring growth using optical density has severe limitations when studying multistrain interactions as it is not possible to measure the growth of individual strains within mixed cultures. Here we demonstrated that constitutively expressed fluorescent proteins can be used to track the growth of individual strains in different liquid media. Fluorescence measurements were highly correlated with optical density measurements and cell counts. This allowed us to assess bacterial growth not only in pure cultures, but also in mixed bacterial cultures and determine the impact of competitors on a focal strain, thereby assessing relative fitness. Furthermore, we were able to track the growth of two different strains simultaneously by using fluorescent proteins with differential excitation and emission wavelengths. Bacterial densities measured by fluorescence yielded more consistent data between technical replicates than optical density measurements. Our setup employs fluorescent microplate readers that allow for high throughput and replication.

ImportanceWe expand on an important limitation of the concept of measuring bacterial growth which is classically limited to one strain at a time. By adopting this approach, it is possible to measure growth of several bacterial strains simultaneously in high temporal resolution and in a high throughput manner. This is important to investigate bacterial interactions such as competition and facilitation.

sentences: Synergy of color and motion vision for detecting approaching objects in Drosophila Color and motion are used by many species to identify salient moving objects. They are processed largely independently, but color contributes to motion processing in humans, for example, enabling moving colored objects to be detected when their luminance matches the background. Here, we demonstrate an unexpected, additional contribution of color to motion vision in Drosophila. We show that behavioral ON-motion responses are more sensitive to UV than for OFF-motion, and we identify cellular pathways connecting UV-sensitive R7 photoreceptors to ON and OFF-motion-sensitive T4 and T5 cells, using neurogenetics and calcium imaging. Remarkably, the synergy of color and motion vision enhances the detection of approaching UV discs, but not green discs with the same chromatic contrast, and we show how this generalizes for visual systems with ON and OFF pathways. Our results provide a computational and circuit basis for how color enhances motion vision to favor the detection of saliently colored objects.

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Analysis of human brain tissue derived from DBS surgery BackgroundTranscriptomic and proteomic profiling of human brain tissue is hindered by availability of fresh samples from living patients. Postmortem samples usually represent the advanced disease stage of the patient. Furthermore, the postmortem interval affects the observed transcriptomic and proteomic profiles. Therefore, access to fresh brain tissue samples from living patients is valuable resource to obtain information on metabolically intact tissue. The implantation of deep brain stimulation (DBS) electrodes into the human brain is a neurosurgical treatment for, e.g., movement disorders. Here, we describe an improved approach to collect brain tissue from surgical instruments used in implantation of DBS device for transcriptomics and proteomics analyses.

MethodsSamples were extracted from guide tubes and recording electrodes used in routine DBS implantation procedure that was carried out to treat patients with Parkinsons disease, genetic dystonia and tremor. RNA sequencing was carried out to tissue extracted from the recording microelectrodes and liquid chromatography-mass spectrometry was carried out to analyze tissue from guide tubes. To assess the performance of the current approach, obtained datasets were compared with previously published datasets representing brain tissue.

ResultsIn RNA sequencing, altogether 32,034 transcripts representing unique Ensembl gene identifiers were detected from eight samples representing both hemispheres of four patients. By using liquid chromatography-mass spectrometry, we identified 734 unique proteins from 31 samples collected from 14 patients. Comparison with previously published brain derived data indicated that both of our datasets reflected the expected brain tissue specific features. The datasets are available via BioStudies database (accession number S-BSST667).

ConclusionsSurgical instruments used in DBS installation retain enough brain material for protein and gene expression studies. Analysis of the datasets indicated that hemisphere-specific expression data can be obtained from individual patients without any sample pooling and without any modifications to the standard surgical protocol. Comparison with previously published datasets obtained with similar approach proved the robustness and reproducibility of the current improved protocol. This approach overcomes the issues that arise from using postmortem tissue, such as effect of postmortem interval, on proteomic and transcriptomic landscape of the brain and can be used for studying molecular aspects of DBS-treatable diseases.

sentences: Long-term potentiation at pyramidal cell to somatostatin interneuron synapses controls hippocampal network plasticity and memory Hippocampal somatostatin (SOM) cells are dendrite-projecting inhibitory interneurons. CA1 SOM cells receive major excitatory inputs from pyramidal cells (PC-SOM synapses) which show mGluR1a- and mTORC1-mediated long-term potentiation (LTP). PC-SOM synapse LTP contributes to CA1 network metaplasticity and memory consolidation, but whether it is sufficient to regulate these processes remains unknown. Here we used optogenetic stimulation of CA1 pyramidal cells and whole cell recordings in slices to show that optogenetic theta burst stimulation (TBSopto) produces LTP at PC-SOM synapses. At the network level, we found that TBSopto differentially regulates metaplasticity of pyramidal cell inputs: enhancing LTP at Schaffer collateral synapses and depressing LTP at temporo-ammonic synapses. At the behavioral level, we uncovered that in vivo TBSopto regulates learning-induced LTP at PC-SOM synapses, as well as contextual fear memory. Thus, LTP of PC-SOM synapses is a long-term feedback mechanism controlling pyramidal cell synaptic plasticity, sufficient to regulate memory consolidation.

sentences: Sinking a giant: quantitative macroevolutionary comparative methods debunk qualitative assumptions Myhrvold et al.1 suggest that our inference of subaqueous foraging among spinosaurids2 is undermined by selective bone sampling, inadequate statistical procedures, and use of inaccurate ecological categorizations. Myhrvold et al.1 ignore major details of our analyses and results, and instead choose to portray our inferences as if they were based on qualitative interpretations of our plots, without providing additional analyses to support their claims. In this manuscript, we thoroughly discuss all the concerns exposed by Myhrvold et al.1. Additional analyses based on our original datasets2 and novel data presented by Myhrvold et al.1 do not change our original interpretations: while the spinosaurid dinosaurs Spinosaurus and Baryonyx are recovered as subaqueous foragers, Suchomimus is inferred as a non-diving animal.

sentences: Metagenomics Study of Bacterial Communities from Yamuna River water of city of Taj, Agra Yamuna river water of Agra city is heavily contaminated with toxic pollutants including heavy metal that causes severe damage to ecological and social aspects. At present, the direct use of river water for the purpose of drinking causes severe hazards due to anthropogenic activities causing environmental pollution in rivers. In present study, Yamuna River water was collected from three different sites of Agra city. Various physico-chemical parameters were estimated by following the standard methods of APHA and the concentration of heavy metals were measured using Atomic Absorption Spectrophotometer (AAS). In case of physico-chemical parameters characterization, the obtained values were extremely above the permissible limits. On account of the research work; the Yamuna river water samples contains heavy metal concentrations (Cadmium, Chromium, Copper, Lead and Nickel) above the desirable and admissible levels except for Zinc. During analysis of non-culturable bacteria a substantial bacterial diversity was observed in the Yamuna river water samples collected from different sites. The water samples were subjected to metagenomic analysis using Illumina platform which revealed that Proteobacteria (phylum), Betaproteobacteria (class), Burkholderiales (order), Comamonadaceae (family), Hydrogenophaga (genus) and Chloroflexi bacterium OLB 14 (species) were found as the most dominant bacterial taxonomic abundance in the river water samples. The presence of such bacterial communities in water indicates the availability of pollutants and suggests the futuristic use in the field of bioremediation.

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Structural mechanism for bi-directional actin crosslinking by T-plastin To fulfill the cytoskeletons diverse functions in cell mechanics and motility, actin networks with specialized architectures are built by crosslinking proteins, which bridge filaments to control micron-scale network geometry through nanoscale binding interactions via poorly defined structural mechanisms. Here, we introduce a machine-learning enabled cryo-EM pipeline for visualizing active crosslinkers, which we use to analyze human T-plastin, a member of the evolutionarily ancient plastin/fimbrin family of tandem calponin-homology domain (CHD) proteins. We define a sequential bundling mechanism which enables T-plastin to bridge filaments in both parallel and anti-parallel orientations. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable crosslinking which are likely to be disrupted by mutations causing hereditary bone diseases. Beyond revealing how plastins are evolutionary optimized to crosslink dense actin networks with mixed polarity, our cryo-EM workflow will broadly enable analysis of the structural mechanisms underlying cytoskeletal network construction.

One sentence summaryCryo-EM, biochemical, and cellular studies reveal how the crosslinking protein T-plastin bridges actin filaments in two opposing orientations.

sentences: Comparing host module activation patterns and temporal dynamics in infection by influenza H1N1 viruses Influenza is a serious global health threat that shows varying pathogenicity among different virus strains. Understanding similarities and differences among activated functional pathways in the host responses can help elucidate therapeutic targets responsible for pathogenesis. To compare the types and timing of functional modules activated in host cells by four influenza viruses of varying pathogenicity, we developed a new DYNAmic MOdule (DYNAMO) method that addresses the need to compare functional module utilization over time. This integrative approach overlays whole genome time series expression data onto an immune-specific functional network, and extracts conserved modules exhibiting either different temporal patterns or overall transcriptional activity. We identified a common core response to influenza virus infection that is temporally shifted for different viruses. We also identified differentially regulated functional modules that reveal unique elements of responses to different virus strains. Our work highlights the usefulness of combining time series gene expression data with a functional interaction map to capture temporal dynamics of the same cellular pathways under different conditions. Our results help elucidate conservation of the immune response both globally and at a granular level, and provide mechanistic insight into the differences in the host response to infection by influenza strains of varying pathogenicity.

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Trisomy 21 increases microtubules and disrupts centriolar satellite localization Trisomy 21, the cause of Down syndrome, causes a 0.5-fold protein increase of the chromosome 21-resident gene Pericentrin (PCNT) and reduces primary cilia formation and signaling. Here we investigate the mechanisms by which PCNT imbalances disrupt cilia. Using isogenic RPE-1 cells with increased chromosome 21 dosage, we find PCNT protein accumulates around the centrosome as a pericentrosomal cluster of enlarged cytoplasmic puncta that localize along and at MT ends. Cytoplasmic PCNT puncta impact the intracellular MT trafficking network required for primary cilia, as the PCNT puncta sequester cargo peripheral to centrosomes in what we call pericentrosomal crowding. The centriolar satellite proteins, PCM1, CEP131 and CEP290, important for ciliogenesis, accumulate at sites of enlarged PCNT puncta in trisomy 21 cells. Reducing PCNT when chromosome 21 ploidy is elevated is sufficient to decrease PCNT puncta, reestablish a normal density of MTs around the centrosome, restore ciliogenesis to wild type levels and decrease pericentrosomal crowding. A transient reduction in MTs also decreases pericentrosomal crowding and partially rescues ciliogenesis in trisomy 21 cells, indicating that increased PCNT leads to defects in the microtubule network deleterious to normal centriolar satellite distribution. We propose that chromosome 21 aneuploidy disrupts MT-dependent intracellular trafficking required for primary cilia.

TOCMcCurdy et al explore why elevated Pericentrin in trisomy 21 negatively impacts primary cilia formation and signaling. They find that elevated Pericentrin produces more pericentrosomal puncta that associates with and increases microtubules. Changes to Pericentrin and microtubules mislocalizes centriolar satellites in a pericentrosomal crowd.

sentences: A practical modification to a resting state fMRI protocol for improved characterization of cerebrovascular function Cerebrovascular reactivity (CVR), defined here as the Blood Oxygenation Level Dependent (BOLD) response to a CO2 pressure change, is a useful metric of cerebrovascular function. Both the amplitude and the timing (hemodynamic lag) of the CVR response can bring insight into the nature of a cerebrovascular pathology and aid in understanding noise confounds when using functional Magnetic Resonance Imaging (fMRI) to study neural activity. This research assessed a practical modification to a typical resting-state fMRI protocol, to improve the characterization of cerebrovascular function. In 9 healthy subjects, we modelled CVR and lag in three resting-state data segments, and in data segments which added a 2-3 minute breathing task to the start of a resting-state segment. Two different breathing tasks were used to induce fluctuations in arterial CO2 pressure: a breath-hold task to induce hypercapnia (CO2 increase) and a cued deep breathing task to induce hypocapnia (CO2 decrease). Our analysis produced voxel-wise estimates of the amplitude (CVR) and timing (lag) of the BOLD-fMRI response to CO2 by systematically shifting the CO2 regressor in time to optimize the model fit. This optimization inherently increases grey matter CVR values and fit statistics. The inclusion of a simple breathing task, compared to a resting-state scan only, increases the number of voxels in the brain that have a significant relationship between CO2 and BOLD-fMRI signals, and improves our confidence in the plausibility of voxel-wise CVR and hemodynamic lag estimates. We demonstrate the clinical utility and feasibility of this protocol in an incidental finding of Moyamoya disease, and explore the possibilities and challenges of using this protocol in younger populations. This hybrid protocol has direct applications for CVR mapping in both research and clinical settings and wider applications for fMRI denoising and interpretation.

sentences: Altered effective connectivity from the posterior insula to the amygdala mediates the relationship between psychopathic traits and concern with the Harm foundation Psychopathic traits have been demonstrated to be associated with different types of morality; however, the neuropsychological mechanism underlying the relationship between psychopathic traits and morality remains unclear. Our study examined the effective connectivity (EC) of psychopathic traits-related brain regions and its association to concern with different moral foundations by combining behavioral measures with resting-state fMRI. We administered the Levenson Self-Report Psychopathy Scale (LSRP) and Moral Foundation Questionnaire (MFQ) to 78 college students after resting-state fMRI scanning. Our results showed that total and primary psychopathic traits score predicted concern with the Harm foundation. The EC from the posterior insula to the amygdala was negatively correlated with psychopathic traits and positively with concern with the Harm foundation. Altered posterior insula-amygdala EC partially mediated the relationship between psychopathic traits and concern with the Harm foundation. Our findings indicated that individuals with elevated psychopathic traits may have atypical processes in recognizing and integrating bodily state information into emotional responses, leading to less concern for harm-related morality. The study deepened our understanding of the neuropsychological mechanism underlying the relationship between psychopathic traits and morality and may have implications for the prevention of higher psychopathic traits individuals from committing serious transgressions.

sentences: Endocytosis-mediated vitellogenin absorption and lipid metabolism in the hindgut-derived pseudoplacenta of the viviparous teleost Xenotoca eiseni Certain viviparous animals possess mechanisms for mother-to-embryo nutrient transport during gestation. Xenotoca eiseni is one such viviparous teleost species in which the mother supplies proteins and other components to the offspring developing in the ovary. The embryo possesses trophotenia, a hindgut-derived pseudoplacenta to receive the maternal supplement. However, the molecular mechanisms underlying viviparous non-mammalian animals remain elusive. We conducted this study to investigate the mechanism for nutrient absorption and degradation in trophoenia of X. eiseni. The tracer assay indicated that a lipid transfer protein, vitellogenin (Vtg), was absorbed into the epithelial layer cells of trophotaenia. Vtg uptake was significantly suppressed by Pitstop-2, an inhibitor of clathrin-mediated endocytosis. Gene expression analysis indicated that the genes involved in endocytosis-mediated lipolysis and lysosomal cholesterol transport were expressed in trophotaenia. In contrast, plasma membrane transporters expressed in the intestinal tract were not functional in trophotaenia. Our results suggested that endocytosis-mediated lysosomal lipolysis is one of the mechanisms underlying maternal component metabolism. Thus, our study demonstrated how viviparous teleost species have acquired a unique developmental system that is based on the hindgut-derived pseudoplacenta.

sentences: N-terminally truncated helper NLR NRG1C antagonizes immunity mediated by its full-length neighbors NRG1A and NRG1B Both animals and plants utilize nucleotide-binding leucine-rich repeat immune receptors (NLRs) to perceive the presence of pathogen-derived molecules and induce immune responses. NLR genes are far more abundant and diverse in higher plants. Interestingly, truncated NLRs, which lack one or more of the canonical domains, are also commonly encoded in plant genomes. However, little is known about their functions, especially regarding the N-terminally truncated ones. Here, we show that Arabidopsis thaliana (A. thaliana) N-terminally truncated helper NLR gene NRG1C (N REQUIREMENT GENE 1) is highly induced upon pathogen infection and in autoimmune mutants. The immune response and cell death conferred by some TIR (Toll/interleukin-1 receptor)-type NLRs (TNLs) are compromised in the NRG1C overexpression lines. Detailed genetic analysis revealed that NRG1C antagonizes the immunity mediated by its full-length neighbors NRG1A and NRG1B. Biochemical tests indicate that NRG1C possibly interferes with the EDS1-SAG101 complex, which likely signals together with NRG1A/1B. Interestingly, Brassicaceae NRG1Cs are functionally exchangeable, and the Nicotiana benthamiana (N. benthamiana) N-terminally truncated helper NLR NRG2 antagonizes NRG1 in tobacco. Together, our study uncovers an unexpected negative role of N-terminally truncated helper NLRs in different plants.

sentences: Synergism between Chromatin Dynamics and Gene Transcription Enhances Robustness and Stability of Epigenetic Cell Memory Apart from carrying hereditary information inherited from their ancestors and being able to pass on the information to their descendants, cells can also inherit and transmit information that is not stored as changes in their genome sequence. Such epigenetic cell memory, which is particularly important in multicellular organisms, involves multiple biochemical modules mainly including chromatin organization, epigenetic modification and gene transcription. The synergetic mechanism among these three modules remains poorly understood and how they collaboratively affect the robustness and stability of epigenetic memory is unclear either. Here we developed a multiscale model to address these questions. We found that the chromatin organization driven by long-range epigenetic modifications can significantly enhance epigenetic cell memory and its stability in contrast to that driven by local interaction and that chromatin topology and gene activity can promptly and simultaneously respond to changes in nucleosome modifications while maintaining the robustness and stability of epigenetic cell memory over several cell cycles. We concluded that the synergism between chromatin dynamics and gene transcription facilitates the faithful inheritance of epigenetic cell memory across generations.

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Imaging sensory transmission and neuronal plasticity in primary sensory neurons with genetically-encoded voltage indicators Detection of somatosensory inputs requires conversion of external stimuli into electrical signals by activation of primary sensory neurons. The mechanisms by which heterogeneous primary sensory neurons encode different somatosensory inputs remains unclear. In vivo dorsal root ganglia (DRG) imaging using genetically-encoded Ca2+ indicators (GECIs) is currently the best technique for this purpose by providing an unprecedented spatial and populational resolution. It permits the simultaneous imaging of >1800 neurons/DRG in live mice. However, this approach is not ideal given that Ca2+ is a second messenger and has inherently slow response kinetics. In contrast, genetically-encoded voltage indicators (GEVIs) have the potential to track voltage changes in multiple neurons in real time but often lack the brightness and dynamic range required for in vivo use. Here, we used soma-targeted ASAP4.4-Kv, a novel GEVI, to dissect the temporal dynamics of noxious and non-noxious neuronal signals during mechanical, thermal, or chemical stimulation in DRG of live mice. ASAP4.4-Kv is sufficiently bright and fast enough to optically characterize individual neuron coding dynamics. Notably, using ASAP4.4-Kv, we uncovered cell-to-cell electrical synchronization between adjacent DRG neurons and robust dynamic transformations in sensory coding following tissue injury. Finally, we found that a combination of GEVI and GECI imaging empowered in vivo optical studies of sensory signal processing and integration mechanisms with optimal spatiotemporal analysis.

HighlightsO_LIIn vivo ultra fast and sensitive dynamic voltage imaging of peripheral primary sensory neurons by a newly generated genetically-encoded voltage indicator.
C_LIO_LIIdentification of mechanical, thermal, or chemical stimuli-evoked voltage signals with superior temporal resolution.
C_LIO_LISingle-cell detection of changes in sub- and suprathreshold voltage dynamics across different disease conditions.
C_LIO_LICombination of voltage (by ASAP4.4-Kv) and Ca2+ (by Pirt-GCaMP3) signals to facilitate the understanding of signal processing and integration of primary sensory neurons, especially for noxious versus non-noxious sensation.
C_LI

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Identification and quantification of α- and β-amanitin in wild mushrooms by HPLC-UV-EC and HPLC-DAD-MS detection Amatoxins are a group of highly toxic peptides, which include - and {beta}-amanitin, found in several species of mushrooms (e.g. Amanita phalloides). Due to their high hepatotoxicity, they account for most deaths occurring after mushrooms ingestion. The determination of - and {beta}- amanitin content in wild mushrooms is invaluable for treating cases involving poisoning. In the present study, we have developed and validated an analytical method based on high-performance liquid chromatography, with in-line ultraviolet and electrochemical detection (HPLC-UV-EC), for the rapid quantification of - and {beta}-amanitin in wild mushroom samples collected from the Inner Center of Portugal. A reproducible and simple solid-phase extraction (SPE) using OASIS(R) PRIME HLB cartridges was used for sample pre-treatment, followed by chromatographic separation based on the RP-C18 column. The UV and EC chromatograms of - and {beta}-amanitin were recorded at 305 nm and +0.600 V vs. Ag/AgCl, respectively. The linear quantification for both amanitins was in the range of 0.5-20.0 g{middle dot}mL-1 (R2 > 0.999). The LOD, calculated based on the calibration curve, was similar for UV and EC detection (0.12-0.33 g ml.-1). Intra-day and inter-day precision were less than 13%, and the recovery ratios ranged from 89% to 117%. Nine Amanita species and five edible mushrooms were analysed by HPLC-UV-EC, and HPLC-DAD-MS confirmed the identification of amatoxins. We find high - and {beta}-amanitin content in A. phalloides and not in the other species analysed. In sum, the developed and validated method provides a simple and fast analysis of - and {beta}-amanitins contents in wild mushrooms and is suitable for screening and routine assessment of mushroom intoxication.

HighlightsO_LINew validated method using HPLC-UV-EC to determine - and {beta}-amanitin in wild mushrooms.
C_LIO_LIReproducible and fast SPE procedure for small samples.
C_LIO_LIEffective sample pre-treatment with the OASIS(R) PRIME HLB SPE cartridge.
C_LIO_LIIdentification and quantification of - and {beta}-amanitin in wild mushroom samples from Portugal.
C_LIO_LIHPLC-DAD-MS confirmation of amatoxins present in mushroom samples.
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A eukaryote-wide perspective on the diversity and evolution of the ARF GTPase protein family The evolution of eukaryotic cellular complexity is interwoven with the extensive diversification of many protein families. One key family is the ARF GTPases that act in eukaryote-specific processes, including membrane traffic, tubulin assembly, actin dynamics, and cilia-related functions. Unfortunately, our understanding of the evolution of this family is limited. Sampling an extensive set of available genome and transcriptome sequences, we have assembled a dataset of over 2,000 manually curated ARF family genes from 114 eukaryotic species, including many deeply diverged protist lineages, and carried out comprehensive molecular phylogenetic analyses. These reconstructed as many as 16 ARF family members present in the last eukaryotic common ancestor (LECA), nearly doubling the previously inferred ancient system complexity. Evidence for the wide occurrence and ancestral origin of Arf6, Arl13 and Arl16 is presented for the first time. Moreover, Arl17, Arl18 and SarB, newly described here, are absent from well-studied model organisms and as a result their function(s) remain unknown. Analyses of our dataset revealed a previously unsuspected diversity of membrane association modes and domain architectures within the ARF family. We detail the step-wise expansion of the ARF family in the metazoan lineage, including discovery of several new animal-specific family members. Delving back to its earliest evolution in eukaryotes, the resolved relationship observed between the ARF family paralogs sets boundaries for scenarios of vesicle coat origins during eukaryogenesis. Altogether, our work fundamentally broadens the understanding of the diversity and evolution of a protein family underpinning the structural and functional complexity of the eukaryote cells.

SignificanceARF Family GTPases are crucial regulations of a diversity of cellular compartments and processes and as such the extent of this system in eukaryotes reflects both cellular complexity in modern eukaryotes and its evolution. Strikingly, a comprehensive comparative genomic analysis of the protein family is lacking, leaving its recent and ancient evolution poorly resolved. We performed a comprehensive molecular evolutionary analysis, reconstructing a highly complex ARF family complement in the Last Eukaryotic Common Ancestor, including a number of paralogs never before identified as such, and we find resolved relationships between the paralogs. This work has implications for cellular evolution from eukaryogenesis to cellular complexity in metazoans.

sentences: Minimal DNA Electron Transfer Catalysts Switched by a Chaotropic Ion Here we demonstrate that a DNA nanodevice can perform switchable electron transfer. The nanodevice is comprised of two strands, one of which can be selectively switched between a G-quadruplex and duplex or single-stranded conformations. In the G-quadruplex state, it binds the cofactor hemin, enabling peroxidase activity. This switching ability arises from our discovery that perchlorate, a chaotropic Hofmeister ion, selectively destabilizes duplex over G-quadruplex DNA. By varying perchlorate concentration, we show that the device can be switched between states that do and do not catalyze electron transfer catalysis. State switching can be achieved in three ways: thermally, by dilution, or by concentration. In each case, when operated in the presence of the cofactor hemin, the device catalyzes electron transfer in only the G-quadruplex state.

sentences: Raabin-WBC: a large free access dataset of white blood cells from normal peripheral blood Accurate and early detection of peripheral white blood cell anomalies plays a crucial role in the evaluation of an individuals well-being. The emergence of new technologies such as artificial intelligence can be very effective in achieving this. In this regard, most of the state-of-the-art methods use deep neural networks. Data can significantly influence the performance and generalization power of machine learning approaches, especially deep neural networks. To that end, we collected a large free available dataset of white blood cells from normal peripheral blood samples called Raabin-WBC. Our dataset contains about 40000 white blood cells and artifacts (color spots). To reassure correct data, a significant number of cells were labeled by two experts, and the ground truth of nucleus and cytoplasm were extracted by experts for some cells (about 1145), as well. To provide the necessary diversity, various smears have been imaged. Hence, two different cameras and two different microscopes were used. The Raabin-WBC dataset can be used for different machine learning tasks such as classification, detection, segmentation, and localization. We also did some primary deep learning experiments on Raabin-WBC, and we showed how the generalization power of machine learning methods, especially deep neural networks, was affected by the mentioned diversity.

sentences: Global Constraints within the Developmental Program of the Drosophila Wing Organismal development is a complex process, involving a vast number of molecular constituents interacting on multiple spatio-temporal scales in the formation of intricate body structures. Despite this complexity, development is remarkably reproducible and displays tolerance to both genetic and environmental perturbations. This robust-ness implies the existence of hidden simplicities in developmental programs. Here, using the Drosophila wing as a model system, we develop a new quantitative strategy that enables a robust description of biologically salient phenotypic variation. Analyzing natural phenotypic variation across a highly outbred population, and variation generated by weak perturbations in genetic and environmental conditions, we observe a highly constrained set of wing phenotypes. Remarkably, the phenotypic variants can be described by a single integrated mode that corresponds to a non-intuitive combination of structural variations across the wing. This work demonstrates the presence of constraints that funnel environmental inputs and genetic variation into phenotypes stretched along a single axis in morphological space. Our results provide quantitative insights into the nature of robustness in complex forms while yet accommodating the potential for evolutionary variations. Methodologically, we introduce a general strategy for finding such invariances in other developmental contexts.

sentences: CoLoRd: Compressing long reads The costs of maintaining exabytes of data produced by sequencing experiments every year has become a major issue in todays genomics. In spite of the increasing popularity of the third generation sequencing, the existing algorithms for compressing long reads exhibit minor advantage over general purpose gzip. We present CoLoRd, an algorithm able to reduce 3rd generation sequencing data by an order of magnitude without affecting the accuracy of downstream analyzes.

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Complete pan-plastome sequences enable high resolution phylogenetic classification of sugar beet and closely related crop wild relatives BackgroundAs the major source of sugar in moderate climates, sugar-producing beets (Beta vulgaris subsp. vulgaris) have a high economic value. However, the low genetic diversity within cultivated beets requires introduction of new traits, for example to increase their tolerance and resistance attributes - traits that often reside in the crop wild relatives. For this, genetic information of wild beet relatives and their phylogenetic placements to each other are crucial. To answer this need, we sequenced and assembled the complete plastome sequences from a broad species spectrum across the beet genera Beta and Patellifolia, both embedded in the Betoideae (order Caryophyllales). This pan-plastome dataset was then used to determine the wild beet phylogeny in high-resolution.

ResultsWe sequenced the plastomes of 18 closely related accessions representing 11 species of the Betoideae subfamily and provided high-quality plastome assemblies which represent an important resource for further studies of beet wild relatives and the diverse plant order Caryophyllales. Their assembly sizes range from 149,723 bp (Beta vulgaris subsp. vulgaris) to 152,816 bp (Beta nana), with most variability in the intergenic sequences. Combining plastome-derived phylogenies with read-based treatments based on mitochondrial information, we were able to suggest a unified and highly confident phylogenetic placement of the investigated Betoideae species.

Our results show that the genus Beta can be divided into the two clearly separated sections Beta and Corollinae. Our analysis confirms the affiliation of B. nana with the other Corollinae species, and we argue against a separate placement in the Nanae section. Within the Patellifolia genus, the two diploid species Patellifolia procumbens and Patellifolia webbiana are, regarding the plastome sequences, genetically more similar to each other than to the tetraploid Patellifolia patellaris. Nevertheless, all three Patellifolia species are clearly separated.

ConclusionIn conclusion, our wild beet plastome assemblies represent a new resource to understand the molecular base of the beet germplasm. Despite large differences on the phenotypic level, our pan-plastome dataset is highly conserved. For the first time in beets, our whole plastome sequences overcome the low sequence variation in individual genes and provide the molecular backbone for highly resolved beet phylogenomics. Hence, our plastome sequencing strategy can also guide genomic approaches to unravel other closely related taxa.

sentences: Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. Oxidative modifications were precisely localized on specific residues using a timsTOF Pro mass spectrometer. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Data are available via ProteomeXchange with identifier PXD021621.

sentences: Chloroplast protein import determines plant proteostasis and retrograde signaling Proteins containing polyglutamine (polyQ) repeats are prone to aggregation and can lead to distinct human pathologies. For instance, Huntingtons disease is caused by an abnormal expansion of the polyQ stretch (> Q35) of Huntingtin (HTT) protein. However, plants express hundreds of proteins containing polyQ regions, but no pathologies arising from these factors have been reported to date. Here, we ask how plants maintain the proteostasis of polyQ-containing proteins, which are intrinsically enriched in the plant proteomes. To this end, we overexpressed an aggregation-prone fragment of human HTT (Q69) in plant cells. In contrast to invertebrate and mammalian transgenic models, we find that Arabidopsis thaliana plants suppress Q69 aggregation. This elevated proteostasis ability is mediated through the import and degradation of Q69 in chloroplasts. Conversely, inhibition of chloroplast protein import either genetically or pharmacologically reduces the capacity of plant cells to prevent Q69 aggregation. We find that Q69 interacts with the chloroplast stromal processing peptidase (SPP). Notably, expression of synthetic Arabidopsis SPP is sufficient to suppress aggregation of polyQ-expanded HTT in human cells. Beyond ectopically expressed Q69-HTT, endogenous polyQ-containing proteins also aggregate in Arabidopsis upon inhibition of chloroplast import. Among them, the plastid casein kinase 2 (pCK2), which contains a polyQ region next to the chloroplast targeting sequence motif, can also be localized into the nucleus. Upon inhibition of chloroplast import, pCK2 accumulates at higher levels in the nucleus and forms diamond-shaped amyloid-like fibrils surrounding the chloroplasts. These results indicate that the differential conformation and redistribution of pCK2 to the nucleus depends on chloroplast import efficiency, providing a role of polyQ repeats in chloroplast to nucleus communication (i.e. retrograde signaling). Together, our findings establish chloroplast protein import and proteases as determinants of polyQ proteostasis, with important implications for plant biology that can also lead to therapeutic approaches for human diseases that involve protein aggregation.

sentences: Microbowls with controlled concavity for accurate microscale mass spectrometry Patterned surfaces can enhance the sensitivity of laser desorption ionization mass spectrometry by segregating and concentrating analytes, but their fabrication can be challenging. Here, we describe a simple method to fabricate substrates patterned with micron-scale wells that yield more accurate and sensitive mass spectrometry measurements compared to flat surfaces. The wells can also concentrate and localize cells and beads for cell-based assays.

sentences: Anatomical and Palynological Investigation within flora of Family Asteraceae A comprehensive microscopic investigation of the leaf epidermis, as well as anatomical and palynological research of selected Asteraceae species from the flora of Havali (Kahutta) Azad Jammu and Kashmir (AJK) Pakistan, was carried out. This study includes 11 plant species comprising of Senecio jacobea, Leucanthemum vulgare, Halianthus annuus, Erigeron bonariensis, Achillea millifolium, Halianthus Linnaeus, Taraxacum officinale, Anaphalius nepalensis, Erigeron Canadensis, and Tagetes erecta. All the species studied were amphistomatic, with four different forms of stomata i.e tetracytic, anomocytic, anisocytic, and tricytic. The main stomatal type was tetracytic, followed by anomocytic. The abaxial epidermis has a higher stomatal density than the adaxial epidermis. Highest stomatal density was present Tagetes erecta while lowest is present in Conyza canadensis. In lower epidermis stomatal index was higher in Taraxacum officinale followed by Halianthus annuus and Tagetes erecta while in upper epidermis highest index was shown by Halianthus annuus. For this study species of family Acteraceae were properly collected. Furthermore, variation in pollen can be seen. List of palynomorph which includes family name, botanical name, local name, English name, flower colour, season and pollen description for the logical arrangement of these species. The logical ordering of these species was guided by dust characteristics such as form and pollen morphology. Palynological data has been found to be too big for taxonomists to calculate and make appropriate observations on their findings.

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Environmental changes dictate selection and nonspecific epistasis in an empirical phenotype-environment-fitness landscape Fitness landscapes, mappings of genotype/phenotype to their effects on fitness, are invaluable concepts in evolutionary biochemistry. Though widely discussed, measurements of phenotype-fitness landscapes in proteins remain scarce. Here, we quantify all single mutational effects on fitness and phenotype of VIM-2 {beta}-lactamase across a 64-fold range of ampicillin concentrations. We then construct a phenotype-fitness landscape that takes variations in environmental selection pressure into account. We found that a simple, empirical landscape accurately models the ~39,000 mutational data points, suggesting the evolution of VIM-2 can be predicted based on the selection environment. Our landscape provides new quantitative knowledge on the evolution of the {beta}-lactamases and proteins in general, particularly their evolutionary dynamics under sub-inhibitory antibiotic concentrations, as well as the mechanisms and environmental dependence of nonspecific epistasis.

One Sentence SummaryAn empirical fitness landscape discloses the environmental dependence of mutational effects in VIM-2 {beta}-lactamase.

sentences: A fast, general synteny detection engine The increasingly widespread availability of genomic data has created a growing need for fast, sensitive and scalable comparative analysis methods. A key aspect of comparative genomic analysis is the study of synteny, co-localized gene clusters shared among genomes due to descent from common ancestors. Synteny can provide unique insight into the origin, function, and evolution of genome architectures, but methods to identify syntenic patterns in genomic datasets are often inflexible and slow, and use diverse definitions of what counts as likely synteny. Moreover, the reliable identification of putatively syntenic regions (i.e., whether they are truly indicative of homology) with different lengths and signal to noise ratios can be difficult to quantify. Here, we present Mology, a fast, flexible, alignment-free, nonparametric method to detect regions of syntenic elements among genomes or other datasets. The core algorithm operates on consecutive, rank-ordered elements, which could be genes, operons, motifs, sequence fragments, or any other orderable element. It is agnostic to the physical distance between distinct elements and also to directionality and order within syntenic regions, although such considerations can be addressed post hoc. We describe the underlying statistical theory behind our analysis method, and employ a Monte Carlo approach to estimate the false positive rate and positive predictive values for putative syntenic regions. We also evaluate how varying amounts of noise affect recovery of true syntenic regions among Saccharomycetaceae yeast genomes with up to ~100 million years of divergence. We discuss different strategies for recursive application of our method on syntenic regions with sparser signal than considered here, as well as the general applicability of the core algorithm.

sentences: Face dissimilarity judgements are predicted by representational distance in deep neural networks and principal-component face space Human vision is attuned to the subtle differences between individual faces. Yet we lack a quantitative way of predicting how similar two face images look, or whether they appear to show the same person. Principal-components-based 3D morphable models are widely used to generate stimuli in face perception research. These models capture the distribution of real human faces in terms of dimensions of physical shape and texture. How well does a "face space" defined to model the distribution of faces as an isotropic Gaussian explain human face perception? We designed a behavioural task to collect dissimilarity and same/different identity judgements for 232 pairs of realistic faces. The stimuli densely sampled geometric relationships in a face space derived from principal components of 3D shape and texture (Basel Face Model, BFM). We then compared a wide range of models in their ability to predict the data, including the BFM from which faces were generated, a 2D morphable model derived from face photographs, and image-computable models of visual perception. Euclidean distance in the BFM explained both similarity and identity judgements surprisingly well. In a comparison against 14 alternative models, we found that BFM distance was competitive with representational distances in state-of-the-art image-computable deep neural networks (DNNs), including a novel DNN trained on BFM identities. Models describing the distribution of facial features across individuals are not only useful tools for stimulus generation. They also capture important information about how faces are perceived, suggesting that human face representations are tuned to the statistical distribution of faces.

sentences: Klebsiella pneumoniae hijacks the Toll-IL-1R protein SARM1 in a type I IFN-dependent manner to antagonize host immunity. Many bacterial pathogens antagonize host defence responses by translocating effector proteins into cells. It remains an open question how those pathogens not encoding effectors counteract anti-bacterial immunity. Here, we show that Klebsiella pneumoniae hijacks the evolutionary conserved innate immune protein SARM1 to control cell intrinsic immunity. Klebsiella exploits SARM1 to regulate negatively MyD88 and TRIF-governed inflammation, and the activation of the MAP kinases ERK and JNK. SARM1 is required for Klebsiella induction of IL10 by fine-tuning the p38-type I IFN axis. SARM1 inhibits the activation of Klebsiella-induced absent in melanoma 2 inflammasome to limit IL1{beta} production, suppressing further inflammation. Klebsiella exploits type I IFNs to induce SARM1 in a capsule and LPS O-polysaccharide-dependent manner via TLR4-TRAM-TRIF-IRF3-IFNAR1 pathway. Absence of SARM1 reduces the intracellular survival of K. pneumonaie in macrophages whereas sarm1 deficient mice control the infection. Altogether, our results illustrate a hitherto unknown anti-immunology strategy deployed by a human pathogen.

sentences: Antidepressant Sertraline Hydrochloride Inhibits the Growth of HER2+ AU565 Breast Cancer Cell Line through Induction of Apoptosis, and Arrest of Cell Cycle Breast cancer is one the most aggressive cancer worldwide, especially Pakistan due to limited therapeutic options. This study was conducted to repurpose the use of selective serotonin reuptake inhibitors (SSRIs), in the treatment of breast cancers, and merit to pursue drug re-positioning in oncology. Anti-proliferative activity of SSRIs, such as fluoxetine, paroxetine, and sertraline hydrochloride on the growth of AU-565, MCF-7, MDA-MB-231, and BT-474 breast cancer cell lines, along with human fibroblast BJ cells was determined in vitro. Changes in nuclear morphology (DAPI staining), and induction of apoptosis (flow cytometry, and caspase-3 activation) were also studied. Sertraline hydrochloride most effectively inhibited the growth of breast cancer cells in vitro. Therefore, pharmacological mechanism involved in sertraline mediated cell death was investigated in HER2+ AU565 cell line. Enhanced nuclear fragmentation, increased Annexin (+) cells, and caspase-3/7 activation indicated that sertraline-mediated cell death could be a result of BCl2-independent apoptosis as evidenced by expression of Bax, and BCl2 genes. Taken together, our results identified sertraline hydrochloride, as a potential candidate for the treatment of HER2-positive breast cancer. Even though these are in vitro results, this study opens great opportunity in the field of drug repurposing for the development of chemotherapeutic agents.

sentences: Chlamydia trachomatis suppresses host cell store-operated Ca2+ entry and inhibits NFAT/calcineurin signaling The obligate intracellular bacterium, Chlamydia trachomatis, replicates within a parasitophorous vacuole termed an inclusion. During development, host proteins critical for regulating intracellular calcium (Ca2+) homeostasis interact with the inclusion membrane. The inclusion membrane protein, MrcA, interacts with the inositol-trisphosphate receptor (IP3R), an ER cationic channel that conducts Ca2+. Stromal interaction molecule 1 (STIM1), an ER transmembrane protein important for regulating store-operated Ca2+ entry (SOCE), localizes to the inclusion membrane via an uncharacterized interaction. We therefore examined Ca2+ mobilization in C. trachomatis infected cells. Utilizing a variety of Ca2+ indicators to assess changes in cytosolic Ca2+ concentration, we demonstrate that C. trachomatis impairs host cell SOCE. Ca2+ regulates many cellular signaling pathways. We find that the SOCE-dependent NFAT/calcineurin signaling pathway is impaired in C. trachomatis L2 infected HeLa cells and likely has major implications on host cell physiology as it relates to C. trachomatis pathogenesis.

sentences: Acriflavine, a clinically aproved drug, inhibits SARS-CoV-2 and other betacoronaviruses SO_SCPLOWUMMARYC_SCPLOWThe COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identified acriflavine as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

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Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during egress in both wild-type RH and {Delta}plp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in {Delta}plp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin-induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.

Author SummaryToxoplasma gondii is a single celled intracellular parasite that infects many different animals, and it is thought to infect up to one third of the human population. This parasite must rupture out of its replicative compartment and the host cell to spread from one cell to another. Previous studies indicated that a decrease in pH occurs within the replicative compartment near the time of parasite exit from host cells, an event termed egress. However, it remained unknown whether the decrease in pH is directly tied to egress and, if so, what is responsible for the decrease in pH. Here we used a fluorescent reporter protein to directly measure pH within the replicative compartment during parasite egress. We found that pH decreases immediately prior to parasite egress and that this decrease is linked to parasite disruption of membranes. We also identified a family of transporters that release acidic products from parasite use of glucose for energy as contributing to the decrease in pH during egress. Our findings provide new insight that connects parasite glucose metabolism to acidification of its replicative compartment during egress from infected cells.

sentences: Combining LSD1 and JAK-STAT inhibition targets Down syndrome-associated myeloid leukemia at its core Children with Down syndrome (DS) are predisposed to developing megakaryoblastic leukemia (ML-DS) and often experience severe toxicities from chemotherapy, highlighting the need for targeted therapies with beneficial risk profiles. The genomic landscape of ML-DS is characterized by a combination of mutations in signaling pathway genes and epigenetic modifiers, while aberrant lysine specific demethylase 1 (LSD1) and JAK-STAT activation have both been implicated in leukemogenesis. Here, we demonstrate that combined LSD1 and JAK1/2 inhibition exerts synergistic anti-leukemic effects specifically in ML-DS, both in vitro and in patient derived xenografts in vivo. The JAK1/2 inhibitor ruxolitinib enhanced the LSD1 inhibitor-induced differentiation, proliferation arrest and apoptosis in patient-derived leukemic blasts. At the transcriptional level, the combination synergistically repressed gene expression signatures essential for cell division. We further observed an immunogenic gene expression pattern in the form of increased cytokine signaling, which - by sensitizing ML-DS blasts to the JAK-STAT signaling blockade induced by ruxolitinib - could explain the increased susceptibility of ML-DS blasts to combination therapy. Taken together, we establish combined LSD1 and JAK-STAT inhibition as an efficacious therapeutic regimen specifically designed to target important steps in ML-DS leukemogenesis, paving the way for targeted therapies in this entity.

sentences: Quantifying cell transitions in C. elegans with data-fitted landscape models Increasing interest has emerged in new mathematical approaches that simplify the study of complex differentiation processes by formalizing Waddingtons landscape metaphor. However, a rational method to build these landscape models remains an open problem. Building on pioneering work by Corson and Siggia (2012, 2017) we study vulval development in C. elegans by developing a framework based on Catastrophe Theory (CT) and approximate Bayesian computation (ABC) to build data-fitted landscape models. We first identify the candidate qualitative landscapes, and then use CT to build the simplest model consistent with the data, which we quantitatively fit using ABC. The resulting model suggests that the underlying mechanism is a quantifiable two-step decision controlled by EGF and Notch-Delta signals, where a non-vulval/vulval decision is followed by a bistable transition to the two vulval states. This new model fits a broad set of data and makes several novel predictions.

sentences: Mutagenic distinction between the receptor-binding and fusion subunits of the SARS-CoV-2 spike glycoprotein and its upshot We observe that a residue R of the spike glycoprotein of SARS-CoV-2 which has mutated in one or more of the current Variants of Concern or Interest or under Monitoring rarely participates in a backbone hydrogen bond if R lies in the S1 subunit and usually participates in one if R lies in the S2 subunit. A partial explanation for this based upon free energy is explored as a potentially general principle in the mutagenesis of viral glycoproteins. This observation could help target future vaccine cargos for the evolving coronavirus as well as more generally. A study of the Delta and Omicron variants suggests that Delta was an energetically necessary intermediary in the evolution from Wuhan-Hu-1 to Omicron.

sentences: Note: Updating the metadata of four misidentified samples in the DrosRTEC dataset This note details the consortiums rationale behind its decision to modify the metadata for putatively misidentified European samples in the DrosRTEC dataset. In brief, we use PCA on published datasets from North America and Europe to generate phylogeographic clusters reflective of worldwide D. melanogaster demography. We used this PCA to train a DAPC model in order the predict the group membership. Our results indicate that 4 out of 73 samples were misclassified and the metadata was updated accordingly. These samples are a spring-fall pair from Spain and Austria.

sentences: Ultrastructural analysis of dendritic spine necks reveals a continuum of spine morphologies Dendritic spines are membranous protrusions, with a bulbous head connected to the dendrite by a thin neck, and receive essentially all excitatory inputs in most mammalian neurons. Spines have a wide variety of morphologies that likely have a significant effect on their biochemical and electrical properties. The question of whether spines belong to distinct morphological or functional subtypes or constitute a continuum is still open. To discern this, it is important to measure spine necks objectively. Recent advances in electron microscopy enable automatic reconstructions of 3D spines with nanometer precision. Analyzing ultrastructural reconstructions from mouse neocortical neurons with computer vision algorithms, we demonstrate that the vast majority of spines can be rigorously separated into head and neck components. Analysis of the head and neck morphologies reveals a continuous distribution of parameters. The spine neck diameter, but not the neck length, was correlated with the head volume. Spines with larger head volumes often had a spine apparatus and pairs of spines in a post-synaptic cell contacted by the same axon had similar head volumes. Our data are consistent with a lack of morphological categories of spines and indicate that the morphologies of the spine neck and head are independently regulated. These results have repercussions for our understanding of the function of dendritic spines in neuronal circuits.

sentences: Frequent co-regulation of splicing and polyadenylation by RNA-binding proteins inferred with MAPP Maturation of eukaryotic pre-mRNAs via splicing, 3 end cleavage and polyadenylation is modulated across cell types and conditions by a variety of RNA-binding proteins (RBPs). Although over 1500 proteins are associated with RNAs in human cells, their binding motifs, targets and functions still remain to be elucidated, especially in the complex environment of human tissues and in the context of diseases. To overcome the lack of methods for systematic and automated detection of sequence motif-guided changes in pre-mRNA processing based on RNA sequencing (RNA-seq) data we have developed MAPP (Motif Activity on Pre-mRNA Processing). We demonstrate MAPPs functionality by applying it to RNA-seq data from 284 RBP knock-down experiments in the ENCODE project, from which MAPP not only infers position-dependent impact profiles of known regulators, but also reveals RBPs that modulate both the inclusion of cassette exons and the poly(A) site choice. Among these, the Polypyrimidine Tract Binding Protein 1 (PTBP1) has a similar activity in glioblastoma samples. This highlights the ability of MAPP to unveil global regulators of mRNA processing under physiological and pathological conditions.

sentences: On the generalizability of diffusion MRI signal representations across acquisition parameters, sequences and tissue types: chronicles of the MEMENTO challenge. Diffusion MRI (dMRI) has become an invaluable tool to assess the microstructural organization of brain tissue. Depending on the specific acquisition settings, the dMRI signal encodes specific properties of the underlying diffusion process. In the last two decades, several signal representations have been proposed to fit the dMRI signal and decode such properties. Most methods, however, are tested and developed on a limited amount of data, and their applicability to other acquisition schemes remains unknown. With this work, we aimed to shed light on the generalizability of existing dMRI signal representations to different diffusion encoding parameters and brain tissue types. To this end, we organized a community challenge - named MEMENTO, making available the same datasets for fair comparisons across algorithms and techniques. We considered two state-of-the-art diffusion datasets, including single-diffusion-encoding (SDE) spin-echo data from a human brain with over 3820 unique diffusion weightings (the MASSIVE dataset), and double (oscillating) diffusion encoding data (DDE/DODE) of a mouse brain including over 2520 unique data points. A subset of the data sampled in 5 different voxels was openly distributed, and the challenge participants were asked to predict the remaining part of the data. After one year, eight participant teams submitted a total of 80 signal fits. For each submission, we evaluated the mean squared error, the variance of the prediction error and the Bayesian information criteria. Most predictions predicted either multi-shell SDE data (37%) or DODE data (22%), followed by cartesian SDE data (19%) and DDE (18%). Most submissions predicted the signals measured with SDE remarkably well, with the exception of low and very strong diffusion weightings. The prediction of DDE and DODE data seemed more challenging, likely because none of the submissions explicitly accounted for diffusion time and frequency. Next to the choice of the model, decisions on fit procedure and hyperparameters play a major role in the prediction performance, highlighting the importance of optimizing and reporting such choices. This work is a community effort to highlight strength and limitations of the field at representing dMRI acquired with trending encoding schemes, gaining insights into how different models generalize to different tissue types and fiber configurations over a large range of diffusion encodings.

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'rbioacc': an R-package to analyse toxicokinetic data O_LI rbioacc is an  R-package dedicated to the analysis of experimental data collected from bioaccumulation tests during which organisms are exposed to a chemical (exposure phase) and then put into a clean media (depuration phase). Internal concentrations are regularly measured over time all along the experiment.
C_LIO_LI rbioacc provides ready-to-use functions to visualize and fully analyze such data. Under a Bayesian framework, this package fits a generic one-compartment toxicokinetic (TK) model automatically built from the data. It provides TK parameter estimates (appropriate uptake and elimination rates) and bioaccumulation metrics (e.g., BCF, BSAF, BMF). All parameter estimates, bioaccumulation metrics as well as predictions of internal concentrations into organisms are delivered with their uncertainty.
C_LIO_LIBioaccumulation metrics are primarily provided in support of environmental risk assessment, in full compliance with regulatory requirements required to approve marketing applications of chemical substances.
C_LIO_LIThis paper gives brief worked examples of the use of  rbioacc from data collected through standard bioaccumulation tests, and publicly available within the scientific literature. These examples constitute step-by-step user-guides to analyze any new data set, uploaded in the right format.
C_LI

sentences: A preclinical randomized multicenter trial of anti-IL-17A treatment for acute ischemic stroke Multiple consensus statements have called for preclinical randomized controlled trials (pRCT) to improve translation in stroke research. Here, we investigated the efficacy of IL-17A neutralizing antibodies in a multicentric pRCT using a murine stroke model. C57/Bl.6 mice were subjected to transient middle cerebral artery occlusion (tMCAO). Mice were randomly allocated (1:1). Either anti-IL-17A (500 {micro}g) or isotype antibody (500 {micro}g) were administered 1 h after tMCAO. Primary analysis of infarct volumes was done by MRI after three days. Secondary analysis included mortality, neurological score, neutrophil infiltration and the impact of the gut microbiome on treatment effects. Out of 136 mice, 109 mice were included in the analysis. Mixed model analysis revealed that the IL-17A neutralization significantly reduced infarct sizes (anti IL-17A: 61.77 {+/-} 31.04 mm3; IgG control: 75.66 {+/-} 34.79 mm3; p=0.01). Secondary outcome measures showed a decrease in mortality (Hazard Ratio=3.43, 95% CI = 1.157 - 10.18; p=0.04) and neutrophil invasion into ischemic cortices. There was no difference in the neurological score. The analysis of the gut microbiome showed significant differences between centers. Taken together, this is the first positive pRCT in an ischemia reperfusion model. It suggests IL-17A neutralization as a potential target in stroke.

sentences: NOTCH1 drives immune-escape mechanisms in B cell malignancies NOTCH1 is a recurrently mutated gene in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). Functional studies to investigate its role have been hampered by the inability to genetically manipulate primary human lymphoma cells, attributed to low transduction-efficacy and procedure-associated toxicity. To overcome these limitations, we have developed a novel method to retrovirally transfer genes into malignant human B cells. We generated isogenic human tumor cells from patients with CLL and MCL, differing only in their expression of NOTCH1. Our data demonstrate that NOTCH1 facilitates immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-{gamma} signaling. In addition, NOTCH1 causes silencing of the entire HLA-class II locus via suppression of the transcriptional co-activator CIITA. These NOTCH1-mediated immune escape mechanisms are associated with the expansion of CD4+ T cells in vivo, further contributing to the poor clinical outcome of NOTCH1-mutated CLL and MCL.

sentences: Axon Diameter Measurements using Diffusion MRI are Infeasible The feasibility of non-invasive axonal diameter quantification with diffusion MRI is a strongly debated topic due to the neuroscientific potential of such information and its relevance for the axonal signal transmission speed. It has been shown that under ideal conditions, the minimal diameter producing detectable signal decay is bigger than most human axons in the brain, even using the strongest currently available MRI systems. We show that resolving the simplest situations including multiple diameters is unfeasible even with diameters much bigger than the diameter limit. Additionally, the recently proposed effective diameter resulting from fitting a single value over a distribution is almost exclusively influenced by the biggest axons. We show how impractical this metric is for comparing different distributions. Overall, axon diameters currently cannot be quantified by diffusion MRI in any relevant way.

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Children with amblyaudia show less flexibility in auditory cortical entrainment to periodic non-speech sounds ObjectiveWe investigated auditory temporal processing in children with amblyaudia (AMB), a subtype of auditory processing disorder, via cortical neural entrainment.

Design and study samplesEvoked responses were recorded to click-trains at slow vs. fast (8.5 vs. 14.9/sec) rates in n=14 children with AMB and n=11 age-matched controls. Source and time-frequency analyses decomposed EEGs into oscillations (reflecting neural entrainment) stemming from the bilateral auditory cortex.

ResultsPhase-locking strength in AMB depended critically on the speed of auditory stimuli. In contrast to age-matched peers, AMB responses were largely insensitive to rate manipulations. This rate resistance was seen regardless of the ear of presentation and in both cortical hemispheres.

ConclusionChildren with AMB show a stark inflexibility in auditory cortical entrainment to rapid sounds. In addition to reduced capacity to integrate information between the ears, we identify more rigid tagging of external auditory stimuli. Our neurophysiological findings may account for certain temporal processing deficits commonly observed in AMB and related auditory processing disorders (APDs) behaviorally. More broadly, our findings may inform communication strategies and future rehabilitation programs; increasing the rate of stimuli above a normal (slow) speech rate is likely to make stimulus processing more challenging for individuals with AMB/APD.

sentences: Spinning Disk Multifocal Microscopy for Dynamic Arbitrarily Shaped Surface Imaging at Centimetre Scale and Micrometre Resolution The large-scale fluorescence microscopy has enabled the observation of dynamic physiological activities at the single cellular level across the mouse cortex, such as distributed neuronal population representations. However, video-rate high-resolution microscopy at sophisticated biological surfaces in nature keeps a challenging task for the tradeoff between the speed, resolution, and field of view. Here we propose Spinning Disk Multifocal Microscopy (SDiM) for arbitrarily shaped surfaces, which enables imaging at centimeter field-of-view, micrometer resolution and up to 30 frames per second across the depth range of 450 {micro}m. We apply this technique in various microscopic systems, including customized macroscopic systems and the Real-time Ultra-large-Scale imaging at High resolution macroscopy (RUSH), in both the reflective mode and the fluorescence mode, and in the study of cortex-wide single-neuron imaging and immune cell tracking. SDiM provides an opportunity for studying the cortex-wide multi-scale cellular interactions in biological tissues.

sentences: Temporally extended and reversible neuronal silencing with Aion Optogenetic silencing allows to reveal the necessity of selected neuronal populations for various neurophysiological functions ranging from specific behaviors to neuronal network activity. An ideal optogenetic silencing tool should be switchable between active and inactive states with precise timing while preserving its activity in the absence of light until switched to an inactive state. Although bistable anion-conducting channelrhodopsins (ACRs) were previously engineered to reach this goal, their conducting state life time was limited to only a few minutes and some ACRs were not fully switchable. Here we report Aion, a bistable ACR displaying a long-lasting open state with a spontaneous closing time constant close to 15 min. Moreover, Aion can be switched between the open and closed state with millisecond precision using blue and orange light, respectively. The long conducting state enables overnight silencing of neurons with minimal light exposure. We further generated trafficking-optimized versions of Aion, which show enhanced membrane localization and allow precisely timed, long-lasting all-optical control of nociceptive responses in larvae of Drosophila melanogaster.

sentences: Piperacillin/tazobactam resistant, cephalosporin susceptible Escherichia coli bloodstream infections are driven by multiple acquisition of resistance across diverse sequence types Resistance to piperacillin/tazobactam (TZP) in Escherichia coli has predominantly been associated with mechanisms that confer resistance to third generation cephalosporins. Recent reports have identified E. coli strains with phenotypic resistance to piperacillin/tazobactam but susceptibility to third generation cephalosporins (TZP-R/3GC-S). In this study we sought to determine the genetic diversity of this phenotype in E. coli (n = 58) isolated between 2014-2017 at a single tertiary hospital in Liverpool, UK, as well as the associated resistance mechanisms. We compare our findings to a UK-wide collection of invasive E. coli isolates (n = 1509) with publicly available phenotypic and genotypic data. These data sets included the TZP-R/3GC-S phenotype (n = 68), a piperacillin/tazobactam and third generation cephalosporin-susceptible (TZP-S/3GC-S, n = 1271) phenotypes. The TZP-R/3GC-S phenotype was displayed in a broad range of sequence types which was mirrored in the same phenotype from the UK-wide collection, and the overall diversity of invasive E. coli isolates. The TZP-R/3GC-S isolates contained a diverse range of plasmids, indicating multiple acquisition events of TZP resistance mechanisms rather than clonal expansion of a particular plasmid or sequence type. The putative resistance mechanisms were equally diverse, including hyperproduction of TEM-1, either via strong promoters or gene amplification, carriage of inhibitor resistant {beta}-lactamases, and an S133G b/aCTX-M-15 mutation detected for the first time in clinical isolates. Several of these mechanisms were present at a lower abundance in the TZP-S/3GC-S isolates from the UK-wide collection, but without the associated phenotypic resistance to TZP. Our findings highlight the complexity of this cryptic phenotype and the need for continued phenotypic monitoring, as well as further investigation to improve detection and prediction of the TZP-R/3GC-S phenotype from genomic data.

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Quantifying predator functional responses under field conditions reveals interactive effects of temperature and interference with sex and stage. O_LIPredator functional responses describe predator feeding rates and are a core component of predator-prey theory. Although originally defined as the relationship between predator feeding rates and prey densities, it is now well known that predator functional responses are shaped by a multitude of factors. Unfortunately, how these factors interact with one another remains unclear, as widely used laboratory methods for measuring functional responses are generally logistically constrained to examining a few factors simultaneously. Furthermore, it is also often unclear whether laboratory derived functional responses translate to field conditions.
C_LIO_LIOur goal was to apply an observational approach for measuring functional responses to understand how sex/stage differences, temperature, and predator interference interact to influence the functional response of zebra jumping spiders on midges under natural conditions.
C_LIO_LIWe used field feeding surveys of jumping spiders to infer spider functional responses. We applied a Bayesian model averaging approach to estimate differences among sexes and stages of jumping spiders in their feeding rates and their dependencies on midge densities, temperature, and predator interference.
C_LIO_LIWe find that females exhibit the steepest functional responses on midges, followed by juveniles, and then males, despite males being larger than juveniles. We also find that sexes and stages differ in the temperature dependence of their space clearance (aka attack) rates. We find little evidence of temperature dependence in females, whereas we find some evidence for an increase in space clearance rate at high temperatures in males and juveniles. Interference effects on feeding rates were asymmetric with little effect of interference on male feeding rates, and effects of interference on females and juveniles depending on the stage/sex from which the interference originates.
C_LIO_LIOur results illustrate the multidimensional nature of functional responses in natural settings and reveal how factors influencing functional responses can interact with one another through behavior and morphology. Further studies investigating the influence of multiple mechanisms on predator functional responses under field conditions will provide an increased understanding of the drivers of predator-prey interaction strengths and their consequences for communities and ecosystems.
C_LI

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MetaMutationalSigs: Comparison of mutational signature refitting results made easy SummaryThe analysis of mutational signatures is becoming increasingly common in cancer genetics, with emerging implications in cancer evolution, classification, treatment decision and prognosis. Recently, several packages have been developed for mutational signature analysis, with each using different methodology and yielding significantly different results. Because of the nontrivial differences in tools refitting results, researchers may desire to survey and compare the available tools, in order to objectively evaluate the results for their specific research question, such as which mutational signatures are prevalent in different cancer types. There is a need for a software that can aggregate results from different refitting packages and present them in a user-friendly way to facilitate effective comparison of mutational signatures.

Availability and implementationMetaMutationalSigs is implemented using R and python and is available for installation using Docker and available at: https://github.com/EESI/MetaMutationalSigs

ContactGail Rosen (glr26@drexel.edu).

Supplementary informationMore information about the package including test data and results are available at https://github.com/EESI/MetaMutationalSigs

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Extracellular matrix and spores impart structural hierarchy and compositional heterogeneity in Bacillus subtilis biofilms Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial in agricultural contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity; the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level, however, the molecular structures and elemental distribution across whole biofilms as well as possible linkages between them remain unexplored. Mapping X-ray diffraction (XRD) across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores and water. By simultaneously following the X-ray fluorescence (XRF) signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution and sporulation across whole intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.

Significance StatementBiofilms are multicellular soft microbial communities that are able to colonize synthetic surfaces as well as living organisms. To survive sudden environmental changes and efficiently share their common resources, cells in a biofilm divide into subgroups with distinct functions, leading to phenotypic heterogeneity. Here, by studying intact biofilms by synchrotron X-ray diffraction and fluorescence, we revealed correlations between biofilm macroscopic architectural heterogeneity and the spatio-temporal distribution of extracellular matrix, spores, water and metal ions. Our findings demonstrate that biofilm heterogeneity is not only affected by local genetic expression and cellular differentiation, but also by passive effects resulting from the physicochemical properties of the molecules secreted by the cells, leading to differential distribution of nutrients that propagates through macroscopic length scales.

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Myosin phosphatase target subunit 1 governs integrity of the embryonic gut epithelium to circumvent atresia development in medaka, Oryzias latipes BackgroundIntestinal atresia (IA) is a congenital gut obstruction caused by the absence of gut opening. Genetic factors are assumed to be critical for the development of IA, in addition to accidental vascular insufficiency or mechanical strangulation. However, the molecular mechanism underlying IA remains poorly understood.

ResultsIn this study, to better understand such a mechanism, we isolated a mutant of Oryzias latipes (the Japanese rice fish known as medaka) generated by N-ethyl-N-nitrosourea mutagenesis, in which IA develops during embryogenesis. Positional cloning identified a nonsense mutation in the myosin phosphatase target subunit 1 (mypt1) gene. Consistent with known Mypt1 function, the active form of myosin regulatory light chain (MRLC), which is essential for actomyosin contraction, and F-actin were ectopically accumulated in the intestinal epithelium of mutant embryos, whereas cell motility, proliferation and cell death were not substantially affected. Corresponding to the accumulation site of F-actin/active MRLC, the intestinal epithelium architecture was disordered. Importantly, blebbistatin, a non-muscle myosin inhibitor, attenuated the development of IA in the mutant.

ConclusionsCytoskeletal contraction governed by mypt1 regulates the integrity of the embryonic intestinal epithelium. This study provides new insight into our understanding of the mechanism of IA development in humans.

Bullet PointsO_LIMedaka mypt1 mutants display intestinal atresia.
C_LIO_LIThe level of phosphorylated myosin regulatory light chain was higher in mypt1 mutant embryos than in wild-type embryos.
C_LIO_LIThe levels of F-actin appeared elevated in the intestinal epithelium of mypt1 mutants.
C_LIO_LIBlebbistatin, an inhibitor of non-muscle myosin II, rescued intestinal atresia in mypt1 mutant embryos.
C_LI

sentences: Proprioceptive Re-alignment drives Implicit Sensorimotor Adaptation Multiple learning processes contribute to successful goal-directed actions in the face of changing physiological states, biomechanical constraints, and environmental contexts. Amongst these processes, implicit sensorimotor adaptation is of primary importance, ensuring that movements remain well-calibrated and accurate. A large body of work on reaching movements has emphasized how adaptation centers on an iterative process designed to minimize visual errors. The role of proprioception has been largely neglected, thought to play a passive role in which proprioception is affected by the visual error but does not directly contribute to adaptation. Here we present an alternative to this visuo-centric framework, arguing that that implicit adaptation can be understood as minimizing a proprioceptive error, the distance between the perceived hand position and its intended goal. We use this proprioceptive re-alignment model (PReMo) to re-examine many phenomena that have previously been interpreted in terms of learning from visual errors, as well as offer novel accounts for unexplained phenomena. We discuss potential challenges for this new perspective on implicit adaptation and outline a set of predictions for future experimentation.

sentences: Supplementary motor area contributions to rhythm perception Timing is everything, but our understanding of the neural mechanisms of timing remains limited, particularly for timing of sequences. Temporal sequences can be represented relative to a recurrent beat (beat-based or relative timing), or as a series of absolute durations (non-beat-based or absolute timing). Neuroimaging work suggests involvement of the basal ganglia, supplementary motor area (SMA), the premotor cortices, and the cerebellum in both beat- and non-beat-based timing. Here we examined how beat-based timing and non-beat-based sequence timing were affected by modulating excitability of the supplementary motor area, the right cerebellum, and the bilateral dorsal premotor cortices, using transcranial direct current stimulation (tDCS). Participants were subjected to a sham stimulation session, followed an active stimulation session where anodal or cathodal 2mA tDCS was applied to the SMA, right premotor cortex, left premotor cortex, or the cerebellum. During both sessions, participants discriminated changes in rhythms which differentially engage beat-based or non-beat-based timing. Rhythm discrimination performance was improved by increasing SMA excitability, and impaired by decreasing SMA excitability. This polarity-dependent effect on rhythm discrimination was absent for cerebellar or premotor cortex stimulation, suggesting a crucial role of the SMA and/or its functionally connected networks in rhythmic timing mechanisms.

sentences: MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1. Ribosomal protein S6 kinase 1 (S6K1), a major downstream effector molecule of mTORC1, regulates cell growth and proliferation by modulating protein translation and ribosome biogenesis. We have recently identified eIF4E as an intermediate in transducing signals from mTORC1 to S6K1 and further demonstrated that the role of mTORC1 is restricted to inducing eIF4E phosphorylation and interaction with S6K1.This interaction relieves S6K1 auto-inhibition and facilitates its hydrophobic motif (HM) phosphorylation and activation as a consequence. These observations underscore a possible involvement of mTORC1independent kinase in mediating HM phosphorylation. Here, we report mTORC2 as an in-vivo HM kinase of S6K1. We show that rapamycin resistant S6K1 truncation mutant {Delta}NH{Delta}CT, continues to display HM phosphorylation with selective sensitivity toward torin-1. We also show that HM phosphorylation of wildtype S6K1and {Delta}NH{Delta}CT depends on the presence of mTORC2 regulatory subunit-rictor. Furthermore, truncation mutagenesis analysis highlights the involvement of a conserved 19 amino acid stretch of S6K1 in mediating mTORC2 dependent HM phosphorylation. We finally show that deletion of this amino acid stretch leads to complete loss of HM phosphorylation regardless of the presence of functional TOS motif. Our data identifies mTORC2 as a physiological HM kinase that can activate S6K1 after its auto-inhibition is overcome by mTORC1. We, therefore, propose a novel mechanism for S6K1 regulation where mTOR complex 1 and 2 act in tandem to activate the enzyme.

sentences: The hippocampus as a sorter and reverberatory integrator of sensory inputs In entorhinal-hippocampal networks, the trisynaptic pathway, including the CA3 recurrent circuit, processes episodes of context and space1-3. Recurrent connectivity can generate reverberatory activity4-6, an intrinsic activity pattern of neurons that occurs after sensory inputs have ceased. However, the role of reverberatory activity in memory encoding remains incompletely understood. Here we demonstrate that in mice, synchrony between conditioned stimulus (CS) and unconditioned stimulus (US)-responsible cells occurs during the reverberatory phase, lasting for approximately 15 s, but not during CS and US inputs, in the CA1 and the reverberation is crucial for the linking of CS and US in the encoding of delay-type cued-fear memory. Retrieval-responsive cells developed primarily during the reverberatory phase. Mutant mice lacking N-methyl-D-aspartate receptors (NRs) in CA3 showed a cued-fear memory impairment and a decrease in synchronized reverberatory activities between CS- and US-responsive CA1 cells. Optogenetic CA3 silencing at the reverberatory phase during learning impaired cued-fear memory. Our findings suggest that reverberation recruits future retrieval-responsive cells via synchrony between CS- and US-responsive cells. The hippocampus uses reverberatory activity to link CS and US inputs, and avoid crosstalk during sensory inputs.

sentences: Dynamic interaction of BcsD subunit in type I bacterial cellulose synthase Cellulose is a promising biological material for supporting sustainable human life. This natural polymer is synthesized by cellulose synthase, a protein complex in the cell membrane. Cellulose synthase in bacteria is a hetero-subunit complex, and its subunit organization varies widely depending on the species. In the type I bacterial cellulose synthase complex, the BcsD (bacterial cellulose synthase D) protein is believed to play an important role in producing cellulose with long slender fiber morphology and high crystallinity, given the phenotype of the bcsD-deficient mutant and the specific existence of the type I operon in bacterial species synthesizing crystalline cellulose microfibrils such as Acetobacter. In this study, we successfully established a heterogeneously expressed Bcs protein in Escherichia coli as an experimental model and conducted biochemical studies for the BcsD protein and the other three major subunits of bacterial cellulose synthase, BcsA, BcsB, and BcsC. It has been shown that the BcsD protein interacts with the functionally required minimal subunits of the BcsAB complex, as well as the BcsC protein. Furthermore, it was shown that BcsD interacts with the BcsAB complex in two modes: direct protein-protein interactions and indirect interactions through the product cellulose. The former and latter modes represent the basal and active states of the type I bacterial cellulose synthase, respectively. This dynamic behavior of the BcsD protein is important for the type I bacterial cellulose synthase complex to regulate the crystallization process of cellulose.

sentences: Codon arrangement modulates MHC-I peptides presentation: implications for a SARS-CoV-2 peptide-based vaccine Among various vaccination strategies, peptide-based vaccines appear as excellent candidates because they are cheap to produce, are highly stable and harbor low toxicity. However, predicting which MHC-I Associated Peptide (MAP) will ultimately reach cell surface remains challenging, due to high false discovery rates. Previously, we demonstrated that synonymous codon arrangement (usage and placement) is predictive of, and modulates MAP presentation. Here, we apply CAMAP (Codon Arrangement MAP Predictor), the artificial neural network we used to unveil the role of codon arrangement in MAP presentation, to predict SARS-CoV MAPs. We report that experimentally identified SARS-CoV-1 and SARS-CoV-2 MAPs are associated with significantly higher CAMAP scores. Based on CAMAP scores and binding affinity, we identified 48 non-overlapping MAP candidates for a peptide-based vaccine, ensuring coverage for a high proportion of HLA haplotypes in the US population (>78%) and SARS-CoV-2 strains (detected in >98% of SARS-CoV-2 strains present in the GISAID database). Finally, we built an interactive web portal (https://www.epitopes.world) where researchers can freely explore CAMAP predictions for SARS-CoV-1/2 viruses. Collectively, we present an analysis framework that can be generalizable to empower the rapid identification of virus-specific MAPs, including in the context of an emergent virus, to help accelerate target identification for peptide-based vaccine designs that could be critical in safely attaining group immunity in the context of a global pandemic.

sentences: THz irradiation inhibits cell division by affecting actin dynamics Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.

sentences: Selective metal extraction by biologically produced siderophores during bioleaching from low-grade primary and secondary mineral resources Siderophores are a class of biogenic macromolecules that have high affinities for metals in the environment, thus could be exploited for alternate sustainable metal recovery technologies. Here, we assess the role of siderophores in the extraction and complexation of metals from an iron oxide-rich metallurgical processing residue and a low-grade primary Ni ore. Evaluation of the biological siderophore bioproduction by three pseudomonads, P. fluorescens, P. azotoformans and P. putida identified that P. putida could generate the highest siderophore yield, which was characterized as a hydroxamate and catecholate mixed-type pyoverdine PyoPpC-3B. Key physiochemical parameters involved in raw siderophore mediated metal extraction were identified using a fractional factorial design of experiments (DOE) and subsequently employed in purified PyoPpC-3B leaching experiments. Further targeted experiments with hydroxamate and catecholate functional analogues of PyoPpC-3B confirmed their marked ability to competitively or selectively leach and chelate hard metal ions, including Al(OH)4-, Mn2+ and Zn2+. Interestingly, complexation of Mn and Zn ions exceeded the natural affinity of pyoverdine for Fe3+, thus despite the low metal recoveries from the materials tested in this study, this work provides important new insights in siderophore-metal interactions.

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Genome-wide analyses of histone modifications and chromatin accessibility reveal the distinct genomic compartments in the Irish potato famine pathogen Phytophthora infestans Phytophthora infestans, the causal agent of potato late blight, is a devastating plant disease that leads to Irish potato famine and threatens world-wide food security. Despite the genome of P. infestans has provided fundamental resource for studying the aggressiveness of this pandemic pathogen, the epigenomes remain poorly understood. Here, utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS), we demonstrate post-translational modifications (PTM) at P. infestans core histone H3. The PTMs not only include these prevalent modifications in eukaryotes, and also some novel marks, such as H3K53me2 and H3K122me3. We focused on the trimethylations of H3K4, H3K9 and H3K27 and H3K36, and profiled P. infestans epigenomes employing Native Chromatin Immunoprecipitation followed by sequencing (N-ChIP-seq). In parallel, we mapped P. infestans chromomatin accessibility by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). We found that adaptive genomic compartments display significantly higher levels of H3K9me3 and H3K27me3, and are generally in condense chromatin. Interestingly, we observed that genes encoding virulence factors, such as effectors, are enriched in open chromatin regions that barely have the four histone modifications. With a combination of genomic, epigenomic, transcriptomic strategies, our study illustrates the epigenetic states in P. infestans, which will help to study genomic functions and regulations in this pathogen.

Author summaryEpigenetics play an important role in various biological processes of eukaryotes, including pathogenicity of plant pathogens. However, the epigenetic landscapes are marginally known in oomycetes that are fungal-like organisms and comprise lots of destructive plant pathogens. In this study, using the Irish potato famine pathogen Phytophthora infestans as a model, we conducted genome-wide studies of histone post modifications and chromatin accessibility, and demonstrate the relationship of gene expression and evolution with the epigenetic marks. We found that one of the most important classes of virulence proteins, effectors, are enriched in open chromatin regions that barely have eu- and hetero-chromatic marks. This study provides an overview of the oomycete epigenetic atlas, and advances our understanding of the regulation of virulence factors in plant pathogens.

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Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.

Author SummaryEntamoeba histolytica is a globally important pathogen that is dramatically understudied. One of the major limitations of this organism is its challenging genome. RNAi is the state-of-the-art tool for genetic manipulation in E. histolytica, though the RNAi pathway has several noncanonical features. Here, we harnessed the RNAi pathway to enable RNAi-based forward genetics for the first time in this organism. We validated the RNAi library by performing the first E. histolytica RNAi screen and identified slow growth mutants. We showed that independently-generated mutants also exhibited slow growth phenotypes, and we characterized protein localization and domains for some of the identified slow growth genes. The RNAi library that we constructed enables modern, quantitative Illumina deep sequencing analysis to identify mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify full-length gene fragments inferred from read mapping. Our approach differs from previous approaches for analysis of RNAi screens, and it better represents the actual DNA fragments and their quantities. This study dramatically improves the tractability of this important pathogen. Moreover, the strategies behind this RNAi library, and its analysis, are novel, and can be applied to other organisms.

Author ContributionsA.B. designed and performed the experiments, except fragmentation of gDNA, immunofluorescence, and growth curves with expression of full-length or truncated proteins. R.L.S. performed SG1 and SG2 immunofluorescence experiments. M.C.R. performed growth curves with expression of full-length or truncated proteins. W.H. generated transfectants expressing full-length SG2. C.G.B. fragmented gDNA and processed the fragments to enable subsequent plasmid library construction. T.S.Y.T. and H.W.M. performed Gal/GalNAc lectin immunofluorescence experiments. S.S.H. designed the analysis strategy for Illumina sequencing data, and analyzed all Illumina sequencing data. M.L.S. oversaw the Illumina analysis approach. K.S.R. performed growth curves, conceived of the overall approach and oversaw the design and analysis of the experiments. A.B., S.S.H., and K.S.R. wrote the manuscript.

sentences: A Reproducibility Analysis-based Statistical Framework for Residue-Residue Evolutionary Coupling Detection Direct coupling analysis (DCA) has been widely used to infer evolutionary coupled residue pairs from the multiple sequence alignment (MSA) of homologous sequences. However, effectively selecting residue pairs with significant evolutionary couplings according to the result of DCA is a non-trivial task. In this study, we developed a general statistical framework for significant evolutionary coupling detection, referred to as IDR-DCA, which is based on reproducibility analysis of the coupling scores obtained from DCA on manually created MSA replicates. IDR-DCA was applied to select residue pairs for contact prediction for monomeric proteins, protein-protein interactions and monomeric RNAs, in which three different versions of DCA were applied. We demonstrated that with the application of IDR-DCA, the residue pairs selected using a universal threshold always yielded stable performance for contact prediction. Comparing with the application of carefully tuned coupling score cutoffs, IDR-DCA always showed better performance. The robustness of IDR-DCA was also supported through the MSA down-sampling analysis. We further demonstrated the effectiveness of applying constraints obtained from residue pairs selected by IDR-DCA to assist RNA secondary structure prediction.

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Quantitative modeling of regular retinal microglia distribution 1Microglia are resident immune cells in the central nervous system (CNS), showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglias morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated.

First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell-cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia.

Author summaryMicroglia are resident immune cells in the central nervous system. It is known that the microglia cells show a regular distribution, but the mechanism underlying the regular distribution remains to be elucidated. In the present study, we quantitatively assayed the regularity of the microglia soma distribution using the image processing technique. Next, we formulated a mathematical model of cell distribution that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters using organ culture. As a result, we obtained parameters for cell migration, cell process dynamics, and extracellular ATP dynamics. Then we undertook numerical simulation of the mathematical model using the parameters obtained by the experiments. The resulting model simulations showed that direct cell-cell contact is the most important factor in generating regular spacing. Finally, we screened possible molecular pathways involved in the regular spacing of microglia, which confirmed the validity of the model.

sentences: Quantitative Mapping of Human Hair Graying and Reversal in Relation to Life Stress Hair graying is a universal hallmark of aging believed to be linked to psychological stress. Here we develop a novel approach to quantitatively profile natural graying events along individual human hair shafts, resulting in a quantifiable physical timescale of hair pigmentation patterns (HPPs). Using this approach, we quantify rare events of white/gray hairs that naturally regain pigmentation within days to weeks, thereby quantitatively defining the reversibility of graying in healthy, unmedicated individuals. Proteomic analysis shows that graying is marked by the upregulation of proteins related to energy metabolism, mitochondria, and antioxidant defenses. Combining hair pigmentation profiling and proteomics at the single hair level, we also report hair graying and its reversal occurring in parallel with behavioral and psychological stressors. A computational simulation suggests a threshold-based mechanism for the temporary reversibility of graying. Quantitatively mapping HPPs in humans provides an opportunity to longitudinally examine the influence of life exposures on biological aging.

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Virtual Parts Repository 2: Model-driven design of genetic regulatory circuits Engineering genetic regulatory circuits is key to the creation of biological applications that are responsive to environmental changes. Computational models can assist in understanding especially large and complex circuits where manual analysis is infeasible, permitting a model-driven design process. However, there are still few tools that offer the ability to simulate the system under design. One of the reasons for this is the lack of accessible model repositories or libraries that cater for the modular composition of models of synthetic systems that do not yet exist in nature. Here, we present the Virtual Parts Repository 2, a resource to facilitate the model-driven design of genetic regulatory circuits, which provides reusable, modular and composable models. The repository is service-oriented and can be utilized by design tools in computational workflows. Designs provided in Synthetic Biology Open Language documents are used to derive system-scale and hierarchical Systems Biology Markup Language models. We also present a rule-based modeling abstraction based on reaction networks to facilitate scalable and modular modeling of complex and large designs. This modeling abstraction incorporates design patterns such as roadblocking, distributed deployment of genetic circuits using plasmids and cellular resource dependency. The computational resources and the modeling abstraction presented in this paper allow computational design tools to take advantage of computational simulations and ultimately help facilitate more predictable applications.

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sentences: Comparative metabolomics of fruits and leaves in a hyperdiverse lineage suggests fruits are a key incubator of phytochemical diversification Interactions between plants and leaf herbivores have long been implicated as the major driver of plant secondary metabolite diversity. However, other plant-animal interactions, such as those between fruits and frugivores, may also be involved in phytochemical diversification. Using 12 species of Piper, we conducted untargeted metabolomics and molecular networking with extracts of fruits and leaves. We evaluated organ-specific secondary metabolite composition and compared multiple dimensions of phytochemical diversity across organs, including richness, structural complexity, and variability across samples at multiple scales within and across species. Plant organ identity significantly influenced secondary metabolite composition, both independent of and in interaction with species identity. Leaves and fruit shared a majority of compounds, but fruits contained more unique compounds and had higher total estimated chemical richness. While organ-level chemical richness and structural complexity varied substantially across species, fruit diversity exceeded leaf diversity in more species than the reverse. Furthermore, the variance in chemical composition across samples was higher for fruits than leaves. By documenting a broad pattern of high phytochemical diversity in fruits relative to leaves, this study lays groundwork for incorporating fruit into a comprehensive and integrative understanding of the ecological and evolutionary factors shaping secondary metabolite composition at the whole-plant level.

sentences: Dopamine and serotonin interplay for valence-based spatial learning Dopamine and serotonin are important modulators of synaptic plasticity and their action has been linked to our ability to learn the positive or negative outcomes or valence learning. In the hippocampus, both neuromodulators affect long-term synaptic plasticity but play different roles in the encoding of uncertainty or predicted reward. Here, we examine the differential role of these modulators on learning speed and cognitive flexibility in a navigational model. We compare two reward-modulated spike time-dependent plasticity (R-STDP) learning rules to describe the action of these neuromodulators. Our results show that the interplay of dopamine (DA) and serotonin (5-HT) improves overall learning performance and can explain experimentally reported differences in spatial task performance. Furthermore, this system allows us to make predictions regarding spatial reversal learning.

sentences: Implicit versus explicit timing - separate or shared mechanisms? AO_SCPLOWBSTRACTC_SCPLOWTime implicitly shapes cognition, but time is also explicitly represented, for instance in the form of durations. Parsimoniously, the brain could use the same mechanisms for implicit and explicit timing. Yet, the evidence has been equivocal, revealing both joint versus separate signatures of timing. Here, we directly compared implicit and explicit timing using magnetoencephalography, whose temporal resolution allows investigating the different stages of the timing processes. Implicit temporal predictability was induced in an auditory paradigm by a manipulation of the foreperiod. Participants received two consecutive task instructions: discriminate pitch (indirect measure of implicit timing) or duration (direct measure of explicit timing). The results show that the human brain efficiently extracts implicit temporal statistics of sensory environments, to enhance the behavioral and neural responses to auditory stimuli, but that those temporal predictions did not improve explicit timing. In both tasks, attentional orienting in time during predictive foreperiods was indexed by an increase in alpha power over visual and parietal areas. Furthermore, pre-target induced beta power in sensorimotor and parietal areas increased during implicit compared to explicit timing, in line with the suggested role for beta oscillations in temporal prediction. Interestingly, no distinct neural dynamics emerged when participants explicitly paid attention to time, compared to implicit timing. Our work thus indicates that implicit timing shapes the behavioral and sensory response in an automatic way, and is reflected in oscillatory neural dynamics, while the translation of implicit temporal statistics to explicit durations remains somewhat inconclusive, possibly due to the more abstract nature of this task.

sentences: Large-scale societal dynamics are reflected in human mood and brain The stock market is a bellwether of socio-economic changes that may directly affect individual well-being. Using large-scale UK-biobank data generated over 14 years, we applied specification curve analysis to rigorously identify significant associations between the local stock market index (FTSE100) and 479,791 UK residents mood, as well as their alcohol intake and blood pressure adjusting the results for a large number of potential confounders, including age, sex, linear and non-linear effects of time, research site, other stock market indexes. Furthermore, we found similar associations between FTSE100 and volumetric measures of affective brain regions in a subsample (n=39,755; measurements performed over 5.5 years), which were particularly strong around phase transitions characterized by maximum volatility in the market. The main findings did not depend on applied effect-size estimation criteria (linear methods or mutual information criterion) and were replicated in two independent US-based studies (Parkinsons Progression Markers Initiative; n=424; performed over 2,5 years and MyConnectome; n=1; 81 measurements over 1,5 years). Our results suggest that phase transitions in the society, indexed by stock market, exhibit close relationships with human mood, health and the affective brain from an individual to population level.

sentences: Subjectivity of time perception alters choice preference for future rewards through fronto-striatal value signal dynamics Our preference for a reward depends on the time of delay for its delivery. Here we show that changes in the external environment can manipulate the perceived duration of time, which alters the formation of the choice preference through value signals in the cortical and subcortical brain regions. Humans anticipated a real liquid reward delayed by tens of seconds, during which colors of visually presented panels gradually changed. The color-change delay was perceived as shorter than a control color-constant delay. Interestingly, participants with greater perceptual bias of the delay showed stronger preference for rewards with the color-change delay. The ventrolateral prefrontal cortex (vlPFC) and ventral striatum (VS) showed dynamic neural signatures of value components that were modulated by subjectively perceived duration. Crucially, these effects were specifically observed while a future reward was anticipated, and moreover, the vlPFC activity was weaker in participants with greater bias in the duration perception. These results demonstrate that subjective time experience leads to biased choice preference of delayed rewards, which is regulated by dynamic value signals of future rewards and accurate perception of the external world in the vlPFC-VS systems.

sentences: The Borrelia burgdorferi adenylyl cyclase, CyaB, is important for virulence factor production and mammalian infection Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3 end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full length SR0623 to evaluate the influence on transcriptional and post-transcriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.

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Progesterone supplementation in mice leads to microbiome alterations and weight gain in a sex-specific manner BackgroundProgesterone is a steroid hormone produced by the ovaries, involved in pregnancy progression and necessary for successful gestation. We have previously shown that progesterone affects gut microbiota composition and leads to increased relative abundance of Bifidobacterium.

ResultsIn non-pregnant female GF mice, levels of progesterone were significantly higher than in SPF mice of the same status. However, no significant differences were observed between GF and SPF males. Females treated with progesterone gained more weight than females treated with a placebo. In contrast to female mice, males treated with progesterone did not gain significantly more weight than males treated with a placebo. Progesterone supplementation led to microbial changes in females but not in males (16S rRNA sequencing). Accordingly, the weight gain observed in female mice treated with progesterone was fully transferable to both male and female germ-free mice via fecal transplantation.

ConclusionsWe demonstrate that bacteria play a role in regulating progesterone levels in a female-specific manner. Furthermore, weight gain and metabolic changes associated with progesterone may be mediated by the gut microbiota.

sentences: Extracellular vesicles induce aggressive phenotype of luminal breast cancer cells by PKM2 phosphorylation The aerobic glycolysis is a hallmark of cancer glucose metabolism. Several studies have suggested that cancer-derived extracellular vesicles (EVs) can modulate glucose metabolism in adjacent cells and promote disease progression. Here we suggest that EVs originated from cancer cell with highly glycolytic activity can modulate glucose metabolism in the recipient cancer cells with relative low glycolytic activity, and further induce cell proliferation. Two types of breast cancer cell lines with different levels of glycolytic activity, MDA-MB-231 of a claudin low-type breast cancer cell and MCF7 of luminal type breast cancer cell, were selected and co-cultured using indirect co-culture system such as transwell system or microfluidic system. Glucose uptake of the recipient MCF7 cells was markedly increased after co-culture with MDA-MB-231 cells. MCF7 cells after co-culture with MDA-MB-231-tdTomato cells represented multiple tdTomato signal inside the cell, which proved that EVs originated from MDA-MB-231-tdTomato were transferred to MCF7 cell. In addition, serine phosphorylation of PKM2 necessary for tumorigenesis was highly activated, and tyrosine phosphorylation of PKM2 suggesting activated aerobic glycolysis was also increased in the co-cultured MCF7 cells. Proteomic profiling of the co-cultured MCF7 cells revealed the proliferation and dedifferentiation of MCF7 cells, and further confirmed epithelial-mesenchymal transition (EMT), which is a key phenomenon for cancer metastasis. In the transcriptomic analysis, glycolysis increased in co-cultured MCF7 cells, and the component analysis of genes associated with glycolysis revealed that the next major component after cytoplasm was extracellular exosome. Proteomic analysis of EVs revealed that there were important proteins in the EV such as EGFR, ERBB2 and MAPK for phosphorylating PKM2. This phenomenon suggests the potential for aggressive cancer cells to affect other cancer cells through EV mediators.

sentences: LRP-1 Commands Two Parallel Signalling Mechanisms to Support Constitutive Tumour Cell Motility in the Absence of Blood Supply Tumour cells often face the stress of ischemic (nutrient paucity and hypoxia) environment and must act self-sufficient to migrate toward the nearest blood supply or die. The mechanism that supports the constitutive motility of tumour cells under stress is poorly understood. We and others have previously shown that the low-density lipoprotein receptor-related protein 1 (LRP-1) plays a critical role in tumour cell migration and invasion in vitro and tumour formation in mice. Herein we show that depletion of LRP-1 completely abolishes the self-supported and serum-independent tumour cell motility. More intriguingly, we demonstrate that LRP-1 commands the full tumour cell motility by connecting with two independent cell surface signalling pathways. First, LRP-1 mediates secreted Hsp90 signalling via the "Hsp90 > LRP-1 receptor autocrine loop" for a half of tumour cell motility. Second, LRP1 stabilizes constitutively activated EGFR signalling that contributes the other half of tumour cell motility. Only combined inhibitions of the secreted Hsp90 autocrine and the EGFR signalling reproduces the effect of LRP1 down-regulation on constitutive tumour cell motility. This study reveals a novel mechanism of how tumour cells migrate in the absence of blood support.

sentences: Unravelling Form and Function: Improved function of engineered cardiac tissue through extra-cellular anisotropy Cardiac tissue engineering is a promising therapeutic option for myocardial repair after injury, however, so far engineered heart patches have shown limited translational utility due to poor electrical integration and tissue contractility. Emerging research suggests that scaffolds that recapitulate the three-dimensional structure of the native myocardium improve physiological function. Complex scaffold fabrication remains a technical challenge and the isolated impact of scaffold architecture on tissue function and cellular physiology is poorly understood. Here, we provided a direct comparison between isotropic and aligned collagen scaffold morphologies where all confounding physio-mechanical features, such as strut wall thickness and surface roughness are conserved. This enabled the independent and systematic assessment of the effects of pore macro-architecture on global tissue function and cellular maturation. We seeded our scaffolds with embryonic stem cell derived cardiomyocytes (hESC-CM) and measured tissue function through calcium signal transduction and dynamic contractile strain. The aligned tissue constructs facilitated improved signalling synchronicity and directional contractility. We further examined the influence of scaffold macrostructure on intercellular organization and intracellular development. Cells on aligned constructs conformed to the orientation of the scaffold macro-structure and were found to have phenotypic and genetic markers of increased maturity. Our results isolate the influence of scaffold macro-structure on engineered tissue function at multiple length scales. These findings inform the design of optimized cardiac tissue and expand the potential for engineered tissue in regenerative and model medical systems by reducing the gaps in tissue functionality that limit their utility.

sentences: Effects of kinship correction on inflation of genetic interaction statistics in commonly used mouse populations It is well understood that variation in relatedness among individuals, or kinship, can lead to false genetic associations. Multiple methods have been developed to adjust for kinship while maintaining power to detect true associations. However, relatively unstudied, are the effects of kinship on genetic interaction test statistics. Here we performed a survey of kinship effects on studies of six commonly used mouse populations. We measured inflation of main effect test statistics, genetic interaction test statistics, and interaction test statistics reparametrized by the Combined Analysis of Pleiotropy and Epistasis (CAPE). We also performed linear mixed model (LMM) kinship corrections using two types of kinship matrix: an overall kinship matrix calculated from the full set of genotyped markers, and a reduced kinship matrix, which left out markers on the chromosome(s) being tested. We found that test statistic inflation varied across populations and was driven largely by linkage disequilibrium. In contrast, there was no observable inflation in the genetic interaction test statistics. CAPE statistics were inflated at a level in between that of the main effects and the interaction effects. The overall kinship matrix overcorrected the inflation of main effect statistics relative to the reduced kinship matrix. The two types of kinship matrices had similar effects on the interaction statistics and CAPE statistics, although the overall kinship matrix trended toward a more severe correction. In conclusion, we recommend using a LMM kinship correction for both main effects and genetic interactions and further recommend that the kinship matrix be calculated from a reduced set of markers in which the chromosomes being tested are omitted from the calculation. This is particularly important in populations with substantial population structure, such as recombinant inbred lines in which genomic replicates are used.

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A unified model for the biogenesis of mitochondrial outer membrane multi-span proteins The mitochondrial outer membrane (MOM) harbors proteins that traverse the membrane via several helical segments, so-called multi-span proteins. Two contradicting mechanisms were suggested to describe their integration into the MOM. The first proposes that the mitochondrial import (MIM) complex facilitates this process and functions as an insertase, whereas the second suggests that such proteins can integrate into the lipid phase without the assistance of import factors in a process that is enhanced by phosphatidic acid. To resolve this discrepancy and obtain new insights on the biogenesis of these proteins, we addressed this issue using yeast mitochondria and the multi-span protein Om14. Testing different truncation variants, we show that only the full-length protein contains all the required information that assure targeting specificity. Employing a specific insertion assay and several single and double deletion strains, we show that neither the import receptor Tom70 nor any other protein with a cytosolically exposed domain have a crucial contribution to the biogenesis process. We further demonstrate that Mim1 and Porin are required for optimal membrane integration of Om14 but none of them is absolutely required. Unfolding of the newly synthesized protein, its optimal hydrophobicity, as well as higher fluidity of the membrane dramatically enhanced the import capacity of Om14. Collectively, our findings suggest that MOM multi-span proteins can follow different biogenesis pathways in which proteinaceous elements and membrane behavior contribute to a variable extent to the combined efficiency.

SummaryZhou et al. provide new insights to the biogenesis of mitochondrial outer membrane proteins. They demonstrate that such proteins can follow various routes where both proteinaceous elements and membrane behavior regulate the efficiency and specificity of this process.

sentences: Widespread translational control regulates retinal development in mouse Retinal development is tightly regulated to ensure the generation of appropriate cell types and the assembly of functional neuronal circuitry. Despite remarkable advances that have been made in understanding the regulation of gene expression during retinal development, how translational regulation guides retinogenesis is less understood. Here, we conduct a comprehensive translatome and transcriptome survey to the mouse retinogenesis from the embryonic to the adult stages. We discover thousands of genes that have dynamic changes at the translational level and pervasive translational regulation in a developmental stage-specific manner with specific biological functions. We further identify genes whose translational efficiencies are frequently controlled by changing usage in the upstream open reading frames during retinal development. These genes are enriched for biological functions highly important to neurons, such as neuron projection organization and microtubule-based protein transport. Surprisingly, we discover hundreds of previously uncharacterized micropeptides, translated from putative long non-coding RNAs and circular RNAs. We validate their protein products in vitro and in vivo and demonstrate their potentials in regulating retinal development. Together, our study presents a rich and complex landscape of translational regulation and provides novel insights into their roles during retinogenesis.

sentences: Beyond fear centers: a distributed fMRI-based neuromarker for the subjective experience of fear The specific neural systems underlying the subjective feeling of fear remain vigorously debated in affective neuroscience. Here, we combined functional MRI with machine learning to identify and evaluate a sensitive and generalizable neural signature predictive of the momentary self-reported subjective fear experience across discovery (n=67), validation (n=20) and generalization (n=31) cohorts. We systematically demonstrate that accurate fear prediction crucially requires distributed brain systems, with important contributions from cortical (e.g., prefrontal, midcingulate and insular cortices) and subcortical (e.g., thalamus, periaqueductal gray, basal forebrain and amygdala) regions. We further demonstrate that the neural representation of subjective fear is distinguishable from the representation of conditioned threat and general negative affect. Overall, our findings suggest that subjective fear, which exhibits distinct neural representation with some other aversive states, is encoded in distributed systems rather than isolated  fear centers. This signature provides a neuromarker for monitoring fear-related neuropathology and evaluating novel treatments targeting pathological fear.

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The homologous recombination complex of the Rad51 paralogs Rad55-Rad57 avoids translesion DNA polymerase recruitment and counterbalances mutagenesis induced by UV radiation The bypass of DNA lesions that block replicative polymerases during DNA replication relies on DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway relies on specialized DNA polymerases that incorporate nucleotides in front of base lesions, potentially inducing mutagenesis. Two error-free pathways can bypass the lesions: the template switching pathway, which uses the sister chromatid as a template, and the homologous recombination pathway (HR), which also can use the homologous chromosome as template. The balance between error-prone and error-free pathways controls the mutagenesis level. Therefore, it is crucial to precisely characterize factors that influence the pathway choice to better understand genetic stability at replication forks. In yeast, the complex formed by the Rad51 paralogs Rad55 and Rad57 promotes HR and template-switching at stalled replication forks. At DNA double-strand breaks (DSBs), this complex promotes Rad51 filament formation and stability, notably by counteracting the Srs2 anti-recombinase. To explore the role of the Rad55-Rad57 complex in error-free pathways, we monitored the genetic interactions between Rad55-Rad57, the translesion polymerases Pol{zeta} or Pol{eta}, and Srs2 following UV radiation that induces mostly single-strand DNA gaps. We found that the Rad55-Rad57 complex was involved in three ways. First, it protects Rad51 filaments from Srs2, as it does at DSBs. Second, it promotes Rad51 filament stability independently of Srs2. Finally, we observed that UV-induced HR is almost abolished in Rad55-Rad57 deficient cells, and is partially restored upon Pol{zeta} or Pol{eta} depletion. Hence, we propose that the Rad55-Rad57 complex is essential to promote Rad51 filament stability on single-strand DNA gaps, notably to counteract the error-prone TLS polymerases and mutagenesis.

Author summaryProcessive and accurate DNA polymerases replicate genomic DNA during the S phase of each cell cycle. DNA base lesions on template DNA block these polymerases and result in an accumulation of single-stranded DNA gaps behind moving replication forks. These gaps are filled-in by error-prone and error-free pathways. In this work, we show that the complex made by the Rad51 paralogs Rad55 and Rad57 is essential for the error-free homologous recombination gap-filling pathway when DNA replication is stalled by UV-induced DNA lesions, but not for DNA double strand break repair. Interestingly, we found that homologous recombination is efficiently outcompeted by error-prone translesion DNA polymerases in Rad55-Rad57-deficient cells. We propose that the Rad55-Rad57 complex is essential for Rad51 filament stability at UV-induced DNA gaps to promote efficient error-free homologous recombination. Furthermore, our study in yeast predicts that inhibitors of error-prone DNA polymerases might selectively target cancer cells in which RAD51 paralogs are mutated.

sentences: The pseudobranch of jawed vertebrates is a mandibular arch-derived gill The pseudobranch is a gill-like epithelial elaboration that sits behind the jaw of most fishes. This structure was classically regarded as a vestige of the ancestral gill-arch like condition of the gnathostome jaw. However, more recently, hypotheses of jaw evolution by transformation of a gill arch have been challenged, and the pseudobranch has alternatively been considered a specialised derivative of the second (hyoid) pharyngeal arch. Here, we demonstrate by cell lineage tracing in a cartilaginous fish, the skate (Leucoraja erinacea), that the pseudobranch does, in fact, derive from the mandibular arch, and that it shares gene expression features and cell types with gills. We also show that the mandibular arch pseudobranch is supported by a spiracular cartilage that is patterned by a shh-expressing epithelial signalling centre. This closely parallels the condition seen in the gill arches, where cartilaginous appendages called branchial rays supporting the respiratory lamellae of the gills are patterned by a shh-expressing gill arch epithelial ridge (GAER). Taken together, these findings support serial homology of the pseudobranch and gills, and an ancestral origin of gill arch-like anatomical features from the gnathostome mandibular arch.

sentences: Optimism and pessimism in optimised replay The replay of task-relevant trajectories is known to contribute to memory consolidation and improved task performance. A wide variety of experimental data show that the content of replayed sequences is highly specific and can be modulated by reward as well as other prominent task variables. However, the rules governing the choice of sequences to be replayed still remain poorly understood. One recent theoretical suggestion is that the prioritization of replay experiences in decision-making problems is based on their effect on the choice of action. We show that this implies that subjects should replay sub-optimal actions that they dysfunctionally choose rather than optimal ones, when, by being forgetful, they experience large amounts of uncertainty in their internal models of the world. We use this to account for recent experimental data demonstrating exactly pessimal replay, fitting model parameters to the individual subjects choices.

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Characterization of retinal development in 13-lined ground squirrels PurposeThe cone-dominant, 13-lined ground squirrel (13-LGS) retina mimics the human foveal region but retinal development in this useful rodent species has not been reported. Here, the embryonic and postnatal development of the 13-LGS retina was studied to further characterize the species as a practical alternative animal model for investigating cone-based vision in health and disease.

MethodsThe spatiotemporal expression of key progenitor and cell type markers was examined in retinas from defined embryonic and postnatal stages using immunohistochemistry. Changes in the postnatal gene expression were also assessed by qPCR.

ResultsThe 13-LGS neuroblastic layer expressed key progenitor markers (Sox2, Vsx2, Pax6, and Lhx2) at E18. Sequential cell fate determination evidenced by the first appearance of cell type-specific marker labeling was: at E18, ganglion cells (Brn-3A, HuC/D) and microglia (Iba1); at E24-25.5 shortly before birth, photoreceptor progenitor (Otx2, Recoverin), horizontal and amacrine cells (Lhx1, Oc1); and at P15, bipolar cells (Vsx1, CaBP5) and Muller glia cells (GS, Rlbp1). Photoreceptor maturation indicated by opsin+ outer segments and PNA labeling of cone sheaths was completed at the time of eye opening, P21-24.

ConclusionsThe timeline and order of retinal cell development in the 13-LGS generally matches that recorded from other mammalian models but with a stark variation in the proportion of various cell types due to cone-dense photoreceptors. This provides a baseline for future examinations of developmental, disease model, and stem cell approach studies employing this emerging rodent model of human vision.

sentences: A multiscale atlas of the molecular and cellular architecture of the human kidney The human kidney is composed of over 26 cell types that actively coordinate with each other to form higher-order structures, such as the nephron. It is not yet understood how these structures vary throughout a single organ or amongst the same organs within the human population. We have developed an extensive lipid and cellular atlas of the human kidney consisting of over 3 million cells comprising 75,000 functional tissue units (i.e., glomeruli, proximal tubules, distal tubules, and collecting ducts) from 13 human subjects. This atlas was developed using spatially registered and integrated technologies consisting of imaging mass spectrometry, multiplexed immunofluorescence, stained microscopy, and autofluorescence microscopy, to comprehensively probe large (i.e., centimeter-sized) areas of tissue. The cellular organization and lipid profiles of glomeruli, proximal tubules, distal tubules, and collecting ducts were discovered through these multimodal imaging data as well as their intra- and inter-subject variance. Relating the lipid profiles obtained from imaging mass spectrometry to distinct cell types obtained from immunofluorescence allowed us to hypothesize the functional role of specific phospholipids that have not previously been described. These hypotheses include subject characteristics, such as BMI and sex. The integrated data from the aforementioned datasets provide a valuable reference for kidney researchers, are publicly available through the NIH Human Biomolecular Atlas Program (https://portal.hubmapconsortium.org/), and discussed below.

sentences: Mixed selectivity coding of sensory and motor social signals in the thalamus of a weakly electric fish Recent studies have shown that high-level neural activity often exhibits mixed selectivity to multivariate signals. How such representations arise and how they modulate natural behavior is poorly understood. The social behavior of weakly electric fish is relatively low-dimensional and easily reproduced in the laboratory. Here we show how electrosensory signals related to courtship and rivalry in Apteronotus leptorhynchus are represented in the preglomerular nucleus, the thalamic region exclusively connecting the midbrain with the pallium. We show that preglomerular cells convert their midbrain inputs into a mixed selectivity code that includes corollary discharge of outgoing communication signals. We discuss how the preglomerular pallial targets might use these inputs to control social behavior and determine dominance in male-male competition and female mate selection during courtship. Our results showcase the potential of the electrocommunication system as an accessible model for studying the neural substrates of social behavior and principles of multi-dimensional neural representation.

sentences: Physiological Substrates and Ontogeny-Specific Expression of the Ubiquitin Ligases MARCH1 and MARCH8 in mice MARCH1 and MARCH8 are ubiquitin ligases that control the expression and trafficking of critical immunoreceptors. Understanding of their function is hampered by three major knowledge gaps: (i) it is unclear which cell types utilize these ligases; (ii) their level of redundancy is unknown; and (iii) most of their putative substrates have been described in cell lines, often overexpressing MARCH1 or MARCH8, and it is unclear which substrates are regulated by either ligase in vivo. Here we address these questions by systematically analyzing the immune cell repertoire of MARCH1- or MARCH8-deficient mice, and applying unbiased proteomic profiling of the plasma membrane of primary cells to identify MARCH1 and MARCH8 substrates. Only CD86 and MHC II were unequivocally identified as immunoreceptors regulated by MARCH1 and MARCH8, but each ligase carried out its function in different tissues. MARCH1 regulated MHC II and CD86 in professional and "atypical" antigen presenting cells of hematopoietic origin, whereas MARCH8 only operated in non-hematopoietic cells. Our results reveal that the range of cells constitutively endowed with antigen-presentation capacity is wider than generally appreciated. They also establish MARCH1 and MARCH8 as specialized regulators of CD4+ T cell immunity in two ontogenically distinct cellular compartments.

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Principled clustering of functional signals reveals gradients in processing both within the anterior hippocampus and across its long axis A particularly elusive puzzle concerning the hippocampus is how the structural differences along its long, anteroposterior axis might beget meaningful functional differences, particularly in terms of the granularity of information processing. One measure posits to quantify this granularity by calculating the average statistical independence of the BOLD signal across neighboring voxels, or inter-voxel similarity (IVS), and has shown the anterior hippocampus to process coarser-grained information than that in the posterior hippocampus. This model of the hippocampus, however, conflicts with a number of task-oriented findings, many of which have varied in their fMRI acquisition parameters and hippocampal parcellation methods. In order to reconcile these findings, we measured IVS across two separate resting-state fMRI acquisitions and compared the results across many of the most widely used parcellation methods in a large young-adult sample (Acquisition 1, N = 253; Acquisition 2, N = 183). Finding conflicting results across acquisitions and parcellations, we reasoned that a principled, data-driven approach to hippocampal parcellation is necessary. To this end, we implemented a group masked independent components analysis (mICA) to identify functional subunits of the hippocampus, most notably separating the anterior hippocampus into separate anterior-medial, anterior-lateral, and posteroanterior-lateral components. Measuring IVS across these components revealed a decrease in IVS along the medial-lateral axis of the anterior hippocampus but an increase from anterior to posterior. We conclude that representational granularity may not change linearly or unidirectionally across the hippocampus, and that moving the study of the hippocampus towards reproducibility requires grounding it in a functionally informed approach.

Significance StatementProcessing information along hierarchical scales of granularity is critical for many of the feats of cognition considered most human. Recently, the changes in structure, cortical connectivity, and apparent functional properties across parcels of the hippocampal long axis have been hypothesized to underlie this hierarchical gradient in information processing. We show here, however, that the choice of parcellation method itself drastically affects the perceived granularity across the hippocampus, and that a principled, functionally informed approach to parcellation reveals gradients both within the anterior hippocampus and in non-linear form across the long axis. These results point to the issue of parcellation as a critical one in the study of the hippocampus and reorient interpretation of existing results.

sentences: Reciprocalspaceship: A Python Library for Crystallographic Data Analysis AO_SCPLOWBSTRACTC_SCPLOWX-ray crystallography is an invaluable technique for studying the atomic structure of macromolecules. Much of crystallographys success is due to the software packages developed to enable the automated processing of diffraction data. However, the analysis of unconventional diffraction experiments can still pose significant challenges--many existing programs are closed-source, sparsely documented, or are challenging to integrate with modern libraries for scientific computing and machine learning. Here we describe reciprocalspaceship, a Python library for exploring reciprocal space. It provides a tabular representation for reflection data from diffraction experiments that extends the widely-used pandas library with built-in methods for handling space group, unit cell, and symmetry-based operations. As we illustrate, this library facilitates new modes of exploratory data analysis while supporting the prototyping, development, and release of new methods.

sentences: Diversity and dynamics of the CRISPR-Cas systems associated with Bacteroides fragilis in human population CRISPR-Cas systems are adaptive immune systems commonly found in prokaryotes that provide sequence-specific defense against invading mobile genetic elements (MGEs). The memory of these immunological encounters are stored in CRISPR arrays, where spacer sequences record the identity and history of past invaders. Analyzing such CRISPR arrays provide insights into the dynamics of CRISPR-Cas systems and the adaptation of their host bacteria to rapidly changing environments such as the human gut. In this study, we utilized 601 Bacteroides fragilis genome isolates from 12 healthy individuals, 6 of which include longitudinal observations, and 222 available B. fragilis reference genomes to update the understanding of B. fragilis CRISPR-Cas dynamics and their differential activities. Analysis of longitudinal genomic data showed that some CRISPR array structures remained relatively stable over time whereas others involved radical spacer acquisition during some periods, and diverse CRISPR arrays (associated with multiple isolates) co-existed in the same individuals with some persisted over time. Furthermore, features of CRISPR adaptation, evolution, and microdynamics were highlighted through an analysis of host-MGE network, such as modules of multiple MGEs and hosts, reflecting complex interactions between B. fragilis and its invaders mediated through the CRISPR-Cas systems. This work demonstrates the power of using culture-based population genomics to reveal the activities and evolution of the CRISPR-Cas systems associated with gut bacteria in human population. We made available of all annotated CRISPR-Cas systems and their target MGEs, and their interaction network as a web resource at https://omics.informatics.indiana.edu/CRISPRone/Bfragilis.

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Chasing the metabolism of novel syntrophic acetate-oxidizing bacteria in thermophilic methanogenic chemostats BackgroundAcetate is the major intermediate of anaerobic digestion of organic waste to CH4. In anaerobic methanogenic systems, acetate degradation is carried out by either acetoclastic methanogenesis or a syntrophic degradation by a syntrophy of acetate oxidizers and hydrogenotrophic methanogens. Due to challenges in isolation of syntrophic acetate-oxidizing bacteria (SAOB), the diversity and metabolism of SAOB, as well as the mechanisms of their interactions with methanogenic partners remain poorly understood.

ResultsIn this study, we successfully enriched previously unknown SAOB by operating continuous thermophilic anaerobic chemostats fed with acetate, propionate, butyrate, or isovalerate as the sole carbon and energy source. They represent novel clades belonging to Clostridia, Thermoanaerobacteraceae, Anaerolineae, and Gemmatimonadetes. In these SAOB, acetate is degraded through reverse Wood-Ljungdahl pathway or an alternative pathway mediated by the glycine cleavage system, while the SAOB possessing the latter pathway dominated the bacterial community. Moreover, H2 is the major product of the acetate degradation by these SAOB, which is mediated by [FeFe]-type electron-confurcating hydrogenases, formate dehydrogenases, and NADPH reoxidation complexes. We also identified the methanogen partner of these SAOB in acetate-fed chemostat, Methanosarcina thermophila, which highly expressed genes for CO2-reducing methanogenesis and hydrogenases to supportively consuming H2 at transcriptional level. Finally, our bioinformatical analyses further suggested that these previously unknown syntrophic lineages were prevalent and might play critical roles in thermophilic methanogenic reactors.

ConclusionThis study expands our understanding on the phylogenetic diversity and in situ biological functions of uncultured syntrophic acetate degraders, and presents novel insights on how they interact with their methanogens partner. These knowledges strengthen our awareness on the important role of SAO in thermophilic methanogenesis and may be applied to manage microbial community to improve the performance and efficiency of anaerobic digestion.

sentences: Multi-Omic Profiling of the Liver Across Diets and Age in a Diverse Mouse Population Systems biology approaches often use inferred networks of gene expression and metabolite data to identify regulatory factors and pathways connected with phenotypic variance. Generally, study-specific multi-layer "Omics" datasets are used to contextualize generic molecular networks. In this regard separating upstream causal mechanisms, downstream biomarkers, and incidental correlations remains a significant challenge, yet it is essential for designing mechanistic experiments. To address this, we designed a study following a population of 2157 individuals from 89 isogenic BXD mouse strains across their lifespan to identify molecular interactions among genotype, environment, age (GxExA) and metabolic fitness. Each strain was separated into two cohorts, one fed low fat (6% cal/fat) and the other high fat (60% cal/fat) diets. Tissues were collected for 662 individuals (309 cohorts) diverging across age (7, 12, 18, and 24 months), diet, sex, and strain. Transcriptome, proteome, and metabolome data were generated for liver. Of these we identified linear relations among these molecular data with lifespan for the same genomes of mice (Roy et al. 2020), and we defined [~]1100 novel protein-coding genes associated with longevity. We knocked down the ortholog of Ctsd in C. elegans. The treatment reduced longevity both in wildtype and in mutant long-lived strains, thus validating the prediction. Next, to assess the molecular impact of GxExA on gene expression, the multi-omics data was parsed into metabolic networks where connectivity varied due to the independent variables. Differences in edge strengths connecting nodes in these molecular networks according to each variable enabled causal inference by using stability selection, with roughly 21% of novel gene-pathway connections being causally affected by diet and/or age. For instance, Chchd2 is activated by aging and drives changes in the proteasome, oxidative phosphorylation, and mitochondrial translation transcriptional networks. Together, we have developed a large multi-omics resource for studying aging in the liver, and a resource for turning standard associations into causal networks.

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Evolving social behaviour through selection of single-cell adhesion in Dictyostelium discoideum The social amoeba Dictyostelium discoideum commonly forms chimeric fruiting bodies by aggregation of different strains. Genetic variants that produce a higher proportion of spores are predicted to undercut multicellular organization unless cooperators assort positively. Cell adhesion is considered a primary factor driving such assortment, but evolution of adhesion has not been experimentally connected to changes in social performance. We modified by experimental evolution the properties of individual cells, selecting for higher and lower adhesion to a surface. We then quantified the effects of these changes on fitness (reproductive success), on development (timing and patterns of aggregation) for clonal and chimeric aggregates. In binary chimeras, both evolved lines produce a smaller fraction of spores than the ancestor. Surprisingly, evolution of a single-cell trait thus appears to have produced social cooperators irrespective of whether stronger or weaker adhesion was selected. Examination of development and cell-cell adhesion however revealed two distinct social behaviours, whereby less adhesive cells behave as cheaters in the multicellular bodies. Such differences are captured by a metric for social success that generalizes the classical measure of spore bias by taking into account clonal spore production. Our work shows that cell mechanical interactions can constrain evolution of development and assortment in chimeras, and that elucidation of proximate mechanisms is necessary in order to understand the ultimate emergence of multicellular organization.

Author summaryThe multicellular life cycle of Dictyostelium discoideum is as amazing as puzzling. Genetically different cells commonly co-aggregate, so that collective functions - that repose on cooperation among cells - are constantly threatened by the presence of exploitative variants, commonly called  cheaters. The evolution of cheating and cooperative behaviours is expected to depend on cell adhesion, which influences assortment and thus the capacity of cells to interact preferentially with those having the same social strategy. Here, we use experimental evolution to explore how selection acting on single-cell adhesion impacts social behaviour. Higher levels of cooperation appear to evolve as a result of both increased and decreased adhesion of single cells to a surface. However, such phenotypes are associated to opposite developmental dynamics in chimeric aggregates. Differences in social behaviour are captured if spore production of isolated strains is also considered, confirming the expectation that within the multicellular body of chimeric aggregates cooperative behaviour is associated to stronger adhesion, while early cell segregation undermines the success of less adhesive cells.

sentences: The Consequences of Differential Origin Licensing Dynamics in Distinct Chromatin Environments MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with under-licensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.

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TAILORED DC INDUCE PROTECTIVE HIV-1 SPECIFIC POLYFUNCTIONAL CD8+ T CELLS IN THE LYMPHOID TISSUE FROM HUMANIZED BLT MICE Effective function of CD8+ T cells and enhanced innate activation of dendritic cells (DC) in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of TBK1-primed DC inducing protective CD8+ T cell responses in lymphoid tissue and peripheral blood and their association with reduced HIV-1 disease progression in vivo in the humanized bone marrow, liver and thymus (hBLT) mouse model. A higher proportion of hBLT-mice vaccinated with TBK1-primed DC exhibited less severe CD4+ T cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to secondary lymphoid organs and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, TBK1-primed DC might be an useful tool for subsequent vaccine studies.

Author summaryEmulating protective immunological characteristics from individuals capable of spontaneously controlling HIV-1 infection might be useful for the development of a protective vaccine. Enhanced function of dendritic cells (DC) in these HIV-1 controllers depends on the activation of TANK-binding Kinase 1 (TBK1) and might associate with protective T cells. Our study evaluated the ability of DCs trained through TBK1 activation inducing protective adaptive immune responses against HIV-1 and reducing disease progression in vivo, using a humanized mouse model. Our data indicate that mice vaccinated with tailored DC exhibit delayed disease progression, increased induction of protective CD8+ T lymphocyte subsets in the lymphoid tissue and blood upon antigen recognition. Therefore, trained-DC might be an useful tool for future HIV-1 vaccine designs.

sentences: Transcriptomic analysis of ribosome biogenesis and pre-rRNA processing during growth stress in Entamoeba histolytica Ribosome biogenesis, a multi-step process involving the transcription, modification, folding and processing of rRNA is the major consumer of cellular energy. It involves the sequential assembly of ribosomal proteins (RP)s via more than 200 ribogenesis factors. Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in Entamoeba histolytica, pre-rRNA synthesis continues, and unprocessed pre-rRNA accumulates. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified the ribosome biogenesis factors in E. histolytica. Of the total [~]279 S. cerevisiae proteins, we could only find 188 proteins in E. histolytica. Some of the proteins missing in E. histolytica were also missing in humans. A number of proteins represented by multiple genes in S. cerevisiae had only a single copy in E. histolytica. It was interesting to note that E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components as compared to S. cerevisiae. Northern hybridization using probes from different spacer regions depicted the accumulation of unprocessed intermediates during stress. Transcriptomic studies revealed the differential regulation of a number of ribosomal factors both in serum-starved and RRP6KD conditions. The ARB1 protein involved at multiple steps of ribosome biogenesis and NEP1 and TSR3 involved in chemical modification of 18S rRNA previously shown to accumulate pre-rRNA precursors upon downregulation in S. cerevisiae and humans were included. The data reveals the importance of some of the major factors required for regulating pre-rRNA processing during stress. This is the first report on the complete repertoire of ribosome biogenesis factors in E. histolytica.

sentences: The proportional recovery rule redux: Arguments for its biological and predictive relevance The proportional recovery rule (PRR) posits that most stroke survivors can expect to reverse a fixed proportion of motor impairment. As a statistical model, the PRR explicitly relates change scores to baseline values - an approach that has the potential to introduce artifacts and flawed conclusions. We describe approaches that can assess associations between baseline and changes from baseline while avoiding artifacts either due to mathematical coupling or regression to the mean due to measurement error. We also describe methods that can compare different biological models of recovery. Across several real datasets, we find evidence for non-artifactual associations between baseline and change, and support for the PRR compared to alternative models. We conclude that the PRR remains a biologically-relevant model of recovery, and also introduce a statistical perspective that can be used to assess future models.

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GRAFIMO: variant and haplotype aware motif scanning on pangenome graphs Transcription factors (TFs) are proteins that promote or reduce the expression of genes by binding short genomic DNA sequences known as transcription factor binding sites (TFBS). While several tools have been developed to scan for potential occurrences of TFBS in linear DNA sequences or reference genomes, no tool exists to find them in pangenome variation graphs (VGs). VGs are sequence-labelled graphs that can efficiently encode collections of genomes and their variants in a single, compact data structure. Because VGs can losslessly compress large pangenomes, TFBS scanning in VGs can efficiently capture how genomic variation affects the potential binding landscape of TFs in a population of individuals. Here we present GRAFIMO (GRAph-based Finding of Individual Motif Occurrences), a command-line tool for the scanning of known TF DNA motifs represented as Position Weight Matrices (PWMs) in VGs. GRAFIMO extends the standard PWM scanning procedure by considering variations and alternative haplotypes encoded in a VG. Using GRAFIMO on a VG based on individuals from the 1000 Genomes project we recover several potential binding sites that are enhanced, weakened or missed when scanning only the reference genome, and which could constitute individual-specific binding events. GRAFIMO is available as an open-source tool, under the MIT license, at https://github.com/pinellolab/GRAFIMO and https://github.com/InfOmics/GRAFIMO.

Author summaryTranscription factors (TFs) are key regulatory proteins and mutations occurring in their binding sites can alter the normal transcriptional landscape of a cell and lead to disease states. Pangenome variation graphs (VGs) efficiently encode genomes from a population of individuals and their genetic variations. GRAFIMO is an open-source tool that extends the traditional PWM scanning procedure to VGs. By scanning for potential TBFS in VGs, GRAFIMO can simultaneously search thousands of genomes while accounting for SNPs, indels, and structural variants. GRAFIMO reports motif occurrences, their statistical significance, frequency, and location within the reference or alternative haplotypes in a given VG. GRAFIMO makes it possible to study how genetic variation affects the binding landscape of known TFs within a population of individuals.

sentences: Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DC) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.

sentences: Mutual Regulation between PFKP and VEGF Promotes Glioblastoma Tumor Growth Glioblastoma (GBM) is highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, it remains unknown whether PFKP and VEGF are reciprocally regulated during GBM tumor growth. Here, we show that PFKP promotes EGFR activation-induced VEGF expression in HIF-1-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in the AKT/SP1-mediated transcriptional expression of HIF-1 and in the AKT-mediated {beta}-catenin S552 phosphorylation, to fully enhance VEGF transcription and subsequent blood vessel formation and brain tumor growth. The levels of PFKP Y64 phosphorylation in human GBM specimens positively correlate with HIF-1 expression, {beta}-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth and provide the therapeutic potential of targeting the PFKP/VEGF regulatory loop for GBM patients.

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CRAM 3.1: Advances in the CRAM Format MotivationCRAM has established itself as a high compression alternative to the BAM file format for DNA sequencing data. We describe updates to further improve this on modern sequencing instruments.

ResultsWith Illumina data CRAM 3.1 is 7 to 15% smaller than the equivalent CRAM 3.0 file, and 50 to 70% smaller than the corresponding BAM file. Long-read technology shows more modest compression due to the presence of high-entropy signals.

AvailabilityThe CRAM 3.0 specification is freely available from https://samtools.github.io/hts-specs/CRAMv3.pdf. The CRAM 3.1 improvements are available from https://github.com/samtools/hts-specs/pull/433, with OpenSource implementations in HTSlib and HTScodecs.

Contactjkb@sanger.ac.uk

Supplementary informationSupplementary data are available online

sentences: RNA inhibits dMi-2/CHD4 chromatin binding and nucleosome remodelling The ATP-dependent nucleosome remodeller Mi-2/CHD4 broadly modulates epigenetic landscapes to repress transcription and to maintain genome integrity. Here we use individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to show that Drosophila Mi-2 associates with thousands of mRNA molecules in vivo. Biochemical data reveal that recombinant dMi-2 preferentially binds to G-rich RNA molecules using two intrinsically disordered regions of previously undefined function. Pharmacological inhibition of transcription and RNase digestion approaches establish that RNA inhibits the association of dMi-2 with chromatin. We also show that RNA inhibits dMi-2-mediated nucleosome mobilization by competing with the nucleosome substrate. Importantly, this activity is shared by CHD4, the human homolog of dMi-2, strongly suggesting that RNA-mediated regulation of remodeller activity is an evolutionary conserved mechanism. Our data support a model in which RNA serves to protect actively transcribed regions of the genome from dMi-2/CHD4- mediated establishment of repressive chromatin structures.

sentences: Single-cell RNA sequencing uncovers the excitatory/inhibitory synaptic unbalance in the retrosplenial cortex after peripheral nerve injury Nerve injury in the somatosensory pathway may induce maladaptive changes at the transcriptional or protein level, contributing to the development and maintenance of neuropathic pain. In contrast to the retrosplenial cortex (RSC), which processes nociceptive information and exhibits structural and molecular changes after nerve injury, detailed transcriptional changes in the RSC are not yet known. Here we confirm the involvement of the RSC in regulating pain sensation and observe that the same peripheral stimulation activates more retrosplenial neurons after nerve injury; reducing the activities of CaMKII+ splenial cells relieves peripheral pain hypersensitivity after nerve injury. Using a single-cell RNA sequencing (scRNA-seq) approach, we identified cell-type-specific gene expression changes after nerve injury, and the gene set enrichment analysis results revealed suppressed ion homeostasis in CaMKII+ neurons. Furthermore, examination of the expression of genes encoding ligand-gated ion channels showed a decrease in Gabar1a but an increase in Gria1 in CaMKII+ neurons; consistently, we confirmed the unbalanced excitatory/inhibitory synaptic transmission by using the electrophysiological recording approach. Moreover, micro-infusion of 1-Naphthyl acetyl spermine in the RSC to reduce excitatory synaptic transmission alleviated peripheral pain hypersensitivity. Our data confirm the involvement of the RSC in pain regulation and provide information on cell type-dependent transcriptomic changes after nerve injury, which will contribute to the understanding of the mechanisms mediating neuropathic pain.

sentences: Host ecology regulates interspecies recombination in bacteria of the genus Campylobacter Horizontal gene transfer (HGT) can allow traits that have evolved in one bacterial species to transfer to another. This has potential to rapidly promote new adaptive trajectories such as zoonotic transfer or antimicrobial resistance. However, for this to occur requires gaps to align in barriers to recombination within a given time frame. Chief among these barriers is the physical separation of species with distinct ecologies in separate niches. Within the genus Campylobacter there are species with divergent ecologies, from rarely isolated single host specialists to multi-host generalist species that are among the most common global causes of human bacterial gastroenteritis. Here, by characterising these contrasting ecologies, we can quantify HGT among sympatric and allopatric species in natural populations. Analysing recipient and donor population ancestry among genomes from 30 Campylobacter species we show that cohabitation in the same host can lead to a 6-fold increase in HGT between species. This accounts for up to 30% of all SNPs within a given species and identifies highly recombinogenic genes with functions including host adaptation and antimicrobial resistance. As described in some animal and plant species, ecological factors are a major evolutionary force for speciation in bacteria and changes to the host landscape can promote partial convergence of distinct species through HGT.

sentences: The effect of a canine semen activator supplementation or addiction on the long-term refrigeration quality of dog spermatozoa Within modern biotechnology, different tools have been developed to maximize canine semen conservation protocol to optimizing reproductive results and making their handling more flexible. In the last decades, the survival of refrigerated semen has been prolonged from 2-3d with the first basic diluents, to 10-14d using the most modern extenders. However, their main limitation is that sperm quality decreases during cold storage. Semen activators (SA) have been produced to provide the molecules necessary to maximize the sperm survival and quality with the aim to enhance fertility and prolificacy. In this study, the effect of SA was recorded by daily evaluation of chilled semen 14d. For this experiment, six adult healthy Neapolitan Mastiff dogs, were used as donors and the semen was manually collected. Spermatozoa-rich fractions of each suject was chilled using a new generation extender for long periods of time (d0) starting from the d1 to d14, different aliquot, with (experimental trial) and without SA (control trial), were evaluated daily for motility vigor, morphology and membrane integrity. The initial sperm concentration of extended semen was 417.3{+/-}170.4x106/mL (mean {+/-} SEM) with 85.89{+/-}4.76% of MNS (morphologically normal sperm), 84.47{+/-}5.22 % vital sperm and a pH of 6.2{+/-}2.8. The initial vigor was 3.83{+/-}0.48, but after one min with SA, it rose to 4.45 {+/-} 0.45 (P<0.001). The semen motility parameter increase significantly (P<0.05) in experimental trial, respect to control, starting to d2 at finish (except for d7). The vigor analysis significantly increase in experimental trial (P<0.05) during the most day of the study with the exclusion of d3 and d14. For evaluate the semen characteristics over time, the experiment was divided into T1 (d0-d5), T2 (d6-d10) and T3 (d11-d14) (P<0.001) in evaluation of morphology and membrane stability. The MNS reached 70% at d10 and finally 65% at d14, being considered normal and possibly fertile. With Host-s, 65% of MNS were also achieved at d14. The presence of glucose and fructose in the diluents used for refrigeration can exert very important effects given the fact that metabolic routes have been found in both sugars, providing both different and complementing effects. It can be concluded that the use of SA prior to artificial insemination improves the quality of chilled semen significantly, although it does not reverse the effects of deterioration due to cellular metabolism over time.

sentences: Habitat Protection Indexes - new monitoring measures for the conservation of coastal and marine habitats A worldwide call to implement habitat protection aims to halt biodiversity loss. To monitor the extent of coastal and marine habitats within protected areas (PAs) in a standardized, open source, and reproducible way, we constructed the Local and the Global Habitat Protection Indexes (LHPI and GHPI, respectively). The LHPI pinpoints the jurisdictions with the greatest opportunity to expand their own PAs, while the GHPI showcases which jurisdictions contribute the most in area to the protection of these habitats globally. Jurisdictions were evaluated to understand which have the highest opportunity to contribute globally to the protection of habitats by meeting a target of 30% coverage of PAs with Areas Beyond National Jurisdiction (ABNJ) having the greatest opportunity to do so. While we focus on marine and coastal habitats, our workflow can be extended to terrestrial and freshwater habitats. These indexes are useful to monitor aspects of Sustainable Development Goal 14 and the emerging post-2020 Global Biodiversity Framework, to understand the current status of international cooperation on coastal and marine habitats conservation.

sentences: A chromosome-level, fully phased genome assembly of the oat crown rust fungus Puccinia coronata f. sp. avenae: a resource to enable comparative genomics in the cereal rusts Advances in sequencing technologies as well as development of algorithms and workflows have made it possible to generate fully phased genome references for organisms with non-haploid genomes such as dikaryotic rust fungi. To enable discovery of pathogen effectors and further our understanding of virulence evolution, we generated a chromosome-scale assembly for each of the two nuclear genomes of the oat crown rust pathogen, Puccinia coronata f. sp. avenae (Pca). This resource complements two previous released partially phased genome references of Pca, which display virulence traits absent in the isolate of historic race 203 (isolate Pca203) which was selected for this genome project. A fully phased, chromosome-level reference for Pca203 was generated using PacBio reads and Hi-C data and a recently developed pipeline named NuclearPhaser for phase assignment of contigs and phase switch correction. With 18 chromosomes in each haplotype and a total size of 208.10 Mbp, Pca203 has the same number of chromosomes as other cereal rust fungi such as Puccinia graminis f. sp. tritici and Puccinia triticina, the causal agents of wheat stem rust and wheat leaf rust, respectively. The Pca203 reference marks the third fully-phased chromosome-level assembly of a cereal rust to date. Here, we demonstrate that the chromosomes of these three Puccinia species are syntenous and that chromosomal size variations are primarily due to differences in repeat element content.

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The structural coverage of the human proteome before and after AlphaFold The protein structure field is experiencing a revolution. From the increased throughput of techniques to determine experimental structures, to developments such as cryo-EM that allow us to find the structures of large protein complexes or, more recently, the development of artificial intelligence tools, such as AlphaFold, that can predict with high accuracy the folding of proteins for which the availability of homology templates is limited.

Here we quantify the effect of the recently released AlphaFold database of protein structural models in our knowledge on human proteins. Our results indicate that our current baseline for structural coverage of 48%, considering experimentally-derived or template-based homology models, elevates up to 76% when including AlphaFold predictions. At the same time the fraction of dark proteome is reduced from 26% to just 10% when AlphaFold models are considered. Furthermore, although the coverage of disease-associated genes and mutations was near complete before AlphaFold release (69% of Clinvar pathogenic mutations and 88% of oncogenic mutations), AlphaFold models still provide an additional coverage of 3% to 13% of these critically important sets of biomedical genes and mutations. Finally, we show how the contribution of AlphaFold models to the structural coverage of non-human organisms, including important pathogenic bacteria, is significantly larger than that of the human proteome.

Overall, our results show that the sequence-structure gap of human proteins has almost disappeared, an outstanding success of direct consequences for the knowledge on the human genome and the derived medical applications.

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Spatial and temporal control of expression with light-gated LOV-LexA The ability to drive expression of exogenous genes in different tissues and cell types, under control of specific enhancers, has catapulted discovery in biology. While many enhancers drive expression broadly, several genetic tricks have been developed to obtain access to isolated cell types. However, studies of topographically organized neuropiles, such as the optic lobe in fruit flies, have raised the need for a system that can access subsets of cells within a single neuron type, a feat currently dependent on stochastic flip-out methods. To access the same subsets of cells consistently across flies, we developed LOV-LexA, a light-gated expression system based on the bacterial LexA transcription factor and the plant-derived LOV photosensitive domain. Expression of LOV-Lex in larval fat body as well as pupal and adult neurons enables spatial and temporal control of expression of transgenes under LexAop sequences with blue light. The LOV-LexA tool thus provides another layer of intersectional genetics, allowing for light-controlled genetic access to the same subsets of cells within an expression pattern across individual flies.

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sentences: Evaluation of in vitro and in vivo toxicity of pristine molybdenum disulphide nanosheets in Swiss albino mice The Molybdenum disulfide nanosheets (MoS2-NSs) thin films has received increasing attention recently due to their versatile multi functionality including catalytic properties, photoluminescence and flexibility, which suggests their future, uses for biomedical applications. However, there are no studies in detail related with biocompatibility of MoS2 thin sheets. Here, weevaluated the dose-dependent effects of MoS2-NSs on cell viability (MTT assay) and release of lactate dehydrogenase (LDH) into culture media using MG-63 cells, as well as haemolysis, hematological, serum biochemical, antioxidants and histopathological parameters in Swiss albino mice. The MoS2-NSs was synthesized via facile hydrothermal method and characterized using XRD, Raman, SEM, TEM and HRTEM. The in vitro study results suggest that at lower concentration MoS2-NSs does not causes any toxicity. The lethal dose (LD50) was evaluated by intraperitoneal administration with different concentrations and estimated as ~1.0 mg kg-1. The higher dose (1.5 mg kg-1) of MoS2-NSs showed significant alteration in hematological markers and serum biochemical enzymes, as compared to control. Lipid peroxidation also shows significant alteration with respect to the control. Histopathological, hematological and biochemical examination, revealed no remarkable changes at lower concentration (less than 1.0 mg kg-1), however, higher concentration (1.5 mg kg-1) causes significant histopathological, antioxidants and biochemical alterations in tissues and serum, respectively. The results suggest that the lower concentration of MoS2-NSs can be used in future biomedical applications.

sentences: Synergistic interferon alpha-based drug combinations inhibit SARS-CoV-2 and other viral infections in vitro There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. In the present study, we found that recombinant human interferon-alpha (IFNa) triggers intrinsic and extrinsic cellular antiviral responses, as well as reduces replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. Although IFNa alone was insufficient to completely abolish SARS-CoV-2 replication, combinations of IFNa with remdesivir or other antiviral agents (EIDD-2801, camostat, cycloheximide, or convalescent serum) showed strong synergy and effectively inhibited SARS-CoV-2 infection in human lung epithelial Calu-3 cells. Furthermore, we showed that the IFNa-remdesivir combination suppressed virus replication in human lung organoids, and that its single prophylactic dose attenuated SARS-CoV-2 infection in lungs of Syrian hamsters. Transcriptome and metabolomic analyses showed that the combination of IFNa-remdesivir suppressed virus-mediated changes in infected cells, although it affected the homeostasis of uninfected cells. We also demonstrated synergistic antiviral activity of IFNa2a-based combinations against other virus infections in vitro. Altogether, our results indicate that IFNa2a-based combination therapies can achieve higher efficacy while requiring lower dosage compared to monotherapies, making them attractive targets for further pre-clinical and clinical development.

sentences: Mitochondrial unfolded protein response transcription factor ATFS-1 increases resistance to exogenous stressors through upregulation of multiple stress response pathways The mitochondrial unfolded protein response (mitoUPR) is an evolutionarily conserved pathway that responds to various insults to the mitochondria through transcriptional changes that restore mitochondrial homeostasis in order to facilitate cell survival. Gene expression changes resulting from the activation of the mitoUPR are mediated by the transcription factor ATFS-1/ATF-5. To further define the mechanisms through which the mitoUPR protects the cell during mitochondrial dysfunction, we characterized the role of ATFS-1 in responding to organismal stress. We found that activation of ATFS-1 is sufficient to cause upregulation of genes involved in multiple stress response pathways, including the DAF-16-mediated stress response pathway, the SKN-1-mediated oxidative stress response pathway, the HIF-mediated hypoxia response pathway, the p38-mediated innate immune response pathway, and antioxidant genes. Moreover, ATFS-1 is required for the upregulation of stress response genes after exposure to exogenous stressors, especially oxidative stress and bacterial pathogens. Constitutive activation of ATFS-1 increases resistance to multiple acute exogenous stressors, while disruption of atfs-1 decreases stress resistance. Although ATFS-1-dependent genes are upregulated in multiple long-lived mutants, constitutive activation of ATFS-1 in wild-type animals results in decreased lifespan. Overall, our work demonstrates that ATFS-1 serves a vital role in organismal survival of acute stresses through its ability to activate multiple stress response pathways, but that chronic ATFS-1 activation is detrimental for longevity.

sentences: Flexible control of representational dynamics in a disinhibition-based model of decision making Current models utilize two primary circuit motifs to replicate neurobiological decision making. Recurrent gain control implements normalization-driven relative value coding, while recurrent excitation and competitive inhibition together implement winner-take-all (WTA) dynamics. Despite evidence for concurrent valuation and selection computations in individual brain regions, existing models focus selectively on either normalization or WTA dynamics and how both arise in a single circuit architecture is unknown. Here we show that a novel hybrid motif unifies both normalized representation and WTA competition, with dynamic control of circuit state governed by local disinhibition. In addition to capturing empirical psychometric and chronometric data, the model produces persistent activity consistent with working memory. Furthermore, disinhibition provides a simple mechanism for flexible top-down control of network states, enabling the circuit to capture diverse task-dependent neural dynamics. These results suggest a new biologically plausible mechanism for decision making and emphasize the importance of local disinhibition in neural processing.

sentences: Microtubule retrograde flow retains neuronal polarization in a fluctuating state In developing vertebrate neurons, a neurite is formed by more than a hundred microtubules. While individual microtubules are dynamic, the microtubule array itself has been regarded as stationary. Using live cell imaging in combination with photoconversion techniques and pharmacological manipulations, we uncovered that the microtubule array flows retrogradely within neurites to the soma. This microtubule retrograde flow drives cycles of microtubule density, a hallmark of the fluctuating state before axon formation. Shortly after axon formation, microtubule retrograde flow slows down in the axon, which stabilizes microtubule density cycles and thereby functions as a molecular wedge to enable axon extension. We propose microtubule retrograde flow and its specific slowdown in the axon to be the long-sought mechanism to single one neurite out to drive neuronal polarization.

sentences: The evolution of stomatal traits along the trajectory towards C4 photosynthesis C4 photosynthesis optimizes plants carbon and water relations, allowing high photosynthetic rate with low stomatal conductance. Stomata have long been believed as a part of C4 syndrome. However, it remains unclear how stomata traits evolved along the path from C3 to C4. Stomatal patterning was examined in Flaveria genus, a model for studying C4 evolution. Comparative, transgenic and semi-in-vitro experiments were used to study molecular basis that underlies stomatal traits along C4 evolution. Novel results: the evolution from C3 to C4 species through intermediate species is accompanied by a stepwise rather than an abrupt change in the stomatal traits. The initial change occurs near Type II and dramatic change occurs at the C4-like species. On the road to C4, stomata become less in number but bigger in size and changes in stomatal density dominates changes in maximum stomatal conductance (gsmax). The reduction of FSTOMAGEN expression underlies altered gsmax between Flaveria species with different photosynthetic pathways and likely occurs in other C4 lineages. Our study provides insight into the pattern, mechanism and role of stomatal evolution along the road towards C4. This work highlights the stomatal traits in the current C4 evolutionary model and the co-evolution of photosynthetic pathway and stomata.

sentences: Non-coding RNAs Associated with Prader-Willi Syndrome Regulate Transcription of Neurodevelopmental Genes in                   Human Induced Pluripotent Stem Cells Mutations and aberrant gene expression during cellular differentiation lead to neurodevelopmental disorders such as Prader-Willi syndrome (PWS) which results from the deletion of an imprinted locus on chromosome 15. We analysed chromatin-associated RNA in human induced pluripotent cells (iPSCs) upon depletion of hybrid small nucleolar long non-coding RNAs (sno-lncRNAs) and 5 snoRNA capped and polyadenylated long non-coding RNAs (SPA-lncRNAs) transcribed from the locus deleted in PWS. We found that rapid ablation of these lncRNAs affects transcription of specific gene classes. Downregulated genes contribute to neurodevelopment and neuronal maintenance while genes that are upregulated are predominantly involved in the negative regulation of cellular metabolism and apoptotic processes. Our data revealed the importance of SPA-lncRNAs and sno-lncRNAs in controlling gene expression in iPSCs and provided a platform for synthetic experimental approaches in PWS studies. We conclude that ncRNAs transcribed from the PWS locus are critical regulators of a transcriptional signature important for neuronal differentiation and development.

sentences: A deep dive into VDAC1 conformational diversity using all-atom simulations provides new insights into the structural origin of the closed states The voltage-dependent anion channel 1 (VDAC1) is a crucial mitochondrial transporter which controls the flow of ions and respiratory metabolites entering or exiting mitochondria. As a voltage-gated channel, VDAC1 can switch between a high conducting "open" state and low conducting "closed" states emerging at high transmembrane potential. Although cell homeostasis depends on channel gating to regulate the transport of ions and metabolites, structural hallmarks characterizing the closed states remain unknown. Here we performed microsecond accelerated molecular dynamics to highlight a vast region of VDAC1 conformational landscape accessible at typical voltage known to promote closure. Conformers exhibiting stable subconducting properties inherent to closed states were identified. In all cases, the low conductance was due to the particular positioning of an unfolded part of the N-terminus which obstructed the channel pore. While the N-terminal tail was found to be sensitive to voltage orientation, our low-conducting models suggest that closed states predominantly take place from disordered events and do not result from the displacement of a voltage sensor or a significant change in the pore. In addition, our results were consistent with conductance jumps observed in experiments and corroborates a recent study describing entropy as a key factor for VDAC gating.

sentences: The payoffs and tradeoffs of hygienic behavior: A large-scale field study on a local population of honey bees Honey bees (Apis mellifera) are exposed to a variety of risk factors, but the ectoparasitic mite Varroa destructor and its associated viruses are considered to be the most significant problem worldwide. It has been widely recognized that honey bee stocks resistant to the mites are an essential part of any sustainable long-term management of Varroa. The aim of this study was to evaluate the efficacy of hygienic behavior in a local population of honey bees in order to reduce Varroa infestation. A bi-directional selection for high and low rates of hygienic behavior was carried out in Israel using either queen artificially inseminated or naturally mated. Colonies were screened for performance: population size, honey production, control of Varroa infestation, and the level of hygienic behavior. Furthermore, we examined the costs and benefits of selection using measurements of colony performance. Either way, selected lines should be tested for trade-offs and benefits to ensure their productivity. The selection process revealed that the trait is heritable. Maternal phenotype has a significant effect on Varroa load, as colonies founded by hygienic daughter queens showed a significantly lower parasite load. No major trade-offs were found between the rate of hygienic behavior, honey yield, and population size. Measuring the direct benefits of hygienic behavior through colony performance suggests that breeding for this trait makes bees more resistant to Varroa destructor. These results are promising for our successful local bee breeding programs in a Mediterranean climate.

sentences: Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using anHb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

sentences: Predicting compound activity from phenotypic profiles and chemical structures Recent advances in deep learning enable using chemical structures and phenotypic profiles to accurately predict assay results for compounds virtually, reducing the time and cost of screens in the drug-discovery process. We evaluate the relative strength of three high-throughput data sources--chemical structures, images (Cell Painting), and gene-expression profiles (L1000)--to predict compound activity using a sparse historical collection of 16,170 compounds tested in 270 assays for a total of 585,439 readouts. All three data modalities can predict compound activity with high accuracy in 6-10% of assays tested; replacing million-compound physical screens with computationally prioritized smaller screens throughout the pharmaceutical industry could yield major savings. Furthermore, the three profiling modalities are complementary, and in combination they can predict 21% of assays with high accuracy, and 64% if lower accuracy is acceptable. Our study shows that, for many assays, predicting compound activity from phenotypic profiles and chemical structures might accelerate the early stages of the drug-discovery process.

sentences: Deep Mutational Scanning of Dynamic Interaction Networks in the SARS-CoV-2 Spike Protein Complexes: Allosteric Hotspots Control Functional Mimicry and Resilience to Mutational Escape We develop a computational approach for deep mutational scanning of residue interaction networks in the SARS-CoV-2 spike protein complexes to characterize mechanisms of functional mimicry and resilience to mutational escape by miniprotein inhibitors. Using a dynamic mutational profiling and sensitivity analysis of protein stability, binding interactions and global network parameters describing allosteric signaling, we identify regulatory hotspots in the SARS-CoV-2 S complexes with the ACE2 host receptor and ultra-potent miniproteins. The results revealed that global circulating variants are associated with allosteric control points that are dynamically coupled to structural stability hotspots. In this mechanism, variant-induced perturbations of flexible allosteric sites can result in global network changes and elicit specific protein responses. The binding affinity fingerprints and allosteric signatures of the SARS-CoV-2 complexes with miniproteins are determined by a dynamic cross-talk between regulatory control points and conformationally adaptable allosteric hotspots that collectively control structure-functional mimicry, signal transmission and resilience to mutational escape.

sentences: Linear B-cell epitope prediction for in silico vaccine design: a performance review of methods available via command-line interface Linear B-cell epitope prediction research has received a steadily growing interest ever since the first method was developed in 1981. B-cell epitope identification with the help of an accurate prediction method can lead to an overall faster and cheaper vaccine design process, a crucial necessity in the covid-19 era. Consequently, several B-cell epitope prediction methods have been developed over the past few decades, but without significant success. In this study, we review the current performance and methodology of some of the most widely used linear B-cell epitope predictors which are available via a command-line interface, namely BcePred, BepiPred, ABCpred, COBEpro, SVMTriP, LBtope, and LBEEP. Additionally, we attempted to remedy performance issues of the individual methods by developing a consensus classifier, which combines the separate predictions of these methods into a single output, accelerating the epitope-based vaccine design. While the method comparison was performed with some necessary caveats and individual methods might perform much better for specialized datasets, we hope that this update in performance can aid researchers towards the choice of a predictor, for the development of biomedical applications such as designed vaccines, diagnostic kits, immunotherapeutics, immunodiagnostic tests, antibody production, and disease diagnosis and therapy.

sentences: Ignoring errors causes inaccurate timing of single-cell phylogenies Single-cell sequencing provides a new way to explore the evolutionary history of cells. Compared to traditional bulk sequencing, where a population of heterogeneous cells is pooled to form a single observation, single-cell sequencing isolates and amplifies genetic material from individual cells, thereby preserving the information about the origin of the sequences. However, single-cell data is more error-prone than bulk sequencing data due to the limited genomic material available per cell. Here, we present error and mutation models for evolutionary inference of single-cell data within a mature and extensible Bayesian framework, BEAST2. Our framework enables integration with biologically informative models such as relaxed molecular clocks and population dynamic models. Our simulations show that modeling errors increase the accuracy of relative divergence times and substitution parameters. We reconstruct the phylogenetic history for a colorectal cancer patient and a healthy patient from single-cell DNA sequencing data. We find that the estimated times of terminal splitting events are shifted forward in time compared to models which ignore errors. We observed that not accounting for errors can overestimate the phylogenetic diversity in single-cell DNA sequencing data. We estimate that 30-50% of the apparent diversity can be attributed to error. Our work enables a full Bayesian approach capable of accounting for errors in the data within the integrative Bayesian software framework BEAST2.

sentences: Flexibility of Functional Neuronal Assemblies Supports Human Memory Summary ParagraphEpisodic memories, or consciously accessible memories of unique events, represent a key aspect of human cognition. Evidence from rodent models suggests that the neural representation of these complex memories requires cooperative firing of groups of neurons on short time scales1, organized by gamma oscillations2, 3. These co-firing groups, termed "neuronal assemblies,"4 represent a fundamental neurophysiological unit supporting memory. Using microelectrode data from neurosurgical patients5, we identify neuronal assemblies in the human MTL and show that they exhibit consistent organization in their firing pattern based on gamma phase information. We connect these properties to memory performance across recording sessions. Finally, we describe how human neuronal assemblies flexibly adjust6-9 over longer time scales. Our findings provide key evidence linking assemblies to human episodic memory for the first time.

sentences: Interpreting the contributions of transcript features to protein abundance prediction models Protein and mRNA levels correlate only moderately across samples. The availability of proteogenomics data sets with protein and transcript measurements from matching samples is providing new opportunities to assess the degree to which protein levels in a system can be predicted from mRNA information. Here we revisited the protein level prediction problem and analyzed large proteogenomics data from 8 cancer types within the Clinical Proteomic Tumor Analysis Consortium (CPTAC) data set. We trained models to predict the abundance of 13,637 proteins using matching transcriptome data from up to 958 tumor or normal adjacent tissue samples each, and compared predictive performances across algorithms and input features. As data depth increased, the incorporation of additional transcripts as input features increasingly improved the ability to predict sample protein abundance. We found widespread occurrences where the abundance of a protein is considerably less well explained by its own cognate transcript level than that of one or more trans locus transcripts, suggesting post-transcriptional regulations broadly influence protein and mRNA correlation. Transcripts that contribute to non-cognate protein abundance primarily involve those encoding known interaction partners and protein complex members of the protein of interest. Network analysis reveals a proteome-wide interdependency of protein abundance on the transcript levels of interacting proteins. Proteogenomic co-expression analysis may have utility for finding gene interactions and predicting expression changes in biological systems.

sentences: A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel. Here, we used this approach to study the human oncogenic Kaposis sarcoma-associated herpesvirus (KSHV). We designed a sgRNA library containing all possible ~22,000 guides targeting the genome of KSHV - one cut site approximately every 8 base pairs - enabling the pooled screening of the entire genome. We used this tool to phenotype all possible Cas9-targeted viruses for transcription of KSHV late genes, which is required to produce structural components of the viral capsid. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we discovered a novel hit, ORF46 - and more specifically its DNA binding domain - is required for viral DNA replication. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.

sentences: Spatio-temporal analysis identifies hotspots of marine mammal strandings along the Indian coastline: implications for developing a National Marine Mammal Stranding Response and Management policy Marine mammal strandings provide vital information on their life histories, population health and status of marine ecosystems. Opportunistic reporting of strandings serve as a potent low-cost tool for conservation monitoring of these highly mobile species. We present the results of spatio-temporal analyses of marine mammal stranding events to identify hotpots along Indian coastline. We collated data over a long-time frame (~270 years) available from various open access databases, reports and publications. Given the inadequacy in data collection over these years, we grouped data into four major groups viz. baleen whales, toothed whales, small cetaceans and dugongs. Further, we described the trends in data for marine mammal sightings, incidental mortalities, induced mortalities and stranding events using the last group for spatio-temporal analysis. Annual strandings along the Indian coast has increased considerably in the recent years (11.25 {+/-} 9.10 strandings/ year), peaking in the last two years (2015-17, mean = 27.66{+/-}12.03 strandings /year). We found that number of strandings spiked in June- September along the west coast and December- January along the east coast. We identified several sections of coastline which have consistently received comparatively higher number of stranded animals (0.38 - 1.82 strandings/km) throughout the study period. Use of novel geospatial tool  Emerging Hotspot Analysis revealed new and consecutive hotspots along the north-west coast, and sporadic hotspots along the south-east coast. Despite the challenges of working with an opportunistic database, this study highlights critical areas to be prioritized for monitoring marine mammal strandings in the country. We recommend establishing regional marine mammal stranding response centres at the identified hotspots coordinated by a National Stranding Monitoring Centre with adequate funding support. Regular conduct of stranding response programs for field veterinarians, frontline personnel focused around identified stranding hotspots would help develop a comprehensive picture of marine mammal populations in Indian waters.

sentences: Mitochondrial phenotypes in purified human immune cell subtypes and cell mixtures Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among molecularly-defined immune cell subtypes, we quantify the natural variation in citrate synthase, mitochondrial DNA copy number (mtDNAcn), and respiratory chain enzymatic activities in human neutrophils, monocytes, B cells, and naive and memory T lymphocyte subtypes. In mixed peripheral blood mononuclear cells (PBMCs) from the same individuals, we show to what extent mitochondrial measures are confounded by both cell type distributions and contaminating platelets. Cell subtype-specific measures among women and men spanning 4 decades of life indicate potential age- and sex-related differences, including an age-related elevation in mtDNAcn, which are masked or blunted in mixed PBMCs. Finally, a proof-of-concept, repeated-measures study in a single individual validates cell type differences and also reveals week-to-week changes in mitochondrial activities. Larger studies are required to validate and mechanistically extend these findings. These mitochondrial phenotyping data build upon established immunometabolic differences among leukocyte sub-populations, and provide foundational quantitative knowledge to develop interpretable blood-based assays of mitochondrial health.

sentences:
Survival mechanism of a novel marine multistress-tolerant Meyerozyma guilliermondii GXDK6 under high NaCl stress as revealed by integrative omics analysis A novel strain named Meyerozyma guilliermondii GXDK6 was provided in this work, which was confirmed to survive independently under high salt stress (12% NaCl) or co-stress condition of strong acid (pH 3.0) and high salts (10% NaCl) without sterilization. Its survival mechanism under high salt stress was revealed by integrated omics for the first time. Whole-genome analysis showed that 14 genes (e.g., GPD1 and FPS1) of GXDK6 relevant to salt tolerance were annotated and known to belong to various salt-resistant mechanisms (e.g., regulation of cell signal transduction and glycerol metabolism controls). Transcriptome sequencing results indicated that 1220 genes (accounting for 10.15%) of GXDK6 were differentially transcribed (p < 0.05) when GXDK6 growth was under 10% stress for 16 h, including important novel salt-tolerant-related genes (e.g., RTM1 and YHB1). Proteomics analysis demonstrated that 1005 proteins (accounting for 27.26%) of GXDK6 were differentially expressed (p < 0.05) when GXDK6 was stressed by 10% NaCl. Some of the differentially expressed proteins were defined as the novel salt-tolerant related proteins (e.g., sugar transporter STL1 and NADPH-dependent methylglyoxal reductase). Metabolomic analysis results showed that 63 types of metabolites (e.g., D-mannose, glycerol and inositol phosphate) of GXDK6 were up- or downregulated when stressed by 10% NaCl. Among them, D-mannose is one of the important metabolites that could enhance the salt-tolerance survival of GXDK6.

IMPORTANCEMicrobial contamination is a huge obstacle in industrial fermentation. The emergence of multistress-tolerant microorganism is expected to realize industrial fermentation without sterilization by controlling specific conditions. However, microorganisms eligible for non-sterile fermentation are required to survive independently under the selected special conditions for the fermentation conditions to be controlled to avoid microbial contamination. Here, a novel marine Meyerozyma guilliermondii was presented, which is able to survive well under high salt stress, its survival mechanism was systematically revealed by integrated omics technology. In addition, finding that NaCl stress could also stimulate the biosynthesis of functional metabolites from GXDK6 (e.g., calcitriol and didemnin B). Among the functional metabolites, calcitriol biosynthesis via microbial method was rarely reported. Thus, its biosynthetic mechanism was further revealed. The findings in this study contributed to understanding the survival mechanism of M. guilliermondii under high salt stress, and the development of new molecular drugs from M. guilliermondii GXDK6.

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sentences: Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling To understand the function of neuronal circuits it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low-throughput, low-selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotrophic RVdGenvA-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of "starter" AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo.

sentences: Strain-level variation influences Methanothermococcus fitness in marine hydrothermal systems Hydrogenotrophic methanogens are ubiquitous chemoautotrophic archaea inhabiting globally distributed deep-sea hydrothermal vent ecosystems and associated subseafloor niches within the rocky subseafloor, yet little is known about how they adapt and diversify in these habitats. To determine genomic variation and selection pressure within methanogenic populations at vents, we examined five Methanothermococcus single cell amplified genomes (SAGs) in conjunction with 15 metagenomes and 10 metatranscriptomes from venting fluids at two geochemically distinct hydrothermal vent fields on the Mid-Cayman Rise in the Caribbean Sea. We observed that some Methanothermococcus strains and their transcripts were more abundant than others in individual vent sites, and among the genes differentiating the Methanothermococcus SAGs were those related to nitrogen fixation and the CRISPR/Cas immune system. SAGs possessing these genes were less abundant than those missing that genomic region. Overall, patterns in nucleotide variation indicated that the population dynamics of Methanothermococcus were not governed by clonal expansions or selective sweeps, at least in the habitats and sampling times included in this study. Together, our results show that although specific lineages of Methanothermococcus co-exist in these habitats, some outcompete others, and possession of accessory metabolic functions does not necessarily provide a fitness advantage in these habitats in all conditions. This work highlights the power of combining single-cell, metagenomic, and metatranscriptomic datasets to determine how evolution shapes microbial abundance and diversity in hydrothermal vent ecosystems.

sentences: Lipid-polymer hybrid nanoparticles utilize B cells and dendritic cells to elicit distinct antigen-specific CD4+ and CD8+ T cell responses Antigen presenting cells (APCs) have been extensively studied for treating cancers and autoimmune diseases. Dendritic cells (DCs) are potent APCs that uptake and present antigens (Ags) to activate immunity or tolerance. Despite their active use in cellular immunotherapies, DCs face several challenges that hinder clinical translation, such as inability to control Ag dosing for tuning immune responses and low abundance in peripheral blood. B cells are a potential alternative to DCs, but their poor non-specific Ag uptake capabilities compromise controllable priming of T cells. We developed phospholipid-conjugated Ags (L-Ags) and lipid-polymer hybrid nanoparticles (L/P-Ag NPs) as Ag delivery platforms to expand the range of accessible APCs for use in priming CD4+ and CD8+ T cells. These delivery platforms were evaluated using DCs, CD40-activated B cells, and resting B cells as a diverse set of APCs to understand the impact of various Ag delivery mechanisms for generation of Ag-specific T cell responses. L-Ag delivery (termed depoting) of MHC class I and II-restricted Ags successfully loaded all APC types in a tunable manner and primed both Ag-specific CD8+ and CD4+ T cells, respectively. Incorporating L-Ags and polymer-conjugated Ags (P-Ag) into NPs can direct Ags to different uptake pathways to engineer the dynamics of presentation and shape T cell responses. DCs were capable of processing and presenting Ag delivered from both L- and P-Ag NPs yet B cells could only utilize Ag delivered from L-Ag NPs. Multivariate analysis of cytokines secreted from APC:T cell co-cultures indicated that L-Ag NPs primed different T cell responses than P-Ag NPs. Altogether, we show that L-Ags and P-Ags can be rationally paired within a single NP to leverage distinct delivery mechanisms to access multiple Ag processing pathways in two APC types, offering a modular delivery platform for engineering immunotherapies.

sentences:
Test-retest reliability of tone- and 40 Hz train-evoked gamma oscillations in female rats and their sensitivity to low-dose NMDA channel blockade RationaleSchizophrenia patients consistently show deficits in sensory-evoked broadband gamma oscillations and click-evoked entrainment at 40 Hz, called the 40 Hz auditory steady-state response (ASSR). Since such evoked oscillations depend on cortical N-methyl D-aspartic acid (NMDA)-mediated network activity, they can serve as pharmacodynamic biomarkers in the preclinical development of drug candidates engaging these circuits. However, there is little test-retest reliability data in preclinical species, a prerequisite for within-subject testing paradigms.

ObjectivesWe investigated the long-term stability of these measures in a rodent model.

MethodsFemale rats with chronic epidural implants were used to record tone- and 40 Hz click-evoked responses at multiple time points and across six sessions, spread over 3 weeks. We assessed reliability using intraclass correlation coefficients (ICC). Separately, we used mixed-effects ANOVA to examine time and session effects. Individual subject variability was determined using the coefficient of variation (CV). Lastly, to illustrate the importance of long-term measure stability for within-subject testing design, we used low to moderate doses of an NMDA antagonist MK801 (0.025-0.15 mg/kg) to disrupt the evoked responses.

ResultsWe found that 40 Hz ASSR showed good reliability (ICC=0.60-0.75) while the reliability of tone-evoked gamma ranged from fair to good (0.33-0.67). We noted time but no session effects. Subjects showed a lower variance for ASSR over tone-evoked gamma. Both measures were dose-dependently attenuated by NMDA antagonism.

ConclusionOverall, while both measures use NMDA transmission, 40 Hz ASSR showed superior psychometric properties of higher ICC and lower CV, relative to tone-evoked gamma.

sentences: The genome of the CTG(Ser1) yeast  Scheffersomyces stipitis  is plastic Microorganisms need to adapt to environmental changes, and genome plasticity can lead to rapid adaptation to hostile environments by increasing genetic diversity. Here, we investigate genome plasticity in the CTG(Ser1) yeast Scheffersomyces stipitis, an organism with an enormous potential for second-generation biofuel production. We demonstrate that S. stipitis has an intrinsically plastic genome and that different S. stipitis isolates have genomes with distinct chromosome organisation. Real-time evolution experiments show that S. stipitis genome plasticity is common and rapid as extensive genomic changes with fitness benefits are detected following in vitro evolution experiments. Hybrid MinION Nanopore and Illumina genome sequencing identifies retrotransposons as major drivers of genome diversity. Indeed, the number and position of retrotransposons is different in different S. stipitis isolates, and retrotransposon-rich regions of the genome are sites of chromosome rearrangements. Our findings provide important insights into the adaptation strategies of the CTG (Ser1) yeast clade and have critical implications in the development of second-generation biofuels. These data highlight that genome plasticity is an essential factor to be considered for the development of sustainable S. stipitis platforms for second-generation biofuels production.

sentences: A universal probabilistic spike count model reveals ongoing modulation of neural variability Neural responses are variable: even under identical experimental conditions, single neuron and population responses typically differ from trial to trial and across time. Recent work has demonstrated that this variability has predictable structure, can be modulated by sensory input and behaviour, and bears critical signatures of the underlying network dynamics and computations. However, current methods for characterising neural variability are primarily geared towards sensory coding in the laboratory: they require trials with repeatable experimental stimuli and behavioural covariates. In addition, they make strong assumptions about the parametric form of variability, rely on assumption-free but data-inefficient histogram-based approaches, or are altogether ill-suited for capturing variability modulation by covariates. Here we present a universal probabilistic spike count model that eliminates these shortcomings. Our method builds on sparse Gaussian processes and can model arbitrary spike count distributions (SCDs) with flexible dependence on observed as well as latent covariates, using scalable variational inference to jointly infer the covariate-to-SCD mappings and latent trajectories in a data efficient way. Without requiring repeatable trials, it can flexibly capture covariate-dependent joint SCDs, and provide interpretable latent causes underlying the statistical dependencies between neurons. We apply the model to recordings from a canonical non-sensory neural population: head direction cells in the mouse. We find that variability in these cells defies a simple parametric relationship with mean spike count as assumed in standard models, its modulation by external covariates can be comparably strong to that of the mean firing rate, and slow low-dimensional latent factors explain away neural correlations. Our approach paves the way to understanding the mechanisms and computations underlying neural variability under naturalistic conditions, beyond the realm of sensory coding with repeatable stimuli.

sentences: Antagonism of D1, but not D2, dopamine receptors inhibits cued sucrose seeking by decreasing arousal Dopamine facilitates approach to reward via its actions on dopamine receptors in the nucleus accumbens. For example, blocking either D1 or D2 dopamine receptors in the accumbens reduces the proportion of reward- predictive cues to which rats respond with cued approach. Recent evidence indicates that accumbens dopamine also promotes wakefulness and arousal, but the relationship between dopamines roles in arousal and reward seeking remains unexplored. Here, we show that the ability of systemic or intra-accumbens injections of the D1 antagonist SCH23390 to reduce cued approach to reward depends on the animals state of arousal. Handling the animal, a manipulation known to increase arousal, was sufficient to reverse the behavioral effects of the antagonist. In addition, SCH23390 reduced spontaneous locomotion and increased time spent in sleep postures, both consistent with reduced arousal, but also increased time spent immobile in postures inconsistent with sleep. In contrast, the ability of the D2 antagonist haloperidol to reduce cued approach was not reversible by handling. Haloperidol reduced spontaneous locomotion but did not increase sleep postures, instead increasing immobility in non-sleep postures. We place these results in the context of the extensive literature on dopamines contributions to behavior, and propose the arousal-motor hypothesis, a novel synthesis that accounts both for our findings and many previous results that have drawn disparate and conflicting conclusions.

sentences: sstar: A Python package for detecting archaic introgression from population genetic data with S* S* is a widely used statistic for detecting archaic admixture from population genetic data. To apply S*, several previous studies used the freezing-archer repository, which is a compilation of Python 2 and R scripts originally developed to detect Neanderthal and Denisovan introgression into modern humans. When studying other species or populations, however, freezing-archer is not directly applicable. Here, we implemented a new Python 3 package named sstar, with better performance than freezing-archer. Reliability was also improved after fixing bugs in freezing-archer. Compared with freezing-archer, sstar is more user-friendly, versatile, and well-documented.

sentences: Dendritic GABAergic inhibition controlled by Shh signaling-dependent stellate cell pool is critical for motor learning Cerebellar inhibitory interneurons are important regulators of neural circuit activity for diverse motor and non-motor functions. The molecular layer interneurons (MLI), consisting of basket cells (BCs) and stellate cells (SCs), provide dendritic and somatic inhibitory synapses onto Purkinje cells, respectively. They are sequentially generated in an inside-out pattern from Pax2+ immature interneurons which migrate from the prospective white matter to the ML of the cortex. However, little is known as to how MLI subtype identities and pool sizes are determined, nor are their contributions to motor learning well understood. Here, we show that GABAergic progenitors fated to generate both BCs and SCs respond to the Shh signal. Conditional abrogation of Shh signaling inhibited proliferation of GABAergic progenitors and reduced the number of Pax2+ cells, whereas persistent Shh pathway activation increased their numbers. These changes, however, did not affect early-born BC numbers but selectively altered the SC pool size. Moreover, genetic depletion of GABAergic progenitors when BCs are actively generated also resulted in a specific reduction of SCs, suggesting that the specification of MLI subtypes is independent of Shh signaling and their birth order and likely occurs after Pax2+ cells settle into their laminar positions in an inside-out sequence. Mutant mice with reduced SC numbers displayed decreased dendritic inhibitory synapses and neurotransmission onto Purkinje cells, resulting in an impaired acquisition of eyeblink conditioning. These findings also reveal an essential role of Shh signaling-dependent SCs in regulating inhibitory dendritic synapses and motor learning.

sentences: Circulating CD138 (syndecan-1) enhances APRIL-mediated autoreactive B cell survival and differentiation in MRL/Lpr mice High levels of serum CD138, a heparan sulfate-bearing proteoglycan, correlates with increased disease activity in systemic lupus erythematosus (SLE) patients. Mechanisms responsible for serum CD138 production and its biological function in SLE disease remain poorly understood. In this study, corroborating patient data, we detected an increase in serum CD138 in MRL/Lpr mice parallel to disease activity. Although TCR{beta}+CD138+ T cells expand in MRL/Lpr mice as the disease progresses, surprisingly, TCR{beta}+CD138- cells were the primary source of circulating CD138 as the transfer of TCR{beta}+CD138- cells to young MRL/Lpr mice, but not TCR{beta}+CD138+ cells, resulted with higher serum CD138 in the recipient mice. We found that elevated trypsin, expressed by TCR{beta}+CD138- cells, was able to cleave CD138 from T cells. Moreover, suggesting the contribution of cleaved CD138 to the increase in blood CD138, trypsin inhibitors  defined trypsin inhibitor (DTI) or leupeptin increased CD138 expression on TCR{beta}+CD138- cells. Furthermore, soluble CD138 was able to bind  a proliferation inducing ligand (APRIL) and enhanced APRIL-mediated plasma cell generation and autoreactive antibody production through the phosphorylation of extracellular-signal-regulated kinase (ERK) in B cells. APRIL receptor,  transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) was involved in the enhancement of APRIL activity by CD138, as the synergistic effect of APRIL and CD138 was ablated on TACI deficient B cells. These findings indicate a regulatory role for soluble CD138 in B cell differentiation and autoreactive antibody secretion in SLE disease.

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Heterogeneous pdgfrβ+ cells regulate coronary vessel development and revascularization during heart regeneration Endothelial cells emerge from the atrioventricular canal (AVC) to form nascent coronary blood vessels in the juvenile zebrafish heart. We found that pdgfr{beta} is first expressed in the epicardium around the AVC and later becomes localized mainly in the mural cells. pdgfr{beta} mutant fish display severe defects in mural cell recruitment and coronary vessel development. pdgfr{beta}+ mural cells are heterogeneous and those associated with coronary arteries also express cxcl12b. Mural cells positive for both pdgfr{beta} and cxcl12b transgenic reporters had elevated expression of smooth muscle cell genes. Interestingly, these mural cells were associated with coronary arteries even in the absence of Pdgfr{beta}, although smooth muscle gene expression was downregulated in these cells. We found that pdgfr{beta} expression dynamically changes in the epicardium derived cells, which we found to be a heterogeneous population. mdka was identified as a gene upregulated in subpopulations of pdgfr{beta}+ cells during heart regeneration. However, pdgfr{beta} but not mdka mutants showed defects in heart regeneration. Our results demonstrated that pdgfr{beta}+ cells and Pdgfr{beta} signaling are essential for coronary development and heart regeneration.

SUMMARY STATEMENTHeterogeneous pdgfr{beta} positive cells are present in developing and regenerating zebrafish hearts and are required for development of mural cells and their association with the nascent coronary vessels during zebrafish heart development and regeneration.

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On slope factor of the operational model of agonism Background and PurposeAlthough being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1985, Black et al. introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves.

Experimental ApproachFunctional analysis of OMA demonstrates that the slope factor implemented by Black et al. affects relationships among parameters of the OMA, leading to wrong estimates of operational efficacy and affinity of an agonist. In contrast, functional analysis of OMA with Hill coefficient as a slope factor has not this flaw.

Key ResultsFitting of the OMA with Black et al. slope factor to concentration-response curves of several models (homotropic allosteric modulation, substrate inhibition and non-competitive auto-inhibition) gives wrong estimates of operational efficacy and affinity. In contrast, fitting of the OMA modified by the Hill coefficient to concentration-response curves of these several models gives correct estimates of operational efficacy and affinity.

Conclusion and ImplicationsThe OMA modified by the Hill coefficient can be used for correct ranking of agonism and subsequent analysis, like quantification of signalling bias.

sentences: Single-cell analyses reveal a continuum of cell state and composition changes in the malignant transformation of polyps to colorectal cancer To chart cell composition and cell state changes that occur during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC), we generated 451,886 single-cell chromatin accessibility profiles and 208,557 single-cell transcriptomes from 48 polyps, 27 normal tissues, and 6 CRCs collected from patients with and without germline APC mutations. A large fraction of polyp and CRC cells exhibit a stem-like phenotype, and we define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from normal to CRC. Advanced polyps contain increasing numbers of stem-like cells, regulatory T-cells, and a subtype of FOX-regulated pre-cancer associated fibroblasts. In the cancerous state, we observe T-cell exhaustion, RUNX1-regulated cancer associated fibroblasts, and increasing accessibility associated with HNF4A motifs in epithelia. Methylation changes in sporadic CRC are strongly anti-correlated with accessibility changes along this continuum, further identifying regulatory markers for molecular staging of polyps.

sentences: NeurotoxKb: compilation, curation and exploration of a knowledgebase of environmental neurotoxicants specific to mammals Exposure to environmental neurotoxicants is a significant concern due to their potential to cause permanent or irreversible damage to the human nervous system. Here, we present the first dedicated knowledgebase, NeurotoxKb, on environmental neurotoxicants specific to mammals. Using a detailed workflow, we have compiled 475 potential non-biogenic neurotoxicants from 835 published studies with evidence of neurotoxicity specific to mammals. A unique feature of NeurotoxKb is the manual curation effort to compile and standardize the observed neurotoxic effects for the potential neurotoxicants from 835 published studies. For the 475 potential neurotoxicants, we have compiled diverse information such as chemical structures, environmental sources, chemical classification, physicochemical properties, molecular descriptors, predicted ADMET properties, and target human genes. To better understand the prospect of human exposure, we have explored the presence of potential neurotoxicants in external exposomes via two different analyses. By analyzing 55 chemical lists representing global regulations and guidelines, we reveal potential neurotoxicants both in regular use and produced in high volume. By analyzing human biospecimens, we reveal potential neurotoxicants detected in them. Lastly, a construction of the chemical similarity network and ensuing analysis revealed the diversity of the toxicological space of 475 potential neurotoxicants. NeurotoxKb is accessible online at: https://cb.imsc.res.in/neurotoxkb/.

sentences: Embryo development in Carica papaya Linn Papaya (Carica papaya Linn.) is a tropical plant whose draft genome has been sequenced. Papaya produces large fruits rich in vitamins A and C and is an important cash crop in developing countries. Nonetheless, little is known about how the female gametophyte develops, how it is fertilized and how it develops into a mature seed containing an embryo and an endosperm. The Papaya female gametophyte displays a Polygonum-type architecture consisting of two synergid cells, an egg cell, a central cell, and three antipodal cells. Reports are available of the presumed existence of varieties in which cross fertilization is bypassed and autonomous development of embryos occurs (e.g., apomixis). In this study, we analyzed the development of female gametophytes in a commercial Hawaiian parental line and in the presumed apomictic Costa Rican line L1. Samples were collected before and after anthesis to compare the overall structure, size and transcriptional patterns of several genes that may be involved in egg and endosperm cell fate and proliferation. These genes were the putative papaya homologs of ARGONAUTE9 (AGO9), MEDEA (MEA), RETINOBLASTOMA RELATED-1 (RBR1), and SLOW WALKER-1 (SWA1). Our results suggest that its feasibly to identify structural features of Polygonum-type development, and that in bagged female flowers of line L1 we might have observed autonomous development of embryo-like structures. Possible downregulation of papaya homologs for AGO9, MEA, RBR1 and SWA1 was observed in embryo sacs from line L1 before and after anthesis, which may suggest a link between suspected apomixis and transcriptional downregulation of genes for RNA-directed DNA methylation, histone remodelers, and rRNA processing. Most notably, the large size of the papaya embryo sac suggests that it could be a cytological alternative to Arabidopsis thaliana for study. Significant variation in embryo sac size was observed between the varieties under study, suggesting wide differences in the genetic regulation of anatomical features.

sentences: Noise-robust recognition of objects by humans and deep neural networks Deep neural networks (DNNs) for object classification have been argued to provide the most promising model of the visual system, accompanied by claims that they have attained or even surpassed human-level performance. Here, we evaluated whether DNNs provide a viable model of human vision when tested with challenging noisy images of objects, sometimes presented at the very limits of visibility. We show that popular state-of-the-art DNNs perform in a qualitatively different manner than humans - they are unusually susceptible to spatially uncorrelated white noise and less impaired by spatially correlated noise. We implemented a noise-training procedure to determine whether noise-trained DNNs exhibit more robust responses that better match human behavioral and neural performance. We found that noise-trained DNNs provide a better qualitative match to human performance; moreover, they reliably predict human recognition thresholds on an image-by-image basis. Functional neuroimaging revealed that noise-trained DNNs provide a better correspondence to the pattern-specific neural representations found in both early visual areas and high-level object areas. A layer-specific analysis of the DNNs indicated that noise training led to broad-ranging modifications throughout the network, with greater benefits of noise robustness accruing in progressively higher layers. Our findings demonstrate that noise-trained DNNs provide a viable model to account for human behavioral and neural responses to objects in challenging noisy viewing conditions. Further, they suggest that robustness to noise may be acquired through a process of visual learning.

sentences: Cellular heterogeneity in DNA alkylation repair as a trade-off between cell survival and genetic plasticity DNA repair mechanisms fulfil a dual role, as they are essential for cell survival and genome maintenance. Here, we studied how cells regulate the interplay between DNA repair and mutation. We focused on the Escherichia coli adaptive response that increases resistance to DNA alkylation damage. Combination of single-molecule imaging and microfluidic-based single-cell microscopy showed that noise in the gene activation timing of the master regulator Ada is accurately propagated to generate a distinct subpopulation of cells in which all proteins of the adaptive response are absent. Although lack of these proteins causes extreme sensitivity to alkylation stress, cellular heterogeneity in DNA alkylation repair provides a functional benefit by increasing the evolvability of the whole population. We demonstrated this by monitoring the dynamics of nascent mutations during alkylation stress as well as the frequency of fixed mutations that are generated by the distinct subpopulations of the adaptive response. This highlighted that evolvability is a trade-off between mutability and cell survival. Stochastic modulation of DNA repair capacity by the adaptive response solves this trade-off through the generation of a viable hypermutable subpopulation of cells that acts as a source of genetic diversity in a clonal population.

sentences: The OCT2 Transporter Regulates Dopamine D1 Receptor Signaling at the GolgiApparatus Dopamine is the main catecholamine in the brain and the kidney, where it controls a number of physiological functions such as locomotion, cognition, emotion, endocrine regulation and renal function. As a membrane impermeant hormone and neurotransmitter, dopamine is thought to signal by binding and activating dopamine receptors, members of the G protein couple receptor (GPCR) family, only on the plasma membrane. Here, using novel nanobody-based biosensors, we demonstrate for the first time that the dopamine D1 receptor (D1DR), the primary mediator of dopaminergic signaling in the brain and kidney, not only functions on the plasma membrane but becomes activated at the Golgi apparatus in the presence of its ligand. We present evidence that activation of the Golgi pool of D1DR is dependent on Organic Cation Transporter 2 (OCT2), a dopamine transporter, providing an explanation for how the membrane impermeant dopamine accesses subcellular pools of D1DR. We further demonstrate that dopamine activates Golgi-D1DR in the striatal medium spiny neurons (MSN) and this activity depends on OCT2 function. We also introduce a new approach to selectively interrogate compartmentalized D1DR signaling by inhibiting Gs coupling, using a nanobody-based chemical recruitment system. Using this strategy, we show that Golgi-localized D1DRs regulate cAMP production and mediate local protein kinase A activation. Together, our data suggest that spatially compartmentalized signaling hubs are previously unappreciated regulatory aspects of D1DR signaling. Our data also provide further evidence for the role of transporters in regulating subcellular GPCR activity.

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The Structure of ChAdOx1/AZD-1222 Reveals Interactions with CAR and PF4 with Implications for Vaccine-induced Immune Thrombotic Thrombocytopenia Adenovirus derived vectors, based on chimpanzee adenovirus Y25 (ChAdOx1) and human adenovirus type 26 are proving critical in combatting the 2019 SARS-CoV-2 pandemic. Following emergency use authorisation, scale up in vaccine administration has inevitably revealed vaccine related adverse effects; too rare to observe even in large Phase-III clinical trials. These include vaccine-induced thrombotic thrombocytopenia (VITT), an ultra-rare adverse event in which patients develop life-threatening blood clots 5-24 days following vaccination.

To investigate vector-host interactions of ChAdOx1 underpinning VITT we solved the structure of the ChAdOx1 capsid by CryoEM, and the structure of the primary receptor tropism determining fiber-knob protein by crystallography. These structural insights have enabled us to unravel key protein interactions involved in ChAdOx1 cell entry and a possible means by which it may generate misplaced immunity to platelet factor 4 (PF4), a protein involved in coagulation.

We use in vitro cell binding assays to show that the fiber-knob protein uses coxsackie and adenovirus receptor (CAR) as a high affinity binding partner, while it does not form a stable interface with CD46. Computational simulations identified a putative mechanism by which the ChAdOx1 capsid interacts with PF4 by binding in the spaces between hexon proteins, with downstream implications for the causes of VITT.

SummaryWe present the structure of the ChAdOx1 viral vector, derived from chimpanzee adenovirus Y25 at 4.2[A] resolution1. ChAdOx1 is in global use in the AstraZeneca vaccine, ChAdOx1 nCoV-19/AZD-1222, to combat the SARS-CoV-2 coronavirus pandemic. Recently observed, rare, adverse events make detailed mechanistic understanding of this vector key to informing proper treatment of affected patients and the development of safer viral vectors.

Here, we determine a primary mechanism ChAdOx1 uses to attach to cells is coxsackie and adenovirus receptor (CAR), a protein which is identical in humans and chimpanzees. We demonstrate the vector does not form a stable CD46 interaction, a common species B adenovirus receptor, via its primary attachment protein.

Further, we reveal the surface of the ChAdOx1 viral capsid has a strong electronegative potential. Molecular simulations suggest this charge, together with shape complementarity, are a mechanism by which an oppositely charged protein, platelet factor 4 (PF4) may bind the vector surface. PF4 is a key protein involved in the formation of blood clots2, and the target of auto-antibodies in heparin-induced immune thrombotic thrombocytopenia (HITT)3, an adverse reaction to heparin therapy which presents similarly to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare complication of ChAdOx1 nCoV-19 vaccination4-6. We propose a mechanism in which the ChAdOx1-PF4 complex may stimulate the production of antibodies against PF4, leading to delayed blood clot formation, as observed in VITT.

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Checkpoint proteins in patients with precancer and cervical cancer The efficiency of immuno-oncological drugs in treatment cervical cancer is not high. The research of new immunological targets in cervical cancer is actual.

The aim was to study the local level of checkpoint proteins of immune cycle in patients with cervical cancer of different stages (sCD25, 4-1BB, B7.2, TGF-b1, CTLA-4, PD-L1, PD-1, Tim-3, LAG-3, Galectin-9, sCD27, PD-L2).

Materials and research methods. The objects of study were the patients with precancer (n=13) and cervical cancer (n=49). The control group consisted of 13 gynecologically healthy women. The material of study was cervical epithelium obtained by cervical brush. The research method was flow cytometry. Test parameters: sCD25, 4-1BB, B7.2, TGF-b1, CTLA-4, PD-L1, PD-1, Tim-3, LAG-3, Galectin-9, sCD27, PD-L2. Statistical analysis of the data was carried out using nonparametric statistics using the Mann - Whitney U-test. The results of the study.

The levels of sCD25, B7.2, PDL1, Tim-3, sCD27, PD-L2 were increased in the comparison groups relation to the control (p <0.05). The differential criterion for invasive cervical cancer may be an increase in the level of LAG-3 in the cervical epithelium by 43% (p <0.05).The levels of PD-L1, PD-1, sCD27 and PD-L2 were increased opposite the levels LAG-3 and Galectin-9 were decreased in patients with metastatic cervical cancer(p <0.05).

sentences: Kinesin-binding protein remodels the kinesin motor to prevent microtubule-binding Kinesins are tightly regulated in space and time to control their activation in the absence of cargo-binding. Kinesin-binding protein (KIFBP) was recently discovered to bind the catalytic motor heads of 8 of the 45 known kinesin superfamily members and inhibit binding to microtubules. In humans, mutation of KIFBP gives rise to Goldberg-Shprintzen syndrome (GOSHS), but the kinesin(s) that is misregulated to produce clinical features of the disease is not known. Understanding the structural mechanism by which KIFBP selects its kinesin binding partners will be key to unlocking this knowledge. Using a combination of cryo-electron microscopy and crosslinking mass spectrometry, we determined structures of KIFBP alone and in complex with two mitotic kinesins, revealing regions of KIFBP that participate in complex formation. KIFBP adopts an alpha-helical solenoid structure composed of TPR repeats. We find that KIFBP uses a 2-pronged mechanism to remodel kinesin motors and block microtubule-binding. First, KIFBP engages the microtubule-binding interface and sterically blocks interaction with microtubules. Second, KIFBP induces allosteric conformational changes to the kinesin motor head that displace a key structural element in the kinesin motor head (-helix 4) required for microtubule binding. We identified two regions of KIFBP necessary for in vitro kinesin-binding as well as cellular regulation during mitosis. Taken together, this work establishes the mechanism of kinesin inhibition by KIFBP and provides the first example of motor domain remodeling as a means to abrogate kinesin activity.

sentences: Single-cell resolution analysis of the crosstalk between chemogenically activated astrocytes and microglia Astrocytes are the most common glial cell type in the brain, yet, it is unclear how their activation affects the transcriptome of neighboring cells. Engineered G protein-coupled receptors (GPCRs) called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) enable selective activation of specific cell types, such as astrocytes. Here, we combine activation of astrocytes in the hippocampus and cortex of healthy mice with single-cell RNA sequencing. Our data show that long-term activation of astrocytes dramatically alters the transcriptome of astrocytes and microglia. Genes that were differentially expressed in Gq-DREADD-activated astrocytes are involved in neurogenesis and low-density lipoprotein particle biology, while those in the microglia were involved in lipoprotein handling, purinergic receptor activity, and immune cell migration and chemotaxis. Furthermore, network analysis showed that Gq-DREADD-mediated activation in astrocytes resulted in an upregulation of genes involved in the GPCR signaling pathways and calcium ion homeostasis, confirming astrocyte activation. This dataset will serve as a resource for the broader neuroscience community, and our findings highlight the importance of studying transcriptomic alterations in microglia after astrocyte activation in vivo.

sentences: Placental uptake and metabolism of 25(OH)Vitamin D determines its activity within the fetoplacental unit Pregnancy 25-hydroxyvitamin D (25(OH)D) concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy.

sentences: Neighbouring modifications interfere with the detection of phosphorylated alpha-synuclein at Serine 129: Revisiting the specificity of pS129 antibodies Alpha-synuclein (aSyn) within Lewy bodies, Lewy neurites, and other pathological hallmarks of Parkinsons disease and synucleinopathies have consistently been shown to accumulate in aggregated and phosphorylated forms of the protein, predominantly at Serine 129 (S129). Antibodies against phosphorylated S129 (pS129) have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues. However, most of the antibodies and immunoassays aimed at detecting pS129-aSyn were developed based on the assumption that neighbouring post-translational modifications (PTMs) either do not co-occur with pS129 or do not influence its detection. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal PTMs (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighbouring PTMs. However, consistent with previous reports, most pS129 antibodies showed cross-reactivity towards other proteins and often detected low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or High Molecular Weight aggregates of aSyn. Our observations suggest that the pS129 antibodies do not capture the biochemical and morphological diversity of aSyn pathology. They also underscore the need for more specific pS129 antibodies, more thorough characterization and validation of existing antibodies, and the use of the appropriate protein standards and controls in future studies.

sentences: The shifting epigenetic landscape and roles of key transcription factors during early retinal cell differentiation During development, all retinal cell types emerge from multipotent retinal progenitor cells (RPCs) following distinct trajectories. Each lineage is subject to regulation by multiple intrinsic and extrinsic factors, which converge onto the epigenetic landscape to dictate the collective gene expression profile and thereby the phenotypes of individual cell states/types. Recent scRNA-seq studies have further delineated these trajectories, providing a general picture of the relationships between the different cell states/types and revealing a transitional RPC state shared by all the early lineages. Nevertheless, how the epigenetic landscape shifts along individual trajectories is only beginning to be disseminated, and the roles of key transcription factors in these shifts are not known. In this study, we dissect the changes of the epigenetic landscape along these trajectories in the early developing retina by scATAC-seq and identify globally the enhancers, enriched motifs, and potential interacting transcription factors underlying the cell state/type specific gene expression in individual lineages, with a focus on retinal ganglion cells (RGCs). Using CUT&Tag-seq, we further identify the enhancers bound directly by four key transcription factors, Otx2, Atoh7, Pou4f2, and Isl1, and uncover their roles in shaping the epigenetic landscape to control gene expression in a sequential and combinatorial fashion during RGC genesis. These results provide experimental support for a general paradigm in which transcription factors collaborate and compete to regulate the emergence of distinct retinal cell types such as RGCs from the multipotent transitional RPCs.

sentences: Dysregulation of Hepatitis B Virus Nucleocapsid Assembly with RNA-directed Small Ligands RNA sequences/motifs dispersed across the pre-genomic copy of the Hepatitis B Virus genome regulate formation of nucleocapsids in vitro in an epsilon/polymerase independent fashion. These multiple RNA Packaging Signals (PSs) form stem-loops presenting in each loop a core protein recognition motif, -RGAG-. Small, drug-like molecules binding these motifs were identified by screening an immobilized library with a fluorescently-labelled RNA oligonucleotide encompassing the most conserved of these sites. This identified 66 "hits", with affinities ranging from low nanomolar to high micromolar in SPR assays. High affinity ligand binding is dependent on the presence of the -RGAG-motif, which also appears to be the common element in cross-binding to other PS sites. Some of these compounds are potent inhibitors of in vitro core protein assembly around the HBV pre-genome. Mathematical modelling confirms the potential of these novel anti-viral drug targets for disrupting replication of this major human pathogen. Preliminary structure-activity relationships of the highest affinity compound reveal critical functional groups for PS-binding. PS-regulated assembly is easily adapted to high-throughput screening allowing future development of pharmacologically active compounds.

sentences: Patient-associated mutations in Drosophila Alk perturb neuronal differentiation and promote survival Activating Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) mutations occur in pediatric neuroblastoma and are associated with poor prognosis. To study ALK-activating mutations in a genetically controllable system we employed CRIPSR/Cas9, incorporating orthologues of the human oncogenic mutations ALKF1174L and ALKY1278S in the Drosophila Alk locus. AlkF1251L and AlkY1355S mutant Drosophila exhibit enhanced Alk signaling phenotypes, but unexpectedly depend on the Jelly belly (Jeb) ligand for activation. Both AlkF1251L and AlkY1355S mutant larval brains display hyperplasia, represented by increased numbers of Alk-positive neurons. Despite this hyperplasic phenotype, no brain tumors were observed in mutant animals. We show that hyperplasia in Alk mutants was not caused by significantly increased rates of proliferation, but rather by decreased levels of apoptosis in the larval brain. Using single-cell RNA sequencing (scRNA-seq), we identify perturbations during temporal fate specification in AlkY1355S mutant mushroom body lineages. These findings shed light on the role of Alk in neurodevelopmental processes and highlight the potential of activating Alk mutations to perturb specification and promote survival in neuronal lineages.

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Cell Centred Finite Element Model for Intestinal Organoids Shape Analysis: From tissue architecture to mechanics Organoids, established from stem cells owing to their self-renewal and differentiation capacities, are self-organised three-dimensional tissue culture recapitulating the original cell populations and their associated functions, as well as tissue architecture. The intestinal organoids established from adult stem cells isolated from the intestinal crypts, recreate 3D epithelial mini-intestines. They represent an excellent tool to study intestinal stem cell capacities and their ability to reconstitute a fully polarised and functional epithelium. These 3D cultures recapitulate in vitro the tissue characteristics, including architecture, either in physiological or disease conditions whether they are established from healthy or pathological tissue samples respectively. In this regard, their use for potential treatments screening carries the hopes for a future personalized medicine for which image-analysis such as HCS are increasingly being developed. Numerous numerical models have been developed to study the effects of organoid development on their shape. Most of them remain mainly restricted in their physical description due to the complex inter-relationship between cell physics, phenotypes and behaviors, exploding the number of variables in modeling formulation. Finite Element Method (FEM) is a numerical analysis method employed in mechanics to model deformation and evaluate residual stress of complex structures making it difficult to obtain analytical solutions. Considering epithelial architecture as a homogeneous material where each cell is an elemental equivalent part of the problem, FEM allows a direct link between tissue architecture deformations and local mechanical constraints. Here we formalise a new organoid cell centred FEM with a physical description borrowed from the engineering world. This model can allow a better understanding of the individual contribution of physical/mechanical properties of individual cells on general tissue architecture.

Author SummaryStem cell research has experienced a breakthrough with the development of powerful tools, namely, organoids. The possibility to recreate in vitro from adult stem cells a fully functional adult tissue with its actual 3D architecture made accessible poorly defined steps in tissue morphogenesis or pathophysiology. If recent efforts have focused on studies of tissue specific organoids functions, modelling a generic bio-mechanic model of organoid remains necessary to decipher the mechanical biodiversity of organoid structures.

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MTG16 (CBFA2T3) regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors Aberrant epithelial differentiation and regeneration contribute to colon pathologies including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). MTG16 (CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 deficiency in mice exacerbates colitis and increases tumor burden in CAC, though the underlying mechanisms remain unclear. Here, we identified MTG16 as a central mediator of epithelial differentiation, promoting goblet and restraining enteroendocrine cell development in homeostasis and enabling regeneration following dextran sulfate sodium (DSS)-induced colitis. Transcriptomic analyses implicated increased E box-binding transcription factor (E protein) activity in MTG16-deficient colon crypts. Using a novel mouse model with a point mutation that disrupts MTG16:E protein complex formation (Mtg16P209T), we established that MTG16 exerts control over colonic epithelial differentiation and regeneration by repressing E protein-mediated transcription. Mimicking murine colitis, MTG16 expression was increased in biopsies from patients with active IBD compared to unaffected controls. Finally, uncoupling MTG16:E protein interactions only partially phenocopied the enhanced tumorigenicity of Mtg16-/- colon in the azoxymethane(AOM)/DSS-induced model of CAC, indicating that MTG16 protects from tumorigenesis through additional mechanisms. Collectively, our results demonstrate that MTG16, via its repression of E protein targets, is a key regulator of cell fate decisions during colon homeostasis, colitis, and cancer.

Graphical Abstract

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org.highwire.dtl.DTLVardef@21948borg.highwire.dtl.DTLVardef@9f60eeorg.highwire.dtl.DTLVardef@194b9fdorg.highwire.dtl.DTLVardef@13eb1a7_HPS_FORMAT_FIGEXP  M_FIG C_FIG

sentences: Recruitment of CTCF to an Fto enhancer is responsible for transgenerational inheritance of obesity Transgenerational transmission of epiphenotypes is poorly understood. Here we show that exposure of pregnant mouse F0 females to BPA results in obesity in the F2 progeny due to increased food intake and leptin resistance. This epiphenotype can be transmitted up to the F6 generation and disappears in F7. Analyses of chromatin accessibility in sperm of the F1-F6 generations reveals alterations in the binding of CTCF at two enhancers of the Fto gene in obese but not control animals that correlates with transmission of obesity. Deletion of the CTCF site in Fto results in mice that fail to become obese when exposed to BPA. These Fto enhancers show increased interactions in sperm of obese mice with the Irx3 and Irx5 genes, which are involved in the differentiation of appetite controlling AgRP/NPY neurons. Single-nucleus and immunofluorescence analyses in the arcuate nucleus of the hypothalamus suggest that exposure to BPA results in expansion of the number of orexigenic AgRP neurons. This expansion correlates with increased accessibility of the Fto proximal enhancer in radial glia-like neural stem cells (RG-NSCs), which give rise to AgRP/NPY neurons, and in mature oligodendrocytes. The results provide a molecular mechanism for transgenerational inheritance in mammals and suggest that both genetic and epigenetic alterations of Fto can lead to the same phenotypic outcomes.

sentences: Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), supplemented by a single further redshifted fluorochrome (670/701-750) were depleted with 775 nm. To the best of our knowledge, these are the first published five color STED images. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.

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Teasing apart the host-related, nutrient-related and temperature-related effects shaping the phenology and microbiome of the tropical seagrass Halophila stipulacea BackgroundHalophila stipulacea seagrass meadows are an ecologically important and threatened component of the ecosystem in the Gulf of Aqaba. Recent studies have demonstrated correlated geographic patterns for leaf endophytic community composition and leaf morphology, also coinciding with different levels of water turbidity and nutrient concentrations. Based on these observations, workers have suggested an environmental microbial fingerprint, which may reflect various environmental stress factors seagrasses have experienced, and may add a holobiont level of plasticity to seagrasses, assisting their acclimation to changing environments and through range expansion. However, it is difficult to tease apart environmental effects from host-diversity dependent effects, which have covaried in field studies, although this is required in order to establish that differences in microbial community compositions among sites are driven by environmental conditions rather than by features governed by the host.

ResultsIn this study we carried out a mesocosm experiment, in which we studied the effects of warming and nutrient stress on the composition of epiphytic bacterial communities and on some phenological traits. We studied H. stipulacea collected from two different meadows in the Gulf of Aqaba, representing differences in the host and the environment alike. We found that the source site from which seagrasses were collected was the major factor governing seagrass phenology, although heat increased shoot mortality and nutrient loading delayed new shoot emergence. Bacterial diversity, however, mostly depended on the environmental conditions. The most prominent pattern was the increase in Rhodobacteraceae under nutrient stress without heat stress, along with an increase in Microtrichaceae. Together, the two taxa have the potential to maintain nitrate reduction followed by an anammox process, which can together buffer the increase in nutrient concentrations across the leaf surface.

ConclusionsOur results thus corroborate the existence of environmental microbial fingerprints, which are independent from the host diversity, and support the notion of a holobiont level plasticity, both important to understand and monitor H. stipulacea ecology under the changing climate.

sentences: Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a critical regulator of cytokinesis predominantly in transformed cells and its deregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals have not been clearly defined. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated perinatal lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3{beta}/{beta}-Catenin/Myc axis in an Akt-dependent manner. The phenotype was recapitulated using human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3{beta}. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting its novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.

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Towards a genome sequence for every animal: where are we now? In less than 25 years, the field of animal genome science has transformed from a discipline seeking its first glimpses into genome sequences across the Tree of Life to a global enterprise with ambitions to sequence genomes for all of Earths eukaryotic diversity (1). As the field rapidly moves forward, it is important to take stock of the progress that has been made to best inform the disciplines future. In this Perspective, we provide a contemporary, quantitative overview of animal genome sequencing. We identified the best available genome assemblies on GenBank, the worlds most extensive genetic database, for 3,278 unique animal species across 24 phyla. We assessed taxonomic representation, assembly quality, and annotation status for major clades. We show that while tremendous taxonomic progress has occurred, stark disparities in genomic representation exist, highlighted by a systemic overrepresentation of vertebrates and underrepresentation of arthropods. In terms of assembly quality, long-read sequencing has dramatically improved contiguity, whereas gene annotations are available for just 34.3% of taxa. Furthermore, we show that animal genome science has diversified in recent years with an ever-expanding pool of researchers participating. However, the field still appears to be dominated by institutions in the Global North, which have been listed as the submitting institution for 77% of all assemblies. We conclude by offering recommendations for how we can collectively improve genomic resource availability and value while also broadening global representation.

Significance statementThe field of animal genome science is rapidly developing, and efforts are underway to sequence genomes for all of Earths eukaryotic biodiversity. Here, we provide an overview of animal genome sequencing, with emphases on taxonomic representation, assembly quality, and geographic representation. We show that while a staggering 3,278 unique animal species have had their genomes sequenced, massive disparities exist in terms of the taxonomic groups receiving attention, the quality of the resources being produced, and the institutions driving the field. We highlight areas where improvements can be made, notably by continuing to increase the quality of genome assemblies, including by improving metadata and voucher specimen associations, and actively developing meaningful collaborations between researchers form the Global North and South.

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Disturbance by soil mixing decreases microbial richness and supports homogenizing community assembly processes The spatial heterogeneity of soils microhabitats warrants the study of ecological patterns and community assembly processes in the context of community coalescence, or the combining and restructuring of communities and their environment. By mixing soil at various frequencies in a 16-week lab incubation, we explored the effects of mixing disturbance on soil bacterial richness, community composition, and community assembly processes. We hypothesized that well-mixed soil would harbor less richness, dominated by homogenizing dispersal and homogeneous selection. Using 16S rRNA gene sequencing, we inferred ecological processes, estimated richness and differential abundance, calculated compositional dissimilarity, and constructed co-occurrence networks. Findings supported our hypotheses, with >20% decrease in soil bacterial richness in well-mixed soil. While soil mixing resulted in increasingly dissimilar communities compared to unmixed soil (Bray-Curtis dissimilarity; 0.75 vs. 0.25), well-mixed soil communities were increasingly self-similar. Our results imply that vast soil diversity may be attributed to the unmixed and spatially heterogeneous nature of soil, and also provide insight into soil communities following coalescence events. By isolating and better understanding the effect of spatial heterogeneity and dysconnectivity on soil microbial communities, we may better extrapolate how anthropogenic disturbances, such as climate change or land use change, may affect broad soil functions.

One sentence summarySoil mixing decreases bacterial richness as several taxa dominate the community, providing evidence for homogenizing community assembly processes.

sentences: FLAMs: A self-replicating ex vivo model of alveolar macrophages for functional genetic studies Alveolar macrophages (AMs) are tissue resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. While the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. Here, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGF{beta}. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.

sentences: A normative framework for learning top-down predictions through synaptic plasticity in apical dendrites Predictive coding has been identified as a key aspect of computation and learning in cortical microcircuits. But we do not know how synaptic plasticity processes install and maintain predictive coding capabilites in these neural circuits. Predictions are inherently uncertain, and learning rules that aim at discriminating linearly separable classes of inputs - such as the perceptron learning rule - do not perform well if the goal is learning to predict. We show that experimental data on synaptic plasticity in apical dendrites of pyramidal cells support another learning rule that is suitable for learning to predict. More precisely, it enables a spike-based approximation to logistic regression, a well-known gold standard for probabilistic prediction. We also show that data-based interactions between apical dendrites support learning of predictions for more complex probability distributions than those that can be handled by single dendrites. The resulting learning theory for top-down inputs to pyramidal cells provides a normative framework for evaluating experimental data, and suggests further experiments for tracking the emergence of predictive coding through synaptic plasticity in apical dendrites.

sentences: Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending  tug-of-war that controls a temporally shifting window of accessibility for the transcription initiation machinery.

sentences: Striatal dopamine encodes the relationship between actions and reward Despite the well-described role of dopamine in the striatum for skill learning and movement, its function in encoding actions and their outcomes for goal-directed action is less clear. Here, rats acquired two actions for distinct outcomes while we simultaneously recorded dopamine release in the dorsomedial striatum as these action-outcome associations were incremented and selectively degraded. Goal-directed actions generated a lateralized dopamine signal that reflected the strength of the action-outcome association and tracked increments and decrements in the specific action-outcome relationship, which was sensitive to changes in both contingency and outcome identity. This lateralized signal was updated by a dopamine action value signal that was broadcast bilaterally during the action and updated by a reward prediction error following exposure to the outcome. Our results establish, therefore, a dual role for striatal dopamine signaling during goal-directed actions: bilateral trial-by-trial burst signaling encoding action values, which modifies the long-term action-outcome association, reflected in emergent lateralization of dopamine release during actions.

sentences: Human single-chain antibodies neutralize SARS-CoV-2 variants by engaging an essential epitope of the spike: a new weapon against COVID-19 As of October 2021, coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Due to high efficiency, remarkable stability, resilience to nebulization and low production cost, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status.

sentences: A ChIP-exo screen of 887 PCRP transcription factor antibodies in human cells Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability are problematic and epitope tagging can be impractical in many cases. In an effort to improve this situation, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human-presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. 887 unique antibodies against 681 unique human transcription factors (TFs), were assayed by ultra-high resolution ChIP-exo/seq, primarily in a single pass in one cell type (K562). Deep systematic analyses of the resulting [~]1,200 ChIP-exo datasets can be found at www.PCRPvalidation.org. Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that was distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We demonstrate and discuss the metrics and challenges to antibody validation in chromatin-based assays.

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Social selection and the evolution of maladaptation Evolution by natural selection is often viewed as a process that inevitably leads to adaptation, or an increase in population fitness over time. However, maladaptation, an evolved decrease in fitness, may also occur in response to natural selection under some conditions. Social selection, which arises from the effects of social partners on fitness, has been identified as a potential cause of maladaptation, but we lack a general rule identifying when social selection should lead to a decrease in population mean fitness. Here we use a quantitative genetic model to develop such a rule. We show that maladaptation is most likely to occur when social selection is strong relative to nonsocial selection and acts in an opposing direction. In this scenario, the evolution of traits that impose fitness costs on others may outweigh evolved gains in fitness for the individual, leading to a net decrease in population mean fitness. Further, we find that maladaptation may also sometimes occur when phenotypes of interacting individuals negatively covary. We outline the biological situations where maladaptation in response to social selection can be expected, provide both quantitative genetic and phenotypic versions of our derived result, and suggest what empirical work would be needed to test it. We also consider the effect of social selection on inclusive fitness and support previous work showing that inclusive fitness cannot suffer an evolutionary decrease. Taken together, our results show that social selection may decrease population mean fitness when it opposes individual-level selection, even as inclusive fitness increases.

Subject areaQuantitative genetics and Mendelian inheritance

sentences: GproDIA enables data-independent acquisition glycoproteomics with comprehensive statistical control Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics. We propose GproDIA, a framework for the proteome-wide characterization of intact glycopeptides from DIA data with comprehensive statistical control by a 2-dimentional false discovery rate approach and a glycoform inference algorithm, enabling accurate identification of intact glycopeptides using wide isolation windows. We further adapt a semi-empirical spectrum prediction strategy to expand the coverage of spectral libraries of glycopeptides. We benchmark our method for N-glycopeptide profiling on DIA data of yeast and human serum samples, demonstrating that DIA with GproDIA outperforms the data dependent acquisition (DDA) based methods for glycoproteomics in terms of capacity and data completeness of identification, as well as accuracy and precision of quantification. We expect that this work can provide a powerful tool for glycoproteomic studies.

sentences: Reduction of HDAC2 expression in human induced pluripotent stem cell derived neurons improves neuronal maturation, mitochondrial dynamics and cellular neurodegenerative disease phenotypes. Histone deacetylase 2 (HDAC2) is a major HDAC protein in the adult brain and has been shown to regulate many neuronal genes. Aberrant expression of HDAC2 and subsequent dysregulation of neuronal gene expression is implicated in neurodegeneration and brain aging. Human induced pluripotent stem cell-derived neurons (hiPSC-Ns) are widely used models for studying neurodegenerative disease mechanisms, but the role of HDAC2 in hiPSC-N differentiation and maturation has not been explored. In this study, we show that levels of HDAC2 progressively decrease as hiPSCs are differentiated towards neurons. This suppression of HDAC2 inversely corresponds to an increase in neuron-specific isoforms of Endophilin-B1, a multifunctional protein involved in mitochondrial dynamics. Expression of neuron-specific isoforms of Endophilin-B1 is accompanied by concomitant expression of a neuron-specific alternative splicing factor, SRRM4. Manipulation of HDAC2 and Endophilin-B1 using lentiviral approaches shows that knock-down of HDAC2 or overexpression of a neuron-specific Endophilin-B1 isoform promotes mitochondrial elongation and protects against cytotoxic stress in hiPSC-Ns, while HDAC2 knock-down specifically influences genes regulating mitochondrial dynamics and synaptogenesis. Furthermore, HDAC2 knock-down promotes enhanced mitochondrial respiration and reduces levels of neurotoxic amyloid beta peptides. Collectively, our study demonstrates a role for HDAC2 in hiPSC-neuronal differentiation, highlights neuron-specific isoforms of Endophilin-B1 as a marker of differentiating hiPSC-Ns, and demonstrates that HDAC2 regulates key neuronal and mitochondrial pathways in hiPSC-Ns.

sentences: Predicting and interpreting large scale mutagenesis data using analyses of protein stability and conservation Understanding and predicting the functional consequences of single amino acid is central in many areas of protein science. Here we collected and analysed experimental measurements of effects of >150,000 variants in 29 proteins. We used biophysical calculations to predict changes in stability for each variant, and assessed them in light of sequence conservation. We find that the sequence analyses give more accurate prediction of variant effects than predictions of stability, and that about half of the variants that show loss of function do so due to stability effects. We construct a machine learning model to predict variant effects from protein structure and sequence alignments, and show how the two sources of information are able to support one another. Together our results show how one can leverage large-scale experimental assessments of variant effects to gain deeper and general insights into the mechanisms that cause loss of function.

sentences: Informing shigellosis prevention and control through pathogen genomics Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMIC), are increasingly antimicrobial resistant and have no licensed vaccine. We performed genomic analyses of 1246 systematically collected shigellae from seven LMIC to inform control and identify factors that could limit the effectiveness of current approaches. We found that S. sonnei contributes [≥]20-fold more disease than other Shigella species relative to its genomic diversity and highlight existing diversity and adaptative capacity among S. flexneri that may generate vaccine escape variants in <6 months. Furthermore, we show convergent evolution of resistance against the current recommended antimicrobial among shigellae. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, and other pathogens.

sentences: Survival of a threatened salmon is linked to spatial variability in river conditions Extirpation of the Central Valley spring-run Chinook Salmon ESU (Oncorhynchus tshawytscha) from the San Joaquin River is emblematic of salmonid declines across the Pacific Northwest. Habitat restoration and fish reintroduction efforts are ongoing, but recent telemetry studies have revealed low outmigration survival of juveniles to the ocean. Previous investigations have focused on modeling survival relative to river discharge and geographic regions, but have largely overlooked the effects of habitat variability. To evaluate the link between environmental conditions and survival of juvenile spring-run Chinook Salmon, we combined high spatial resolution habitat mapping approaches with acoustic telemetry along a 150 km section of the San Joaquin River during the spring of 2019. While overall outmigration survival was low (5%), our habitat-based classification scheme described variation in survival of acoustic-tagged smolts better than other candidate models based on geography or distance. There were two regional mortality sinks evident along the longitudinal profile of the river, revealing poor survival in areas that shared warmer temperatures but that diverged in chlorophyll-, fDOM, turbidity and dissolved oxygen levels. These findings demonstrate the value of integrating river habitat classification frameworks to improve our understanding of survival dynamics of imperiled fish populations. Importantly, our data generation and modeling methods can be applied to a wide variety of fish species that transit heterogeneous and diverse habitat types.

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Binocular Mirror-Symmetric Microsaccadic Sampling Enables Drosophila Hyperacute 3D-Vision Neural mechanisms behind stereopsis, which requires simultaneous disparity inputs from two eyes, have remained mysterious. Here we show how ultrafast mirror-symmetric photomechanical contractions in the frontal forward-facing left and right eye photoreceptors give Drosophila super-resolution 3D-vision. By interlinking multiscale in vivo assays with multiscale simulations, we reveal how these photoreceptor microsaccades - by verging, diverging and narrowing the eyes overlapping receptive fields - channel depth information, as phasic binocular image motion disparity signals in time. We further show how peripherally, outside stereopsis, microsaccadic sampling tracks a flying flys optic flow field to better resolve the world in motion. These results change our understanding of how insect compound eyes work and suggest a general dynamic stereo-information sampling strategy for animals, robots and sensors.

Significance statementTo move efficiently, animals must continuously work out their x,y,z-positions in respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a new morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila super-resolution 3D-vision and proposes neural computations for accurately predicting these flies depth-perception dynamics, limits, and visual behaviors.

sentences: Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drive rapid and potent immunogenicity capable of single-dose protection Antibodies from SARS-CoV-2 vaccines may target epitopes which reduce durability or increase the potential for escape from vaccine-induced immunity. Using a novel synthetic vaccinology pipeline, we developed rationally immune focused SARS-CoV-2 Spike-based vaccines. N-linked glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and comprehensive in vitro screening, we incorporated glycans into the Spike Receptor-Binding Domain (RBD) and assessed antigenic profiles. We developed glycan coated RBD immunogens and engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicited potent neutralizing antibodies in guinea pigs, hamsters and multiple mouse models, including human ACE2 and human B cell repertoire transgenics. RBD nanoparticles encoding wild-type and the P.1 SARS-CoV-2 variant induced high levels of cross-neutralizing antibodies. Single, low dose immunization protected against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of novel, potent coronavirus vaccines.

sentences: Gradual Compaction of the Central Spindle Decreases its Dynamicity as Revealed in PRC1 and EB1 Gene-Edited Human Cells During mitosis the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using gene editing, we generated human cells that express from their endogenous locus fluorescent PRC1 and EB1 to quantify their native spindle distribution and binding/unbinding turnover. EB1 plus end tracking revealed a general slowdown of microtubule growth, while PRC1, similar to its yeast orthologue Ase1, binds increasingly strongly to compacting antiparallel microtubule overlaps. KIF4A and CLASP1 bind more dynamically to the central spindle, but also show slowing down turnover. These results show that the central spindle gradually becomes more stable during mitosis, in agreement with a recent  bundling, sliding and compaction model of antiparallel midzone bundle formation in the central spindle during late mitosis.

sentences: A computational probe into the behavioral and neural markers of atypical facial emotion processing in autism. Despite ample behavioral evidence of atypical facial emotion processing in individuals with autism (IwA), the neural underpinnings of such behavioral heterogeneities remain unclear. Here, I have used brain-tissue mapped artificial neural network (ANN) models of primate vision to probe candidate neural and behavior markers of atypical facial emotion recognition in IwA at an image-by-image level. Interestingly, the ANNs image-level behavioral patterns better matched the neurotypical subjects behavior than those measured in IwA. This behavioral mismatch was most remarkable when the ANN behavior was decoded from units that correspond to the primate inferior temporal (IT) cortex. ANN-IT responses also explained a significant fraction of the image-level behavioral predictivity associated with neural activity in the human amygdala -- strongly suggesting that the previously reported facial emotion intensity encodes in the human amygdala could be primarily driven by projections from the IT cortex. Furthermore, in silico experiments revealed how learning under noisy sensory representations could lead to atypical facial emotion processing that better matches the image-level behavior observed in IwA. In sum, these results identify primate IT activity as a candidate neural marker and demonstrate how ANN models of vision can be used to generate neural circuit-level hypotheses and guide future human and non-human primate studies in autism.

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Dimerization of a membrane transporter is driven by differential energetics of lipid solvation of dissociated and associated states Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect caused by their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium towards the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the proteins biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.

IMPACT STATEMENTDifferences in the lipid solvation energetics of associated and dissociated states is a primary driving force for membrane protein oligomerization, presenting a molecular mechanism for lipid regulation in biology.

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Integrin signaling is critical for myeloid-mediated support of T-cell acute lymphoblastic leukemia We previously found that T-cell acute lymphoblastic leukemia (T-ALL) requires support from tumor-associated myeloid cells, which activate IGF1R signaling in the leukemic blasts. However, IGF1 is not sufficient to sustain T-ALL survival in vitro, implicating additional myeloid-mediated signals in T-ALL progression. Here, we find that T-ALL cells require close contact with myeloid cells to survive. Transcriptional profiling and in vitro assays demonstrate that integrin-mediated cell adhesion and activation of the downstream FAK/PYK2 kinases are required for myeloid-mediated support of T-ALL cells and promote IGF1R activation. Consistent with these findings, inhibition of integrins or FAK/PYK2 signaling diminishes leukemia burden in multiple organs and confers a survival advantage in a mouse model of T-ALL. Inhibiting integrin-mediated cell adhesion or FAK/PYK2 also diminishes survival of primary patient T-ALL cells co-cultured with myeloid cells. Furthermore, elevated integrin pathway gene signatures correlate significantly with myeloid enrichment and an inferior prognosis in pediatric T-ALL patients.

Statement of significanceAlthough tumor-associated myeloid cells provide critical support for T-ALL, our understanding of the underlying mechanisms remains limited. This study reveals that integrin-mediated adhesion and signaling are key mechanisms by which myeloid cells promote survival and progression of T-ALL blasts in the leukemic microenvironment.

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The impact of sparsity in low-rank recurrent neural networks Neural population dynamics are often highly coordinated, allowing task-related computations to be understood as neural trajectories through low-dimensional subspaces. How the network connectivity and input structure give rise to such activity can be investigated with the aid of low-rank recurrent neural networks, a recently-developed class of computational models which offer a rich theoretical framework linking the underlying connectivity structure to emergent low-dimensional dynamics. This framework has so far relied on the assumption of all-to-all connectivity, yet cortical networks are known to be highly sparse. Here we investigate the dynamics of low-rank recurrent networks in which the connections are randomly sparsified, which makes the network connectivity formally full-rank. We first analyse the impact of sparsity on the eigenvalue spectrum of low-rank connectivity matrices, and use this to examine the implications for the dynamics. We find that in the presence of sparsity, the eigenspectra in the complex plane consist of a continuous bulk and isolated outliers, a form analogous to the eigenspectra of connectivity matrices composed of a low-rank and a full-rank random component. This analogy allows us to characterise distinct dynamical regimes of the sparsified low-rank network as a function of key network parameters. Altogether, we find that the low-dimensional dynamics induced by low-rank connectivity structure are preserved even at high levels of sparsity, and can therefore support rich and robust computations even in networks sparsified to a biologically-realistic extent.

Author summaryIn large networks of neurons, the activity displayed by the population depends on the strength of the connections between each neuron. In cortical regions engaged in cognitive tasks, this population activity is often seen to be highly coordinated and low-dimensional. A recent line of theoretical work explores how such coordinated activity can arise in a network of neurons in which the matrix defining the connections is constrained to be mathematically low-rank. Until now, this connectivity structure has only been explored in fully-connected networks, in which every neuron is connected to every other. However, in the brain, network connections are often highly sparse, in the sense that most neurons do not share direct connections. Here, we test the robustness of the theoretical framework of low-rank networks to the reality of sparsity present in biological networks. By mathematically analysing the impact of removing connections, we find that the low-dimensional dynamics previously found in dense low-rank networks can in fact persist even at very high levels of sparsity. This has promising implications for the proposal that complex cortical computations which appear to rely on low-dimensional dynamics may be underpinned by a network which has a fundamentally low-rank structure, albeit with only a small fraction of possible connections present.

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ROCK1 regulates insulin secretion from β-cells ObjectiveThe endocrine pancreatic {beta}-cells play a pivotal role in the maintenance of whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate {beta}-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homoeostasis.

MethodsMice lacking ROCK1 in pancreatic {beta}-cells (RIP-Cre; ROCK1loxP/loxP, {beta}-ROCK1-/-) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. Insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from {beta}-ROCK1-/- mice or {beta}-cell lines with knockdown of ROCK1 were also evaluated. Proximity ligation assay was performed to determine the physical interactions between PK and ROCK1.

ResultsMice with a deficiency of ROCK1 in pancreatic {beta}-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, {beta}-ROCK1-/- mice displayed progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS was markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium levels, ATP levels, and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown {beta}-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that ROCK1 binding to PK is greatly enhanced by glucose stimulation in {beta}-cells.

ConclusionsOur findings demonstrate that {beta}-cell ROCK1 is essential for glucose-stimulated insulin secretion and maintenance of glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.

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Deficiency of Rho Kinase 1 (ROCK1) attenuates neutrophil NETosis and ameliorates UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines.

sentences: Suppressed prefrontal neuronal firing variability and impaired social representation in IRSp53-mutant mice Social deficit is a major feature of neuropsychiatric disorders, including autism spectrum disorders, schizophrenia, and attention-deficit/hyperactivity disorder, but its neural mechanisms remain unclear. Here, we examined neuronal discharge characteristics in the medial prefrontal cortex (mPFC) of IRSp53-mutant mice, which show social deficits, during social approach. IRSp53-mutant excitatory mPFC neurons displayed an increase in baseline neuronal firing and decreases in variability and dynamic range of firing rates and burst firing during social and non-social target approaches compared to wild-type controls. As a consequence, their firing activity was less differential between social and non-social targets. In addition, there was a decrease in the proportion of excitatory mPFC neurons encoding social information but not that of those encoding non-social information. These results suggest that insufficient neuronal activity dynamics may underlie impaired cortical encoding of social information and social behaviors in IRSp53-mutant mice.

sentences: Unifying Single-Channel Permeability from Rare-Event Sampling and Steady-State Flux Various all-atom molecular dynamics (MD) simulation methods have been developed to compute free energies and crossing rates of ions and small molecules through ion channels. However, a systemic comparison across different methods is scarce. Using a carbon nanotube as a model of small conductance ion channel, we computed the single-channel permeability for potassium ion using umbrella sampling, Markovian milestoning, and steady-state flux under applied voltage. We show that a slightly modified inhomogeneous solubility-diffusion equation yields a single-channel permeability consistent with the mean first passage time (MFPT)-based method. For milestoning, applying cylindrical and spherical bulk boundary conditions yield consistent MFPT if factoring in the effective bulk concentration. The sensitivity of the MFPT to the output frequency of collective variables is highlighted using the convergence and symmetricity of the inward and outward MFPT profiles. The consistent transport kinetic results from all three methods demonstrated the robustness of MD-based methods in computing ion channel permeation. The advantages and disadvantages of each technique are discussed, focusing on the future applications of milestoning in more complex systems.

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DNA metabarcoding Uncover the Diet of Subterranean Rodents in China ObjectiveIn the past, the zokor, which lived in northern China, caused great harm to agriculture and forestry production due to its large and sophisticated diet. Since the rat lives underground for most of its life, researchers know little about its dietary habits. Further understanding of its diet in the field is of important meaning for developing green and sustainable control strategies for the rat.

MethodsLongde County in Liupan Mountain area of Ningxia Hui Autonomous Region was selected as the interest area to capture zokor and investigate the species of habitat plants.We selected chloroplast trnL gene and eukaryotic internal transcription spacer 1 (ITS 1) primers to amplify DNA from the gastric contents of zokor,and then sequenced on Illumina Miseq PE300 platform.

ResultsThe gastric contents of Eospalax smithii (n=16) and E.cansus(n=9) were analyzed by operational taxonomic units (OTU) clustering and amplicon sequence variants(ASVs).The OTU clustering method obtained 2,995 OTUs, and the ASV method obtained 4,657 ASVs. The ASV method was better than the OTU clustering method, and the ASV method was adopted in the subsequent analysis. The food list of 32 families, 80 genera and 154 species was obtained by ASV method after the error was removed. The food composition of zokor was evaluated by relative abundance(%RA) method and frequency of occurrence(%ROO) method. At the Family level, it was found that zokor mainly fed on Asteraceae, Poaceae, Rosaceae, Pinaceae, Brassicaceae, Apiaceae, etc. At the Genus level, they are mainlyEchinops, Littledalea,Artemisia,Picea, Cirsium, Elymus and so on. The diet alpha diversity of E.cansus was slightly higher than that of E.smithii (P > 0.05). The correlation coefficient between Sobs index of alpha diversity and body weight of zokor was -0.382 (P = 0.059). The diet beta diversity proved that most zokors (22/25) clustered together, with low heterogeneity. They fed positively on Calamagrostis, Cirsium, Echinops, Medicago, Sanguisorba and Taraxacum. We found that zokor mainly fed on the roots of perennial herbs(PH), which were rich in water, carbohydrate, fat and protein, which provided an important energy source for its survival.

ConclusionHigh-throughput sequencing(HTS) based DNA metabarcoding technology has well revealed the diet of zokor, which are generalist.

sentences: Visuomotor mismatch responses as a hallmark of explaining away in causal inference How are visuomotor mismatch responses in visual cortex embedded into cortical processing? We here argue that mismatch responses are best understood as the result of a cooperation of motor and visual areas to jointly explain optic flow. This cooperation ensures that optic flow is not explained redundantly by both areas, which in the language of causal inference is termed explaining away. This improves the efficiency of the resulting neural code, and allows the animal to easily detect movements that are independent of its own locomotion. We demonstrate the emergence of mismatch responses from explaining away in simulations, where spiking neurons learn to encode optic flow stimuli and locomotion. We furthermore lay out arguments against the prevailing idea that mismatch signals are the result of a dedicated error computation in a hierarchical model. These results provide a new perspective on several recent experiments of cross-modal neural interactions in cortex.

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Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single cell RNA sequencing Single cell RNA sequencing (scRNAseq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNAseq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. The performance of different scRNAseq methods to study SARS-CoV-2 RNAs has not been thoroughly evaluated. Here, we compare different scRNAseq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs), which are produced only during active viral replication and not present in viral particles. We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') and Chromium Next GEM Single Cell V(D)J (10X 5') sequencing, we find that 10X 5' with an extended R1 sequencing strategy maximizes the unambiguous detection of sgmRNAs by increasing the number of reads spanning leader-sgmRNA junction sites. Differential gene expression testing and KEGG enrichment analysis of infected cells compared with bystander or mock cells showed an enrichment for COVID19-associated genes, supporting the ability of our method to accurately identify infected cells. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.

ImportanceSingle cell RNA sequencing (scRNAseq) has emerged as a valuable tool to study host-viral interactions particularly in the context of COronaVIrus Disease-2019 (COVID-19). scRNAseq has been developed and optimized for analyzing eukaryotic mRNAs, and the ability of scRNAseq to measure RNAs produced by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has not been fully characterized. Here we compare the performance of different scRNAseq methods to detect and quantify SARS-CoV-2 RNAs and develop an analysis workflow to specifically quantify unambiguous reads derived from SARS-CoV-2 genomic RNA and subgenomic mRNAs. Our work demonstrates the strengths and limitations of scRNAseq to measure SARS-CoV-2 RNA and identifies experimental and analytical approaches that allow for SARS-CoV-2 RNA detection and quantification. These developments will allow for studies of coronavirus RNA biogenesis at single-cell resolution to improve our understanding of viral pathogenesis.

sentences: MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N=10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase (www.facebase.org, doi.org/10.25550/3-HXMC) and GitHub (https://github.com/jaydevine/MusMorph).

sentences: The role of network connectivity on epileptiform activity A number of potentially important mechanisms have been identified as key players to generate epileptiform activity, such as genetic mutations, activity-dependent alteration of synaptic functions, and functional network reorganization at the macroscopic level. Here we study how network connectivity at cellular level can affect the onset of epileptiform activity, using computational model networks with different wiring properties. The model suggests that networks connected as in real brain circuits are more resistant to generate seizure-like activity. The results suggest new experimentally testable predictions on the cellular network connectivity in epileptic individuals, and highlight the importance of using the appropriate network connectivity to investigate epileptiform activity with computational models.

sentences: Effect of cellular rearrangement time delays on the rheology of vertex models for confluent tissues Large-scale tissue deformation during biological processes such as morphogenesis requires cellular rearrangements. The simplest rearrangement in confluent cellular monolayers involves neighbor exchanges among four cells, called a T1 transition, in analogy to foams. But unlike foams, cells must execute a sequence of molecular processes, such as endocytosis of adhesion molecules, to complete a T1 transition. Such processes could take a long time compared to other timescales in the tissue. In this work, we incorporate this idea by augmenting vertex models to require a fixed, finite time for T1 transitions, which we call the "T1 delay time". We study how variations in T1 delay time affect tissue mechanics, by quantifying the relaxation time of tissues in the presence of T1 delays and comparing that to the cell-shape based timescale that characterizes fluidity in the absence of any T1 delays. We show that the molecular-scale T1 delay timescale dominates over the cell shape-scale collective response timescale when the T1 delay time is the larger of the two. We extend this analysis to tissues that become anisotropic under convergent extension, finding similar results. Moreover, we find that increasing the T1 delay time increases the percentage of higher-fold coordinated vertices and rosettes, and decreases the overall number of successful T1s, contributing to a more elastic-like - and less fluid-like - tissue response. Our work suggests that molecular mechanisms that act as a brake on T1 transitions could stiffen global tissue mechanics and enhance rosette formation during morphogenesis.

sentences: Bidirectional, unlike unidirectional transport, allows transporting axonal cargos against their concentration gradient Even though most axonal cargos are synthesized in the soma, the concentration of many of these cargos is larger at the presynaptic terminal than in the soma. This requires transport of these cargos from the soma to the presynaptic terminal or other active sites in the axon. Axons utilize both bidirectional (for example, slow axonal transport) and unidirectional (for example, fast anterograde axonal transport) modes of cargo transport. Bidirectional transport seems to be less efficient because it requires more time and takes more energy to deliver cargos. In this paper, we studied a family of models which differ by the modes of axonal cargo transport (such as anterograde and retrograde motor-driven transport and passive diffusion) as well as by the presence or absence of pausing states. The models are studied to investigate their ability to describe axonal transport against the cargo concentration gradient. We argue that bidirectional axonal transport is described by a higher-order mathematical model, which allows imposing cargo concentration not only at the axon hillock but also at the axon terminal. The unidirectional transport model allows only for the imposition of cargo concentration at the axon hillock. Due to the great lengths of the axons, anterograde transport mostly relies on molecular motors, such as kinesins, to deliver cargos synthesized in the soma to the terminal and other active sites in the axon. Retrograde transport can be also motor-driven, in which case cargos are transported by dynein motors. If cargo concentration at the axon tip is higher than at the axon hillock, retrograde transport can also occur by cargo diffusion. However, because many axonal cargos are large or they assemble in multiprotein complexes for axonal transport, the diffusivity of such cargos is very small. We investigated the case of a small cargo diffusivity using a perturbation technique and found that for this case the effect of diffusion is limited to a very thin diffusion boundary layer near the axon tip. If cargo diffusivity is decreased in the model, we show that without motor-driven retrograde transport the model is unable to describe a high cargo concentration at the axon tip. To the best of our knowledge, our paper presents the first explanation for the utilization of seemingly inefficient bidirectional transport in neurons.

sentences: Cell type profiling in salamanders identifies innovations in vertebrate forebrain evolution The evolution of advanced cognition in vertebrates is associated with two independent innovations in the forebrain: the six-layered neocortex in mammals and the dorsal ventricular ridge (DVR) in sauropsids (reptiles and birds). How these novelties arose in vertebrate ancestors remains unclear. To reconstruct forebrain evolution in tetrapods, we built a cell type atlas of the telencephalon of the salamander Pleurodeles waltl. Our molecular, developmental, and connectivity data indicate that parts of the sauropsid DVR trace back to tetrapod ancestors. In contrast, the salamander dorsal pallium is devoid of cellular and molecular characteristics of the mammalian neocortex, yet shares similarities with entorhinal cortex and subiculum. Our findings chart the series of innovations that resulted in the emergence of the sauropsid DVR, and the mammalian six-layered neocortex.

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Post-stroke administration of the p75 neurotrophin receptor modulator, LM11A-31, attenuates chronic changes in brain metabolism, increases neurotransmitter levels, and improves recovery The aim of this study was to test whether post-stroke oral administration of a small molecule p75 neurotrophin receptor (p75NTR) modulator (LM11A-31) can augment neuronal survival and improve recovery in a mouse model of stroke. Mice were administered LM11A-31 for up to 12 weeks, beginning 1 week after stroke. Metabolomic analysis revealed that after 2 weeks of daily treatment, mice that received LM11A-31 were distinct from vehicle treated mice by principal component analysis and had higher levels of serotonin, acetylcholine, and dopamine in their ipsilateral hemisphere. LM11A-31 treatment also improved redox homeostasis by restoring reduced glutathione. It also offset a stroke induced reduction in glycolysis by increasing acetyl-CoA. There was no effect on cytokine levels in the infarct. At 13 weeks following stroke, adaptive immune cell infiltration in the infarct was unchanged in LM11A-31 treated mice, indicating that LM11A-31 does not alter the chronic inflammatory response to stroke at the site of the infarct. However, LM11A-31 treated mice had less brain atrophy, neurodegeneration, tau pathology, and microglial activation in other regions of the ipsilateral hemisphere. These findings correlated with improved recovery of motor function on a ladder test, improved sensorimotor and cognitive abilities on a nest construction test, and less impulsivity in an open field test. These data support small molecule modulation of the p75 neurotrophin receptor for preserving neuronal health and function during stroke recovery.

SIGNIFICANCE STATEMENTThe findings from this study introduce the p75 neurotrophin receptor as a novel small molecule target for promotion of stroke recovery. Given that LM11A-31 is in clinical trials as a potential therapy for Alzheimers disease, it could be considered as a candidate for assessment in stroke or vascular dementia studies.

sentences: Pout2Prot: an efficient tool to create protein (sub)groups from Percolator output files In metaproteomics, the study of the collective proteome of microbial communities, the protein inference problem is more challenging than in single-species proteomics. Indeed, a peptide sequence can not only be present in multiple proteins or protein isoforms of the same species, but also in homologous proteins from closely related species. To assign the taxonomy and functions of the microbial species, specialized tools have been developed, such as Prophane. This tool, however, is not directly compatible with post-processing tools such as Percolator. In this manuscript we therefore present Pout2Prot, which takes Percolator Output (.pout) files from multiple experiments and creates protein group and protein subgroup output files (.tsv) that can be used directly with Prophane. We investigated different grouping strategies, and compared existing protein grouping tools to develop an advanced protein grouping algorithm that offers a variety of different approaches, allows grouping for multiple files, and uses a weighted spectral count for protein (sub)groups to reflect abundance. Pout2Prot is available as a web application at https://pout2prot.ugent.be and is installable via pip as a standalone command line tool and reusable software library. All code is open source under the Apache License 2.0 and is available at https://github.com/compomics/pout2prot.

sentences: Functional differentiation in the language network revealed by lesion-symptom mapping Theories of language organization in the brain commonly posit that different regions underlie distinct linguistic mechanisms. However, such theories have been criticized on the grounds that many neuroimaging studies of language processing find similar effects across regions. Moreover, condition by region interaction effects, which provide the strongest evidence of functional differentiation between regions, have rarely been offered in support of these theories. Here we address this by using lesion-symptom mapping in three large, partially-overlapping groups of aphasia patients with left hemisphere brain damage due to stroke (N=121, N=92, N= 218). We identified multiple measure by region interaction effects, associating damage to the posterior middle temporal gyrus with syntactic comprehension deficits, damage to posterior inferior frontal gyrus with expressive agrammatism, and damage to inferior angular gyrus with semantic category word fluency deficits. Our results are inconsistent with recent hypotheses that regions of the language network are undifferentiated with respect to high-level linguistic processing.

sentences: A genetic bottleneck of mitochondrial DNA during human lymphocyte development Mitochondria are essential organelles in eukaryotic cells that provide critical support for energetic and metabolic homeostasis. Mutations that accumulate in mitochondrial DNA (mtDNA) in somatic cells have been implicated in cancer, degenerative diseases, and the aging process. However, the mechanisms used by somatic cells to maintain proper functions despite their mtDNA mutation load are poorly understood. Here, we analyzed somatic mtDNA mutations in more than 30,000 human single peripheral and bone marrow mononuclear cells and observed a significant overrepresentation of homoplastic mtDNA mutations in B, T and NK lymphocytes despite their lower mutational burden than other hematopoietic cells. The characteristic mutational landscape of mtDNA in lymphocytes were validated with data from multiple platforms and individuals. Single-cell RNA-seq and computational modeling demonstrated a stringent mitochondrial bottleneck during lymphocyte development likely caused by lagging mtDNA replication relative to cell proliferation. These results illuminate a potential mechanism used by highly metabolically active immune cells for quality control of their mitochondrial genomes.

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Comparison of brain activity while tasting of passion fruit juice sweetened with caloric and non-caloric sweeteners Sweetener type can influence sensory properties and consumers acceptance and preference for low-calorie products. An ideal sweetener does not exist, and each sweetener must be used in situations to which it is best suited. Aspartame and sucralose can be good substitutes for sucrose in passion fruit juice. Despite the interest in artificial sweeteners, little is known about how artificial sweeteners are processed in the human brain. Here, we evaluated brain signals of 11 healthy subjects when they tasted passion fruit juice equivalently sweetened with sucrose (9.4 g/100 g), sucralose (0.01593 g/100 g), or aspartame (0.05477 g/100 g). Electroencephalograms were recorded for two sites in the gustatory cortex (i.e., C3 and C4). Data with artifacts were disregarded, and the artifact-free data were used to feed a CNN. Our results indicated that the brain responses distinguish juice sweetened with different sweeteners with an average accuracy of 0.823.

Practical ApplicationsFinding sweeteners that best fit consumer preferences evolves understanding how the gustatory cortex processes sweeteners. Ideal equivalence will occur when the brain is no longer able to distinguish stimuli that are consciously perceived. This study presents a method of signal acquisition using a single channel and an open-source processing environment. This would allow, for example, to disregard the use of a commercial electroencephalograph and expand the studies in this area and offering to food industry additional tools in the development of products sweetened with non-caloric sweeteners.

sentences: A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression Few studies so far have investigated the impact of different cell migration traits on tumor progression. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the migration and invasion traits of Giantin depleted cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.

sentences: CRISPR Knock-in Designer: automatic oligonucleotide design software to introduce point mutations using CRISPR/Cas9 Knock-in of precise point mutations into protein-coding genes has been one of the earliest and most important applications of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9. The ability to perform such precise gene editing is crucial to interrogate the function of specific protein residues and to create models of human diseases caused by protein amino acid changes. The homologous protein residues can be mutated in model animal species, and the consequences of these mutations can be studied, leading to a better understanding of the disease in question. Design of point mutation knock-in strategies has been a combination of manual steps assisted by several computational tools resulting in a time-consuming process and preventing a single rapid and integrated solution. We have therefore designed CRISPR Knock-in Designer, which can perform rapid and automatic design of point mutation knock-in DNA oligonucleotides upon provision of the mutation, a guide RNA, and essential identifier or sequence information. The tool supports most experimentally established CRISPR types and has multiple options for the resulting oligonucleotides to satisfy the needs of most users. We also provide allele-specific PCR-based and restriction enzyme-based genotyping strategies as part of the program output. CRISPR Knock-in Designer adjusts to the genomic context of any target codon and tries to design knock-in strategies when a codon straddles two exons, a situation we explored in whole genomes of several model species. CRISPR Knock-in Designer output can also be adapted for use with some of the newer Prime Editing design tools to facilitate the introduction of a specific mutation sequence using this advanced technology.

sentences: Cell transcriptomic atlas of the non-human primate Macaca fascicularis Studying tissue composition and function in non-human primates (NHP) is crucial to understand the nature of our own species. Here, we present a large-scale single-cell and single-nucleus transcriptomic atlas encompassing over one million cells from 43 tissues from the adult NHP Macaca fascicularis. This dataset provides a vast, carefully annotated, resource to study a species phylogenetically close to humans. As proof of principle, we have reconstructed the cell-cell interaction networks driving Wnt signalling across the body, mapped the distribution of receptors and co-receptors for viruses causing human infectious diseases and intersected our data with human genetic disease orthologous coordinates to identify both expected and unexpected associations. Our Macaca fascicularis cell atlas constitutes an essential reference for future single-cell studies in human and NHP.

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DeepDDS: deep graph neural network with attention mechanism to predict synergistic drug combinations MotivationDrug combination therapy has become a increasingly promising method in the treatment of cancer. However, the number of possible drug combinations is so huge that it is hard to screen synergistic drug combinations through wet-lab experiments. Therefore, computational screening has become an important way to prioritize drug combinations. Graph neural network have recently shown remarkable performance in the prediction of compound-protein interactions, but it has not been applied to the screening of drug combinations.

ResultsIn this paper, we proposed a deep learning model based on graph neural networks and attention mechanism to identify drug combinations that can effectively inhibit the viability of specific cancer cells. The feature embeddings of drug molecule structure and gene expression profiles were taken as input to multi-layer feedforward neural network to identify the synergistic drug combinations. We compared DeepDDS with classical machine learning methods and other deep learning-based methods on benchmark data set, and the leave-one-out experimental results showed that DeepDDS achieved better performance than competitive methods. Also, on an independent test set released by well-known pharmaceutical enterprise AstraZeneca, DeepDDS was superior to competitive methods by more than 16% predictive precision. Furthermore, we explored the interpretability of the graph attention network, and found the correlation matrix of atomic features revealed important chemical substructures of drugs. We believed that DeepDDS is an effective tool that prioritized synergistic drug combinations for further wet-lab experiment validation.

Availability and implementationSource code and data are available at https://github.com/Sinwang404/DeepDDS/tree/master

sentences: HIV-specific CD8 T cells from elite controllers have an epigenetic imprint that preserves effector functions Several lines of evidence support a central role for CD8 T cells as key determinants in the control of HIV, particularly in rare "elite controllers" who control the virus to undetectable levels in the blood in the absence of antiretroviral therapy (ART). While HIV-specific CD8 T cells isolated from elite controllers have enhanced antiviral cytokine production and proliferative capacity in response to antigen stimulation when compared to cells isolated from viremic or even aviremic ART-suppressed non-controllers, the cell-intrinsic mechanisms underlying the enhanced T cell memory-like function of HIV-specific CD8 T cells in elite controllers remain largely undefined. To identify the transcriptional and epigenetic pathways that regulate functional capacity in HIV-specific CD8 T cells in elite controllers, we performed genome-wide transcriptional and DNA methylation analysis of MHC Class I multimer+ CD8 T cells sorted from aviremic elite controllers compared to aviremic non-controllers on suppressive ART. Co-omics analysis revealed enrichment for gene signatures that support a multipotent differentiation state, cell survival, and a long-lived effector cell fate in HIV-specific CD8 T cells from elite controllers. Specifically, we observed DNA methylation programs at the transcription factor binding sites of the stem-associated factors TCF-1 and LEF1 that delineate HIV-specific CD8 T cells from elite controllers versus ART-treated individuals. HIV-specific CD8 T cells in elite controllers also maintain T cell receptor and IL-12/STAT4 pathway signaling and have suppressed pro-apoptotic TNF pathway signaling. These findings show that HIV-specific CD8 T cells from elite controllers have enhanced expression and DNA methylation programs that maintain developmental potential and in turn promote long-term survival, proliferative potential, and effector capacity. These data also provide new insights into the relationship between stem-associated transcription factors and stable epigenetic restriction of T cell developmental capacity.

sentences: Study of growth characteristics of callus, suspension and root crops Scutellaria baicalensis Scutellaria baicalensis is a popular traditional plant in Chinese medicine. Widely distributed biologically active substances of Scutellaria baicalensis are flavonoids (baicalin, baicalein, vogonin and vogonoside), which are responsible for the antitumor activity. Their antitumor effect is due to the absorption of oxidative radicals, the weakening of the activity of NF-kB (nuclear factor-kB), the suppression of the expression of the COX-2 gene and the regulation of the cell cycle. In addition, baicalein, baicalin, and vogonin showed strong antioxidant activity. The root culture in vitro of the medicinal plant Scutellaria baicalensis is characterized by intensive growth. The growth index at the end of the cultivation cycle was 40. The growth curve has a standard S-shape, with pronounced growth phases. The stationary phase was observed from 5-7 weeks of cultivation. The root culture growth index was 22.

sentences: Simulations on the efficacy of radiotherapy with different time schemes of antiangiogenic therapy The combination of radiotherapy and antiangiogenic agents has been suggested to be potent in tumor growth control compared to the application of antiangiogenic therapy or radiotherapy alone. Since radiotherapy is highly dependent on the oxygen level of the tumor area, antiangiogenic agents are utilized for the reoxygenation of tumor vasculature. We present a mathematical framework to investigate the efficacy of radiotherapy combined with antiangiogenic treatment. The framework consists of tumor cells, vasculature, and oxygenation levels evolving with time to mimic a tumor microenvironment. Non-linear partial differential equations (PDEs) are employed to simulate each component of the framework. Different treatment schemes are investigated to see the changes in tumor growth and oxygenation. To test combination schedules, radiation monotherapy, neoadjuvant, adjuvant, and concurrent cases are simulated. The efficiency of each therapy scheme on tumor growth control, the changes in tumor cell density, and oxygen levels shared by tumor cells are represented. The simulation results indicate that the application of radiotherapy after antiangiogenic treatment is more efficient in tumor growth control compared to other therapy schemes. The present study gives an insight into the possible interaction and timing of the combination of radiotherapy and antiangiogenic drug treatment.

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Vcflib and tools for processing the VCF variant call format Since its introduction in 2011 the variant call format (VCF) has been widely adopted for processing DNA and RNA variants in practically all population studies -- as well as in somatic and germline mutation studies. VCF can present single nucleotide variants, multi-nucleotide variants, insertions and deletions, and simple structural variants called against a reference genome. Here we present over 125 useful and much used free and open source software tools and libraries, part of vcflib tools and bio-vcf. We also highlight cyvcf2, hts-nim and slivar tools. Application is typically in the comparison, filtering, normalisation, smoothing, annotation, statistics, visualisation and exporting of variants. Our tools run daily and invisibly in pipelines and countless shell scripts. Our tools are part of a wider bioinformatics ecosystem and we consider it very important to make these tools available as free and open source software to all bioinformaticians so they can be deployed through software distributions, such as Debian, GNU Guix and Bioconda. vcflib, for example, was installed over 40,000 times and bio-vcf was installed over 15,000 times through Bioconda by December 2020. We shortly discuss the design of VCF, lessons learnt, and how we can address more complex variation that can not easily be represented by the VCF format. All source code is published under free and open source software licenses and can be downloaded and installed from https://github.com/vcflib.

Author summaryMost bioinformatics workflows deal with DNA/RNA variations that are typically represented in the variant call format (VCF) -- a file format that describes mutations (SNP and MNP), insertions and deletions (INDEL) against a reference genome. Here we present a wide range of free and open source software tools that are used in biomedical sequencing workflows around the world today.

sentences: Synthetic glycans that control gut microbiome structure mitigate colitis in mice Relative abundances of bacterial species in the gut microbiome have been linked to many diseases. Species of gut bacteria are ecologically differentiated by their abilities to metabolize different glycans, making glycan delivery a powerful way to alter the microbiome to promote health. We describe the properties and therapeutic potential of chemically diverse synthetic glycans (SGs). Fermentation of SGs by gut microbiome cultures resulted in compound-specific shifts in taxonomic and metabolite profiles not observed with reference glycans, including prebiotics. Model enteric pathogens grow poorly on most SGs, potentially increasing their safety for at-risk populations. SGs increased survival, reduced weight loss, and improved clinical scores in mouse models of colitis. Synthetic glycans are thus a promising modality to improve health through selective changes to the gut microbiome.

sentences: Femtosecond laser microdissection isolating regenerating C. elegans neurons for single cell RNA sequencing Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single neuron resolution in small model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate regenerating neurons from the nematode. We present femtosecond laser microdissection (fs-LM), a new single cell isolation method that dissects intact cells directly from living tissue by leveraging the micron-scale precision of fs-laser ablation. We show that fs-LM facilitated sensitive and specific gene expression profiling by single cell RNA-sequencing, while mitigating the stress related transcriptional artifacts induced by tissue dissociation. Single cell RNA-sequencing of fs-LM isolated regenerating C. elegans neurons revealed transcriptional program leading to successful regeneration in wild-type animals or regeneration failure in animals lacking DLK-1/p38 kinase. The ability of fs-LM to isolate specific neurons based on phenotype of interest allowed us to study the molecular basis of regeneration heterogeneity displayed by neurons of the same type. We identified gene modules whose expression patterns were correlated with axon regrowth rate at a single neuron level. Our results establish fs-LM as a highly specific single cell isolation method ideal for precision and phenotype-driven studies.

sentences: Activation of T cell checkpoint pathways during beta-cell antigen presentation by engineered dendritic cells promotes protection of non-obese diabetic mice from type 1 diabetes Defective immune regulation has been recognized in type 1 diabetes (T1D). Immune regulatory T cell check-point receptors, which are generally upregulated on activated T cells, have been the molecules of attention as therapeutic targets for enhancing immune response in tumor therapy. We reported that dendritic cells (DCs) that are engineered to express selective ligands for checkpoint receptors can induce effective T cell tolerance in antigen-immunization models. Here, we show that pancreatic {beta} -cell antigen (BcAg) presentation by engineered tolerogenic-DCs (tDCs) that express CTLA4 selective ligand (B7.1wa) or a combination of CTLA4, PD1 and BTLA selective ligands (B7.1wa, PD-L1, and HVEM-CRD1 respectively; multiligand-DCs) causes an increase in regulatory cytokine and T cell (Treg) responses and suppression of the effector T cell function as compared to engineered control-DCs. Non-obese diabetic (NOD) mice treated with BcAg-pulsed CTLA4-ligand-DCs and multiligand-DCs at pre-diabetic and early-hyperglycmic stages showed significantly lower degree of insulitis, higher frequencies of insulin-positive islets, profound delay in, and reversal of, hyperglycemia for a significant duration. Immune cells from the tDC treated mice not only produced lower amounts of IFN{gamma} and higher amounts of IL10 and TGF{beta}1 upon BcAg challenge, but also failed to induce hyperglycemia upon adoptive transfer. While both CTLA4-ligand-DCs and multiligand-DCs were effective in inducing tolerance, multiligand-DC treatment produced an overall higher suppressive effect on effector T cell function and disease outcome. These studies show that enhanced engagement of T cell checkpoint receptors during BcAg presentation can modulate T cell function and suppress autoimmunity and progression of the disease in T1D.

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Brain virtual histology with X-ray phase-contrast tomography Part I: whole-brain myelin mapping in white-matter injury models White-matter injury leads to severe functional loss in many neurological diseases. Myelin staining on histological samples is the most common technique to investigate white-matter fibers. However, tissue processing and sectioning may affect the reliability of 3D volumetric assessments. The purpose of this study was to propose an approach that enables myelin fibers to be mapped in the whole rodent brain with microscopic resolution and without the need for strenuous staining. With this aim, we coupled inline (propagation-based) X-ray phase-contrast tomography (XPCT) to ethanol-induced brain sample dehydration. We here provide the proof-of-concept that this approach enhances myelinated axons in rodent and human brain tissue. In addition, we demonstrated that white-matter injuries could be detected and quantified with this approach, using three animal models: ischemic stroke, premature birth and multiple sclerosis. Furthermore, in analogy to diffusion tensor imaging (DTI), we retrieved fiber directions and DTI-like diffusion metrics from our XPCT data to quantitatively characterize white-matter microstructure. Finally, we showed that this non-destructive approach was compatible with subsequent complementary brain sample analysis by conventional histology. In-line XPCT might thus become a novel gold-standard for investigating white-matter injury in the intact brain. This is Part I of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part II shows how in-line XPCT enables the whole-brain 3D morphometric analysis of amyloid-{beta} (A{beta}) plaques.

HighlightsO_LIX-ray phase-contrast tomography (XPCT) enables myelin mapping of the whole brain
C_LIO_LIXPCT detects and quantifies white-matter injuries in a range of diseases
C_LIO_LIFiber directions and anisotropy metrics can be retrieved from XPCT data
C_LIO_LIXPCT is compatible with subsequent conventional histology of brain samples
C_LIO_LIXPCT is a powerful virtual histology tool that requires minimal sample preparation
C_LI

Graphical Abstract

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sentences: A new analytical pipeline reveals metatranscriptomic changes upon high-fat diet in a Down syndrome mouse model. The existing methods designated for metatranscriptomic studies are still rare and being developed. In this paper we present a new analytical pipeline combining contig assembly, gene selection and functional annotation. This pipeline allowed us to reconstruct contigs with very high unique mappability (83%) and select sequences encoding putative bacterial genes reaching also a very high (66%), unique mappability of the NGS sequencing reads. Then, we have applied our pipeline to study faecal metatranscriptome of a Down syndrome (DS) mouse model, the Ts65Dn mice, in order to identify the differentially expressed transcripts. Recent studies have implicated dysbiosis of gut microbiota in several central nervous system (CNS) disorders, including DS. Given that DS individuals have an increased prevalence of obesity, we also studied the effects of a high-fat diet (HFD) on the transcriptomic changes of mice gut microbiomes, as the complex symbiotic relationship between the gut microbiome and its host is strongly influenced by diet and nutrition. Using our new pipeline we found that compared to wild type (WT), Ts65Dn mice showed an elevated expression levels of genes involved in hypoxanthine metabolism, which contributes to oxidative stress, and a down-regulated expression of genes involved in interactions with host epithelial cells and virulence. Microbiomes of mice fed HFD showed significantly higher expression levels of genes involved in membrane lipopolysaccharides / lipids biosynthesis, and decreased expression of osmoprotection and lysine fermentation genes, among others. We also found evidence that mice microbiota is capable of expressing genes encoding for neuromodulators, which may play a role in development of compulsive overeating and obesity. Our results show a DS-specific metatranscriptome profile and show that a high-fat diet affects the metabolism of mice gut microbiome by changing activity of genes involved in lipids, sugars, proteins and amino acids metabolism and cell membranes turnover. Our new analytical pipeline combining contig assembly, gene selection and functional annotation provides new insights into the metatranscriptomic studies.

sentences: Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domain Cytoplasmic dynein is activated by dynactin and cargo adapters in vitro, and the activation also needs LIS1 (Lissencephaly 1) in vivo. How this process is regulated remains unclear. Here we found in Aspergillus nidulans that a dynein AAA4 arginine-finger mutation bypasses the requirement of LIS1 for dynein activation driven by the early endosomal adapter HookA. As the AAA4 arginine-finger is implicated in AAA3 ATP hydrolysis, we examined AAA3 mutants defective in ATP binding and hydrolysis respectively. Astonishingly, blocking AAA3 ATP hydrolysis allows dynein activation by dynactin in the absence of LIS1 or HookA. As a consequence, dynein accumulates at microtubule minus ends while early endosomes stay near the plus ends. On the other hand, blocking AAA3 ATP binding abnormally prevents LIS1 from being dissociated from dynein upon motor activation. Thus, the AAA3 ATPase cycle regulates the coordination between dynein activation and cargo binding as well as the dynamic dynein-LIS1 interaction.

sentences: Pronounced genetic separation among varieties of the Primula cusickiana species complex, a Great Basin endemic Distinguishing between unique species and populations with strong genetic structure is a common challenge in population genetics, especially in fragmented habitats where allopatric speciation may be widespread and distinct groups may be morphologically similar. Such is often the case with species complexes across sky island environments. In these scenarios, biogeography may help to explain the relations between species complex members, and RADseq methods are commonly used to compare closely related species across thousands of genetic loci. Here we use RADseq to clarify the relations between geographically distinct but morphologically similar varieties of the Primula cusickiana species complex, and to contextualize past findings of strong genetic structure among populations within varieties. Our genomic analyses demonstrate pronounced separation between isolated populations of this Great Basin endemic, indicating that the current varietal classification of complex members is inaccurate and emphasizing their conservation importance. We discuss how these results correspond to recent biogeographical models used to describe the distribution of other sky island taxa in western North America. Our findings also fit into a wider trend observed for alpine Primula species complexes, and we consider how heterostylous breeding systems may be contributing to frequent diversification via allopatric speciation in this genus.

sentences: Gut commensal bacteria enhance pathogenesis of a tumorigenic murine retrovirus The influence of the microbiota on viral transmission and replication is well appreciated. However, its impact on retroviral pathogenesis outside of transmission/replication control remained unknown. Using Murine Leukemia Virus (MuLV), we found that some commensal bacteria promoted the development of leukemia induced by this retrovirus. The promotion of leukemia development by commensals was due to suppression of the adaptive immune response through upregulation of several negative regulators of immunity. These negative regulators included Serpinb9b and Rnf128, which are associated with a poor prognosis of some spontaneous human cancers. Upregulation of Serpinb9b was mediated by sensing of bacteria by NOD1/NOD2/RIPK2 pathway. This work describes a novel mechanism by which the microbiota enhances tumorigenesis within gut-distant organs and points at potential new targets for cancer therapy.

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Genome-wide H3K9 Acetylation Level Increases with Age-Dependent Senescence of Flag Leaf in Rice (Oryza sativa) Flag leaf senescence is an important biological process that drives the remobilization of nutrients to the growing organs of rice. Leaf senescence is controlled by genetic information via gene expression and epigenetic modification, but the precise mechanism is as of yet unclear. Here, we analyzed genome-wide acetylated lysine residue 9 of histone H3 (H3K9ac) enrichment by chromatin immunoprecipitation-sequencing (ChIP-seq) and examined its association with transcriptomes by RNA-seq during flag leaf aging in rice (Oryza sativa). We found that genome-wide H3K9 acetylation levels increased with age-dependent senescence in rice flag leaf, and there was a positive correlation between the density and breadth of H3K9ac and gene expression and transcript elongation. A set of 1,249 up-regulated, differentially expressed genes (DEGs) and 996 down-regulated DEGs showing a strong relationship between temporal changes in gene expression and gain/loss of H3K9ac was observed during rice flag leaf aging. We produced a landscape of H3K9 acetylation-modified gene expression targets that includes known senescence-associated genes, metabolism-related genes, as well as miRNA biosynthesis-related genes. Our findings reveal a complex regulatory network of metabolism- and senescence-related pathways mediated by H3K9ac and also elucidate patterns of H3K9ac-mediated regulation of gene expression during flag leaf aging in rice.

Significance statementGenome-wide H3K9 acetylation levels increased with age-dependent senescence in rice flag leaf, and positively correlation the density and breadth of H3K9ac with transcript elongation and expression. Identified numerous H3K9 acetylation-modified gene expression targets reveal a complex regulatory network and metabolism-mediated senescence network that are associated with H3K9ac during leaf aging in rice.

sentences: Transfer of Knowledge from Model Organisms to Evolutionarily Distant Non-Model Organisms: The Coral Pocillopora damicornis Membrane Signaling Receptome With the ease of gene sequencing and the technology available to study and manipulate non-model organisms, the need to translate our understanding of model organisms to non-model organisms has become an urgent problem. For example, mining of large coral and their symbiont sequence data is a challenge, but also provides an opportunity for understanding functionality and evolution of these and other non-model organisms. Much more information than for any other eukaryotic species is available for humans, especially related to signal transduction and diseases. However, the coral cnidarian host and human have diverged over 700 million years ago and homologies between proteins are therefore often in the gray zone or undetectable with traditional BLAST searches. We introduce a two-stage approach to identifying putative coral homologues of human proteins. First, through remote homology detection using Hidden Markov Models, we identify candidate human homologues in the cnidarian genome. However, for many proteins, the human genome alone contains multiple family members with similar or even more divergence in sequence. In the second stage, therefore, we filter the remote homology results based on the functional and structural plausibility of each coral candidate, shortlisting the coral proteins likely to be true human homologues. We demonstrate our approach with a pipeline for mapping membrane receptors in humans to membrane receptors in corals, with specific focus on the stony coral, P. damicornis. More than 1000 human membrane receptors mapped to 335 coral receptors, including 151 G protein coupled receptors (GPCRs). To validate specific sub-families, we chose opsin proteins, representative GPCRs that confer light sensitivity, and Toll-like receptors, representative non-GPCRs, which function in the immune response, and their ability to communicate with microorganisms. Through detailed structure-function analysis of their ligand-binding pockets and downstream signaling cascades, we selected those candidate remote homologues likely to carry out related functions in the corals. This pipeline may prove generally useful for other non-model organisms, such as to support the growing field of synthetic biology.

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A De Novo regulation design shows an effectiveness in altering plant secondary metabolism IntroductionTranscription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants.

ObjectivesWe selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering.

MethodsG-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism.

ResultsPAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P+T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P+T and PAP1 genotypes.

ConclusionG-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

sentences: Increased intestinal permeability in an orally-reactive peanut allergy model identifies Angiopoietin like-4 as a biomarker Peanut allergy reaction severity correlates with increased intestinal epithelial cell (IEC) barrier permeability. CC027/GeniUnc mice develop peanut allergy by intragastric administration of peanut proteins without adjuvant. We report that peanut-allergic CC027/GeniUnc mice showed increased IEC barrier permeability and systemic peanut allergen Ara h 2 after challenge. Jejunal epithelial cell transcriptomics showed effects of peanut allergy on IEC proliferation, survival, and metabolism, and revealed IEC-predominant angiopoietin like-4 (Angptl4) as a unique feature of CC027/GeniUnc peanut allergy. CC027/GeniUnc mice and peanut-allergic pediatric patients demonstrated significantly higher serum Angptl4 and ANGPTL4 compared to control C3H/HeJ mice and non-peanut-allergic but atopic patients, respectively, highlighting its potential as a biomarker of peanut allergy.

sentences: A complex interplay between balancing selection and introgression maintains a genus-wide alternative life-history strategy. Alternative life-history strategies (ALHS) are genetic polymorphisms generating phenotypes differing in life histories that generally arise due to metabolic resource allocation tradeoffs. Althouigh ALHS are often be limited to a single sex or populations of a species, they can, in rare cases, be found among several species across a genus. In the butterfly genus Colias, at least a third of the species have a female limited ALHS called Alba. While many females develop brightly pigmented wings, Alba females reallocate nitrogen resources used in pigment synthesis to reproductive development, producing white-winged, more fecund females. Whether this ALHS evolved once or many times, and whether it has moved among species via introgression or been maintained via long-term balancing selection, has not been established. Answering these questions presents an opportunity to investigate the genetic basis and evolutionary forces acting upon ALHS, which have rarely been studied at a genus level. Here we identify the genetic locus of Alba in a second Colias species, allowing us to compare this with previous results in a larger phylogenetic context. Our findings suggest Alba has a singular origin and has been maintained in Colias through a combination of balancing selection and introgression for nearly one million years and at least as many generations. Finally, using CRISPR/Cas9 deletions in the cis-regulatory region of the Alba allele, we demonstrate that the Alba allele is a modular enhancer for the BarH1 gene and is necessary for the induction of the ALHS, which potentially facilitates its long-term persistence in the genus.

sentences: Untargeted 1H NMR Metabolomics and Pathway Analysis Reveals Dysregulated Proteostasis in Cyclophilin D (CypD)-deficient Mice Tissues Cyclophilin D (CypD) is a nuclear-encoded mitochondrial protein. Although best known as a regulator of the mitochondrial permeability transition pore (MPTP), it is also implicated in the regulation of cellular bioenergetics and extramitochondrial activities. In addition, being a peptidyl prolyl cis-trans isomerase (PPIase), deletion of CypD is likely to affect protein stability in the mitochondria; however, there is little direct evidence of this. In this study, untargeted 1H NMR metabolomics, coupled with multivariate analysis, was used to describe simultaneous changes in the metabolic system of CypD-deficient mice liver, heart, and pancreas, with data from the serum to identify systematic changes. Metabolomics Pathway Analyses (MetPA) revealed commonly perturbed metabolites in the different mouse tissues lacking CypD, with significantly enriched pathways that are related to amino acid, glucose and purine metabolisms, and bioenergetics. Serum from CypD-deficient mice confirmed changes in tissue urea cycle, lipid metabolism and ketogenesis. Overall this study reveals the role of CypD in maintaining protein homeostasis, concurring with its biochemical property as a peptidyl-prolyl isomerase and also demonstrates the wider metabolic adaptations induced by the deletion of the ppif gene, resulting in a CypD-deficient mice metabolome that is different from the wild-type system.

sentences: Diagnostic Evidence GAuge of Single cells (DEGAS): A flexible deep-transfer learning framework for prioritizing cells in relation to disease We propose DEGAS (Diagnostic Evidence GAuge of Single cells), a novel deep transfer learning framework, to transfer disease information from patients to cells. We call such transferrable information "impressions," which allow individual cells to be associated with disease attributes like diagnosis, prognosis, and response to therapy. Using simulated data and ten diverse single cell and patient bulk tissue transcriptomic datasets from Glioblastoma Multiforme (GBM), Alzheimers Disease (AD), and Multiple Myeloma (MM), we demonstrate the feasibility, flexibility, and broad applications of the DEGAS framework. DEGAS analysis on newly generated myeloma single cell transcriptomics led to the identification of PHF19high myeloma cells associated with progression.

sentences: Phase-based cortical synchrony is affected by prematurity Inter-areal synchronization by phase-phase correlations (PPC) of cortical oscillations mediates many higher neurocognitive functions, which are often affected by prematurity, a globally prominent neurodevelopmental risk factor. Here, we used electroencephalography (EEG) to examine brain-wide cortical PPC networks at term-equivalent age, comparing human infants after early prematurity to a cohort of healthy controls. We found that prematurity affected these networks in a sleep state-specific manner, and the differences between groups were also frequency-selective, involving brain-wide connections. The strength of synchronization in these networks was predictive of clinical outcomes in the preterm infants. These findings show that prematurity affects PPC networks in a clinically significant manner suggesting early functional biomarkers of later neurodevelopmental compromise to be used in clinical and translational studies after early neonatal adversity.

sentences: Global mapping of RNA homodimers in living cells RNA homodimerization is important for various physiological processes, including the assembly of membraneless organelles, RNA subcellular localization, and packaging of viral genomes. However, understanding of RNA homodimerization has been hampered by the lack of systematic in vivo detection methods. Here we show that PARIS, COMRADES, and other RNA proximity ligation methods can detect RNA homodimers transcriptome-wide as "overlapping" chimeric reads that contain more than one copy of the same sequence. Analysing published proximity ligation datasets, we show that RNA:RNA homodimers mediated by direct base-pairing interactions are rare across the transcriptome, but highly enriched in specific transcripts, including U8 snoRNA, U2 snRNA and a subset of tRNAs. Analysis of data from virus-infected cells reveals homodimerization of SARS-CoV-2 and Zika genomes, mediated by specific palindromic sequences located within protein-coding regions of N protein in SARS-CoV-2 and NS2A gene in Zika. We speculate that regions of viral genomes involved in homodimerization may constitute effective targets for antiviral therapies.

sentences: Evaluation of seasonal dynamics of fungal DNA assemblages in a flow-regulated stream in a restored forest using eDNA metabarcoding Investigation of seasonal variation in fungal communities is essential for understanding biodiversity and ecosystem functions. However, the conventional sampling method, with substrate removal and high spatial heterogeneity of community composition, makes surveying the seasonality of fungal communities challenging. Recently, water environmental DNA (eDNA) analysis has been explored for its utility in biodiversity surveys. In this study, we assessed whether the seasonality of fungal communities can be detected by monitoring eDNA in a forest stream. We conducted monthly water sampling in a forest stream over two years and used DNA metabarcoding to identify fungal eDNA. The stream water contained DNA from functionally diverse aquatic and terrestrial fungi, such as plant decomposers, parasites, and mutualists. The variation in the fungal assemblage showed a regular annual periodicity, meaning that the assemblages in a given season were similar, irrespective of the year or sampling. Furthermore, the strength of the annual periodicity varied among functional groups. Our results suggest that forest streams may act as a "trap" for terrestrial fungal DNA derived from different habitats, allowing the analysis of fungal DNA in stream water to provide information about the temporal variation in fungal communities in both the aquatic and the surrounding terrestrial ecosystems.

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Performance of three freely available methods for extracting white matter hyperintensities: FreeSurfer, UBO Detector and BIANCA White matter hyperintensities (WMH) of presumed vascular origin are frequently found in MRIs of healthy older adults. WMH are also associated with aging and cognitive decline. Here, we compared and validated three freely available algorithms for WMH extraction: FreeSurfer, UBO Detector, and BIANCA (Brain Intensity AbNormality Classification Algorithm) using a longitudinal dataset comprising MRI data of cognitively healthy older adults (baseline N = 231, age range 64-87 years). As reference we manually segmented WMH in T1w, 3D FLAIR, 2D FLAIR images. These manual segmentations were then used to assess the segmentation accuracy of the different automated algorithms. Further, we assessed the relationships of WMH volume estimates provided by the algorithms with Fazekas scores and age. FreeSurfer underestimated the WMH volumes and scored worst in Dice Similarity Coefficient (DSC = 0.434) but its WMH volumes strongly correlated with the Fazekas scores (rs = 0.73). BIANCA accomplished the highest DCS (0.602) with 3D FLAIR images. However, the relations with the Fazekas scores were only moderate, especially in the 2D FLAIR images (rs = 0.41), and many outlier WMH volumes were detected when exploring within-person trajectories (2D FLAIR: ~30%). UBO Detector performed similarly to BIANCA in DSC with both modalities and reached the best DSC in 2D FLAIR (0.531) without requiring a tailored training dataset. In addition, it achieved very high associations with the Fazekas scores (2D FLAIR: rs = 0.80).

In summary, our results emphasize the importance of carefully contemplating the choice of the WMH segmentation algorithm and MR-modality.

sentences: MLC Seq: De novo sequencing of full-length tRNA isoforms by mass ladder complementation tRNAs can exist in distinct isoforms because of different chemical modifications, which confounds attempts to accurately sequence individual tRNA species using next generation sequencing approaches or to quantify different RNA modifications at specific sites on a tRNA strand. Herein, we develop a mass spectrometric (MS) ladder complementation sequencing (MLC-Seq), allowing for direct and simultaneous sequencing of full-length tRNA molecules, including those with low abundance. MLC-seq is achieved by improved instrumentation, and advanced algorithms that identify each tRNA species and related isoforms in an RNA mixture, and assemble full MS ladders from partial ladders with missing ladder components. Using MLC-Seq, we successfully obtained the sequence of tRNA-Phe from yeast and tRNA-Glu from mouse hepatocytes, and simultaneously revealed new tRNA isoforms derived from nucleotide modifications. Importantly, MLC-Seq pinpointed the location and stoichiometry changes of RNA modifications in tRNA-Glu upon the treatment of dealkylated enzyme AlkB, which confirmed its known enzymatic activity and suggested previously unidentified effects in RNA editing.

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Stand-alone lipoylated H-protein of the glycine cleavage system enables glycine cleavage and the synthesis of glycine from one-carbon compounds in vitro H-protein, one of the four component proteins (H, T, P and L) of glycine cleavage system (GCS), is generally considered a shuttle protein interacting with the other three GCS-proteins via a lipoyl swinging arm. We report that without P-, T- and L-proteins, lipoylated H-protein (Hlip) enables GCS reactions in both glycine cleavage and synthesis directions in vitro. This apparent catalytic activity is closely related to the cavity on the H-protein surface where the lipoyl arm is attached. Heating or mutation of selected residues in the cavity destroys or reduces the stand-alone activity of Hlip, which can be restored by adding the other three GCS-proteins. Systematic study of the Hlip-catalyzed overall GCS reactions and the individual reaction steps provides a first step towards understanding the stand-alone function of Hlip. The results in this work provide some inspiration for further understanding the mechanism of the GCS and give some interesting implications on the evolution of the GCS.

Significance statementGlycine cleavage system (GCS) plays central roles in C1 and amino acids metabolisms and the biosynthesis of purines and nucleotides. Manipulations of GCS are desired to promote plant growth or to treat serious pathophysiological processes such as aging, obesity and cancers. Reversed GCS reactions form the core of the reductive glycine pathway (rGP), one of the most promising pathway for the assimilation of formate and CO2 in the emerging C1-synthetic biology. H-protein, one of the four GCS component proteins (H, T, P and L) is generally considered a shuttle protein interacting with the other three proteins via a lipoyl swinging arm. Here, we discovered that without P-, T- and L-proteins, H-protein alone can catalyze GCS reactions in both glycine cleavage and synthesis directions in vitro. The surprising catalytic activities are related to a structural region of H-protein which can be manipulated. The results have impacts on engineering GCS to treat related diseases, to improve photorespiration, and to efficiently use C1-carbon for biosynthesis.

sentences: Sensory evidence accumulation using optic flow in a naturalistic navigation task Sensory evidence accumulation is considered a hallmark of decision-making in noisy environments. Integration of sensory inputs has been traditionally studied using passive stimuli, segregating perception from action. Lessons learned from this approach, however, may not generalize to ethological behaviors like navigation, where there is an active interplay between perception and action. We designed a sensory-based sequential decision task in virtual reality in which humans and monkeys navigated to a memorized location by integrating optic flow generated by their own joystick movements. A major challenge in such closed-loop tasks is that subjects actions will determine future sensory input, causing ambiguity about whether they rely on sensory input rather than expectations based solely on a learned model of the dynamics. To test whether subjects performed sensory integration, we used three independent experimental manipulations: unpredictable optic flow perturbations, which pushed subjects off their trajectory; gain manipulation of the joystick controller, which changed the consequences of actions; and manipulation of the optic flow density, which changed the reliability of sensory evidence. Our results suggest that both macaques and humans relied heavily on optic flow, thereby demonstrating a critical role for sensory evidence accumulation during naturalistic action-perception closed-loop tasks.

sentences: In silico approach on drug repurposing - Antimalarial drugs against HIV-1 protease Acquired Immunodeficiency Syndrome (AIDS), belonging to the retrovirus family is one of the most devastating contagious diseases of this century. Most of the available approved drugs are small molecules which are used in antiretroviral therapy (ART) that trigger the therapeutic response through binding to a targeted protein, HIV-1 protease (PR). This protein represents the most important antiretroviral drug target due to its key role in viral development inhibition. Computational tools using computer-aided technologies have proven useful in accelerating the drug discovery. In this study we evaluated selected FDA (USA) approved antimalarial drugs against HIV-1 protease to find a potential inhibitor candidate for HIV-1 PR (PDB 6DJ1). Binding affinities and Ki inhibition constant of an AutoDock 4.2 study suggest that of all assessed antimalarial agents, Lumefantrine (LUM) would be a most promising HIV-1 PR inhibitor.

sentences: SARS-CoV-2 triggers DNA damage response in Vero E6 cells The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and general malaise may continue long after the infection has ended in recovered patients, suggesting that SARS-CoV-2 infection has profound consequences in the host cells. Here we report that SARS-CoV-2 infection can trigger a DNA damage response (DDR) in African green monkey kidney cells (Vero E6). We observed a transcriptional upregulation of the Ataxia telangiectasia and Rad3 related protein (ATR) in infected cells. In addition, we observed enhanced phosphorylation of CHK1, a downstream effector of the ATR DNA damage response, as well as H2AX. Strikingly, SARS-CoV-2 infection lowered the expression of TRF2 shelterin-protein complex, and reduced telomere lengths in infected Vero E6 cells. Thus, our observations suggest SARS-CoV-2 may have pathological consequences to host cells beyond evoking an immunopathogenic immune response.

sentences: A weakly structured stem for human origins in Africa While it is now broadly accepted that Homo sapiens originated within Africa, considerable uncertainty surrounds specific models of divergence and migration across the continent. Progress is hampered by a paucity of fossil and genomic data, as well as variability in prior divergence time estimates. Here we use linkage disequilibrium and diversity-based statistics, optimized for rapid, complex demographic inference to discriminate among such models. We infer detailed demographic models for populations across Africa, including representatives from eastern and western groups, as well as 44 newly whole-genome sequenced individuals from the Nama (Khoe-San). Despite the complexity of African population history, contemporary population structure dates back to Marine Isotope Stage (MIS) 5. The earliest population divergence among contemporary populations occurs 120-135ka, between the Khoe-San and other groups. Prior to the divergence of contemporary African groups, we infer long-lasting structure between two or more weakly differentiated ancestral Homo populations connected by gene flow over hundreds of thousands of years (i.e. a weakly structured stem). We find that weakly structured stem models provide more likely explanations of polymorphism that had previously been attributed to contributions from archaic hominins in Africa. In contrast to models with archaic introgression, we predict that fossil remains from coexisting ancestral populations should be morphologically similar. Despite genetic similarity between these populations, an inferred 1-4% of genetic differentiation among contemporary human populations can be attributed to genetic drift between stem populations. We show that model misspecification explains variation in previous divergence time estimates and argue that studying a suite of models is key to robust inferences about deep history.

sentences: Gastric Neurons in the Nucleus Tractus Solitarius are Selective to the Orientation of Gastric Electrical Stimulation Gastric electrical stimulation (GES) is a bioelectric intervention for gastroparesis, obesity, and other functional gastrointestinal disorders. In a potential mechanism of action, GES activates the nerve endings of vagal afferent neurons and induces the vago-vagal reflex through the nucleus tractus solitarius (NTS) in the brainstem. However, it is unclear where and how to stimulate in order to optimize the vagal afferent responses. To address this question with electrophysiology in rats, we applied mild electrical currents to two serosal targets on the distal forestomach with dense distributions of vagal intramuscular arrays that innervated the circular and longitudinal smooth muscle layers. During stimulation, we recorded single and multi-unit responses from gastric neurons in NTS and evaluated how the recorded responses depended on the stimulus orientation and amplitude. We found that NTS responses were highly selective to the stimulus orientation for a range of stimulus amplitudes. The strongest responses were observed when the applied current flowed in the same direction as the intramuscular arrays in parallel with the underlying smooth muscle fibers. Our results suggest that gastric neurons in NTS may encode the orientation-specific activity of gastric smooth muscles relayed by vagal afferent neurons. This finding suggests that the orientation of GES is critical to effective engagement of vagal afferents and should be considered in light of the structural phenotypes of vagal terminals in the stomach.

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Microbial association networks in cheese: a meta-analysis Interactions among starter and non-starter microorganisms (starter bacteria, naturally occurring or intentionally added non-starter bacteria, yeasts and filamentous fungi, spoilage and pathogenic microorganisms and, finally bacteriophages and even arthropods) deeply affect the dynamics of cheese microbial communities and, as a consequence, multiple aspects of cheese quality, from metabolites affecting the taste, aroma and flavour, to body, texture and colour. Understanding and exploiting microbial interactions is therefore key to managing cheese quality. This is true for the simplest systems (fresh cheeses produced from pasteurized milk using defined starters composed solely of Lactic Acid Bacteria) and the more so for complex, dynamic systems, like surface ripened cheese produced from raw milk, in which a dynamic succession of diverse microorganisms is essential for obtained the desired combination of sensory properties while guaranteeing safety. Positive (commensalism, protocooperation) and negative (competition, amensalism, predation and parasitism) among members of the cheese biota have been reviewed multiple times. Although the complex, multidimensional datasets generated by multi-omic approaches to cheese microbiology and biochemistry are ideally suited for the representation of biotic and metabolic interactions as networks, network science concepts and approaches are rarely applied to cheese microbiology.

In this review we first illustrate concepts relevant to the description of microbial interaction networks using network science concepts. Then, we briefly review methods used for the inference and analysis of microbial association networks and their potential use in the interpretation of the cheese interactome. Since these methods can only be used for mining microbial associations, a review of the experimental methods used to confirm the nature of microbial interactions among cheese microbes. Finally, we demonstrate the potential of microbial association network inference by mining metataxonomic data stored in the public database DairyFMBN, a specialized version of FoodMicrobionet which collates data on 74 metataxonomic studies on dairy products. Microbial association networks were inferred from 34 studies on cheese with up to 4 different methods and the results discussed to evaluate several aspects (choice of method, level of taxonomic resolution for the analysis, network, node and edge properties) which provide insight on the usefulness of this approach as explorative tool in the detection of microbial interactions in cheese.

HighlightsO_LIApproaches for inference of association networks from metataxonomic data were reviewed
C_LIO_LIA metastudy on association networks in cheese was carried out using 34 recent studies
C_LIO_LIInference method and taxonomic resolution should be chosen carefully
C_LIO_LISPIEC-EASI may be used as a conservative method for microbial association inference
C_LIO_LIEdge and node properties support the formulation of testable hypotheses for microbial interactions
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sentences: Revisiting Ancient Polyploidy in Leptosporangiate Ferns Ferns, and particularly homosporous ferns, have long been assumed to have experienced recurrent whole-genome duplication (WGD) events because of their substantially large genome sizes, surprisingly high chromosome numbers, and high degrees of polyploidy among many extant members. Although, consequently, the number of sequenced fern genomes is very limited, recent studies using transcriptome data to find evidence for WGDs in ferns reached conflicting results concerning the occurrence of ancient polyploidy, for instance, in the lineage of leptosporangiate ferns. Because identifying WGDs in a phylogenetic context is the foremost step in studying the contribution of ancient polyploidy to evolution, we revisited earlier identified WGDs in leptosporangiate ferns, mainly the core leptosporangiate ferns, by building age distributions and applying substitution rate corrections and by conducting statistical gene tree - species tree reconciliation analyses. Our integrative analyses confidently identified four ancient WGDs in the sampled core leptosporangiates and suggest both false positives and false negatives for the WGDs that recent studies have reported earlier. In conclusion, we underscore the significance of substitution rate corrections and uncertainties in gene tree - species tree reconciliations in calling WGD events, and that failing to do so likely leads to incorrect conclusions.

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Methionine Metabolism Controls the B-cell EBV Epigenome and Viral Latency Epstein-Barr virus (EBV) subverts host epigenetic pathways to switch between viral latency programs, colonize the B-cell compartment and reactivate. Within memory B-cells, the reservoir for lifelong infection, EBV genomic DNA and histone methylation marks restrict gene expression. But, this epigenetic strategy also enables EBV-infected tumors, including Burkitt lymphomas to evade immune detection. Little is known about host cell metabolic pathways that support EBV epigenome landscapes. We therefore used amino acid restriction, metabolomic and CRISPR approaches to identify that an abundant methionine supply, and interconnecting methionine and folate cycles, maintain Burkitt EBV gene silencing. Methionine restriction, or methionine cycle perturbation, hypomethylated EBV genomes, de-repressed latent membrane protein and lytic gene expression. Methionine metabolism also shaped EBV latency gene regulation required for B-cell immortalization. Dietary methionine restriction altered murine Burkitt xenograft metabolomes and de-repressed EBV immunogens in vivo. These results highlight epigenetic/immunometabolism crosstalk supporting the EBV B-cell lifecycle and suggest therapeutic approaches.

HighlightsO_LIMethionine metabolism is critical for Epstein-Barr virus B-cell latency
C_LIO_LIExtensive cross-talk enables methionine metabolism to control the EBV epigenome
C_LIO_LIMethionine restriction also impairs EBV-driven human B-cell immortalization
C_LIO_LIDietary methionine restriction unmasks EBV antigens in Burkitt xenografts in vivo
C_LI

sentences: Forward genetics in Wolbachia: Regulation of Wolbachia proliferation by the amplification and deletion of an addictive genomic island Wolbachia is one of the most prevalent bacterial endosymbionts, infecting approximately 40% of terrestrial arthropod species. Wolbachia is often a reproductive parasite but can also provide fitness benefits to its host, as, for example, protection against viral pathogens. This protective effect is currently being applied to fight arboviruses transmission by releasing Wolbachia-transinfected mosquitoes. Titre regulation is a crucial aspect of Wolbachia biology. Higher titres can lead to stronger phenotypes and fidelity of transmission but can have a higher cost to the host. Since Wolbachia is maternally transmitted, its fitness depends on host fitness, and, therefore, its cost to the host may be under selection. Understanding how Wolbachia titres are regulated and other aspects of Wolbachia biology has been hampered by the lack of genetic tools. Here we developed a forward genetic screen to identify new Wolbachia over-proliferative mutant variants. We characterized in detail two new mutants, wMelPop2 and wMelOctoless, and show that the amplification or loss of the Octomom genomic region lead to over-proliferation. These results confirm previous data and expand on the complex role of this genomic region in the control of Wolbachia proliferation. Both new mutants shorten the host lifespan and increase antiviral protection. Moreover, we show that Wolbachia proliferation rate in Drosophila melanogaster depends on the interaction between Octomom copy number, the host developmental stage, and temperature. Our analysis also suggests that the life shortening and antiviral protection phenotypes of Wolbachia are dependent on different, but related, properties of the endosymbiont; the rate of proliferation and the titres near the time of infection, respectively. Altogether, we demonstrate the feasibility of a novel and unbiased experimental approach to study Wolbachia biology, which can be further adapted to characterize other genetically intractable bacterial endosymbionts.

sentences: Fast leaps between millisecond confinements govern Ase1 diffusion along microtubules Diffusion is the most fundamental mode of protein translocation within cells. Confined diffusion of proteins along the electrostatic potential constituted by the surface of microtubules, although modeled meticulously in molecular dynamics simulations, has not been experimentally observed in real-time. Here, we used interferometric scattering microscopy to directly visualize the movement of the microtubule-associated protein Ase1 along the microtubule surface at nanometer and microsecond resolution. We resolved millisecond confinements of Ase1 and fast leaps between these positions of dwelling preferentially occurring along the microtubule protofilaments, revealing Ase1s mode of diffusive translocation along the microtubules periodic surface. The derived interaction potential closely matches the tubulin-dimer periodicity and the distribution of the electrostatic potential on the microtubule lattice. We anticipate that mapping the interaction landscapes for different proteins on microtubules, finding plausible energetic barriers of different positioning and heights, will provide valuable insights into regulating the dynamics of essential cytoskeletal processes, such as intracellular cargo trafficking, cell division, and morphogenesis, all of which rely on diffusive translocation of proteins along microtubules.

sentences: Accurate and sensitive mutational signature analysis with MuSiCal Mutational signature analysis is a recent computational approach for interpreting somatic mutations in the genome. Its application to cancer data has enhanced our understanding of mutational forces driving tumorigenesis and demonstrated its potential to inform prognosis and treatment decisions. However, methodological challenges remain for discovering new signatures and assigning proper weights to existing signatures, thereby hindering broader clinical applications. We present MuSiCal (Mutational Signature Calculator), a rigorous analytical framework with novel algorithms that solves fundamental problems in the standard workflow. Our simulation studies demonstrate that MuSiCal outperforms state-of-the-art algorithms for both signature discovery and assignment. By reanalyzing over 2,700 cancer genomes, we provide an improved reference catalog of signatures and their assignments, discover nine indel signatures absent in the current catalog, resolve long-standing issues with the ambiguous  flat signatures, and give insights into signatures with unknown etiologies. We expect MuSiCal and the improved catalog to be a step towards establishing best practices for mutational signature analysis.

sentences: The histone variant H2A.W and linker histone H1 co-regulate heterochromatin accessibility and DNA methylation In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. To analyze the in vivo function of H2A.W, we created a h2a.w null mutant via CRISPR-Cas9, h2a.w-2. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.

sentences: Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of canonical RAS GTPases. Here, we have analyzed the phenotypes of LZTR1 loss-of-function mutants in both fruit flies and mice and have demonstrated biochemical dependency on their RIT1 orthologs. Moreover, we show that LZTR1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of RIT1.

sentences: Structural Characterization of the PawL-Derived Peptide Family, an Ancient Subfamily of Orbitides Plants are an excellent source of bioactive peptides, often with disulfide bonds and/or a cyclic backbone. While focus has predominantly been directed at disulfide-rich peptides, a large family of small, cyclic but non-disulfide bonded plant peptides, known as orbitides, has been relatively ignored. A recently discovered subfamily of orbitides is the PawL-Derived Peptides (PLPs), produced during the maturation of precursors for seed storage albumins. Although their evolutionary origins have been dated, in-depth exploration of the familys structural characteristics and potential bioactivities remains to be conducted. Here we present an extensive and systematic characterization of the PLP family. Nine PLPs were chosen and prepared by solid phase peptide synthesis. Their structural features were studied using solution NMR spectroscopy and seven were found to possess regions of backbone order. Ordered regions consist of {beta}-turns, with some PLPs adopting two well-defined {beta}-turns within sequences as short as seven residues, which are largely the result of side chain interactions. Our data highlight that the sequence diversity within this family results in equally diverse molecular scaffolds. None of these nine PLPs showed antibacterial or antifungal activity.

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The cytolethal distending toxin modulates cell differentiation and elicits epithelial to mesenchymal transition We are frequently exposed to bacterial genotoxins, such as cytolethal distending toxin (CDT), a prevalent heterotrimeric toxin whose active moiety is its CdtB subunit. CdtB triggers potent DNA damage, predisposing factors in the development of cancers, in host cells. CDT from Helicobacter hepaticus, a mouse pathogen, was shown to be directly involved in the development of murine hepatocarcinoma. Preliminary studies have shown that CDT induces certain phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process by which cells lose their epithelial characteristics in favor of mesenchymal ones, conducive to cell motility. In the present study, we investigated the different steps of EMT using liver tissues of mice infected with H. hepaticus, as well as human epithelial cell lines and xenograft mouse models following H. hepaticus CdtB expression. Most of the different steps of the EMT process were reproduced throughout the studied models. Indeed, microarray data showed a CdtB- dependent regulation of EMT-related transcripts. The key transcriptional regulators of EMT (SNAIL1 and ZEB1) and EMT markers (Vimentin, Fibronectin and 5{beta}1 integrin) were upregulated both at RNA and protein levels in response to CdtB. It also induced cell-cell junctions disassembly, causing individualization of cells and acquisition of a spindle-like morphology. CdtB activated the expression and activity of matrix metalloproteases and increased cell motility. This study demonstrated that CDT/CdtB elicits EMT process activation, supporting the idea that infection with genotoxin-producing bacteria can promote malignant transformation.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/487255v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@f94756org.highwire.dtl.DTLVardef@1ba7d59org.highwire.dtl.DTLVardef@7b80ccorg.highwire.dtl.DTLVardef@44d56d_HPS_FORMAT_FIGEXP  M_FIG C_FIG Author SummaryWe are frequently exposed to infection with genotoxin--producing bacterial from the gut microbiota, such as cytolethal distending toxin (CDT). CDT, via its active CdtB subunit, causes severe DNA damage in host cells, well-known risk factor of cancer development and progression. Chronic infection with CDT-producing bacteria is thus involved in cancer development. CDT is widespread among many bacteria and its impact in human cancer seems likely underestimated. Despite its major significance, CDT remains little studied. Here, we showed that cells exposed to CdtB are no longer cohesive, become individualized and acquire a spindle-shaped morphology known to be conducive to migration. These cells also express increased level of mesenchymal markers, as well as increased level of SNAIL1 and ZEB1, two key transcription factors orchestrating a crucial mechanism for cancer initiation and progression: epithelial to mesenchymal transition. These effects induced by CdtB were associated with increased matrix metalloproteinases degrading activity and emergence of cellular motility. Collectively, these data showed that CdB activates epithelial to mesenchymal transition, supporting the role of CDT in tumorigenesis.

sentences: An interpretable meta-clustering framework for single-cell RNA-Seq data integration and evaluation Single-cell RNA sequencing (scRNA-Seq) datasets that are produced from clinical samples are often confounded by batch effects and inter-patient variability. Existing batch effect removal methods typically require strong assumptions on the composition of cell populations being near identical across patients. Here we present a novel meta-clustering workflow, CIDER, based on inter-group similarity measures. We demonstrate that CIDER outperforms other scRNA-Seq clustering methods and integration approaches in both simulated and real datasets. Moreover, we show that CIDER can be used to assess the biological correctness of integration in real datasets, while it does not require the existence of prior cellular annotations.

sentences: MinION™, a portable long-read sequencer, enables rapid vaginal microbiota analysis in a clinical setting It has been suggested that the local microbiota in the reproductive organs is relevant to womens health and may also affect pregnancy outcomes. Analysis of partial 16S ribosomal RNA (rRNA) gene sequences generated by short-read sequencers has been used to identify vaginal and endometrial microbiota, but it requires a long time to obtain the results, making it unsuitable for the rapid analysis of small samples in a clinical context. We demonstrated a simple workflow using the nanopore sequencer MinION that allows high-resolution and rapid differentiation of vaginal microbiota. Vaginal samples collected from 18 participants were subjected to DNA extraction and full-length 16S rRNA gene sequencing with MinION. The principal coordinate analysis showed no differences in the bacterial compositions regardless of the sample collection method. The vaginal microbiota results could be reported within 2 days of specimen receipt. Although bacterial vaginosis (BV) was not diagnosed by the Nugent score in any cases, groups with both healthy and BV-like vaginal microbiota were clearly characterized by MinION sequencing. We conclude that full-length 16S rRNA gene sequencing analysis with MinION provides a rapid means for identifying vaginal bacteria with higher resolution. Species-level profiling of human vaginal microbiota by MinION sequencing can allow the analysis of associations with conditions such as genital infections, endometritis, and threatened miscarriage.

sentences: Towards a global investigation of transcriptomic signatures through co-expression networks and pathway knowledge for the identification of disease mechanisms In this work, we attempt to address a key question in the joint analysis of transcriptomic data: can we correlate the patterns we observe in transcriptomic datasets to known molecular interactions and pathway knowledge to broaden our understanding of disease pathophysiology? We present a systematic approach that sheds light on the patterns observed in hundreds of transcriptomic datasets from over sixty indications by using pathways and molecular interactions as a template. Our analysis employs transcriptomic datasets to construct dozens of disease specific co-expression networks, alongside a human interactome network of protein-protein interactions described in the literature. Leveraging the interoperability between these two network templates, we explore patterns both common and particular to these diseases on three different levels. Firstly, at the node-level, we identify the most and least common proteins in these diseases and evaluate their consistency against the interactome as a proxy for their prevalence in the scientific literature. Secondly, we overlay both network templates to analyze common correlations and interactions across diseases at the edge-level. Thirdly, we explore the similarity between patterns observed at the disease level and pathway knowledge to identify pathway signatures associated with specific diseases and indication areas. Finally, we present a case scenario in the context of schizophrenia, where we show how our approach can be used to investigate disease pathophysiology.

sentences: A viral toolbox for conditional and transneuronal gene expression in zebrafish The zebrafish is an important model in systems neuroscience but a key limitation is the lack of viral tools to dissect the structure and function of neuronal circuitry. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 can be combined with the Gal4/UAS system to target cell types with high spatial, temporal and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches.

sentences: Production of adaptive movement patterns via an insect inspired Spiking Neural Network Central Pattern Generator. Navigation in ever-changing environments requires effective motor behaviours. Many insects have developed adaptive movement patterns which increase their success in achieving navigational goals. A conserved brain area in the insect brain, the Lateral Accessory Lobe, is involved in generating small scale search movements which increase the efficacy of sensory sampling. When the reliability of an essential navigational stimulus is low, searching movements are initiated whereas if the stimulus reliability is high, a targeted steering response is elicited. Thus the network mediates an adaptive switching between motor patterns. We developed Spiking Neural Network models to explore how an insect inspired architecture could generate adaptive movements in relation to changing sensory inputs. The models are able to generate a variety of adaptive movement patterns, the majority of which are of the zig-zagging kind, as seen in a variety of insects. Furthermore, these networks are robust to noise. Because a large spread of network parameters lead to the zig-zagging movement dynamics, we conclude that the investigated network architecture is inherently well suited to generating adaptive movement patterns.

sentences: PERK Activation by SB202190 Attenuates Amyloidogenesis via TFEB-induced Autophagy-Lysosomal Pathway The protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK), a key ER stress sensor of the unfolded protein response (UPR), can confer beneficial effects by facilitating the removal of cytosolic aggregates through the autophagy-lysosome pathway (ALP). In neurodegenerative diseases, the ALP ameliorates the accumulation of intracellular protein aggregates in the brain. Transcription factor-EB (TFEB), a master regulator of the ALP, positively regulates key genes involved in the cellular degradative pathway. However, in neurons, the role of PERK activation in mitigating amyloidogenesis by ALP remains unclear. In this study, we found that SB202190 selectively activates PERK independently of its inhibition of p38 mitogen-activated protein kinase, but not inositol-requiring transmembrane kinase/endoribonuclease-1 (IRE1) or activating transcription factor 6 (ATF6), in human neuroblastoma cells. PERK activation by SB202190 was dependent on mitochondrial ROS production and promoted Ca2+-calcineurin activation. The activation of the PERK-Ca2+-calcineurin axis by SB202190 positively affects TFEB activity to increase ALP in neuroblastoma cells. Collectively, our study reveals a novel physiological mechanism underlying ALP activation, dependent on PERK activation, for ameliorating amyloidogenesis in neurodegenerative diseases.

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CryoGrid-PIXUL-RNA: High throughput RNA isolation platform for tissue transcript analysis Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in life sciences analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such specimens remain, in comparison, inefficient. To mitigate this bottleneck, we have developed a low-cost user-friendly system, the CryoGrid, that consists of CryoBlock, thermometer/thermocouple, and QR coded CryoTrays to freeze and store frozen tissue fragments, and hand-held CryoCore tool for tissue sampling supported by iPad and Google apps to display tissues, direct coring and share metadata.

RNA is one of the most studied analytes. There is a decades-long history of developing methods to isolate and analyze RNA. Still, the throughput of sampling and RNA extraction from tissues has not matched that of the high throughput transcriptome analytical platforms. To address this need, we have integrated the CryoGrid system with PIXUL-based methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. TRIzol is commonly used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. We developed a PIXUL-based TRIzol-free RNA isolation fast protocol that uses a buffer containing proteinase K (PK). Virtually every disease (and often therapeutic agents toxicity) is a systemic syndrome but often only one organ is examined. CryoGrid-PIXUL, integrated with either TRIzol or PK buffer RNA isolation protocols, yielded similar RNA profiles in a multiorgan (brain, heart, kidney and liver) mouse model of sepsis. Thus, RNA isolation using the CryoGrid-PIXUL system combined with the PK buffer offers an inexpensive user-friendly workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

sentences: Darwin's finches can have taste preferences, but not for human foods Urbanization is rapidly changing ecological niches. On the inhabited Galapagos Islands, Darwins finches consume human-introduced foods preferentially; however, it remains unclear why. Here we presented pastry with flavour profiles typical of human foods (oily, salty, sweet) to small ground finches (Geospiza fuliginosa) and medium ground finches (Geospiza fortis) to test if latent taste preferences might drive selection of human foods. If human-food flavours were consumed more than a neutral or bitter control only at sites with human foods, then we predicted tastes were acquired after urbanization; however, if no site-differences were found then this would indicate latent taste preferences. Contrary to both predictions, we found little evidence that human-food flavours were preferred compared to control flavours at any site. Instead, finches showed a weak aversion to oily foods, but only at remote (no human foods present) sites. This was further supported by behavioural responses, with beak wiping occurring more often at remote sites after finches tasted flavours associated with human foods. Our results suggest, therefore, that while Darwins finches may have acquired a tolerance to the flavours of human food, latent taste preferences are unlikely to have played a major role in their dietary response to increased urbanization.

sentences: 3'UTR-directed, kinase proximal mRNA decay inhibits C/EBPβ phosphorylation/activation to suppress senescence in tumor cells C/EBP{beta} is a potent regulator of RAS-induced senescence (RIS) and the SASP. C/EBP{beta} is post- translationally activated in RIS cells by the effector kinases ERK1/2 and CK2, but in tumor cells activation is suppressed by the CEBPB 3UTR. 3'UTR regulation of protein activity (UPA) requires a G/U-rich element (GRE) and its cognate binding protein, HuR. These components segregate CEBPB transcripts away from a perinuclear compartment harboring ERK1/2 and CK2, restricting C/EBP{beta} from its activating kinases. We report here that the mRNA decay proteins UPF1 and Staufen1/2 are essential UPA factors enriched within the perinuclear cytoplasm. STAU1/2 colocalize with CK2 on perinuclear signaling endosomes where they promote localized CEBPB mRNA decay. UPF1 or STAU1/2 depletion in tumor cells increased CEBPB transcripts adjacent to CK2 foci, coinciding with C/EBP{beta} activation and senescence. The GRE and an adjacent STAU binding site act to suppress C/EBP{beta}-mediated senescence, while a separate 3UTR region inhibits SASP induction. Accordingly, KrasG12D-driven lung tumors in mice carrying a Cebpb GRE deletion rarely progressed to malignant adenocarcinomas. Our findings identify kinase-proximal mRNA decay as a novel mechanism to inhibit C/EBP{beta} activating modifications in tumor cells to facilitate senescence bypass.

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Pre-vaccination Frequency of Circulatory Tfh is associated with Robust Immune Response to TV003 Dengue Vaccine It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents.

Author summaryDengue virus (DENV) is a worldwide threat that causes significant health and economic burden. Currently, there are several challenges in the development of a DENV vaccine including the existence of four different serotypes all; capable of causing disease and antibody dependent enhancement (ADE). For complete protection, a vaccine must be able to generate neutralizing antibodies against all 4 serotypes to avoid ADE. Currently, there is one licensed DENV vaccine, CYD-TDV (DENGVAXIATM). However, this vaccine is only efficacious in protecting against severe disease in DENV seropositive individuals therefore serostatus effect must be further studied for optimal vaccine design. A subset of CD4+ T cells called T-follicular helper (Tfh) cells have been well known to play a major role in aiding high affinity antibody production. Therefore, we chose to look at subsets of Tfh and the cytokines they produce in human blood that can serve as biomarkers for effective vaccine design. We found that DENV sero-positive participants had increased pre-vaccination frequencies of Tfh cells and higher levels of the Tfh related chemokine CXCL13/BLC that plays a role in directing antigen-specific responses. This pre-vaccination Tfh profile and CXCL13/BLC are then correlated positively with the vaccinees ability to produce neutralizing antibody against all four sero-types (breadth of the Response) of DENV, an important goal for all DENV vaccine trials.

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Beamforming seizures from the temporal lobe BackgroundSurgical treatment of drug-resistant temporal lobe epilepsy (TLE) depends on proper identification of the seizure onset zone (SOZ), and differentiation of mesial, temporolimbic seizure onsets from temporal neocortical seizure onsets. Non-invasive source imaging using electroencephalography (EEG) and magnetoencephalography (MEG) can provide accurate information on interictal spike localization; however, EEG and MEG have low sensitivity for epileptiform activity restricted to deep temporolimbic structures. Moreover, in mesial temporal lobe epilepsy (MTLE), interictal spikes frequently arise in neocortical foci distant from the SOZ, rendering interictal spike localization potentially misleading for presurgical planning.

MethodsIn this study, we used two different beamformer techniques applied to the MEG signal of ictal events acquired during EEG-MEG recordings in six patients with TLE (three neocortical, three MTLE). The ictal source localization results were compared to the patients ground truth SOZ localizations determined from intracranial EEG and/or clinical, neuroimaging and postsurgical outcome evidence.

ResultsBeamformer analysis proved to be highly accurate in all cases and able to reliably identify focal seizure onsets localized to mesial, temporolimbic structures. In three patients, interictal spikes were either absent, too complex for inverse dipole modeling, or localized to anterolateral temporal neocortex distant to a mesial temporal SOZ.

ConclusionsThis report demonstrates the suitability of MEG beamformer analysis of ictal events in TLE, which can supersede or complement the traditional analysis of interictal spikes. The method outlined is applicable to any type of epileptiform event, greatly expanding the information value of MEG and broadening its utility for presurgical recording in epilepsy.

sentences: Suppression of cdc13-2-associated senescence by pif1-m2 requires Ku-mediated telomerase recruitment In Saccharomyces cerevisiae, recruitment of telomerase to telomeres requires an interaction between Cdc13, which binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. A second pathway involving an interaction between the yKu complex and telomerase RNA (TLC1) contributes to telomerase recruitment, but cannot sufficiently recruit telomerase on its own to prevent replicative senescence when the primary Cdc13-Est1 pathway is abolished--for example, in the cdc13-2 mutant. In this study, we find that mutation of PIF1, which encodes a helicase that inhibits telomerase, suppresses the replicative senescence of cdc13-2 increasing reliance on the yKu-TLC1 pathway for telomerase recruitment. Our findings reveal new insight into telomerase-mediated telomere maintenance.

sentences: Relationship between bacterial phylotype and specialized metabolite production in the culturable microbiome of two freshwater sponges Microbial drug discovery programs rely heavily on accessing bacterial diversity from the environment to acquire new specialized metabolite (SM) lead compounds for the therapeutic pipeline. Therefore, knowledge of how certain bacterial taxa are distributed in nature, in addition to the degree of variation of SM production within those taxa, is critical to informing these front-end discovery efforts and making the overall sample collection and bacterial library creation process more efficient. In the current study we employed MALDI-TOF mass spectrometry and the bioinformatics pipeline IDBac to analyze diversity within phylotype groupings and SM profiles of hundreds of bacterial isolates from two Eunapius fragilis freshwater sponges, collected 1.5 km apart. We demonstrated that within two sponge samples of the same species, the culturable bacterial populations contained significant overlap in approximate genus-level phylotypes but mostly non-overlapping populations of isolates when grouped lower than the level of genus. Further, correlations between bacterial phylotype and SM production varied at the species level and below, suggesting SM distribution within bacterial taxa must be analyzed on a case-by-case basis. Our results suggest that two E. fragilis freshwater sponges collected in similar environments can exhibit large culturable diversity on a species-level scale, thus researchers should scrutinize the isolates with analyses that take both phylogeny and SM production into account in order to optimize the chemical space entering into a downstream bacterial library.

sentences: The peroxisomal transporter ABCD3 plays a major role in dicarboxylic fatty acid metabolism Peroxisomes metabolize a specific subset of fatty acids, which include dicarboxylic fatty acids (DCAs) generated by {omega}-oxidation. Data obtained in vitro suggest that the peroxisomal transporter ABCD3 (also known as PMP70) mediates the transport of DCAs into the peroxisome, but in vivo evidence to support this role is lacking. In this study, we studied an Abcd3 KO mouse model generated by CRISPR-Cas9 technology using targeted and untargeted metabolomics, histology, immunoblotting, and stable isotope tracing technology. We show that ABCD3 functions in DCA metabolism and uncover a novel role for this peroxisomal transporter in lipid metabolic homeostasis. The Abcd3 KO mouse presents with lipodystrophy, increased circulating free fatty acids, decreased ketone bodies, enhanced hepatic cholesterol synthesis and decreased hepatic de novo lipogenesis. Moreover, our study suggests that DCAs are metabolized by mitochondrial {beta}-oxidation when ABCD3 is not functional, reflecting the importance of the metabolic compartmentalization and communication between peroxisomes and mitochondria. In summary, this study provides data on the role of the peroxisomal transporter ABCD3 in hepatic lipid homeostasis and DCA metabolism, and the consequences of peroxisomal dysfunction for the liver.

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BleTIES: Annotation of natural genome editing in ciliates using long read sequencing SummaryCiliates are single-celled eukaryotes that eliminate specific, interspersed DNA sequences (internally eliminated sequences, IESs) from their genomes during development. These are challenging to annotate and assemble because IES-containing sequences are much less abundant in the cell than those without, and IES sequences themselves often contain repetitive and low-complexity sequences. Long read sequencing technologies from Pacific Biosciences and Oxford Nanopore have the potential to reconstruct longer IESs than has been possible with short reads, and also the ability to detect correlations of neighboring element elimination. Here we present BleTIES, a software toolkit for detecting, assembling, and analyzing IESs using mapped long reads.

Availability and implementationBleTIES is implemented in Python 3. Source code is available at https://github.com/Swart-lab/bleties (MIT license), and also distributed via Bioconda.

ContactContact: kb.seah@tuebingen.mpg.de

Supplementary informationBenchmarking of BleTIES with published sequence data.

sentences: Leveraging spiking deep neural networks to understand the neural mechanisms underlying selective attention Spatial attention enhances sensory processing of goal-relevant information and improves perceptual sensitivity. Yet, the specific neural mechanisms underlying the effects of spatial attention on performance are still contested. Here, we examine different attention mechanisms in spiking deep convolutional neural networks. We directly contrast effects of precision (internal noise suppression) and two different gain modulation mechanisms on performance on a visual search task with complex real-world images. Unlike standard artificial neurons, biological neurons have saturating activation functions, permitting implementation of attentional gain as gain on a neurons input or on its outgoing connection. We show that modulating the connection is most effective in selectively enhancing information processing by redistributing spiking activity, and by introducing additional task-relevant information, as shown by representational similarity analyses. Precision only produced minor attentional effects in performance. Our results, which mirror empirical findings, show that it is possible to adjudicate between attention mechanisms using more biologically realistic models and natural stimuli.

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Plasmid characterization in bacterial isolates of public health relevance in a tertiary healthcare facility in Kilimanjaro, Tanzania Plasmids are infectious double stranded DNA molecules that are found within bacteria. Horizontal gene transfer promotes successful spread of different types of plasmids within or among bacteria species, making their detection an important task for guiding clinical treatment. We used whole genome sequenced data to determine the prevalence of plasmid replicons in clinical bacterial isolates, the presence of resistance and virulence genes in plasmids, and the relationship between resistance and virulence genes within each plasmid. All bacterial sequences were de novo assembled using Unicycler before extraction of plasmids. Assembly graphs were submitted to Gplas+plasflow for plasmid prediction. The predicted plasmid components were validated using PlasmidFinder.

A total of 159 (56.2%) out of 283 bacterial isolates were found to carry plasmids, with E. coli, K. pneumoniae and S. aureus being the most prevalent plasmid carriers. A total of 27 (87.1%) combined plasmids were found to carry both resistance and virulence genes compared to 4 (12.9%) single plasmids. No statistically significant correlation was found between the number of antimicrobial resistance and virulence genes in plasmids (r =-0.25, p > 0.05). Our findings show a relatively high proportion of plasmid-carrying isolates suggesting selection pressure due to antibiotic use in the hospital. Co-occurrence of antibiotic resistance and virulence genes in clinical isolates is a public health relevant problem needing attention.

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Sensory system-specific associations between brain structure and balance Nearly 75% of older adults in the United States report balance problems. Balance difficulties are more pronounced during sensory feedback perturbation (e.g., standing with the eyes closed or on foam). Although it is known that aging results in widespread brain atrophy, less is known about how brain structure relates to balance performance under varied sensory conditions in older age. We measured postural sway of 36 young (18-34 years) and 22 older (66-84 years) adults during four conditions: eyes open, eyes closed, eyes open on foam, and eyes closed on foam. We calculated three summary measures indicating visual, proprioceptive, and vestibular contributions to balance. We also collected T 1-weighted and diffusion-weighted anatomical MRI scans. We aimed to: 1) test for age group differences in brain structure-balance relationships across a range of structural brain measures (i.e., volumetric, surface, and white matter microstructure); and 2) assess how brain structure measures relate to balance, regardless of age. Across both age groups, thinner cortex in multisensory integration regions was associated with greater reliance on visual inputs for balance. Greater gyrification within sensorimotor and parietal cortices was associated with greater reliance on proprioceptive inputs for balance. Poorer vestibular function was correlated with thinner vestibular cortex, greater gyrification within sensorimotor, parietal, and frontal cortices, and lower free water-corrected axial diffusivity in the superior-posterior corona radiata and across the corpus callosum. These results contribute to our scientific understanding of how individual differences in brain structure relate to balance. This has implications for developing brain stimulation interventions to improve balance.

Significance StatementOlder age is associated with greater postural sway, particularly when sensory information is perturbed (e.g., by closing ones eyes). Our work contributes to the field by identifying how individual differences in regional brain structure relate to balance under varying sensory conditions in young and older adults. Across both age groups, lower cortical thickness in sensory integration and vestibular regions, greater gyrification within sensorimotor, parietal, and temporal regions, and lower free water-corrected axial diffusivity in the corpus callosum and corona radiata were related to individual differences in balance scores. We identified brain structures that are associated with specific sensory balance scores; therefore, these results have implications for which brain regions to target in future interventions for different populations.

sentences: A Whole-Brain Map and Assay Parameter Analysis of Mouse VTA Dopaminergic Activation Ascending dopaminergic projections from neurons located in the Ventral Tegmental Area (VTA) are key to the etiology, dysfunction, and control of motivation, learning, and addiction. Due to evolutionary conservation of this nucleus and the extensive use of mice as disease models, establishing an assay for VTA dopaminergic signalling in the mouse brain is crucial for the translational investigation of motivational control as well as of neuronal function phenotypes for diseases and interventions. In this article we use optogenetic stimulation directed at VTA dopaminergic neurons in combination with functional Magnetic Resonance Imaging (fMRI), a method widely used in human deep brain imaging. We present a comprehensive assay producing the first whole-brain opto-fMRI map of dopaminergic activation in the mouse, and show that VTA dopaminergic system function is consistent with its structural VTA projections, diverging only in a few key aspects. While the activation map predominantly highlights target areas according to their relative projection densities (e.g. strong activation of the nucleus accumbens and low activation of the hippocampus), it also includes areas for which a structural connection is not well established (such as the dorsomedial striatum). We further detail the variability of the assay with regard to multiple experimental parameters, including stimulation protocol and implant position, and provide evidence-based recommendations for assay reuse, publishing both reference results and a reference analysis workflow implementation.

sentences: Appraisal of Cell Cycle Delay and Cytotoxicity Inducing Potential of 3-epicaryoptin in Allium Cepa L. Diterpenoid 3-epicaryoptin (C26H36O9) is abundant in leaves of Clerodendrum inerme, a traditionally used medicinal plant, having insect antifeedant activities. Here, we aim to explore its cell cycle delay and cytotoxic effects in Allium cepa root apical meristem cells. Allium cepa roots were treated with 3-epicaryoptin (100, 150 & 200 {micro}g mL-1 concentration) and colchicine (200 {micro}g mL-1 concentration) for 2, 4, 4+16 h (4 h treatment followed by 16 h recovery) and the induced cell cycle delay and cytotoxic effects were analyzed. The highest metaphase cells frequency for 3-epicaryoptin (150 {micro}g mL-1) and colchicine (200 {micro}g mL-1) treated samples were found to be 66.2{+/-}0.58 % and 82.35{+/-}0.74 % respectively at 4 h treatment. Treatment of 3-epicaryoptin and colchicine increased the aberrant cells %, CA, MN, c-metaphase, and PP cells. Therefore, indiscriminate use of C. inerme in traditional medicine should be restricted and the cell cycle delay and cytotoxicity inducing effects of 3-epicaryoptin needs further exploration for its cancer chemotherapeutic applications.

sentences: Ecological specialization, rather than the island rule, explains morphological diversification in an ancient radiation of geckos Island colonists are often assumed to experience higher levels of phenotypic diversification than continental taxa. However, empirical evidence has uncovered exceptions to this "island effect". Here, we tested this pattern using the geckos of the genus Pristurus from continental Arabia and Africa and the Socotra Archipelago. Using a recently published phylogeny and an extensive morphological dataset, we explore the differences in phenotypic evolution between Socotran and continental taxa. Moreover, we reconstructed ancestral habitat occupancy to examine if ecological specialisation is correlated with morphological change, comparing phenotypic disparity and trait evolution between habitats. We found a heterogeneous outcome of island colonisation. Namely, only one of the three colonisation events resulted in a body size increase. However, in general, Socotran species do not present higher levels or rates of morphological diversification than continental groups. Instead, habitat specialisation explains better the body size and shape evolution in Pristurus. Particularly, the colonisation of ground habitats appears as the main driver of morphological change, producing the highest disparity and evolutionary rates. Additionally, arboreal species show very similar body size and head proportions. These results reveal a determinant role of ecological mechanisms in morphological evolution and corroborate the complexity of ecomorphological dynamics in continent-island systems.

sentences: MaPPeRTrac: A Massively Parallel, Portable, and Reproducible Tractography Pipeline Large-scale diffusion MRI tractography remains a significant challenge. Users must orchestrate a complex sequence of instructions that requires many software packages with complex dependencies and high computational cost. We developed MaPPeRTrac, a probabilistic tractography pipeline that simplifies and vastly accelerates this process on a wide range of high performance computing (HPC) environments. It fully automates the entire tractography pipeline, from management of raw MRI machine data to edge density imaging (EDI) of the structural connectome. Dependencies are containerized with Docker or Singularity and de-coupled from code to enable rapid proto-typing and modification. Data artifacts are strictly organized with the Brain Imaging Data Structure (BIDS) to ensure that they are findable, accessible, interoperable, and reusable following FAIR principles. The pipeline takes full advantage of HPC resources using the Parsl parallel programming frame-work, resulting in the creation of connectome datasets of unprecedented size. MaPPeRTrac is publicly available and tested on commercial and scientific hardware, so that it may accelerate brain connectome research for a broader user community.

sentences: Auto-qPCR: A Python based web app for automated and reproducible analysis of qPCR data Quantifying changes in DNA and RNA levels is essential in numerous molecular biology protocols. Quantitative real time PCR (qPCR) techniques have evolved to become commonplace, however, data analysis includes many time-consuming and cumbersome steps, which can lead to mistakes and misinterpretation of data. To address these bottlenecks, we have developed an open-source Python software to automate processing of result spreadsheets from qPCR machines, employing calculations usually performed manually. Auto-qPCR is a tool that saves time when computing qPCR data, helping to ensure reproducibility of qPCR experiment analyses. Our web-based app (https://auto-q-pcr.com/) is easy to use and does not require programming knowledge or software installation. Using Auto-qPCR, we provide examples of data treatment, display and statistical analyses for four different data processing modes within one program: (1) DNA quantification to identify genomic deletion or duplication events; (2) assessment of gene expression levels using an absolute model, and relative quantification (3) with or (4) without a reference sample. Our open access Auto-qPCR software saves the time of manual data analysis and provides a more systematic workflow, minimizing the risk of errors. Our program constitutes a new tool that can be incorporated into bioinformatic and molecular biology pipelines in clinical and research labs.

sentences: Evaluating Differences in Elastic Modulus of Regenerated and Uninjured Mouse Digit Bone through microCT Density-Elasticity Calculation and Nanoindentation Testing Bone is an essential, healing structure in vertebrates that ensures daily function. However, the regenerative capacity of bone declines with age, compromising quality of life in the elderly and increasing cost of care. Here, for the first time, the elasticity of regenerated bone in a mouse digit amputation model is evaluated in order to better investigate biomechanics of skeletal regeneration. Amputation of the distal one third of the digit (third phalangeal element - P3) results in de novo regeneration of the digit, where analyzing the structural quality of this regenerated bone is a challenging task due to its small scale and triangular shape. To date, the evaluation of structural quality of the P3 bone has primarily focused on mineral density and bone architecture. This work describes an image-processing based method for assessment of elasticity in the whole P3 bone by using microcomputed tomography-generated mineral density data to calculate spatially discrete elastic modulus values across the entire P3 bone volume. Further, we validate this method through comparison to nanoindentation-measured values for elastic modulus. Application to a set of regenerated and unamputated digits shows that regenerated bone has a lower elastic modulus compared to the uninjured digit, with a similar trend for experimental hardness values. This method will be impactful in predicting and evaluating the regenerative outcomes of potential treatments and heightens the utility of the P3 regenerative model.

sentences: Penetrance estimation of SORL1 loss-of-function variants using a family-based strategy adjusted on APOE genotypes suggest a non-monogenic inheritance For complex disorders, estimating the age-related penetrance associated with rare variants of strong effect is essential before a putative use for genetic counseling or disease prevention. However, rarity and co-occurrence with other risk factors make such estimations difficult. In the context of Alzheimer disease, we present a survival model to estimate the penetrance of SORL1 rare (allele frequency< 1%) Loss-of-Function variants (LoF) while accounting for APOE-{varepsilon}4, the main risk factor (allele frequency[~] 14% in Caucasians). We developed an efficient strategy to compute penetrance estimates accounting for both common and rare genetic variants based on available penetrance curves associated with common risk factors and using incomplete pedigree data to quantify the additional risk conferred by rare variants. Our model combines: (i) a baseline for non-carriers of SORL1 LoF variants, stratified by APOE genotypes derived from the Rotterdam study and (ii) an age-dependent proportional hazard effect for SORL1 LoF variants estimated from pedigrees with a proband carrying such a variant. We embed this model into an Expectation-Maximisation algorithm to accommodate for missing genotypes. Confidence intervals were computed by bootstraps. To correct for ascertainment bias, proband phenotypes were omitted. We obtained penetrance curves associated with SORL1 LoF variants at the digenic level. By age 70, we estimate a 100% penetrance of SORL1 LoF variants only among APOE-{varepsilon}4{varepsilon}4 carriers, while penetrance is 56%[40% - 72%] among {varepsilon}4 heterozygous carriers and 37%[26% - 51%] among {varepsilon}4 non-carriers. We conclude that rare SORL1 LoF variants should not be used for genetic counseling regardless of the APOE status.

sentences: Multiple Synaptic Contacts combined with Dendritic Filtering enhance Spatio-Temporal Pattern Recognition capabilities of Single Neurons A cortical neuron typically makes multiple synaptic contacts on the dendrites of a post-synaptic target neuron. The functional implications of this apparent redundancy are unclear. The dendritic location of a synaptic contact affects the time-course of the somatic post-synaptic potential (PSP) due to dendritic cable filtering. Consequently, a single pre-synaptic axonal spike results with a PSP composed of multiple temporal profiles. Here, we developed a "filter-and-fire" (F&F) neuron model that captures these features and show that the memory capacity of this neuron is threefold larger than that of a leaky integrate-and-fire (I&F) neuron, when trained to emit precisely timed output spikes for specific input patterns. Furthermore, the F&F neuron can learn to recognize spatio-temporal input patterns, e.g., MNIST digits, where the I&F model completely fails. Multiple synaptic contacts between pairs of cortical neurons are therefore an important feature rather than a bug and can serve to reduce axonal wiring requirements.

sentences: The language network supports both lexical access and sentence generation during language production A network of left frontal and temporal brain regions has long been implicated in language comprehension and production. However, because of relatively fewer investigations of language production, the precise role of this  language network in production-related cognitive processes remains debated. Across four fMRI experiments using picture naming/description to mimic the translation of conceptual representations into words/sentences, we characterize the response of the language regions to production demands using individual-subject-level analyses. We report three novel results. First, sentence production elicits strong responses in the language network regardless of output modality (speaking vs. typing). Second, the language regions respond to both lexical access and sentence-generation demands, mirroring the strong integration between lexico-semantic and combinatorial processes that has been observed in language comprehension. Finally, we find no evidence of brain regions--including outside the language network--that selectively support sentence generation. Instead, language regions respond overall more strongly during production than during comprehension for similar linguistic strings, suggesting that production incurs a greater cost for the language network. Together, these results align with the idea that language comprehension and production draw on the same knowledge representations, which are stored in the language-selective network and are used both to interpret and generate linguistic utterances.

sentences: Polycomb Repressive Complexes occupancy reveals PRC2-independent PRC1 critical role in the control of limb development The Polycomb Repressive Complexes (PRC) are key players in the regulation of tissue-specific gene expression through their ability to epigenetically silence developmental genes. They are subdivided into two multicomponent complexes, PRC1 and PRC2, functioning through post-translational modifications of histone tails. A large body of work revealed functional interactions between PRC1 and PRC2, whereby trimethylation of lysine 27 on histone H3 (H3K27me3) by PRC2 contributes to the recruitment of canonical PRC1 (cPRC1). In parallel, a PRC2-independent binding of PRC1 has been uncovered and referred to as non-canonical PRC1 or variant PRC1 (vPRC1), in which PRC1-dependent ubiquitination of lysine 119 on histone H2A is involved in recruiting PRC2/propagating PRC2-dependent H3K27 trimethylation. While it was initially assumed that cPRC1 and vPRC1 bind distinct targets, subsequent evidence pointed to cPRC1 and vPRC1 sharing a significant subset of targets. How the functional interplay between PRC2, cPRC1 and vPRC1 contributes to gene regulation remains partially understood. Here, we show that, in the developing limb, PRC2 inactivation barely affects PRC1 occupancy, as the majority of PRC2- bound loci are bound by vPRC1 (RYBP-PRC1), both in wild type and PRC2 mutant limbs. Consistent with this, we found that loci bound by CBX2, a PRC1 subunit involved in the recognition of H3K27me3 and thereby recruitment of cPRC1, are, for the vast majority, also bound by vPRC1. Intriguingly, analysis of PRC2 mutant limbs revealed that while a large part of CBX2 occupancy is lost in absence of PRC2 function, as expected from the absence of H3K27me3, there is a significant number of genes retaining CBX2 occupancy as well as a few genes with apparent gain of CBX2 binding. Importantly, among these genes, 56 of them correspond to developmental genes known for playing a key role in limb morphogenesis. Based on the importance of vPRC1 in gene silencing, our findings emphasize the primary role of PRC2-independent PCR1 function in regulating developmental genes and questions the role of PRC2/cPRC1 in controlling developmental programs.

sentences: RiboTRIBE: Monitoring Translation with ADAR-meditated RNA Editing RNA translation is tightly regulated to ensure proper protein expression in cells and tissues. Translation is often assayed with biochemical assays such as ribosome profiling and TRAP, which are effective in many contexts. These assays are however not ideal with limiting amounts of biological material when it can be difficult or even impossible to make an extract with sufficient signal or sufficient signal:noise. Because of our interest in translational regulation within the few Drosophila adult circadian neurons, we fused the ADAR catalytic domain (ADARcd) to several small subunit ribosomal proteins and assayed mRNA editing in Drosophila S2 cells. The strategy is named RiboTRIBE and is analogous to a recently published APOBEC-based method. The list of RiboTRIBE-edited transcripts overlaps well with ribosome profiling targets, especially with more highly ranked targets. There is also an enriched number of editing sites in ribosome-associated mRNA comparing to total mRNA, indicating that editing occurs preferentially on polyribosome-associated transcripts. The use of cycloheximide to freeze translating ribosomes causes a substantial increase in the number of RiboTRIBE targets, which is decreased by pretreating cells with the chain terminating drug puromycin. NOTE: Additional experiments performed after first submitting this manuscript to BioRxiv estimate that less than 5% of Rps28b-ADAR is ribosome-associated. This is because the vast majority of the fusion protein sediments at the top of a polyribosome gradient. We therefore suggest that most editing reported in the manuscript is not catalyzed by ribosome-associated ADAR (10/2/2021).

sentences: Antimicrobial Effects of Ibuprofen Combined with Standard of Care Antimicrobials against Cystic Fibrosis Pathogens Cystic Fibrosis (CF) is a common fatal genetic disease caused by mutations happened to cystic fibrosis transmembrane conductance regulator (CFTR) gene. Lungs of CF patients are often colonized or infected with microorganisms. Drug resistant bacterial infection has been problematic in cystic fibrosis patient. The chronic bacterial infections and concomitant airway inflammation could damage the lung and lead to respiratory failure. Several clinical trials have demonstrated that high-dose ibuprofen reduces the rate of pulmonary function decline in CF patients. This beneficial effect has been attributed to the anti-inflammatory properties of ibuprofen. Previously, we have confirmed that high-dose ibuprofen demonstrated antimicrobial activity against P. aeruginosa in in vitro and in vivo. However, no study has examined the antimicrobial effect of combining ibuprofen with standard-of-care (SoC) antimicrobials. Here, we evaluated possible synergistic activity of combinations of common nonsteroidal anti-inflammatory drugs (NSAIDs), namely, aspirin, naproxen, and ibuprofen, with commonly used antibiotics for CF patients. The drug combinations were screened against different CF clinical isolates. Drugs that demonstrated efficacy in the presence of ibuprofen were further verified synergistic effects between these antimicrobials and NSAIDs. Finally, the survival analysis of an P. aeruginosa murine infection model was used to demonstrate the efficacy of synergistic combination. Our results suggest that combinations of ibuprofen with commonly used antibiotics demonstrate synergistic antimicrobial activity against drug resistant, clinical bacterial strains in in vitro. The efficacy of combination ceftazidime and ibuprofen was demonstrated in in vivo.

sentences: On the Mental Status of Stargazer Mice In many childhood-onset genetic epilepsies, seizures are accompanied by neurobehavioral impairments and motor disability. In the Stargazer mutant mouse, genetic disruptions of Cacng2 result in absence-like spike-wave seizures, cerebellar gait ataxia and vestibular dysfunction. Here, we combine videotracking and instrumented home-cage monitoring to resolve the neurobehavioral facets of the murine Stargazer syndrome. We find that despite their gait ataxia, stargazer mutants display horizontal hyperactivity and variable rates of repetitive circling behavior. While feeding rhythms, circadian or ultradian oscillations in activity are unchanged, mutants exhibit fragmented sleep bouts, atypical licking dynamics and lowered sucrose preference. Mutants also display an attenuated response to visual and auditory home-cage perturbations, together with profound reductions in voluntary wheel-running. Our results reveal that the seizures and ataxia of Stargazer mutants occur in the context of a more pervasive behavioral syndrome with elements of encephalopathy, repetitive behavior and anhedonia. These findings expand our understanding of the function of Cacng2/CACNG2, variants in which have been identified in patients with bipolar disorder and schizophrenia.

sentences: Quantifying the capacity for assisted migration to achieve conservation and forestry goals under climate change Many tree species might be threatened with extinction because they cannot disperse or adapt quickly enough to keep pace with climate change. One potential, and potentially risky, strategy to mitigate this threat is assisted migration, the intentional movement of species to facilitate population range shifts to more climatically suitable locations under climate change. The ability for assisted migration to minimize risk and maximize conservation and forestry outcomes depends on a multi-faceted decision process for determining, what, where, and how much to move. To quantify how the benefits and risks of assisted migration could affect the decision-making process, we used a dynamical vegetation model parameterized with 23 tree species in the western United States. We found that most of the modeled species are likely to experience a substantial decline in biomass, potentially facing regional extinction by 2100 under the high-emission SSP5-85 climate-change scenario. Though simulations show assisted migration had little effect on the forestry goal of total biomass across all species, its effects on the conservation goal of promoting individual species persistence were far more substantial. Among eight assisted migration strategies we tested that differ in terms of life cycle stage of movement and target destination selection criteria, the approach that conserved the highest biomass for individual species involved relocating target seedlings to areas with the highest canopy openness. Although this strategy significantly reduced extinction risk for six at-risk species compared to no action, it also slightly reduced biomass of four species, due to increasing competition. Species with relatively weak tolerance to drought, fire or high temperature were the most likely candidate groups for assisted migration. This model framework could be applied to other forest ecosystems to evaluate the efficacy of assisted migration globally.

sentences: Differential effects of endogenous and exogenous attention on sensory tuning Covert spatial attention (without concurrent eye movements) improves performance in many visual tasks (e.g., orientation discrimination and visual search). However, both covert attention systems, endogenous (voluntary) and exogenous (involuntary), exhibit differential effects on performance in tasks mediated by spatial and temporal resolution suggesting an underlying mechanistic difference. We investigated whether these differences manifest in sensory tuning by assessing whether and how endogenous and exogenous attention differentially alter the representation of two basic visual dimensions: orientation and spatial frequency (SF). The same human observers detected a vertical grating embedded in noise in two separate experiments (with cues to elicit either endogenous or exogenous attention) and reverse correlation was used to infer the underlying neural representation from behavioral responses. Both endogenous and exogenous attention similarly improved performance at the attended location by enhancing the gain of all orientations without changing tuning width. In the SF dimension, endogenous attention enhanced the gain of SFs above and below the target SF, whereas exogenous attention only enhanced those above. Additionally, exogenous attention shifted peak sensitivity to SFs above the target SF, whereas endogenous attention did not. Both covert attention systems modulated sensory tuning via the same computation (gain changes). However, there were differences in the strength of the gain. Compared to endogenous attention, exogenous attention had a stronger orientation gain enhancement but a weaker overall SF gain enhancement. These differences in sensory tuning may underlie differential effects of endogenous and exogenous attention on performance.

sentences: Context-independent expression of spatial code in hippocampus Hippocampus plays a crucial role in formation and retrieval of spatial memory across mammals and episodic memory in humans. Episodic as well as spatial memories can be retrieved irrespectively of subjects awake behavioral state and independently of its actual spatial context. The nature of hippocampal network activity during such out-context retrieval has not been described so far, though. Theoretically, context-independent spatial memory retrieval suggests a shift from the hippocampal spatial representations coding the actual- to the remembered context. In this study we show in rats that CA3 neuronal population can switch spontaneously across representations and transiently activate another stored familiar spatial pattern without a direct external sensory cuing. This phenomenon qualitatively differs from the well described sharp wave-related pattern reactivations during immobility. Here it occurred under theta oscillatory state during an active exploration and reflected the preceding experience of sudden environment change. The respective out-context coding spikes appeared later in the theta cycle than the in-context ones. Finally, the experience induced as well an emergence of population vectors with a co-expression of both codes segregated into different phases of the theta cycle.

sentences: Real-time single-cell characterizationof the eukaryotic transcription cyclereveals correlations between RNAinitiation, elongation, and cleavage The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

sentences: A data compendium of Mycobacterium tuberculosis antibiotic resistance The Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) presents here a compendium of 15,211 Mycobacterium tuberculosis global clinical isolates, all of which have undergone whole genome sequencing (WGS) and have had their minimum inhibitory concentrations to 13 antitubercular drugs measured in a single assay. It is the largest matched phenotypic and genotypic dataset for M. tuberculosis to date. Here, we provide a summary detailing the breadth of data collected, along with a description of how the isolates were collected and uniformly processed in CRyPTIC partner laboratories across 23 countries. The compendium contains 6,814 isolates resistant to at least one drug, including 2,129 samples that fully satisfy the clinical definitions of rifampicin resistant (RR), multi-drug resistant (MDR), pre-extensively drug resistant (pre-XDR) or extensively drug resistant (XDR). Accurate prediction of resistance status (sensitive/resistant) to eight antitubercular drugs by using a genetic mutation catalogue is presented along with the presence of suspected resistance-conferring mutations for isolates resistant to the newly introduced drugs bedaquiline, clofazimine, delamanid and linezolid. Finally, a case study of rifampicin mono-resistance demonstrates how this compendium could be used to advance our genetic understanding of rare resistance phenotypes. The compendium is fully open-source and it is hoped that the dataset will facilitate and inspire future research for years to come.

sentences: Eliciting a potent antitumor immune response by expressing tumor antigens in a skin commensal Immune modulation has become central to treating cancer. However, global immune stimulation is only effective in a subset of patients and can lead to serious complications, including colitis and type I diabetes. Newer modalities like engineered T cells and tumor vaccines are more specific, but they have shown limited efficacy in solid tumors and are difficult to scale. Bacterial strains from the human microbiome can induce antigen-specific T cells to help maintain barrier function. Here, we redirect CD8+ and CD4+ T cells elicited by the skin commensal Staphylococcus epidermidis to recognize tumor cells by expressing tumor-derived antigens in the bacterial cell. S. epidermidis expressing the model antigen ovalbumin (S. epidermidis-OVA) stimulates antigen-specific CD8+ and CD4+ T cells in vitro. The subcellular localization of the antigen skews the response: cell wall-attached OVA preferentially stimulates CD8+ T cells whereas secreted OVA predominantly induces CD4+ T cells. In a syngeneic tumor model (OVA-expressing B16 melanoma), mice colonized topically with S. epidermidis-OVA exhibit a marked reduction in subcutaneous tumor volume compared to mice colonized with S. epidermidis expressing mCherry; this effect is dependent on live bacteria and a combination of CD8+ and CD4+ T cells. S. epidermidis-OVA also reduces tumor burden when tumor cells are injected intravenously (a model of metastasis), demonstrating that the antitumor effect operates in tissues distant from the site of bacterial colonization. S. epidermidis strains expressing neoantigen peptides from the B16 tumor cell line exhibit potent antitumor efficacy without inducing an autoimmune response against melanocytes in healthy tissue. Antigen-expressing colonists are a simple but powerful strategy to elicit a targeted T cell response in the context of cancer and other diseases.

sentences: Experimentally disambiguating models of sensory cue integration Sensory cue integration is one of the primary areas in which a normative mathematical framework has been used to define the "optimal" way in which to make decisions based upon ambiguous sensory information and compare these predictions to behaviour. The conclusion from such studies is that sensory cues are integrated in a statistically optimal fashion. However, numerous alternative computational frameworks exist by which sensory cues could be integrated, many of which could be described as "optimal" based on different criteria. Existing studies rarely assess the evidence relative to different candidate models, resulting in an inability to conclude that sensory cues are integrated according to the experimenters preferred framework. The aims of the present paper are to summarise and highlight the implicit assumptions rarely acknowledged in testing models of sensory cue integration, as well as to introduce an unbiased and principled method by which to determine, for a given experimental design, the probability with which a population of observers behaving in accordance with one model of sensory integration can be distinguished from the predictions of a set of alternative models.

sentences: Reconstructing genotypes from GWAS Summary Statistics The emergence of genomewide association studies (GWAS) has led to the creation of large repositories of human genetic variation, creating enormous opportunities for genetic research and worldwide collaboration. Methods that are based on GWAS summary statistics seek to leverage such records, overcoming barriers that often exist in individual-level data access while also offering significant computational savings. Here, we propose a novel framework that can reconstruct allelic and genotypic counts/frequencies for each SNP from case-control GWAS summary statistics. Our framework is simple and efficient without the need of any complicated underlying assumptions. Illustrating the great potential of this framework we also propose three summary-statistics-based applications implemented in a new software package (ReACt): GWAS meta-analysis (with and without sample overlap), case-case GWAS, and, for the first time, group polygenic risk score (PRS) estimation. We evaluate our methods against the current state-of-the-art on both synthetic data and real genotype data and show high performance in power and error control. Our novel group PRS method based on summary statistics could not be achieved prior to our proposed framework. We demonstrate here the potential applications and advantages of this approach. Our work further highlights the great potential of summary-statistics-based methodologies towards elucidating the genetic background of complex disease and opens up new avenues for research.

sentences: Dual Dysfunction of Kir2.1 Underlies Conduction and Excitation-Contraction Coupling Defects Promoting Arrhythmias in a Mouse Model of Andersen-Tawil Syndrome Type 1 Andersen-Tawil Syndrome (ATS) is associated with life threatening arrhythmias of unknown mechanism. We report on a mouse model carrying the trafficking-deficient mutant Kir2.1{Delta}314-315. The mouse recapitulates the electrophysiological phenotype of type 1 (ATS1), with slower conduction velocities in response to flecainide, QT prolongation exacerbated by isoproterenol, and increased vulnerability to calcium-mediated arrhythmias resembling catecholaminergic polymorphic ventricular tachycardia (CPVT). Kir2.1{Delta}314-315 expression significantly reduced inward rectifier K+ and Na+ inward currents, depolarized resting membrane potential and prolonged action potential duration. Immunolocalization in wildtype cardiomyocytes and skeletal muscle cells revealed a novel sarcoplasmic reticulum (SR) microdomain of functional Kir2.1 channels contributing to intracellular Ca2+ homeostasis. Kir2.1{Delta}314-315 cardiomyocytes showed defects in SR Kir2.1 localization and function, which contributed to abnormal spontaneous Ca2+ release events. This is the first in-vivo demonstration of a dual arrhythmogenic mechanism of ATS1 defects in Kir2.1 channel function at the sarcolemma and the SR, with overlap between ATS1 and CPVT.

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MinoTour, real-time monitoring and analysis for Nanopore Sequencers. SummaryMinoTour offers a LIMS system for Oxford Nanopore Technology (ONT) sequencers, with real-time metrics and analysis available permanently for review. Integration of unique real-time automated analysis can reduce the time required to answer biological questions, including mapping and classification of sequence whilst a run is in progress. Real-time sequence data requires new methods of analysis which do not wait for the completion of a run and MinoTour provides a framework to allow users to exploit these features.

Availability and ImplementationSource code and documentation are available at https://github.com/LooseLab/minotourcli and https://github.com/LooseLab/minotourapp. Docker images are available from https://hub.docker.com/r/adoni5/, and can be installed using a preconfigured docker-compose script at https://github.com/LooseLab/minotour-docker. An example server is available at http://137.44.59.170.

sentences: An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition Extracellular matrix (ECM) is a dynamic network of proteins, proteoglycans and glycosaminoglycans, providing structure to the tissue and biochemical and biomechanical instructions to the resident cells. In fibrosis, the composition and the organization of the ECM are altered, and these changes influence cellular behaviour. Biochemical (i. e. protein composition) and biomechanical changes in ECM take place simultaneously in vivo. Investigating these changes individually in vitro to examine their (patho)physiological effects has been difficult. In this study, we generated an in vitro model to reflect the altered mechanics of a fibrotic microenvironment through applying fibre crosslinking via ruthenium/sodium persulfate crosslinking on native lung ECM-derived hydrogels. Crosslinking of the hydrogels without changing the biochemical composition of the ECM resulted in increased stiffness and decreased viscoelastic stress relaxation. The altered stress relaxation behaviour was explained using a generalized Maxwell model. Fibre analysis of the hydrogels showed that crosslinked hydrogels had a higher percentage of matrix with a high density and a shorter average fibre length. Fibroblasts seeded on ruthenium-crosslinked lung ECM-derived hydrogels showed myofibroblastic differentiation with a loss of spindle-like morphology together with greater -smooth muscle actin (-SMA) expression, increased nuclear area and circularity without any decrease in the viability, compared with the fibroblasts seeded on the native lung-derived ECM hydrogels. In summary, ruthenium crosslinking of native ECM-derived hydrogels provides an exciting opportunity to alter the biomechanical properties of the ECM-derived hydrogels while maintaining the protein composition of the ECM to study the influence of mechanics during fibrotic lung diseases.

sentences: Development of a Novel Peptide Aptamer that Interacts with the eIF4E Capped-mRNA Binding Site using Peptide Epitope Linker Evolution (PELE). Identifying new binding sites and poses that modify biological function are an important step towards drug discovery. We have identified a novel disulphide constrained peptide that interacts with the cap-binding site of eIF4E, an attractive therapeutic target that is commonly overexpressed in many cancers and plays a significant role in initiating a cancer specific protein synthesis program though binding the 5cap (7methyl-guanoisine) moiety found on mammalian mRNAs. The use of disulphide constrained peptides to explore intracellular biological targets is limited by their lack of cell permeability and the instability of the disulphide bond in the reducing environment of the cell, loss of which results in abrogation of binding. To overcome these challenges, the cap-binding site interaction motif was placed in a hypervariable loop on an VH domain, and then selections performed to select a molecule that could recapitulate the interaction of the peptide with the target of interest in a process termed Peptide Epitope Linker Evolution (PELE). A novel VH domain was identified that interacted with the eIF4E cap binding site with a nanomolar affinity and that could be intracellularly expressed in mammalian cells. Additionally, it was demonstrated to specifically modulate eIF4E function by decreasing cap-dependent translation and cyclin D1 expression, common effects of eIF4F complex disruption.

sentences: Proteomic and single-cell transcriptomic dissection of human plasmacytoid dendritic cell response to influenza virus Plasmacytoid dendritic cells [pDCs] represent a rare innate immune subset uniquely endowed with the capacity to produce substantial amounts of type-I interferons [IFN-I]. This function of pDCs is critical for effective antiviral defenses and has been implicated in autoimmunity. While IFN-I and select cytokines have been recognized as pDC secreted products, a comprehensive agnostic profiling of the pDC secretome in response to a physiologic stimulus has not been reported. We applied LC-MS/MS to catalogue the repertoire of proteins secreted by pDCs in response to challenge with live influenza H1N1. Additionally, using single-cell RNA-seq [scRNA-seq], we perform multidimensional analyses of pDC transcriptional diversification following stimulation. Our data reveal an abundance of protein species released by pDCs in addition to IFN-I, and evidence highly specialized roles within the pDC population ranging from dedicated cytokine super-producers to cells with APC-like functions. Moreover, dynamic expression of transcription factors and surface markers characterize activated pDC fates.

sentences: Mucosal and systemic responses to SARS-CoV-2 vaccination in infection naive and experienced individuals With much of the world infected with or vaccinated against SARS-CoV-2, understanding the immune responses to the SARS-CoV-2 spike (S) protein in different situations is crucial to controlling the pandemic. We studied the clinical, systemic, mucosal, and cellular responses to two doses of SARS-CoV-2 mRNA vaccines in 62 individuals with and without prior SARS-CoV-2 exposure that were divided into three groups based on serostatus and/or degree of symptoms: Antibody negative, Asymptomatic, and Symptomatic. In the previously SARS-CoV-2-infected (SARS2-infected) Asymptomatic and Symptomatic groups, symptoms related to a recall response were elicited after the first vaccination. Anti-S trimer IgA and IgG levels peaked after 1st vaccination in the SARS2-infected groups, and were higher that the in the SARS2-naive group in the plasma and nasal samples at all time points. Neutralizing antibodies titers were also higher against the WA-1 and B.1.617.2 (Delta) variants of SARS-CoV-2 in the SARS2-infected compared to SARS2-naive vaccinees. After the first vaccination, differences in cellular immunity were not evident between groups, but the AIM+ CD4+ cell response correlated with durability of humoral immunity against the SARS-CoV-2 S protein. In those SARS2-infected, the number of vaccinations needed for protection, the durability, and need for boosters are unknown. However, the lingering differences between the SARS2-infected and SARS2-naive up to 10 months post-vaccination could explain the decreased reinfection rates in the SARS2-infected vaccinees recently reported and suggests that additional strategies (such as boosting of the SARS2-naive vaccinees) are needed to narrow the differences observed between these groups.

sentences: DNA-controlled Spatio-temporal Patterning of a Cytoskeletal Active Gel Living cells move and change their shape because signaling chemical reactions modify the state of their cytoskeleton; an active gel that converts chemical energy into mechanical forces. To create life-like materials, it is thus key to engineer chemical pathways that drive active gels. Here, we describe the preparation of DNA-responsive surfaces that control the activity of a cytoskeletal active gel comprised of microtubules: a DNA signal triggers the release of molecular motors from the surface into the gel bulk, generating forces that structure the gel. Depending on the DNA sequence and concentration, the gel forms a periodic band pattern or contracts globally. Finally, we show that the structuration of the active gel can be spatially controlled in the presence of a gradient of DNA concentration. We anticipate that such DNA-controlled active matter will contribute to the development of life-like materials with self-shaping properties.

sentences: The maize gene maternal depression of r1 (mdr1) encodes a DNA glycosylase with maternal and paternal fertility functions Demethylation of transposons can activate expression of nearby genes and cause imprinted gene expression in endosperm, and it is hypothesized to lead to expression of transposon siRNAs that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting are its main function in endosperm.

sentences: Spontaneous DNA synapsis by forming noncanonical intermolecular structures We report the spontaneous formation of DNA-DNA junctions in solution in the absence of proteins visualised using atomic force microscopy. The synapsis position fits with potential G-quadruplex (G4) sites. In contrast to the Holliday structure, these conjugates have affinity for G4 antibodies. Molecular modelling was used to elucidate the possible G4/IM-synaptic complex structures. Our results indicate a new role of the intermolecular noncanonical structures in chromatin architecture and genomic rearrangement.

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Expectation for sweet taste changes peripheral glucose metabolism via basolateral amygdala Anticipatory physiological responses to food were first reported by Ivan Pavlov a century ago but the associated neural mechanism is still ill-defined. Here, we identified two types of neurons in the basolateral amygdala (BLA), which are activated by sweetener (saccharin) or water after sucrose conditioning, representing expected sweet taste and unmet expectation, respectively. Saccharin-induced met-expectation of sweet taste enhances, while H2O-induced unmet-expectation deteriorates, glucose metabolism in peripheral tissues. Deletion of saccharin-responsive neurons in BLA impaired saccharin-induced increase in insulin sensitivity. Deletion of H2O-responsive neurons in BLA improved glucose intolerance by unmet-expectation. Saccharin- and H2O-responsive neurons had different gene expressions. Our data suggest that the gap between the expected incoming sugar and sweet taste is evaluated by distinct BLA neurons to control peripheral glucose metabolism.

One-Sentence SummaryNeurons in the basolateral amygdala control blood glucose levels by comparing anticipated sugar intake and sweet taste

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Structural covariance and heritability of the optic tract and primary visual cortex in living human brains Individual differences among human brains exist at many scales, spanning gene expression, white matter tissue properties, and the size and shape of cortical areas. One notable example is an approximately 3-fold range in the size of human primary visual cortex (V1), a much larger range than is found in overall brain size. A previous study (Andrews et al., 1997) reported a correlation between optic tract cross-section area and V1 size in post-mortem human brains, suggesting that there may be a common developmental mechanism for multiple components of the visual pathways. We evaluated the relationship between properties of the optic tract and V1 in a much larger sample of living human brains by analyzing the Human Connectome Project 7 Tesla Retinotopy Dataset. This dataset includes retinotopic maps measured with functional MRI (fMRI) and fiber tract data measured with diffusion MRI (dMRI). We found a negative correlation between optic tract fractional anisotropy and V1 surface area (r = -0.2). This correlation, though small, was consistent across multiple dMRI datasets differing in acquisition parameters. Further, we found that both V1 size and optic tract properties were correlated among twins, with higher correlations for monozygotic than dizygotic twins, indicating a high degree of heritability for both properties. Together, these results demonstrate covariation across individuals in properties of the retina (optic tract) and cortex (V1) and show that each is influenced by genetic factors.

Significance statementThe size of human primary visual cortex (V1) has large inter-individual differences. These differences cannot be explained by differences in overall brain size. A previous post-mortem study reported a correlation between the size of the human optic tract and V1. In this study, we evaluated the relationship between the optic tract and V1 in living humans by analyzing a neuroimaging dataset that included functional and diffusion MRI data. We found a small, but robust correlation between optic tract tissue properties and V1 size, supporting the existence of structural covariance between the optic tract and V1 in living humans. The results suggest that characteristics of retinal ganglion cells, reflected in optic tract measurements, are related to individual differences in human V1.

sentences: Sex disparities in influenza: a multiscale network analysis Sex differences in the pathogenesis of infectious diseases due to differential immune responses between females and males have been well documented for multiple pathogens. However, the molecular mechanism underlying the observed sex differences in influenza virus infection remains poorly understood. In this study, we used a network-based approach to characterize the blood transcriptome collected over the course of infection with influenza A virus from female and male ferrets to dissect sex-biased gene expression. We identified significant differences in the temporal dynamics and regulation of immune responses between females and males. Our results elucidate sex-differentiated pathways involved in the unfolded protein response (UPR), lipid metabolism, and inflammatory responses, including a female-biased IRE1/XBP1 activation and male-biased crosstalk between metabolic reprogramming and IL-1 and AP-1 pathways. Overall, our study provides molecular insights into sex differences in transcriptional regulation of immune responses and contributes to a better understanding of sex bias in influenza pathogenesis.

sentences: In silico modeling and interactive profiling of BPH resistant R genes with elicitor molecules of rice planthoppers Brown planthopper resistant NBS-LRR specific R genes (Bph9, Bph14, Bph18, Bph26) have been reported in rice. BPH specific R genes were clustered with other R genes of rice on chromosome 12 (Bph9, Bph18, Bph26) and 3 (Bph14). Motif analysis of BPH specific R genes showed the predominant motifs as CC, NBS and LRR regions. Bph9, Bph18 and Bph26 R genes exhibited high degree of sequence similarity in their CC and NBS region and are considered as functional alleles of BPH resistance at chromosome 12. LRR region of BPH genes were interacting with the elicitor molecules of planthoppers and are the potential lignad binding site. Bph14 exhibited more number of LRR repeats and were interacting efficiently with all the tested salivary elictor molecules of planthoppers. Bph18 with no LRR region exhibited reduced interaction efficiency with the tested elicitor molecules of planthoppers. Our in silico studies confirms that Bph14 R gene resistance protein to be a promising candidate for providing broad spectrum resistance against planthoppers of rice. The study further provides new avenues to investigate the mechanism of receptor-ligand recognition and signaling mechanism against rice planthoppers.

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Repeated truncation of a modular antimicrobial peptide gene for neural context Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading pathogens. These genes commonly encode multiple products as post-translationally cleaved polypeptides. Recent studies have highlighted roles for AMPs in neurological contexts suggesting functions for these defence molecules beyond infection. During our immune study characterizing the antimicrobial peptide gene Baramicin, we recovered multiple Baramicin paralogs in Drosophila melanogaster and other species, united by their N-terminal IM24 domain. Not all paralogs were immune-induced. Here, through careful dissection of the Baramicin familys evolutionary history, we find that these non-immune paralogs result from repeated events of duplication and subsequent truncation of the coding sequence from an immune-inducible ancestor. These truncations leave only the IM24 domain as the prominent gene product. Surprisingly, using mutation and targeted gene silencing we demonstrate that two such genes are adapted for function in neural contexts in D. melanogaster. We also show enrichment in the head for independent Baramicin genes in other species. The Baramicin evolutionary history reveals that the IM24 Baramicin domain is not strictly useful in an immune context. We thus provide a case study for how an AMP-encoding gene might play dual roles in both immune and non-immune processes via its multiple peptide products. We reflect on these findings to highlight a blind spot in the way researchers approach AMP research in in vivo contexts.

Significance statementAntimicrobial peptides are immune proteins recently implicated in neurological roles. To date little attention has been paid to the contributions of different gene products in this function. Here we show that an antimicrobial peptide gene encodes multiple products with either immune-specific or neurological roles.

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Methacrylic acid-based biomaterials promote peripheral innervation in the subcutaneous space of mice Peripheral nerve innervation is essential for regulating tissue repair and regeneration. MAA-based biomaterials have been previously shown to promote angiogenesis. Here we show a new role for MAA-based biomaterials in promoting terminal axon nerve growth. Our results demonstrate that MAA-based biomaterials promote peripheral nerve growth in an Igf-1 and Shh dependent manner. The resulting nerves increased the sensitivity of treated mice paws to nociception. iDISCO clearing showed that MAA increased the presence of peripheral nerve structures in whole explants. MAA was also able to increase the expression of key neuronal markers and growth factors in a peripheral neuropathy model, the diabetic db/db mouse, suggesting that MAA-based biomaterials may be relevant to treatment of peripheral neuropathy. Moreover, in a peripheral neuropathy model, MAA was able to up-regulate the expression of growth factors for an extended duration suggesting MAA may prevent degeneration through an effect on factors that promote survival. As all tissues are innervated, MAA-based biomaterials could have broad applications in the promoting regeneration and preventing degeneration of peripheral nerves.

HIGHLIGHTSO_LIMethacrylic acid-based biomaterials promote axon growth in-situ without exogenous growth factors or cells
C_LIO_LIMethacrylic acid-based biomaterial induced terminal axon growth displays nociception, an indicator of functional outgrowth
C_LIO_LIMethacrylic acid-based biomaterials terminal axon growth is Igf-1 and Shh driven
C_LI

sentences: Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations in vitro Micro-electrode arrays (MEAs) are increasingly used to characterize neuronal network activity of human induced pluripotent stem-cell (hiPSC)-derived neurons. Despite their gain in popularity, MEA recordings from hiPSC-derived neuronal networks are not always used to their full potential in respect to experimental design, execution and data analysis. Therefore, we benchmarked the robustness and sensitivity of MEA-derived neuronal activity patterns derived from ten healthy individual control lines. We provide recommendations on experimental design and analysis to achieve standardization. With such standardization, MEAs can be used as a reliable platform to distinguish (disease-specific) network phenotypes. In conclusion, we show that MEAs are a powerful and robust tool to uncover functional neuronal network phenotypes from hiPSC-derived neuronal networks, and provide an important resource to advance the hiPSC field towards the use of MEAs for disease-phenotyping and drug discovery.

sentences: Investigating the mutational landscape of the SARS-CoV-2 Omicron variant via ab initio quantum mechanical modeling SARS-CoV-2 variant "Omicron" B1.1.529 was first identified in South Africa in November 2021. Given the large number of mutations in Omicrons spike protein compared to the original Wuhan strain, its binding efficacy to the ACE2 receptor and its potential to escape antibodies are in the spotlight. Recently, we presented an ab initio quantum mechanical model to characterize the interactions of spike proteins Receptor Binding Domain (RBD) with select antibodies and ACE2 variants. The model identified weak links among the residues constituting interactions with the human ACE2 receptor (hACE2), and also enabled us to characterize in silico mutated RBDs to identify potential Variants of Concern (VOC). In particular, we focused on the role of RBD residue 484 in the interaction of the Delta variant with ACE2 and neutralizing antibodies (nAbs). In this report, we apply our model to the Omicron VOC, and characterize its interaction pattern with hACE2. Our results show that (i) binding affinity with hACE2, compared to Delta, is considerably increased, possibly contributing to increased infectivity. (ii) The interaction pattern between B1.1.529 and hACE2 differs from previous variants by shifting the hot-spot interaction residues on hACE2, and potentially affecting nAbs efficacy. (iii) A K mutation in the RBD residue 484 can further improve Omicrons binding of hACE2 and evasion of nAbs. Finally, we argue that a library of hot-spots for point-mutations can predict binding interaction energies of complex variants.

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YAP restricts renal inflammation and mitigates kidney damage in nephronothisis related kidney disease. Nephronophthisis (NPH) is an orphan recessive kidney disease mostly caused by mutations in NPHP1 and 20 other genes encoding proteins that localize to primary cilia. To date the pathways linking altered primary cilia function to progressive kidney scarring in NPH remain poorly defined and therapeutic options allowing NPH patients to escape end-stage kidney disease are lacking.

Distinct proteins mutated in NPH interact with components of the Hippo pathway, an important regulator of cell fate. YAP (Yes-associated protein) overactivation has been shown to induce renal scarring while YAP inhibition showed protective effect in kidney diseases unrelated to NPH. Yet, the therapeutic potential of YAP inhibition in NPH has not been formerly assessed.

Here we studied the impact of both genetic and pharmacologic YAP inhibition on the NPH-like phenotype caused by a bi-allelic mutation of Lkb1, a ciliary kinase interacting with NPHP1. Contrary to non NPH renal disease, our results reveal an unexpected protective role of YAP in Lkb1 mutant kidneys. Indeed, YAP genetic disruption drastically increase kidney disease burden in Lkb1 deficient mice, while pharmacologic inhibition of YAP failed to improve their phenotype.

Collectively these results suggest that YAP inhibition is not a valid therapeutic strategy in NPH and suggest that LKB1 and YAP are parallel negative regulators of a yet uncharacterized pathway detrimental for kidney health.

sentences: Robust effects of corticothalamic feedback during naturalistic visual stimulation Neurons in the dorsolateral geniculate nucleus (dLGN) of the thalamus receive a substantial proportion of modulatory inputs from corticothalamic (CT) feedback and brain stem nuclei. Hypothesizing that these modulatory influences might be differentially engaged depending on the visual stimulus and behavioral state, we performed in vivo extracellular recordings from mouse dLGN while optogenetically suppressing CT feedback and monitoring behavioral state by locomotion and pupil dilation. For naturalistic movie clips, we found CT feedback to consistently increase dLGN response gain and promote tonic firing. In contrast, for gratings, CT feedback effects on firing rates were mixed. For both stimulus types, the neural signatures of CT feedback closely resembled those of behavioral state, yet effects of behavioral state on responses to movies persisted even when CT feedback was suppressed. We conclude that CT feedback modulates visual information on its way to cortex in a stimulus-dependent manner, but largely independently of behavioral state.

sentences: Image-based taxonomic classification of bulk biodiversity samples using deep learning and domain adaptation Complex bulk samples of invertebrates from biodiversity surveys present a great challenge for taxonomic identification, especially if obtained from unexplored ecosystems. High-throughput imaging combined with machine learning for rapid classification could overcome this bottleneck. Developing such procedures requires that taxonomic labels from an existing source data set are used for model training and prediction of an unknown target sample. Yet the feasibility of transfer learning for the classification of unknown samples remains to be tested. Here, we assess the efficiency of deep learning and domain transfer algorithms for family-level classification of below-ground bulk samples of Coleoptera from understudied forests of Cyprus. We trained neural network models with images from local surveys versus global databases of above-ground samples from tropical forests and evaluated how prediction accuracy was affected by: (a) the quality and resolution of images, (b) the size and complexity of the training set and (c) the transferability of identifications across very disparate source-target pairs that do not share any species or genera. Within-dataset classification accuracy reached 98% and depended on the number and quality of training images and on dataset complexity. The accuracy of between-datasets predictions was reduced to a maximum of 82% and depended greatly on the standardisation of the imaging procedure. When the source and target images were of similar quality and resolution, albeit from different faunas, the reduction of accuracy was minimal. Application of algorithms for domain adaptation significantly improved the prediction performance of models trained by non-standardised, low-quality images. Our findings demonstrate that existing databases can be used to train models and successfully classify images from unexplored biota, when the imaging conditions and classification algorithms are carefully considered. Also, our results provide guidelines for data acquisition and algorithmic development for high-throughput image-based biodiversity surveys.

sentences: Determinants of directionality and efficiency of the ATP synthase Fo motor at atomic resolution Fo subcomplex of ATP synthase is an membraneembedded rotary motor that converts proton motive force into mechanical energy. Despite a rapid increase in the number of high-resolution structures, the mechanism of tight coupling between proton transport and motion of the rotary c-ring remains elusive. Here, using extensive all-atom free energy simulations, we show how the motors directionality naturally arises from the interplay between intra-protein interactions and energetics of protonation of the c-ring. Notably, our calculations reveal that the strictly conserved arginine in the a-subunit (R176) serves as a jack-of-all-trades: it dictates the direction of rotation, controls the protonation state of the proton-release site and separates the two proton-access half-channels. Therefore, arginine is necessary to avoid slippage between the proton flux and the mechanical output and guarantees highly efficient energy conversion. We also provide mechanistic explanations for the reported defective mutations of R176, reconciling the structural information on the Fo motor with previous functional and single-molecule data.

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NOMA: a high-throughput microchip for robust, sequential measurements of secretions from the same single-cells Despite recent advances in single-cell analysis technologies, lacking simple methods to keep the live single-cells traceable for longitudinal detection reliably poses a significant obstacle in single-cell secretion analysis. Here we reported the high-density NOMA (narrow-opening microwell array) microchip that realized the retention of[≥] 97% of trapped single cells in dedicated spatial locations during repetitive detection procedures, verified with both adherent and suspension cells by two researchers independently. We applied it in monitoring single-cell protein secretions sequentially from the same single cells, and we found the digital protein secretion patterns dominate the protein secretion. We also demonstrated the microchip for longitudinally tracking IL-8 and the CD81+EV secretions from the same single-cells over days, which revealed the presence of "super secretors" within the cell population be more persistent to secrete protein or extracellular vesicle for an extended period. The NOMA platform reported here is simple, robust, and easy to operate for tracking sequential measurements from the same single cells, representing a novel and informative tool to inspire new observations in biomedical research.

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sentences: Evaluating Impacts of Syntenic Block Detection Strategies on Rearrangement Phylogeny Using Mycobacterium tuberculosis Isolates AO_SCPLOWBSTRACTC_SCPLOWPhylogenetic inference based on genomic structural variations, that manipulate the gene order and content of whole chromosomes, promises to inform a more comprehensive understanding of evolution. The first challenge in using such data, the incompleteness of available de novo assemblies, is easing as long read technologies enable (near-)complete genome assembly, but methodological challenges remain. To obtain the input to rearrangement-based inference methods, we need to detect syntenic blocks of orthologous sequences, a task that can be accomplished in many ways, none of which are obviously preferable. In this paper, we use 94 reference quality genomes of primarily Mycobacterium tuberculosis (Mtb) isolates as a benchmark to evaluate these methods. The clonal nature of Mtb evolution, the manageable genome sizes, along with substantial levels of structural variation make this an ideal benchmarking dataset. We test several methods for detecting homology and obtaining syntenic blocks, and two methods for inferring phylogenies, comparing them to the standard method that uses substitutions for inferring the tree. We find that not only the choice of methods but also their parameters can impact results, especially among branches with lower support. In particular, a method based on an encoding of adjacencies applied to Cactus-defined blocks was fully compatible with the highly supported branches of the substitution-based tree. Thus, we were able to combine the two trees to obtain a supertree with high resolution utilizing both SNPs and rearrangements. Furthermore, we observed that the results were much less affected by the choice of the tree inference method than by the method used to determine the underlying syntenic blocks. Overall, our results indicate that accurate trees can be inferred using genome rearrangements, but the choice of the methods for inferring the homology matters and requires care.

sentences: Prioritizing and characterizing functionally relevant genes across human tissues Knowledge of genes that are critical to a tissues function remains difficult to ascertain and presents a major bottleneck toward a mechanistic understanding of genotype-phenotype links. Here, we present the first machine learning model - FUGUE - combining transcriptional and network features, to predict tissue-relevant genes across 30 human tissues. FUGUE achieves an average cross-validation auROC of 0.86 and auPRC of 0.50 (expected 0.09). In three independent datasets, FUGUE accurately distinguishes tissue or cell type-specific genes, significantly outperforming the conventional metric based on tissue-specific expression alone. Comparison of tissue-relevant transcription factors across tissue recapitulate their developmental relationships. Interestingly, the tissue-relevant genes cluster on the genome within topologically associated domains and furthermore, are highly enriched for differentially expressed genes in the corresponding cancer type. We provide the prioritized gene lists in 30 human tissues and an open-source software to prioritize genes in a novel context given multi-sample transcriptomic data.

sentences: Dopamine biases sensitivity to personal goals and social influence in self-control over everyday desires People regularly give in to daily temptations in spite of conflict with personal goals. To test hypotheses about neuropharmacological influences on self-control, we used positron emission tomography to measure dopamine D2-like receptors (D2R) and experience sampling surveys to naturalistically track daily desires outside the laboratory in everyday life in a sample of 103 adults. Higher D2R availability in the ventral striatum was associated with increased sensitivity to personal goal conflict but not desire strength in deciding whether to attempt to resist a desire. The influence of D2Rs on sensitivity to personal goal conflict depended on whether desires were experienced in a social context. D2R availability in the midbrain (but not the ventral striatum) influenced whether desires were enacted. These findings provide unique evidence that the dopamine system influences decision making and regulatory behavior and provides new insights into how these mechanisms interact with personal goals and social contexts.

sentences: Effects of substrate stiffness and actin velocity on fibronectin fibril morphometry and mechanics Assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils is a critical step during embryonic development and wound healing; misregulation of FN fibril assembly has been implicated in many diseases, including fibrotic diseases and cancer. We have previously developed a computational model of FN fibril assembly that recapitulates the morphometry and mechanics of cell-derived FN fibrils. Here we use this model to probe two important questions: how is FN fibril formation affected by the contractile phenotype of the cell, and how is FN fibril formation affected by the stiffness of the surrounding tissue? We show that FN fibril formation depends strongly on the contractile phenotype of the cell, but only weakly on tissue stiffness. These results are consistent with previous experimental data and provide a better insight into conditions that promote FN fibril assembly. We have also used the model to look at two distinct phenotypes of FN fibrils that we have previously identified; we show that the ratio of the two phenotypes depends on both tissue stiffness and contractile phenotype, with intermediate contractility and high tissue stiffness creating an optimal condition for stably stretched fibrils. Finally, we have investigated how re-stretch of a fibril affects cellular response. We probed how the contractile phenotype of the re-stretching cell affects the mechanics of the fibril; results indicate that the number of myosin motors only weakly affects the cellular response, but increasing actin velocity results in a decrease in the apparent stiffness of the fibril and a decrease in the stably-applied force to the fibril. Taken together, these results give novel insights into the combinatorial effects of tissue stiffness and cell contractility on FN fibril assembly.

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

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Avoiding Data Loss: Synthetic MRIs Generated from Diffusion Imaging Can Replace Corrupted Structural Acquisitions For Freesurfer-Seeded Tractography 1Magnetic Resonance Imaging (MRI) motion artefacts frequently complicate structural and diffusion MRI analyses. While diffusion imaging is easily  scrubbed of motion affected volumes, the same is not true for structural images. Structural images are critical to most diffusion-imaging pipelines thus their corruption can lead to disproportionate data loss. To enable diffusion-image processing when structural images have been corrupted, we propose a means by which synthetic structural images can be generated from diffusion MRI. This technique combines multi-tissue constrained spherical deconvolution, which is central to many existing diffusion analyses, with the Bloch equations which allow simulation of MRI intensities given scanner parameters and magnetic resonance (MR) tissue properties. We applied this technique to 32 scans, including those acquired on different scanners, with different protocols and with pathology present. The resulting synthetic T1w and T2w images were visually convincing and exhibited similar tissue contrast to acquired structural images. These were also of sufficient quality to drive a Freesurfer-based tractographic analysis. In this analysis, probabilistic tractography connecting the thalamus to the primary sensorimotor cortex was delineated with Freesurfer, using either real or synthetic structural images. Tractography for real and synthetic conditions was largely identical in terms of both voxels encountered (Dice 0.88 - 0.95) and mean fractional anisotropy (intrasubject absolute difference 0.00 - 0.02). We provide executables for the proposed technique in the hope that these may aid the community in analysing datasets where structural image corruption is common, such as studies of children or cognitively impaired persons.

HighlightsO_LIWe propose a simple means of synthesizing T1w and T2w images from diffusion data
C_LIO_LIThe proposed method worked well for a variety of acquisitions
C_LIO_LISynthetic images showed tissue contrast akin to acquired images
C_LIO_LISynthetic images were high enough quality to be used for Freesurfer seeded diffusion tractography
C_LIO_LIThis method enables analysis of datasets where motion has corrupted acquired structural MRIs
C_LI

sentences: Shade-induced transcription of PIF-Direct-Target Genes precedes H3K4-trimethylation chromatin modification rises The phytochrome (phy)-PIF (Phytochrome Interacting Factor) sensory module perceives and transduces light signals to Direct-Target Genes (DTGs), which then drive the adaptational responses in plant growth and development, appropriate to the prevailing environment. These signals include the first exposure of etiolated seedlings to sunlight upon emergence from subterranean darkness, and the change in color of the light that is filtered through, or reflected from, neighboring vegetation ( shade). Previously, we identified three broad categories of rapidly signal-responsive genes: those repressed by light and conversely induced by shade; those repressed by light, but subsequently unresponsive to shade; and those responsive to shade only. Here, we investigate the potential role of epigenetic chromatin modifications in regulating these contrasting patterns of phy-PIF module-induced expression of DTGs. Using RNA-seq and ChlP-seq, time-resolved profiling of transcript and histone 3 lysine 4 trimethylation (H3K4me3) levels, respectively, we show that, whereas the initial dark-to-light transition triggers a rapid, apparently temporally-coincident decline of both parameters, the light-to-shade transition induces similarly rapid increases in transcript levels that precede increases in H3K4me3 levels. Together with other recent findings, these data raise the possibility that, rather than being causal in the shade-induced expression changes, H3K4me3 may function to buffer the rapidly fluctuating shade/light switching that is intrinsic to vegetational canopies under natural sunlight conditions.

sentences: PreDarwinian evolution, prebiotic change: a principle of least selection Evolution prior to its Darwinian era occurred in an age of variation. Events allowed by starting materials, chemistry and physics were realized, directed by intrinsic probabilities. In this landscape, a principle of least selection defines the most probable evolutionary path. For selection from a population where x varies, the rate of x evolution dx/dt = {Delta}.  is the population mean of the selected property x,  a proportionality constant. Explicit equations for selected change {Delta} are presented. Simple results exist for common cases; for example, selecting for x [≥] a threshold. Maximizing {Delta} minimizes need for selection. Least selections for x demand maximal dependence on x, maximal x standard deviation{sigma} , and maximal lower x threshold (consistent with the need for a survivor). Least selection is free of the cost of natural selection, thus can support rapid evolution. Least selection also unites previous seemingly independent optima. As examples, evolution via  starting bloc selection and  chance utility (where selection spans a minimal change) and  near-ideal reactions (where variable reactant supplies recurrently create easy reactions) occur via transitory paths of least selection.  Bayesian convergence is guaranteed by least selections extremum (which sharply defines evolutions path).  Distribution fitness (specific outcomes selected among multiple concurrent variations) is least selection of rare functional minorities. Inheritance of encoded chemical capabilities first evolves because templating and catalysis are related, potentially joined in a single molecular complex. Least selection defines directions, biostructures define destinations for likely pathways of preDarwinian evolution.

sentences: Conserved and unique transcriptional features of pharyngeal arches in the skate (Leucoraja erinacea) and evolution of the jaw The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral patterning mechanisms within the developing mandibular, hyoid and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralising BMP and endothelin signalling pathways and their effectors, the joint markers bapx1 and gdf5 and pro-chondrogenic transcription factors barx1 and gsc, and the dorsalising transcription factor pou3f3. Additionally, we find that mesenchymal expression of eya1/six1 is an ancestral feature of the mandibular arch of jawed vertebrates, while differences in notch signalling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the dorsoventral axis of the pharyngeal arches, including scamp5 as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.

sentences: Co-cultivation is a powerful approach to produce a robust functionally designed synthetic consortium as a live biotherapeutic product (LBP) The successes of fecal microbiota transplants (FMT) have provided the necessary proof-of-concept for microbiome therapeutics. Because of the many risks and uncertainties associated with feces-based therapies, defined microbial consortia that modify the microbiome in a targeted manner have emerged as a promising safer alternative to FMT. The development of such live biotherapeutic products has important challenges, including the selection of appropriate strains and the production of the consortia at scale. Here, we report on an ecology and biotechnology-based approach to microbial consortium design that overcomes these issues. We designed a nine-strain consortium that emulates the central metabolic pathways of carbohydrate fermentation in the healthy human gut microbiota. We show that continuous co-culturing the bacteria produce a stable consortium whose activity is distinct from an equivalent mix of individually cultured strains. Further, we showed that our function-based consortium is as effective as FMT in counteracting dysbiosis in a dextran sodium sulfate mouse model of acute colitis. We propose that combining a bottom-up functional design with continuous co-cultivation is a powerful strategy to produce robust, functionally designed synthetic consortia for therapeutic use.

sentences: Theoretical Quantification of the Polyvalent Binding of Nanoparticles Coated with Peptide-MHC to TCR-Nanoclusters Nanoparticles (NPs) coated with pMHCs can reprogram a specific type of CD4+ T cells into diseasesuppressing T regulatory type 1 cells by binding to their TCRs expressed as TCR-nanoclusters (TCRnc). NP size and number of pMHCs coated on them (called valence) can be adjusted to increase their efficacy. Here we explore how this polyvalent interaction is manifested and examine if it can facilitate T cell activation. This is done by developing a multiscale biophysical model that takes into account the complexity of this interaction. Using the model, we quantify pMHC insertion probabilities, dwell time of NP binding, TCRnc carrying capacity, the distribution of covered and bound TCRs by NPs, and cooperativity in the binding of pMHCs within the contact area. Model fitting and parameter sweeping further reveal that moderate jumps between IFN{gamma} dose-response curves at low valences can occur, suggesting that the geometry of NP binding can prime T cells for activation.

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In vivo engineered B cells retain memory and secrete high titers of anti-HIV antibodies in mice As a potential single-shot HIV therapy, transplanted engineered B cells allow robust secretion of broadly neutralizing antibodies (bNAbs). However, ex vivo engineering of autologous B cells is expensive and requires specialized facilities, while allogeneic B cell therapy necessitates MHC compatibility. Here, we develop in vivo B cell engineering, by injecting two adeno associated viral vectors, one coding for saCas9 and another coding for a bNAb. Following immunizations, we demonstrate memory retention and bNAb secretion at neutralizing titers. We observed minimal CRISPR/Cas9 off-target cleavage, using unbiased CHANGE-Seq analysis, while on-target cleavage at undesired tissues is reduced by expressing saCas9 from a B cell specific promoter. In vivo B cell engineering is thus a safe, potent and scalable method for expressing desired antibodies against HIV and beyond.

One sentence summaryB cells can be engineered in vivo to robustly secrete anti-HIV bNAbs in a safe, durable and scalable manner.

sentences: The impact of rare germline variants on human somatic mutation processes Somatic mutations are an inevitable component of ageing and the most important cause of cancer. The rates and types of somatic mutation vary across individuals, but relatively few inherited influences on mutation processes are known. We performed a comprehensive gene-based rare variant association study with diverse mutational processes, using human cancer genomes from over 11,000 individuals of European ancestry. By combining burden and variance tests, we identify 207 associations involving 15 somatic mutational phenotypes and 42 genes that replicated in an independent data set at a FDR of 1%. We associated rare inherited deleterious variants in novel genes such as MSH3, EXO1, SETD2, and MTOR with two different forms of DNA mismatch repair deficiency, and variants in genes such as EXO1, PAXIP1, and WRN with deficiency in homologous recombination repair. In addition, we identified associations with other mutational processes, such as APEX1 with APOBEC-signature mutagenesis. Many of the novel genes interact with each other and with known mutator genes within cellular sub-networks. Considered collectively, damaging variants in the newly-identified genes are prevalent in the population. We suggest that rare germline variation in diverse genes commonly impacts mutational processes in somatic cells.

sentences: Atomic structure of human sapovirus capsid by single particle cryo-electron microscopy Sapovirus is a cause of acute gastroenteritis in humans and animals. Infants and younger children have the greatest disease burden. Although it shares many similarities with norovirus, the lack of detailed structural information has hampered the development of vaccines and therapeutics. Here, we investigated the human sapovirus VLP by single particle cryo-electron microscopy and are the first to report the atomic structure of the capsid at 2.9 [A] resolution. The atomic model revealed the domain interactions of the capsid protein and functionally important amino acid residues. The extended loop from the P1 subdomain was involved in interactions in the P2 domain, forming unique arch-like dimeric protrusions of capsid proteins. All hypervariable regions that are important candidates for immune response or receptor binding, formed a large cluster at the top of the P domain. These results pave the way for developing vaccines, antiviral drugs, and diagnostic systems for this infectious disease.

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Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E The ARF family of regulatory GTPases is ancient, with 16 members predicted to have been present in the last eukaryotic common ancestor. Our phylogenetic profiling of paralogs in diverse species identified four family members whose presence correlates with that of a cilium/flagellum: ARL3, ARL6, ARL13, and ARL16. No prior evidence links ARL16 to cilia or other cell functions, despite its presence throughout eukaryotes. Deletion of ARL16 in MEFs results in decreased ciliogenesis yet increased ciliary length. We also found Arl16 KO in MEFs to alter ciliary protein content, including loss of ARL13B, ARL3, INPP5E, and the IFT-A core component IFT140. Instead, both INPP5E and IFT140 accumulate at the Golgi in Arl16 KO lines, while other IFT proteins do not, suggesting a specific defect in traffic from Golgi to cilia. We propose that ARL16 regulates a Golgi-cilia traffic pathway and is required specifically in the export of IFT140 and INPP5E from the Golgi.

SummaryPhylogenetic analyses of ARF family GTPases predict that ARL16 is linked to cilia. This was confirmed using MEFs deleted for ARL16, resulting in defects in Golgi to cilium traffic, with accumulation of IFT140 and INPP5E at Golgi.

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Selenium in wheat from farming to food Selenium (Se) plays an important role in human health. Approximately 80% of the worlds population does not consume enough Se which recommended by WHO (World Health Organization). Wheat is an important staple food and Se source for most people in the world. This article summarizes literatures about Se from 1936 to 2020 to investigate Se in wheat farming soil, wheat, and its derived foods. Se fortification and the recommended Se level in wheat were also discussed. Results showed that Se contents in wheat farming soil, grain, and its derived foods around the world were 3.8-552 (mean, 220.99), 0-8,270 (mean, 347.30), and 15-2,372 (mean, 211.86) g{middle dot}kg-1, respectively. Adopting suitable agronomic measures could effectively realize Se fortification in wheat. The contents in grain, flour, and its derived foods could be improved from 93.94 to 1,181.92, 73.06 to 1,007.75, and 86.90 to 587.61 g{middle dot}kg-1 in average after leaf Se fertilizer application in the field. There was a significant positive correlation between Se content in farming soil and grain, and it was extremely the same between foliar Se fertilizer concentration rate and grain Se increased rate. The recommended Se fortification level in cultivation of wheat in China, India, and Spain was 18.53-23.96, 2.65-3.37, and 3.93-9.88 g{middle dot}hm-2 respectively. Milling processing and food type could greatly affect Se content of wheat derived food and should be considered seriously to meet peoples Se requirement by wheat.

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Prediction of DNA from context using neural networks One way to better understand the structure in DNA is by learning to predict the sequence. Here, we train a model to predict the missing base at any given position, given its left and right flanking contexts.

Our best-performing model is a neural network that obtains an accuracy close to 54% on the human genome, which is 2% points better than modelling the data using a Markov model. In likelihood-ratio tests, we show that the neural network is significantly better than any of the alternative models by a large margin. We report on where the accuracy is obtained, observing first that the performance appears to be uniform over the chromosomes. The models perform best in repetitive sequences, as expected, although they are far from random performance in the more difficult coding sections, the proportions being ~ 70:40%. Exploring further the sources of the accuracy, Fourier transforming the predictions reveals weak but clear periodic signals. In the human genome the characteristic periods hint at connections to nucleosome positioning. To understand this we find similar periodic signals in GC/AT content in the human genome, which to the best of our knowledge have not been reported before.

On other large genomes similarly high accuracy is found, while lower predictive accuracy is observed on smaller genomes. Only in mouse did we see periodic signals in the same range as in human, though weaker and of different type. Interestingly, applying a model trained on the mouse genome to the human genome results in a performance far below that of the human model, except in the difficult coding regions.

Despite the clear outcomes of the likelihood ratio tests, there is currently a limited superiority of the neural network methods over the Markov model. We expect, however, that there is great potential for better modelling DNA using different neural network architectures.

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Strategies of tolerance reflected in two North American maple genomes Maples (the genus Acer) represent important and beloved forest, urban, and ornamental trees distributed throughout the Northern hemisphere. They exist in a diverse array of native ranges and distributions, across spectrums of tolerance or decline, and have varying levels of susceptibility to biotic and abiotic stress. Among Acer species, several stand out in their importance to economic interest. Here we report the first two chromosome-scale genomes for North American species, Acer negundo and Acer saccharum. Both assembled genomes contain scaffolds corresponding to 13 chromosomes, with A. negundo at a length of 442 Mb, N50 of 32 Mb and 30,491 genes, and A. saccharum at 626 Mb, N50 of 46 Mb, and 40,074 genes. No recent whole genome duplications were detected, though A. saccharum has local gene duplication and more recent bursts of transposable elements, as well as a large-scale translocation between two chromosomes. Genomic comparison revealed that A. negundo has a smaller genome with recent gene family evolution that is predominantly contracted and expansions that are potentially related to invasive tendencies and tolerance to abiotic stress. Examination of expression from RNA-Seq obtained from A. saccharum grown in long-term aluminum and calcium soil treatments at the Hubbard Brook Experimental Forest, provided insights into genes involved in aluminum stress response at the systemic level, as well as signs of compromised processes upon calcium deficiency, a condition contributing to maple decline.

Significance statementThe first chromosome-scale assemblies for North American members of the Acer genus, sugar maple (Acer saccharum) and boxelder (Acer negundo), as well as transcriptomic evaluation of abiotic stress response in A. saccharum. This integrated study describes in-depth aspects contributing to each species approach to tolerance and applies current knowledge in many areas of plant genome biology with Acer physiology to help convey the genomic complexities underlying tolerance in broadleaf tree species.

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Mitochondrial fission is required for proper nucleoid distribution within mitochondrial networks Mitochondrial DNA (mtDNA) maintenance is essential to sustain a functionally healthy population of mitochondria within cells. Proper mtDNA replication and distribution within mitochondrial networks are essential to maintain mitochondrial homeostasis. However, the fundamental basis of mtDNA segregation and distribution within mitochondrial networks is still unclear. To address these questions, we developed an algorithm, Mitomate tracker to unravel the global distribution of nucleoids within mitochondria. Using this tool, we decipher the semi-regular spacing of nucleoids across mitochondrial networks. Furthermore, we show that mitochondrial fission actively regulates mtDNA distribution by controlling the distribution of nucleoids within mitochondrial networks. Specifically, we found that primary cells bearing disease-associated mutations in the fission proteins DRP1 and MYH14 show altered nucleoid distribution, and acute enrichment of enlarged nucleoids near the nucleus. Further analysis suggests that the altered nucleoid distribution observed in the fission mutants is the result of both changes in network structure and nucleoid density. Thus, our study provides novel insights into the role of mitochondria fission in nucleoid distribution and the understanding of diseases caused by fission defects.

Significance statementMutation or deletion of mitochondrial DNA (mtDNA) is associated with a large number of human diseases. However, the mechanisms controlling mtDNA replication and segregation are still poorly understood. Here, we have developed a new computational method to quantify the distribution of nucleoids (mtDNA with associated proteins) and define how nucleoid distribution is affected by changes in mitochondrial network structure. We demonstrate that mitochondrial fission is required for the proper distribution of nucleoids across mitochondrial networks, cells from patients with fission defects showing irregular nucleoid distribution and perinuclear accumulation. Nonetheless, each fission mutant behaved in a distinct manner, indicating a complex relationship between mitochondrial dynamics and nucleoid distribution.

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Mycobacterium tuberculosis SufR Responds to Nitric oxide via its 4Fe-4S cluster and Regulates Fe-S cluster Biogenesis for Persistence in Mice The persistence of Mycobacterium tuberculosis (Mtb) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtbs physiology are not fully understood. Since NO damages Fe-S clusters of essential enzymes, the mechanism(s) involved in regulating iron-sulfur (Fe-S) cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe-4S cluster and promotes persistence of Mtb by mobilizing the Fe-S cluster biogenesis system; suf operon (Rv1460-Rv1466). Analysis of anaerobically purified SufR by UV-visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe-4S cluster. Atmospheric O2 and H2O2 gradually degrade the 4Fe-4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe-4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA- sequencing of Mtb{Delta}sufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory and redox buffering capacity of Mtb {Delta}sufR. Lastly, Mtb{Delta} sufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics.

HighlightsO_LIMycobacterium tuberculosis (Mtb) induces the expression of suf operon for Fe-S cluster biogenesis in response to nitric oxide (NO).
C_LIO_LIWe found that a transcription factor SufR senses NO via its 4Fe-4S cluster and regulates the expression of the suf operon for Fe-S cluster biogenesis.
C_LIO_LISufR-regulated Fe-S cluster biogenesis confers respiratory and redox features that promote recovery of Mtb from NO stress.
C_LIO_LISufR activity is required to support the NO-dependent persistence of Mtb in macrophages and mice.
C_LI

sentences: Early zygotic gene product Dunk interacts with anillin to regulate Myosin II during Drosophila cleavage Drosophila cellularization is a special form of cleavage that converts syncytial embryos into cellular blastoderms by partitioning the peripherally localized nuclei into individual cells. An early event in cellularization is the recruitment of non-muscle myosin II ("myosin") to the basal tip of cleavage furrows, where myosin forms an interconnected basal array before reorganizing into individual cytokinetic rings. The initial recruitment and organization of basal myosin are regulated by a cellularization-specific gene, dunk, but the underlying mechanism is unclear. Through a genome-wide yeast two-hybrid screen, we identified anillin (Scraps in Drosophila), a conserved scaffolding protein in cytokinesis, as the primary binding partner of Dunk. We show that Dunk regulates the localization of anillin at the cleavage furrows during early cellularization and functionally interacts with anillin in regulating basal myosin. Furthermore, we show that anillin colocalizes with myosin since the onset of cellularization and is required for the initial recruitment and maintenance of myosin at the basal array, prior to the well-documented function of anillin in regulating cytokinetic ring assembly. Based on these results, we propose that Dunk regulates myosin recruitment and organization during early cellularization by interacting with and regulating anillin.

sentences: Long range, high-gamma phase coherence in the human brain during overt and covert speech Using intracranial electroencephalography (iEEG) in patients undergoing diagnostic workup for epilepsy, we show that during reading, phase coherent high-gamma activity emerges between spatially distant regions of proven involvement in vocalization. Using the novel metric of "phase-dependent power-causal correlations", causal interactions were shown to be maximal at high-gamma frequencies, and displayed stronger correlations to power fluctuations near the optimal time delay, with this delay potentially accounted for by axonal conduction. We conclude that high-gamma activity may represents propagated, directed, large-scale integration between task related regions of the human brain.

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A multidrug-resistant Salmonella enterica serotype Typhimurium DT104 lineage circulating among humans and cattle in the United States lost the ability to produce pertussis-like toxin ArtAB in close temporal proximity to the global DT104 epidemic Salmonella enterica subspecies enterica serotype Typhimurium phage type DT104 (DT104) can infect both humans and animals and is often multidrug-resistant (MDR). Previous studies have indicated that, unlike most S. Typhimurium, the overwhelming majority of DT104 strains produce a pertussis-like toxin, ArtAB, via prophage-encoded artAB; however, DT104 that lack artAB have been described on occasion. Here, we identify a MDR DT104 lineage circulating among humans and cattle in the United States, which lacks artAB (i.e., the "U.S. artAB-negative major clade"; n = 41 genomes). Unlike most other bovine- and human-associated DT104 strains from the U.S. (n = 219 total genomes), which harbor artAB on prophage Gifsy-1 (n = 167), members of the U.S. artAB-negative major clade lack Gifsy-1, as well as anti-inflammatory effector gogB. The U.S. artAB-negative major clade was predicted to have lost artAB, Gifsy-1, and gogB circa 1993-1995 (95% highest posterior density interval 1989.5-1997.7), in close temporal proximity to a predicted rapid increase in the U.S. DT104 effective population size (circa 1996). When compared to DT104 genomes from other world regions (n = 752 total genomes), several additional, sporadic artAB, Gifsy-1, and/or gogB loss events among clades encompassing [≤]5 genomes were observed. In phenotypic assays that simulate conditions encountered during human and/or bovine digestion, members of the U.S. artAB-negative major clade did not differ from closely related Gifsy-1/artAB/gogB-harboring U.S. DT104 strains (ANOVA raw P > 0.05); thus, future research is needed to elucidate the roles that artAB, gogB, and Gifsy-1 play in DT104 virulence in humans and animals.

Impact StatementMulti-drug resistant (MDR) Salmonella enterica serotype Typhimurium phage type DT104 (DT104) was responsible for a global epidemic among humans and animals throughout the 1990s and continues to circulate worldwide. Previous studies have indicated that the vast majority of DT104 produce a pertussis-like toxin, ArtAB, via prophage-encoded artAB. However, here we identify a DT104 lineage circulating among cattle and humans across [≥]10 U.S. states, which lacks the ability to produce ArtAB (i.e., the "U.S. artAB-negative major clade"). The common ancestor of all U.S. artAB-negative major clade members lost the ability to produce ArtAB in close temporal proximity to the global MDR DT104 epidemic; however, the reason for this loss-of-function event within this well-established pathogen remains unclear. The role that ArtAB plays in DT104 virulence remains elusive, and phenotypic assays conducted here indicate that members of the U.S. artAB-negative major clade do not have a significant advantage or disadvantage relative to closely related Gifsy-1/artAB/gogB-harboring U.S. DT104 strains when exposed to stressors encountered during human and/or bovine digestion in vitro. However, ArtAB heterogeneity among DT104 suggest clade-specific selection for or against maintenance of ArtAB. Thus, future studies querying the virulence potential of the U.S. artAB- negative major clade are needed.

Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

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Predicting potentially permissive substitutions that improve the fitness of A(H1N1)pdm09 viruses bearing the H275Y NA substitution Oseltamivir-resistant influenza viruses arise due to amino-acid mutations in key residues, but these changes often reduce their replicative and transmission fitness. Widespread oseltamivir-resistance has not yet been observed in A(H1N1)pdm09 viruses. However, it is known that permissive mutations in the neuraminidase (NA) of former seasonal A(H1N1) viruses from 2007-2009 buffered the detrimental effect of the NA H275Y mutation, resulting in fit oseltamivir-resistant viruses that circulated widely. This study explored two approaches to predict permissive mutations that may enable a fit H275Y A(H1N1)pdm09 variant to arise.

A computational approach used phylogenetic and in silico protein stability analyses to predict potentially permissive mutations, which were then evaluated by in vitro NA enzyme activity and expression analysis, followed by in vitro replication. The second approach involved the generation of a virus library which encompassed all possible individual 2.9 x 104 codon mutations in the NA whilst keeping H275Y fixed. To select for variant viruses with the greatest fitness, the virus library was serially passaged in ferrets (via contact and aerosol transmission) and resultant viruses were deep sequenced.

The computational approach predicted three NA permissive mutations, and even though they only offset the in vitro impact of H275Y on NA enzyme expression by 10%, they could restore replication fitness of the H275Y variant in A549 cells. In our experimental approach, a diverse virus library (97% of 8911 possible single amino-acid substitutions were sampled) was successfully transmitted through ferrets, and sequence analysis of resulting virus pools in nasal washes identified three mutations that improved virus transmissibility. Of these, one NA mutation, I188T, has been increasing in frequency since 2017 and is now present in 90% of all circulating A(H1N1)pdm09 viruses.

Overall, this study provides valuable insights into the evolution of the influenza NA protein and identified several mutations that may potentially facilitate the emergence of a fit H275Y A(H1N1)pdm09 variant.

sentences: Long-term in vivo imaging of mouse spinal cord through an optically cleared intervertebral window Spinal cord, as part of the central nervous system, accounts for the main communication pathway between the brain and the peripheral nervous system. Spinal cord injury is a devastating and largely irreversible neurological trauma, and can result in lifelong disability and paralysis with no available cure. In vivo spinal cord imaging in mouse models without introducing immunological artifacts is critical to understand spinal cord pathology and discover effective treatments. We developed a minimal-invasive intervertebral window by retaining ligamentum flavum to protect the underlying spinal cord. By introducing an optical clearing method, we achieved repeated two-photon fluorescence and stimulated Raman scattering imaging at subcellular resolution with up to 16 imaging sessions over 167 days and observed no inflammatory response. Using this optically cleared intervertebral window, we studied the neuron-glia dynamics following laser axotomy and observed strengthened contact of microglia with the nodes of Ranvier during axonal degeneration. By enabling long-term, repetitive, stable, high-resolution and inflammation-free imaging of mouse spinal cord, our method provides a reliable platform in the research aiming at understanding and treatment of spinal cord pathology.

sentences: Annotation of Spatially Resolved Single-cell Data with STELLAR Spatial protein and RNA imaging technologies have been gaining rapid attention but current computational methods for annotating cells are based on techniques established for dissociated single-cell technologies and thus do not take spatial organization into account. Here we present STELLAR, a geometric deep learning method that utilizes spatial and molecular cell information to automatically assign cell types from an annotated reference set as well as discover new cell types and cell states. STELLAR transfers annotations across different dissection regions, tissues, and donors and detects higher-order tissue structures with dramatic time savings.

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Proteomic pipeline to identify age-related compositional and structural changes of the human dentin extracellular matrix ObjectiveDevise a pipeline to investigate the protein composition of the human root dentin extracellular matrix (ECM) from single individuals of different age cohorts.

DesignIndividual cervical root dentin of sound human molars from two age brackets, young (18-25 years old; n=3) and old (75-85 years old; n=3), were cut and pulverized. Protein extraction and fractionation were completed by sequential demineralization with EDTA buffer, chaotropic extraction with guanidine hydrochloride, and urea. The resulting protein extracts of differential solubility were digested into peptides and peptides were analyzed by mass spectrometry. Data generated for this study are available via ProteomeXchange, identifier PXD018320.

ResultsWe found that protein extracts of different solubilities present distinct biochemical compositions. We further define the matrisome of young (48 proteins) and old (50 proteins) human root dentin and report the identification of compositional and structural differences in ECM proteins from young and old teeth.

ConclusionOur study provides a rigorous pipeline, from sample preparation to data analysis, to investigate the ECM composition - or matrisome - of the dentin. This pipeline has the potential to lead to the discovery of biomarkers of tooth aging and health.

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High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19 Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH2-terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting.

Author SummaryMutant sequences of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) arising during any individual case of coronavirus disease 2019 (COVID-19) could theoretically enable the virus to evade immune responses or antiviral therapies that target the predominant infecting virus sequence. However, commonly used sequencing technologies are not optimally designed to detect variant virus sequences within each sample. To address this issue, we developed novel technology for sequencing large numbers of individual SARS-CoV-2 genomic RNA molecules across the region encoding the virus surface proteins. This technology revealed extensive genetic diversity in cultured viruses from a clinical isolate of SARS-CoV-2, but lower diversity in samples from 7 individuals with COVID-19. Importantly, concurrent analysis of paired serum samples in selected individuals revealed relatively low levels of antibody binding to the SARS-CoV-2 spike protein at the time of initial sequencing. With increased serum binding to spike protein, we detected multiple SARS-CoV-2 variants bearing independent mutations in a single epitope, as well as a transient increase in virus burden. These findings suggest that SARS-CoV-2 replication creates sufficient virus genetic diversity to allow immune-mediated selection of variants within the time frame of acute COVID-19. Large-scale studies of SARS-CoV-2 variation and specific immune responses will help define the contributions of intra-individual SARS-CoV-2 evolution to COVID-19 clinical outcomes and antiviral drug susceptibility.

sentences: Recovery of high-qualitied Genomes from a deep-inland Salt Lake Using BASALT Metagenomic binning enables the in-depth characterization of microorganisms. To improve the resolution and efficiency of metagenomic binning, BASALT (Binning Across a Series of AssembLies Toolkit), a novel binning toolkit was present in this study, which recovers, compares and optimizes metagenomic assembled genomes (MAGs) across a series of assemblies from short-read, long-read or hybrid strategies. BASALT incorporates self-designed algorithms which automates the separation of redundant bins, elongate and refine best bins and improve contiguity. Evaluation using mock communities revealed that BASALT auto-binning obtained up to 51% more number of MAGs with up to 10 times better MAG quality from microbial community at low (132 genomes) and medium (596 genomes) complexity, compared to other binners such as DASTool, VAMB and metaWRAP. Using BASALT, a case-study analysis of a Salt Lake sediment microbial community from northwest arid region of China was performed, resulting in 426 non-redundant MAGs, including 352 and 69 bacterial and archaeal MAGs which could not be assigned to any known species from GTDB (ANI < 95%), respectively. In addition, two Lokiarchaeotal MAGs that belong to superphylum Asgardarchaeota were observed from Salt Lake sediment samples. This is the first time that candidate species from phylum Lokiarchaeota was found in the arid and deep-inland environment, filling the current knowledge gap of earth microbiome. Overall, BASALT is proven to be a robust toolkit for metagenomic binning, and more importantly, expand the Tree of Life.

sentences: Genomic comparison of eight closed genomes of multidrug resistant Salmonella enterica strains isolated from broiler farms and processing plants in Trinidad and Tobago Salmonella enterica is an important foodborne pathogen worldwide. We used long and short read sequencing to close genomes of eight multidrug resistant (MDR) Salmonella enterica strains, belonging to serovars Infantis (2), Albany, Oranienburg, I 4,[5],12:i:-, Javiana, Schwarzengrund, and Kentucky from broiler chicken farms and processing plants in Trinidad and Tobago. They also belonged to seven different sequence types (STs- 32, 292, 1510, 19, 24, and 96). Seven of the strains demonstrated multi-drug resistance with the presence of at least three AMR genes. Three isolates contained the quinolone resistance gene qnrB19 in plasmids (CFSAN103840, CFSAN103854, and CFSAN103872). The extended spectrum {beta}-lactamase genes blaCTX-M-65 (CFSAN103796) and blaTEM-1 (CFSAN103852) were detected in this study. The genomes closed in this study will be useful for future source tracking and outbreak investigations in Trinidad and Tobago and worldwide.

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Nonlinear Parameter and State Estimation Approach in End-stage Kidney Disease Patients ObjectiveThere are nearly 786,000 Americans with end-stage renal disease (ESKD), of which 71% rely on life-saving hemodialysis (HD) treatment. HD care costs Medicare annually in excess of $70 billion. One primary goal of HD treatment is to remove excess fluid via ultrafiltration. Intradialytic hypotension (IDH), a significant reduction in blood volume due to an imbalance between ultrafiltration and interstitial to intravascular fluid refilling rates, has severe implications for morbidity and mortality. However, fluid and blood volume are not accessible. It has been suggested that knowledge of extracellular fluid and absolute blood volume should improve HD outcomes.

MethodsThe new estimation approach combines a validated physiological-based fluid volume model during ultrafiltration, the nonlinear least squares method with an uncertainty quantification technique, and the unscented Kalman filter.

ResultsWe develop a novel estimation approach that estimates the underlying model parameters, bounds error estimation due to model uncertainty and measurement noise, reconstructs unmeasured fluid and blood volume, and predicts personalized outcomes (hematocrit), during HD treatment. Finally, we test the performance of our approach using actual HD data.

ConclusionThe extracellular (plasma, interstitial) fluid volume, absolute blood volume, and fluid shift/refill parameters can be estimated and bounded by the Fisher information matrix lower bounds. In addition, fluid volume corrections and personalized predictions for the blood volume evolution can be achieved during ultrafiltration by fitting the underlying parameters to a patients data.

SignificanceThis novel estimation algorithm, readily available for integration in current HD machines, could improve HD treatment outcomes.

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Nanoscopical analysis reveals an orderly arrangement of the presynaptic scaffold protein Bassoon at the Golgi-apparatus 1.Bassoon is a large, 440 kDa, scaffold protein of the specialized sites mediating neurotransmitter release from presynaptic nerve terminals, called active zones. At active zones of the brain, Bassoon is arranged with its C-terminus facing the plasma membrane. In young, cultured neurons Bassoon is associated with the Golgi-apparatus, where it may assemble active zone precursors structures, but whether or not Bassoon is an extended protein at the Golgi-apparatus and whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis.

Here, we generated recombinant Bassoon constructs for expression in neurons, with tags positioned to allow for the specific detection of N- and C-terminal regions of Bassoon by single-domain antibodies, called nanobodies. Using stimulated emission depletion (STED) nanoscopy in cultured hippocampal neurons, we confirmed that recombinant Bassoon is oriented with its C-terminus towards the active zone plasma membrane at synapses. Focusing on the soma, we found that an intramolecular tag located immediately downstream of amino acid 97 of Bassoon, showed prominent colocalization with markers of the trans Golgi network, including TGN38 and syntaxin-6. In contrast, tags located immediately downstream of the C-terminal amino acid 3938 of Bassoon showed significantly less colocalization with these Golgi-markers. The intramolecular N-terminal tag was located between 48 and 69 nm away from TGN38, while C-terminal tags were located between 100 and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon, but not its N-myristoylation sequence, were required for this arrangement.

Our results indicate that at the Golgi-apparatus Bassoon is oriented with its N-terminus towards and its C-terminus away from the trans Golgi network membrane. Moreover, they suggest that Bassoon is an extended molecule at the trans Golgi network with the distance between amino acids 97 and 3938 estimated to be between 46 and 52 nm. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.

sentences: Tight junction ZO proteins maintain tissue fluidity, ensuring efficient collective cell migration Tight junctions are essential components of epithelial tissues connecting neighboring cells to provide protective barriers. Albeit their general function to seal compartments is well understood, their role in collective cell migration is largely unexplored. Here, the importance of the tight junction proteins ZO1 and ZO2 for epithelial migration is investigated employing video microscopy in conjunction with velocimetry, segmentation, cell tracking, and atomic force microscopy/spectroscopy. The results indicate that ZO proteins are necessary for fast and coherent migration. In particular, ZO1 and 2 loss (dKD) induces actomyosin remodeling away from the central cortex towards the periphery of individual cells, resulting in altered viscoelastic properties. A tug-of-war emerges between two subpopulations of cells with distinct morphological and mechanical properties: 1) smaller and highly contractile cells with an outward-bulged apical membrane, and 2) larger, flattened cells, which, due to tensile stress, display a higher proliferation rate. In response, the cell density increases, leading to crowding-induced jamming and more small cells over time. Co-cultures comprising wildtype and dKD cells display phase separation based on differences in contractility rather than differential adhesion. This study shows that ZO proteins are necessary for efficient collective cell migration by maintaining tissue fluidity and controlling proliferation.

sentences: Eurasian lynx populations in Western Europe: What prospects for the next 50 years? Persistence of viable populations may be uncertain for large carnivore species, especially for those established in human-dominated landscapes. Here, we studied the Eurasian lynx in Western Europe established in the Upper Rhine metapopulation (i.e., Jura, Vosges-Palatinian and Black Forest populations) and in the Alpine population. These populations are currently considered as endangered or critically endangered due to high anthropogenic mortality and isolation. We assessed lynx persistence over a 50-year time horizon by implementing a spatially-explicit individual-based model while accounting for road mortality and habitat selection. Forecasts showed a steady growth rapidly reaching a stabilization phase with high resident female occupancy for the Alpine and Jura populations, and a positive growth for the newly re-established Vosges-Palatinian population. Moreover, this population showed a difference in female occupancy between the northern part, where a recent reintroduction program was conducted in the Palatinate Forest, and the southern part. Only the group of individuals in the Black Forest had an irregular growth probably due to the small number of only male lynx at start of the simulation and poor connectivity to surrounding populations. Exchanges of individuals between populations were limited, the Jura population playing the role of a crossroad. Persistence of lynx in Western Europe seems likely on a large scale over the next 50 years. However, lynx persistence in the southern part of the Vosges-Palatinian population and in the Black Forest appears challenging without long-term conservation management.

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Uncertainty in denoising of MRSI using low-rank methods PurposeLow-rank denoising of MRSI data results in an apparent increase in spectral SNR. However, it is not clear if this translates to a lower uncertainty in metabolite concentrations after spectroscopic fitting. Estimation of the true uncertainty after denoising is desirable for downstream analysis in spectroscopy.

In this work the uncertainty reduction from low-rank denoising methods based on spatio-temporal separability and linear predictability in MRSI are assessed. A new method for estimating metabolite concentration uncertainty after denoising is proposed. Finally, automatic rank threshold selection methods are assessed in simulated low SNR regimes.

MethodsAssessment of denoising methods is conducted using Monte Carlo simulation of proton MRSI data, and by reproducibility of repeated in vivo acquisitions in five subjects.

ResultsIn simulated and in vivo data, spatio-temporal based denoising is shown to reduce the concentration uncertainty, but linear prediction denoising increases uncertainty. Uncertainty estimates provided by fitting algorithms after denoising consistently under-estimate actual metabolite uncertainty. However, the proposed uncertainty estimation, based on an analytical expression for entry-wise variance after denoising, is more accurate. Finally, it is shown automated rank threshold selection using Marchenko-Pastur distribution can bias the data in low SNR conditions. An alternative soft-thresholding function is proposed.

ConclusionLow-rank denoising methods based on spatio-temporal separability do reduce uncertainty in MRS(I) data. However, thorough assessment is needed as assessment by SNR measured from residual baseline noise is insufficient given the presence of non-uniform variance. It is also important to select the right rank thresholding method in low SNR cases.

sentences: Genomic sequences and RNA binding proteins predict RNA splicing kinetics in various single-cell contexts RNA splicing is a key step of gene expression in higher organisms. Accurate quantification of the two-step splicing kinetics is of high interests not only for understanding the regulatory machinery, but also for estimating the RNA velocity in single cells. However, the kinetic rates remain poorly understood due to the intrinsic low content of unspliced RNAs and its stochasticity across contexts. Here, we estimated the relative splicing efficiency across a variety of single-cell RNA-Seq data with scVelo. We further extracted three large feature sets including 92 basic genomic sequence features, 65,536 octamers and 120 RNA binding proteins features and found they are highly predictive to RNA splicing efficiency across multiple tissues on human and mouse. A set of important features have been identified with strong regulatory potentials on splicing efficiency. This predictive power brings promise to reveal the complexity of RNA processing and to enhance the estimation of single-cell RNA velocity.

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Novel method for reliably measuring spontaneous postsynaptic potentials/currents in whole-cell patch clamp recordings in the central nervous system In chemical synapses of the central nervous system (CNS) an information unit is transmitted via the presynaptic release of a neurotransmitter vesicle, a quantum, eliciting a postsynaptic membrane electric response of a certain amplitude, a quantal size. This key determinant of neural computation has not been reliably measured due to the nature of synaptic communication and technical limitations of electrophysiological recordings in the CNS. Measuring amplitudes of spontaneous postsynaptic currents (sPSCs) or potentials (sPSPs) at the cell soma potentially offers a technically straight forward way to estimate the quantal size as these miniature responses (minis) are ellicited by the spontaneous release of a single neurotransmitter vesicle. However, somatically recorded minis are massively attenuated and, for the most part, are indistinguishable from background noise fluctuations. For them to be accurately estimated, a revised method would have to isolate the noise component. As the first step in devising such a method, we developed and described a novel sPSP/sPSC detection algorithm as part of our quantal analysis software called  minis. We tested the performance of the algorithm in detecting real and simulated minis in rat cortical slice whole-cell recordings and compared it to other most commonly used similar detection algorithms in the field of the synaptic function research. This benchmarking analysis revealed superior detection performance by our algorithm. The release version of the algorithm also offers great flexibility as it can be controlled through graphical and programming interfaces making it suitable for the needs of most individual researchers studying the central synapse function.

Significance statementBeing able to measure accurately the electrical size of synapses in the brain is critical for understanding neural computations. Offering a novel algorithm for measuring properties of spontaneous synaptic signalling is an important first step in doing so. By its qualities of being transparent and objectively evaluated, this algorithm sets an important methodological standard for comparative assessment of other popular algorithms in the field of the synaptic function research. Because of its superior performance and flexibility, the algorithm also offers a technological improvement greatly useful to individual researchers in the field. Finally, its evaluation process highlighted common methodological pitfalls surrounding the spontaneous synaptic signalling research that hopefully could reduce their future impact.

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Portable low-cost macroscopic mapping system for all-optical cardiac electrophysiology SignificanceAll-optical cardiac electrophysiology enables the visualization and control of key parameters relevant to the detection of cardiac arrhythmias. Mapping such responses in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is of great interest for cardiotoxicity and personalized medicine applications.

AimWe introduce and validate a very low-cost compact mapping system for macroscopic all-optical electrophysiology in layers of hiPSC-CMs.

ApproachThe system uses oblique trans-illumination, low-cost cameras, light-emitting diodes and off-the-shelf components (total < $15,000) to capture voltage, calcium and mechanical waves under electrical or optical stimulation.

ResultsOur results corroborate the equivalency of electrical and optogenetic stimulation of hiPSC-CMs, and Vm - [Ca2+]i similarity in conduction under pacing. Green-excitable optical sensors are combinable with blue optogenetic actuators (Chanelrhodopsin2) only under very low green light (< 0.05mW/mm2). Measurements in warmer culture medium yield larger spread of action potential duration and higher conduction velocities compared to Tyrodes solution at room temperature.

ConclusionsAs multiple optical sensors and actuators are combined, our results can help handle the "spectral congestion" and avoid parameter distortion. We illustrate the utility of the system for uncovering the action of cellular uncoupling agents and show extensibility to an epi-illumination mode for future imaging of thicker native or engineered tissues.

sentences: Allosteric interactions prime androgen receptor dimerization and activation The androgen receptor (AR) is a steroid receptor and master transcription factor that governs gene expression programs required for luminal development of prostate epithelium, formation of muscle tissue and maintenance of the male phenotype. AR misregulation is a hallmark of multiple malignancies, including prostate cancer, where AR hyperactivation and expansion of its transcriptome occur in part through AR gene amplification and interaction with oncoprotein cofactors. Despite its biological importance, how ARs individual domains and its protein cofactors cooperate to bind DNA have remained elusive. Using a combination of reconstitution biochemistry and single particle cryo-electron microscopy (EM), we have isolated three conformational states of AR bound to DNA. We observe that AR forms a non-obligate dimer, with the buried dimer interface utilized by related ancestral nuclear receptors repurposed to facilitate cooperative DNA binding. We identify surfaces bridging ARs domains responsible for allosteric communication, that are compromised in partial androgen insensitivity syndrome (PAIS), and are reinforced by ARs oncoprotein cofactor, ERG, and DNA binding site motifs. Finally, we present evidence that this plastic dimer interface for transcriptional activation may have been adopted by AR at the expense of DNA binding. Our work highlights how fine-tuning of ARs cooperative interactions translate to consequences in development and disease.

sentences: L-citrulline ameliorates pathophysiology in a rat model of superimposed preeclampsia Preeclampsia, characterized by hypertension, proteinuria, and fetal growth restriction, is one of the leading causes of maternal and perinatal mortality. By far, there is no effective pharmacological therapy for preeclampsia. The present study was conducted to investigate the effects of L-citrulline supplementation in Dahl salt-sensitive rat, a model of superimposed preeclampsia. Parental DSSR were treated with L-citrulline (2.5 g/L in drinking water) from the day of mating to the end of lactation period. Blood pressure of the rats was monitored throughout pregnancy and markers of preeclampsia were assessed. Endothelial function of the pregnant DSSR was assessed by wire myograph. L-citrulline supplementation significantly reduced gestational hypertension, proteinuria, and levels of circulating soluble fms-like tyrosine kinase 1 in DSSR. L-citrulline improved maternal endothelial function by augmenting the production of nitric oxide in the aorta and improving endothelium-derived hyperpolarizing factor-mediated vasorelaxation in resistance arteries. L-citrulline supplementation improved placental insufficiency and fetal growth, which were associated with an enhancement of angiogenesis and reduction of fibrosis and senescence in the placentas. In addition, L-citrulline downregulated genes involved in the toll-like receptor 4 and nuclear factor-{kappa}B signaling pathway. In conclusion, this study shows that L-citrulline supplementation reduces gestational hypertension, improves placentation and fetal growth in a rat model of superimposed preeclampsia. L-citrulline supplementation may represent an effective and safe therapeutic strategy for preeclampsia that benefit both the mother and the fetus.

sentences: Loss of tumor suppressor WWOX enhances RAS activity in pancreatic cancer Pancreatic cancer is one of the most lethal cancers, due to late diagnoses and chemotherapy resistance. The tumor suppressor WWOX, spanning one of the most active common fragile sites in the human genome (FRA16D), is commonly altered in pancreatic cancer. However, the direct contribution of WWOX loss to pancreatic cancer development and progression is largely unknown. Here, we report that combined conditional deletion of Wwox and activation of KRasG12D in Ptf1a-CreER-expressing mice resulted in accelerated formation of precursor lesions and pancreatic carcinoma. In addition, these mice displayed enhanced MAPK and IL6/Jak/Stat3 signalling, combined with higher rates of macrophage infiltration and inflammation signature at sites of acinar to ductal metaplasia (ADM). Moreover, accumulated DNA double strand breaks were observed at ADM lesions. Finally, overexpression of WWOX in patient-derived xenografts led to reduction in KRAS MAPK activity and IL6/Jak/Stat3 signalling so diminishing their aggressiveness and in vivo tumor growth. Our data underscore the important role of WWOX in regulating RAS activity in pancreatic carcinogenesis.

sentences: Population structure and genetic connectivity of the scalloped hammerhead shark (Sphyrna lewini) across nursery grounds from the Eastern Tropical Pacific: implications for management and conservation Defining demographically independent units and understanding gene flow between them is essential for managing and conserving exploited populations. The scalloped hammerhead shark, Sphyrna lewini, is a coastal semi-oceanic species found worldwide in tropical and subtropical waters. Pregnant females give birth in shallow coastal estuarine habitats that serve as nursery grounds for neonates and small juveniles, and adults move offshore and become highly migratory. We evaluated the population structure and connectivity of S. lewini in coastal areas across the Eastern Tropical Pacific (ETP) using both sequences of the mitochondrial DNA control region (mtCR) and nuclear-encoded microsatellite loci. The mtCR defined two genetically discrete geographic groups: the Mexican Pacific and the central-southern Eastern Tropical Pacific (Guatemala, Costa Rica, Panama, and Colombia). Overall, the mtCR data showed low levels of haplotype diversity ranged from 0.000 to 0.608, while nucleotide diversity ranged from 0.000 to 0.0015. A more fine-grade population structure analysis was detected using microsatellite loci where Guatemala, Costa Rica, and Panama differed significantly. Genetic diversity analysis with nuclear markers revealed an observed heterozygosity ranging from 0.68 to 0.71 and an allelic richness from 5.89 to 7.00. Relatedness analysis revealed that individuals within nursery areas were more closely related than expected by chance, suggesting that S. lewini may exhibit reproductive philopatric behaviour within the ETP. Findings of at least two different management units, and evidence of philopatric behaviour call for intensive conservation actions for this critically endangered species in the ETP.

sentences: Modelling the future distribution of rare bryophytes in Scotland: is inclusion of habitat loss important? Species distribution models (SDMs) have been widely used to predict species ranges and their future distribution under climate change scenarios. In this study we applied Maxent, one of the most used SDMs, to project the distribution of some rare bryophyte species in Scotland in the 2050s. Most of these species are strongly linked to the blanket bog habitat, which is threatened by climate change in the near future. To assess the extent to which changes in habitat distribution leads to a different modelled distribution of the selected bryophytes, blanket bog distribution was included in the model as one of the explanatory variables for some species, and Maxent was run for three 2050s scenarios: once with the current blanket bog distribution and two other runs using the blanket bog distribution derived from two bioclimatic models (Lindsay modified and Blanket Bog Tree model) under the same climate change scenario. For seven out of nine of our studied bryophyte species, the modelled distribution in Scotland was predicted to decline, with some species retreating towards the north-west and other species almost disappearing. When the change in blanket bog distribution was also accounted for, further areas in the north/centre east of Scotland and in the south were predicted to be unfavourable for many of the species considered. Our findings suggest that when modelling species distributions, habitat distribution also needs to be considered, especially when there is a strong relationship between the species and a particular habitat.

sentences: Triku: a feature selection method based on nearest neighbors for single-cell data Feature selection is a relevant step in the analysis of single-cell RNA sequencing datasets. Triku is a feature selection method that favours genes defining the main cell populations. It does so by selecting genes expressed by groups of cells that are close in the nearest neighbor graph. Triku efficiently recovers cell populations present in artificial and biological benchmarking datasets, based on mutual information and silhouette coefficient measurements. Additionally, gene sets selected by triku are more likely to be related to relevant Gene Ontology terms, and contain fewer ribosomal and mitochondrial genes. Triku is available at https://gitlab.com/alexmascension/triku.

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Local brain-state dependency of effective connectivity: evidence from TMS-EEG BackgroundSpontaneous cortical oscillations have been shown to modulate cortical responses to transcranial magnetic stimulation (TMS). If not controlled for, they might increase variability in responses and mask meaningful changes in the signals of interest when studying the brain with TMS combined with electroencephalography (TMS-EEG). To address this challenge in future closed-loop stimulation paradigms, we need to understand how spontaneous oscillations affect TMS-evoked responses.

ObjectiveTo describe the effect of the pre-stimulus phase of cortical mu (8-13 Hz) and beta (13-30 Hz) oscillations on TMS-induced effective connectivity patterns.

MethodsWe applied TMS to the left primary motor cortex and right pre-supplementary motor area of three subjects while recording EEG. We classified trials off-line into positive- and negative-phase classes according to the mu and beta rhythms. We calculated differences in the global mean-field amplitude (GMFA) and compared the cortical spreading of the TMS-evoked activity between the two classes.

ResultsPhase had significant effects on the GMFA in 11 out of 12 datasets (3 subjects x 2 stimulation sites x 2 frequency bands). Seven of the datasets showed significant differences in the time range 15-50 ms, nine in 50-150 ms, and eight after 150 ms post-stimulus. Source estimates showed complex spatial differences between the classes in the cortical spreading of the TMS-evoked activity.

ConclusionsTMS-evoked effective connectivity appears to depend on the phase of local cortical oscillations at the stimulated site. This may be crucial for efficient design of future brain-state-dependent and closed-loop stimulation paradigms.

sentences: Bi-steric mTORC1-selective Inhibitors activate 4EBP1 reversing MYC-induced tumorigenesis and synergize with immunotherapy The MYC oncogene is causally involved in the pathogenesis of most types of human cancer but it remains therapeutically untargeted. The mTORC1 protein complex regulates cap-dependent translation through 4EBP1 and S6K and thereby, downstream MYC protein expression. However, to date, agents such as rapalogs that selectively target mTORC1 (as compared to mTORC2) fail to reactivate 4EBP1 and thus, to block MYC in vivo. In contrast, agents that nonselectively inhibit both protein complexes of the mTOR pathway, mTORC1 and mTORC2, can activate 4EBP1, but often suffer from a lack of tolerability including in vivo hepatotoxicity and immunosuppression. Here, we report the anti-tumor activity of bi-steric mTORC1-selective inhibitors, including Revolution Medicines clinical candidate RMC-5552, that potently and selectively target mTORC1 over mTORC2. In an autochthonous transgenic mouse model of MYC-amplified and MYC-driven hepatocellular carcinoma (HCC), representative bi-steric mTORC1-selective inhibitors suppress translation initiation via activation of 4EBP1, thereby suppressing MYC protein expression and blocking tumor growth. Furthermore, in human HCC samples, the low levels of 4EBP1 and MYC is correlated with immune reactivation. Immunohistochemistry, CIBERSORT, and CODEX reveal that selective mTORC1 inhibition results in activation of both CD4+ T cell- and NKp46+ NK cell-mediated immune surveillance. Moreover, bi-steric mTORC1-selective inhibitors synergize with -PD-1 to induce sustained tumor regression, with immune cell degranulation and release of perforins and granzyme B. These agents also exhibit anti-tumor activity in human patient-derived xenografts of HCC, colorectal cancer, head and neck cancer, and ovarian cancer harboring genomic amplifications in MYC. We infer that selective mTORC1 inhibition is a potential therapeutic strategy to drive effective MYC inactivation in cancer, and the consequent restoration of immune surveillance against neoplasia.

sentences: The high-throughput gene prediction of more than 1,700 eukaryote genomes using the software package EukMetaSanity Gene prediction and annotation for eukaryotic genomes is challenging with large data demands and complex computational requirements. For most eukaryotes, genomes are recovered from specific target taxa. However, it is now feasible to reconstruct or sequence hundreds of metagenome-assembled genomes (MAGs) or single-amplified genomes directly from the environment. To meet this forth-coming wave of eukaryotic genome generation, we introduce EukMetaSanity, which combines state-of-the-art tools into three pipelines that have been specifically designed for extensive parallelization on high-performance computing infrastructure. EukMetaSanity performs an automated taxonomy search against a protein database of 1,482 species to identify phylogenetically compatible proteins to be used in downstream gene prediction. We present the results for intron, exon, and gene locus prediction for 112 genomes collected from NCBI, including fungi, plants, and animals, along with 1,669 MAGs and demonstrate that EukMetaSanity can provide reliable preliminary gene predictions for a single target taxon or at scale for hundreds of MAGs. EukMetaSanity is freely available at https://github.com/cjneely10/EukMetaSanity.

sentences: Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis As the principal internalization mechanism in mammalian cells, clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of disrupted cholesterol synthesis, sterol specificity, mechanisms involved, and relevance to disease pathology. Using genome-edited cell lines, we demonstrate that inhibition of post-squalene cholesterol biosynthesis as observed in inborn errors of cholesterol metabolism, results in striking immobilization of CME and impaired transferrin uptake. Imaging of membrane bending dynamics and CME pit ultrastructure revealed prolonged clathrin pit lifetimes and accumulation of shallow clathrin-coated structures that scaled with diminishing sterol abundance. Moreover, fibroblasts derived from Smith-Lemli-Opitz syndrome subjects displayed reduced CME function. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest reduced CME contributes to cellular phenotypes observed within disorders of cholesterol metabolism.

sentences: Interferon-γ Exposure of Human iPSC-derived Neurons Alters Major Histocompatibility Complex I and Synapsin I Protein Expression Human epidemiological data links maternal immune activation during gestation with increased risk for neurodevelopmental disorders including schizophrenia. Animal models of maternal immune activation (MIA) provide causal evidence for this association and strongly suggest that inflammatory cytokines act is a critical link between maternal infection and aberrant offspring brain and behavior development. This includes evidence for reduced synapse formation, consistent with post-mortem and in vivo evidence of reduced synaptic density in schizophrenia. However, to what extent specific cytokines are necessary and sufficient for these effects remains unclear. Using a human cellular model, we recently demonstrated that acute exposure to interferon-{square} (IFN{square}) recapitulates molecular and cellular phenotypes associated with neurodevelopmental disorders. Here, we extend this work to test whether IFN{square} affects synapse formation in an induced neuron model that generates forebrain glutamatergic neurons. Using immunocytochemistry and quantitative PCR, we demonstrate that acute IFN{square} exposure results in significantly increased MHCI expression at the mRNA and protein level. Furthermore, acute IFN{square} exposure decreases synapsin I protein in neurons but does not affect synaptic gene mRNA levels. Interestingly, complement component 4A (C4A) mRNA is also significantly increased following acute IFN{square} exposure. This study builds on our previous work by showing that IFN{square}-mediated disruption of relevant synaptic proteins can occur at early stages of synapse formation, potentially contributing to neurodevelopmental disorder phenotypes such as schizophrenia.

sentences: Immediate and sustained effects of cobalt and zinc-containing pigments on macrophages Pigments are among the oldest nanoparticulate products known to mankind, and their use in tattoos is also very old. Nowadays, 25% American people aged 18 to 50 are tattooed, which poses the question of the delayed effects of tattoos. In this article, we investigated three cobalt (Pigment Violet 14 (purple color)) or cobalt alloy pigments (Pigment Blue 28 (blue color), Pigment Green 14 (green color)), and one zinc pigment (Pigment White 4 (white color)) which constitute a wide range of colors found in tattoos. These pigments contain microparticles and a significant proportion of nanoparticles (in either aggregate or free form). Because of the key role of macrophages in the scavenging of particulate materials, we tested the effects of cobalt- and zinc-based pigments on the J774A.1 macrophage cell line. In order to detect delayed effects, we compared two exposure schemes: acute exposure for 24 hours and an exposure for 24 hours followed by a 3-day post-exposure recovery period. The conjunction of these two schemes allowed for the investigation of the delayed or sustained effects of pigments. All pigments induced functional effects on macrophages, most of which were pigment-dependent. For example, Pigment Green 19, Pigment Blue 28, and Pigment White 4 showed a delayed alteration of the phagocytic capacity of cells. Moreover, all the pigments tested induced a slight but significant increase in TNF secretion. This effect, however, was transitory. Conversely, only Pigment Blue 28 induced a short and sustained increase in interleukin 6. Results showed that in response to bacterial stimuli (LPS), the secretion of tumor necrosis factor and interleukin 6 declined after recovery exposure with pigments. The pigments also induced persisting changes in some important macrophage membrane markers such as CD11b, an integrin contributing to cell adhesion and immunological tolerance. In conclusion, the pigments induced functional dis-orders in macrophages, which, in some cases, persist long after exposure, even at non-toxic doses.

sentences: Profiling of the drug resistance of thousands of Src tyrosine kinase mutants uncovers a regulatory network that couples autoinhibition to the dynamics of the catalytic domain Protein kinase inhibitors are effective cancer therapies, but acquired resistance often limits clinical efficacy. Despite the cataloguing of numerous resistance mutations with model studies and in the clinic, we still lack a comprehensive understanding of kinase inhibitor resistance. Here, we measured the resistance of thousands of Src tyrosine kinase mutants to a panel of ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Srcs catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Srcs phosphotransferase activity were prone to the development of resistance. Unexpectedly, a resistance-prone cluster of residues that are on the top face of the N-terminal lobe of the catalytic domain contributes to Src autoinhibition by reducing the dynamics of the catalytic domain, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how comprehensive profiling of drug resistance can be used to understand potential resistance pathways and uncover new mechanisms of kinase regulation.

sentences: Mitochondrial phosphoproteomes are functionally specialized across tissues Mitochondria are essential organelles involved in critical biological processes such as energy metabolism and cell survival. Their dysfunction is linked to numerous human pathologies that often manifest in a tissue-specific manner. Accordingly, mitochondrial fitness depends on versatile proteomes specialized to meet diverse tissue-specific requirements. Furthermore, increasing evidence suggests that phosphorylation may also play an important role in regulating tissue-specific mitochondrial functions and pathophysiology. We hypothesized that recent advances in mass spectrometry (MS)-based proteomics would now enable in-depth measurement to quantitatively profile mitochondrial proteomes along with their matching phosphoproteomes across tissues. We isolated mitochondria from mouse heart, skeletal muscle, brown adipose tissue, kidney, liver, brain, and spleen by differential centrifugation followed by separation on Percoll gradients and high-resolution MS analysis of the proteomes and phosphoproteomes. This in-depth map substantially quantifies known and predicted mitochondrial proteins and provides a resource of core and tissue modulated mitochondrial proteins (mitophos.de). We also uncover tissue-specific repertoires of dozens of kinases and phosphatases. Predicting kinase substrate associations for different mitochondrial compartments indicates tissue-specific regulation at the phosphoproteome level. Illustrating the functional value of our resource, we reproduce mitochondrial phosphorylation events on DRP1 responsible for its mitochondrial recruitment and fission initiation and describe phosphorylation clusters on MIGA2 linked to mitochondrial fusion.

sentences: An intestinally secreted host factor limits bacterial colonization but promotes microsporidia invasion of C. elegans Microsporidia are ubiquitous obligate intracellular pathogens of animals. These parasites often infect hosts through an oral route, but little is known about the function of host intestinal proteins that facilitate microsporidia invasion. To identify such factors necessary for infection by Nematocida parisii, a natural microsporidian pathogen of Caenorhabditis elegans, we performed a forward genetic screen to identify mutant animals that have a Fitness Advantage with Nematocida (Fawn). We isolated four fawn mutants that are resistant to Nematocida infection and contain mutations in T14E8.4, which we renamed aaim-1 (Antibacterial and Aids invasion by Microsporidia). Expression of AAIM-1 in the intestine of aaim-1 animals restores N. parisii infectivity and this rescue of infectivity is dependent upon AAIM-1 secretion. N. parisii spores in aaim-1 animals are improperly oriented in the intestinal lumen, leading to reduced levels of parasite invasion. Conversely, aaim-1 mutants display both increased colonization and susceptibility to the bacterial pathogen Pseudomonas aeruginosa and overexpression of AAIM-1 reduces P. aeruginosa colonization. Competitive fitness assays show that aaim-1 mutants are favoured in the presence of N. parisii but disadvantaged on P. aeruginosa compared to wild type animals. Together, this work demonstrates how microsporidia exploits a secreted protein to promote host invasion. Our results also suggest evolutionary trade-offs may exist to optimizing host defense against multiple classes of pathogens.

sentences: Microglia control glutamatergic synapses in the adult mouse hippocampus Microglial cells are active players in regulating synaptic development and plasticity in the brain. However, how these cells influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological depletion of microglia, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features accompanied by higher levels of plasticity. In addition, microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. Remarkably, microglial repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in synaptic organization and activity of glutamatergic synapses.

sentences:
The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses Th17 cells protect mucosal barriers, but their aberrant activity can cause autoimmunity. Molecular networks dictating human Th17 function are largely unexplored, and this hinders disease-studies. Here, we investigated the roles of the AP-1 factors, FOSL1 and FOSL2, in inducing human Th17 responses. Transient knockdown and over-expression strategies found the two proteins to inhibit Th17-cell identity, while revealing a distinct cooperativity between their functions. Strikingly, FOSL1 plays different roles in human and mouse and FOSL-mediated Th17 regulation is opposed by the AP-1 factor, BATF. Genome-wide occupancy analysis demonstrated the co-localization of FOSL1, FOSL2 and BATF in the vicinity of key Th17 genes. The functional interplay among these factors is possibly governed by sharing interactions with a common set of lineage-associated proteins. We further discovered that the genomic binding sites of these factors harbour a large number of disease-linked SNPs, many of which alter the ability of a given factor to bind DNA. Our findings thus provide crucial insights into the transcriptional regulation of human Th17 function and associated pathologies.

ONE SENTENCE SUMMARYFOSL1- and FOSL2-mediated transcription during early human Th17 differentiation

sentences:
Quantifying Structural Diversity of CNG Trinucleotide Repeats Using Diagrammatic Algorithms Trinucleotide repeat expansion disorders (TREDs) exhibit complex mechanisms of pathogenesis, some of which have been attributed to RNA transcripts of overexpanded CNG repeats, resulting in possibly a gain-of-function. In this paper, we aim to probe the structures of these expanded transcript by analyzing the structural diversity of their conformational ensembles. We used graphs to catalog the structures of an NG-(CNG)16-CN and NG-(CNG)50-CN oligomer and grouped them into sub-ensembles based on their characters and calculated the structural diversity and thermodynamic stability for these ensembles using a previously described graph factorization scheme. Our findings show that the generally assumed structure for CNG repeats--a series of canonical helices connected by two-way junctions and capped with a hairpin loop--may not be the most thermodynamically favorable, and the ensembles are characterized by largely open and less structured conformations. Furthermore, a length-dependence is observed for the behavior of the ensembles diversity as higher-order diagrams are included, suggesting that further studies of CNG repeats are needed at the length scale of TREDs onset to properly understand their structural diversity and how this might relate to their functions.

STATEMENT OF SIGNIFICANCETrinucleotide repeats are DNA satellites that are prone to mutations in the human genome. A family of diverse disorders are associated with an overexpansion of CNG repeats occurring in noncoding regions, and the RNA transcripts of the expanded regions have been implicated as the origin of toxicity. Our understanding of the structures of these expanded RNA transcripts is based on sequences that have limited lengths compared to the scale of the expanded transcripts found in patients. In this paper, we introduce a theoretical method aimed at analyzing the structure and conformational diversity of CNG repeats, which has the potential of overcoming the current length limitations in the studies of trinucleotide repeat sequences.

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Sex and age differences in cognitive bias and neural activation in response to cognitive bias Cognitive symptoms of depression, including negative cognitive bias, are more severe in women than in men. Current treatments to reduce negative cognitive bias are not effective and sex differences in the neural activity underlying cognitive bias may play a role. Here we examined sex and age differences in cognitive bias and functional connectivity in a novel paradigm. Male and female rats underwent an 18-day cognitive bias procedure, in which they learned to discriminate between two contexts (shock paired context A, no-shock paired context B), during either adolescence (postnatal day (PD 40)), young adulthood (PD 100), or middle-age (PD 210). Cognitive bias was measured as freezing behaviour in response to an ambiguous context (context C), with freezing levels akin to the shock paired context coded as negative bias. All animals learned to discriminate between the two contexts, regardless of sex or age. However, adults (young adults, middle-aged) displayed a greater negative cognitive bias compared to adolescents, and middle-aged males had a greater negative cognitive bias than middle-aged females. Females had greater neural activation of the nucleus accumbens, amygdala, and hippocampal regions to the ambiguous context compared to males, and young rats (adolescent, young adults) had greater neural activation in these regions compared to middle-aged rats. Functional connectivity between regions involved in cognitive bias differed by age and sex, and only adult males had negative correlations between the frontal regions and hippocampal regions. These findings highlight the importance of examining age and sex when investigating the underpinnings of negative cognitive bias and lay the groundwork for determining what age- and sex-specific regions to target in future cognitive bias studies.

HighlightsO_LIMiddle-aged males had a greater negative cognitive bias than middle-aged females
C_LIO_LIAdult rats displayed a greater negative cognitive bias compared to adolescents
C_LIO_LIGreater neural activity in females than males in limbic and reward regions
C_LIO_LIGreater role of the frontal cortex activation in the cognitive bias of adults
C_LIO_LIFunctional connectivity in response to cognitive bias differed by age and sex
C_LI

sentences:
An ensemble of toxic channel types underlies the severity of the de novo variant G375R of the human BK channel De novo mutations play a prominent role in neurodevelopmental diseases including autism, schizophrenia, and intellectual disability. Many de novo mutations are dominant and so severe that the afflicted individuals do not reproduce, so the mutations are not passed into the general population. For multimeric proteins, such severity may result from a dominant-negative effect where mutant subunits assemble with WT to produce channels with adverse properties. Here we study the de novo variant G375R heterozygous with the WT allele for the large conductance voltage- and Ca2+-activated potassium (BK channel, Slo1. This variant has been reported to produce devastating neurodevelopmental disorders in three unrelated children. If mutant and WT subunits assemble randomly to form tetrameric BK channels, then ~6% of the assembled channels would be wild type (WT, ~88% would be heteromeric incorporating from 1-3 mutant subunits per channel, and ~6% would be homomeric mutant channels consisting of four mutant subunits. To test this hypothesis, we analyzed the biophysical properties of the single BK channels in the ensemble of channels expressed following a 1:1 injection of mutant and WT cRNA into oocytes. We found ~3% were WT channels, ~85% were heteromeric channels, and ~12% were homomeric mutant channels. All of the heteromeric channels as well as the homomeric mutant channels displayed toxic properties, indicating a dominant negative effect of the mutant subunits. The toxic channels were open at inappropriate negative voltages, even in the absence of Ca2+, which would lead to altered cellular function and decreased neuronal excitability.

SignificanceWe examined the molecular basis for a severe channelopathy associated with developmental and neurological disorders for a de novo variant of the Ca2+-activated potassium (BK) channel, Slo1. To identify the types of BK channels underlying the pathogenicity of the variant, we recorded from BK channels expressed for the G375R mutation heterozygous with the WT allele. We found five different functional types of BK channels, four of which displayed a gain of function toxicity by shifting channel opening to more negative voltages in proportion to the number of mutant subunits per channel and WT. The aberrant channels would disrupt cellular function and the normal electrical activity of neurons, providing a molecular basis for the severity of the phenotype.

sentences: Nutrient availability shifts the biosynthetic potential of soil-derived microbial communities Secondary metabolites produced by microorganisms are the main source of antimicrobials other pharmaceutical drugs. Soil microbes have been the primary discovery source for these secondary metabolites, often producing complex organic compounds with specific biological activities. Research suggests that secondary metabolism broadly shapes microbial ecological interactions, but little is known about the factors that shape the abundance, distribution, and diversity of biosynthetic gene clusters in the context of microbial communities. In this study, we investigate the role of nutrient availability on the abundance of biosynthetic gene clusters in soil-derived microbial consortia. We found that soil microbial consortia enriched in medium with 150 mg/L of glucose and 200 mg/L of trehalose (here defined as high sugar) had more biosynthetic gene cluster and higher inhibitory activity than soil microbial consortia enriched in medium with 15 mg/L of glucose + 20 mg/L of trehalose (here defined as low sugar). Our results demonstrate that laboratory microbial communities are a promising tool to study ecology of specialized metabolites.

sentences:
Does the posteromedial cortex play a primary role for the capacity for consciousness in rats? It remains unclear how specific cortical regions contribute to the brains overall capacity for consciousness. Clarifying this could help distinguish between theories of consciousness. Here, we investigate the association between markers of regionally specific (de)activation and the brains overall capacity for consciousness.

We recorded electroencephalographic (EEG) responses to cortical electrical stimulation in 6 rats, and computed Perturbational Complexity Index state-transition (PCIST), which has been extensively validated as an index of the capacity for consciousness in humans. We also estimated the balance between activation and inhibition of specific cortical areas with the ratio between high and low frequency power (HF/LF) from spontaneous EEG activity at each electrode. We repeated these measurements during wakefulness, and under the influence of ketamine anaesthesia at two doses: the minimal dose needed to induce behavioural unresponsiveness and twice this dose.

We found that PCIST was only slightly reduced from wakefulness to light ketamine anaesthesia, but dropped significantly down with deeper anaesthesia. The high-dose effect was selectively associated with reduced HF/LF ratio in the posteromedial cortex, which strongly correlated with PCIST. Conversely, behavioural unresponsiveness induced by light ketamine anaesthesia, was associated with similar spectral changes in frontal, but not posterior cortical regions.

These findings seem to support the claim that the posteromedial cortex may play a primary role for the capacity for consciousness. Such region-specific associations between cortical activation and the overall capacity for consciousness must be accounted for by theories of consciousness.

sentences: Transcriptional analysis of peripheral memory T cells reveals Parkinson's disease-specific gene signatures Parkinsons disease (PD) is a multi-stage neurodegenerative disorder with largely unknown etiology. Recent findings have identified PD-associated autoimmune features including roles for T cells. To further characterize the role of T cells in PD, we performed RNA sequencing on PBMC and peripheral CD4 and CD8 memory T cell subsets derived from PD patients and age-matched healthy controls. When the groups were stratified by their T cell responsiveness to alpha-synuclein (-syn) as a proxy for ongoing inflammatory autoimmune response, the study revealed a broad differential gene expression profile in memory T cell subsets and a specific PD associated gene signature. We identified a significant enrichment of transcriptomic signatures previously associated with PD, including for oxidative stress, phosphorylation, autophagy of mitochondria, cholesterol metabolism and inflammation, and the chemokine signaling proteins CX3CR1, CCR5 and CCR1. In addition, we identified genes in these peripheral cells that have previously been shown to be involved in PD pathogenesis and expressed in neurons, such as LRRK2, LAMP3, and aquaporin. Together, these findings suggest that features of circulating T cells with -syn-specific responses in PD patients provide insights into the interactive processes that occur during PD pathogenesis and suggest potential intervention targets.

sentences:
Prediction of RNA-protein interactions using a nucleotide language model MotivationThe accumulation of sequencing data has enabled researchers to predict the interactions between RNA sequences and RNA-binding proteins (RBPs) using novel machine learning techniques. However, existing models are often difficult to interpret and require additional information to sequences. Bidirectional encoder representations from Transformer (BERT) is a language-based deep learning model that is highly interpretable. Therefore, a model based on BERT architecture can potentially overcome such limitations.

ResultsHere, we propose BERT-RBP as a model to predict RNA-RBP interactions by adapting the BERT architecture pre-trained on a human reference genome. Our model outperformed state-of-the-art prediction models using the eCLIP-seq data of 154 RBPs. The detailed analysis further revealed that BERT-RBP could recognize both the transcript region type and RNA secondary structure only from sequence information. Overall, the results provide insights into the fine-tuning mechanism of BERT in biological contexts and provide evidence of the applicability of the model to other RNA-related problems.

AvailabilityPython source codes are freely available at https://github.com/kkyamada/bert-rbp.

Contactmhamada@waseda.jp

sentences: DDX3X and DDX3Y are redundant in protein synthesis DDX3 is a DEAD-box RNA helicase that regulates translation and is encoded by the X- and Y-linked paralogs DDX3X and DDX3Y. While DDX3X is ubiquitously expressed in human tissues and essential for viability, DDX3Y is male-specific and shows lower and more variable expression than DDX3X in somatic tissues. Heterozygous genetic lesions in DDX3X mediate a class of developmental disorders called DDX3X syndrome, while loss of DDX3Y is implicated in male infertility. One possible explanation for female-bias in DDX3X syndrome is that DDX3Y encodes a polypeptide with different biochemical activity. In this study, we use ribosome profiling and in vitro translation to demonstrate that the X- and Y-linked paralogs of DDX3 play functionally redundant roles in translation. We find that transcripts that are sensitive to DDX3X depletion or mutation are rescued by complementation with DDX3Y. Our data indicate that DDX3X and DDX3Y proteins can functionally complement each other in human cells. DDX3Y is not expressed in a large fraction of the central nervous system. These findings suggest that expression differences, not differences in paralog-dependent protein synthesis, underlie the sex-bias of DDX3X-associated diseases.

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Feed efficiency of lactating Holstein cows is less reproducible when changing dietary starch and fibre concentrations than within diet over subsequent lactation stages BackgroundImproving feed efficiency has become a common target for dairy farmers to meet the requirement of producing more milk with fewer resources. To improve feed efficiency, a prerequisite is to ensure that the cows identified as most or least efficient will remain as such, independently of diet composition. Therefore, the current research analysed the ability of lactating dairy cows to maintain their feed efficiency while changing the energy density of the diet by changing its concentration in starch and fibre. A total of 60 lactating Holstein cows, including 33 primiparous cows, were first fed a high starch diet-low fibre (diet S+F-), then switched over to a low starch diet-high fibre (diet S-F+). To know if diet affect feed efficiency, we compared the ability of feed efficiency to be maintained within a diet over subsequent lactation stages, known as repeatability of feed efficiency, with its ability to be maintained across diets, known as reproducibility of feed efficiency. To do so we used two indicators: the estimation of the error of repeatability/reproducibility, which is commonly used in metrology, and the coefficient of correlation of concordance (CCC), which is used in biology. The effect of diet change could also lead to a change in cows sorting behaviour which could potentially affect feed efficiency if for example the most efficient cows select more concentrate than the least efficient. We therefore analysed the relationship between the differences in individual feed refusals composition and the differences in feed efficiency. To do so, the composition of each feed refusal was described with its near infra-red (NIR) spectroscopy and was performed on each individual feed ingredient, diet and refusals and used as composition variable. The variability of the NIR spectra of the refusals was described with its principal components thanks to a principal component analysis (PCA). The Pearson correlation was estimated to check the relationship between feed efficiency and refusals composition, i.e. sorting behaviour.

ResultsThe error of reproducibility of feed efficiency across diets was 2.95 MJ/d. This error was significantly larger than the errors of repeatability estimated within diet, which were 2.01 MJ/d within diet S-F+ and 2.40 MJ/d within diet S+F-. The CCC was 0.64 between feed efficiency estimated within diet S+F- and feed efficiency estimated within diet S-F+. This CCC was smaller than the one observed for feed efficiency estimated within diet between two subsequent lactation stages (CCC = 0.72 within diet S+F- and 0.85 within diet S-F+). Feed efficiency was poorly correlated to the first two principal components, which explained 90% of the total variability of the NIR spectra of the individual refusals. This suggests that feed sorting behaviour did not explain differences in feed efficiency.

ConclusionsFeed efficiency was significantly less reproducible across diets than repeatable within the same diet over subsequent lactation stages, but cows ranking for feed efficiency was not significantly affected by diet change. This loss in repeatability across diets could be due to a more pronounced feed sorting subsequent to the change in diet composition. However, the differences in sorting behaviour between cows were not associated to feed efficiency differences in this trial neither with the S+F- diet nor with the S-F+ diet. Those results have to be confirmed on diets having different forage to concentrate ratios to ensure that the least and most efficient cows will not change.

sentences:
Recovery of small plasmid sequences via Oxford Nanopore sequencing Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (<20 kbp) were underrepresented in ONT ligation read sets (by a mean factor of [~]4) but were not underrepresented in ONT rapid read sets. This effect correlated with plasmid size, with the smallest plasmids being the most underrepresented in ONT ligation read sets. We also found lower rates of chimeric reads in the rapid read sets relative to ligation read sets. These results show that when small plasmid recovery is important, ONT rapid library preparations are preferable to ligation-based protocols.

Impact statementResearchers who use Oxford Nanopore Technologies (ONT) platforms to sequence bacterial genomes can currently choose from two library preparation methods. The first is a ligation-based approach, which uses ligase to attach sequencing adapters to the ends of DNA molecules. The second is a rapid approach, which uses a transposase enzyme to cleave DNA and attach adapters in a single step. There are advantages to each preparation, for example ligation can produce better yields but rapid is a simpler procedure. Our study reveals another advantage of rapid preparations: they are more effective at sequencing small plasmids. We show that sequencing of ligation-based libraries yields fewer reads derived from small plasmids, making such plasmids harder to detect in bacterial genomes. Since small plasmids can contain clinically relevant genes, including antimicrobial resistance (AMR) or virulence determinants, their exclusion could lead to unreliable conclusions that have serious consequences for AMR surveillance and prediction. We therefore recommend that researchers performing ONT-only sequencing of bacterial genomes should consider using rapid preparations whenever small plasmid recovery is important.

Data summarySupplementary figures, tables, data and code can be found at: github.com/rrwick/Small-plasmid-Nanopore

sentences: Mixology: a tool for calculating required masses and volumes for laboratory solutions We have created a tool to calculate the amount of each ingredient required to make up a custom solution with defined concentrations. It can convert between many kinds of volumetric, mass and concentration units. This includes the ability to convert between molarities and mass-based concentrations, using molecular masses retrieved from the ChEBI database. Mixology can be accessed at http://mixology.science.

sentences: Comprehensive assessment of functional effects of commonly used sweeteners on ex vivo human gut microbiome The gut microbiome composition and function are associated with health and diseases. Sweeteners are widely used food additives, although many studies using animal models have linked sweetener consumption to gut microbial changes and health issues. Whether sweeteners directly change the human gut microbiome functionality remains largely unknown. In this study, we systematically investigated the responses of five human gut microbiomes to 21 common sweeteners, using an approach combining high-throughput ex vivo microbiome culturing and metaproteomics to quantify functional changes in different taxa. Hierarchical clustering based on metaproteomic responses of individual microbiomes resulted in two clusters. The first cluster was composed of non-caloric artificial sweeteners (NAS) and two sugar alcohols with shorter carbon backbones (4-5 carbon atoms), and the second cluster was composed of sugar alcohols with longer carbon backbones. The metaproteomic functional responses of the second cluster were similar to the prebiotic fructooligosaccharides and kestose, indicating that these sugar alcohol-type sweeteners have potential prebiotic functions. This study provides a comprehensive evaluation of the direct effects of commonly used sweeteners on the functions of the human gut microbiome using a functional metaproteomics approach, improving our understanding of the roles of sweeteners on microbiome-associated human health and disease issues.

sentences: Sensory convergence in the world s largest cavefish diversification: patterns of neuromast evolution, distribution and associated behaviour in Sinocyclocheilus The genus Sinocyclocheilus represents the largest freshwater cavefish genus in the world. This emerging model system is endemic to the southern Chinese karstic landscape, and demonstrates multiple adaptations for caves (troglomorphism), with eye-degeneration being the most pronounced. The less-apparent lateral line system, which is often expanded in cave-dwellers, has been studied in other cavefish systems, but never in the context of this diversification. Here we investigated the distribution and evolution of cephalic neuromasts in 26 Sinocyclocheilus species. We used live-staining and behavioural assays, and interpreted results in a phylogenetic context. We show that asymmetry in neuromast features and the rate of evolution is greater in cave-adapted species. Ancestral state reconstructions show that most Sinocyclocheilus are right-biased with some scatter, and show convergence of neuromast phenotypes. There is substantial variation in cephalic neuromast distribution patterns between and (to a lesser extent) within species. Behavioural assays show blind species have a distinctive wall-following behaviour. We explain these patterns in the context of the deep evolutionary history associated with this karstic region, other organismal traits, and habitat occupation of these remarkable diversifications of fishes. Interestingly, some of these neuromast patterns and behaviour show convergence with other phylogenetically distant cavefish systems.

sentences: Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution Actively maintained close appositions, or contact sites, between organelle membranes, enable the efficient transfer of biomolecules between the various cellular compartments. Several such sites have been described together with their tethering machinery. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize the proteome of contact sites and support the discovery of new tethers and functional molecules, we established a high throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, two of which have never been studied before, on the background of 1165 strains expressing a mCherry-tagged yeast protein that have a cellular punctate distribution (a hallmark of contact sites). By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified one set of hits as previously unrecognized homologs to Vps13 and Atg2. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell.

sentences: Remodeled Connexin 43 hemichannels alter cardiac excitability and promote arrhythmias Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of a dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the {beta}-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pre-treatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability and greater plasma membrane depolarization, which subsequently leads to triggered activity. These cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac stress-induced arrhythmogenicity.

sentences: The role of temporal coherence and temporal stability in the build-up of auditory grouping The cochlea decomposes sounds into separate frequency channels, from which the auditory brain must reconstruct the auditory scene. To do this the auditory system must make decisions about which frequency information should be grouped together, and which should remain distinct. Two key cues for grouping are temporal coherence, resulting from coherent changes in power across frequency, and temporal predictability, resulting from regular or predictable changes over time. To test how these cues contribute to the construction of a sound scene we present listeners with a range of precursor sounds, which act to prime the auditory system by providing information about each sounds structure, followed by a fixed masker in which participants were required to detect the presence of an embedded tone. By manipulating temporal coherence and/or temporal predictability in the precursor we assess how prior sound exposure influences subsequent auditory grouping. In Experiment 1, we measure the contribution of temporal predictability by presenting temporally regular or jittered precursors, and temporal coherence by using either narrow or broadband sounds, demonstrating that both independently contribute to masking/unmasking. In Experiment 2, we measure the relative impact of temporal coherence and temporal predictability and ask whether the influence of each in the precursor signifies an enhancement or disruption of unmasking. We observed that interfering precursors produced the largest changes to thresholds.

sentences: Profiling of human neural crest chemoattractant activity as replacement of fetal bovine serum for in vitro chemotaxis assays Fetal bovine serum (FBS) is the only known stimulus for migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis assays to be incorporated in a test battery for reproductive and developmental toxicity. We confirmed here in an optimized transwell assay that FBS triggers directed migration along a concentration gradient. The responsible factor was found to be a protein in the 30-100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chemotactic for NCCs in animal models (e.g. VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined, whether human cells would produce any factor able to trigger NCC migration in a broad screening approach. We found that HepG2 hepatoma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease- sensitivity suggested also a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants.

sentences: Electrophysiological markers of fairness and selfishness revealed by a combination of Dictator and Ultimatum Games AO_SCPLOWBSTRACTC_SCPLOWEvent Related Potentials (ERPs) were recorded from 39 participants who played the role of Allocators in a Dictator Game (DG) and Responders in an Ultimatum Game (UG). Most participants expressed very low levels of altruistic decision making, and two homogeneous groups could be identified, one formed by fair (N = 10) individuals and another by selfish (N = 8) individuals. At fronto-central cortical sites, the ERP early negativity (N1) was reduced in selfish participants with a latency about 10 ms earlier than in fair participants. In fair DG players, the features of the subsequent positive wave P2 suggested that more cognitive resources were required when they allocated the least gains to the other party. P2 latency and amplitude in the selfish group supported the hypothesis that these participants tended to maximize their profit, as expected by a rational Homo economicus. During UG, we observed that a medial frontal negativity (MFN) occurred earlier and with greater amplitude when selfish participants rejected less favorable endowment shares. In this case, all players received zero payoffs, which showed that MFN in selfish participants was associated with a spiteful punishment. At posterior-parietal sites we found that the greater the selfishness, the greater the amplitude of the late positive component (LPC). Our results bring new evidence to the existence of specific somatic markers associated with the activation of distinct cerebral circuits by the evaluation of fair and unfair proposals in participants characterized by different expressions of perceived fairness, thus suggesting that particular brain dynamics could be associated with moral decisions.

sentences: Viral tolerance in Aedes aegypti relies on the negative cooperativity between Toll5A and the cytokine ligand Spz1C A. aegypti has evolved to become an efficient vector of Dengue viruses among other arboviruses despite Toll-regulated infection levels. Interestingly, both Toll and its ligand Spatzle (Spz) have undergone gene duplication in A. aegypti raising the possibility of neofunctionalization and mutualism to develop between arboviruses and mosquitoes. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A-Spz1C complexes that display transient but specific interactions. Binding of the first ligand alters receptor-receptor interactions and promotes asymmetric contacts in the vicinity of the Z-loop in Toll5A. This conformation then restricts binding of a second ligand, while bridging the C termini that promote signalling. In contrast, increased receptor concentrations promote inactivating head-to-head receptor assemblies. We also found that Spz1C differs from orthologous and paralogous cytokines in their transcriptional responses upon A. aegypti Aag2 cell stimulation. Interestingly, Spz1C uniquely controls genes involved in innate immunity, lipid metabolism and tissue regeneration. Given the remarkable DENV-induced expression patterns of these proteins, our data rationalises how Spz1C upregulation might promote innate immunity in the midgut, and Toll5A upregulation, viral tolerance in the salivary glands.

sentences: Simulating lesion-dependent functional recovery mechanisms Functional recovery after brain damage varies widely and depends on many factors, including lesion site and extent. When a neuronal system is damaged, recovery may occur by engaging residual (e.g., perilesional) components. When damage is extensive, recovery depends on the availability of other intact neural structures that can reproduce the same functional output (i.e., degeneracy). A systems response to damage may occur rapidly, require learning or both. Here, we simulate functional recovery from four different types of lesions, using a generative model of word repetition that comprised a default premorbid system and a less used alternative system. The synthetic lesions (i) completely disengaged the premorbid system, leaving the alternative system intact, (ii) partially damaged both premorbid and alternative systems, and (iii) limited the experience-dependent plasticity of both. The results, across 1000 trials, demonstrate that (i) a complete disconnection of the premorbid system naturally invoked the engagement of the other, (ii) incomplete damage to both systems had a much more devastating long-term effect on model performance and (iii) the effect of reducing learning capacity within each system. These findings contribute to formal frameworks for interpreting the effect of different types of lesions.

sentences: happi: a Hierarchical Approach to Pangenomics Inference Recovering metagenome-assembled genomes (MAGs) from shotgun sequencing data is an increasingly common task in microbiome studies, as MAGs provide deeper insight into the functional potential of both culturable and non-culturable microorganisms. However, metagenome-assembled genomes vary in quality, and may contain omissions and contamination. These errors present challenges for detecting genes and comparing gene enrichment across sample types. To address this, we propose happi, an approach to testing hypotheses about gene enrichment that accounts for genome quality. We illustrate the advantages of happi over existing approaches using published Saccharibacteria MAGs and via simulation.

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Structure of the Shaker Kv channel and mechanism of slow C-type inactivation Voltage-activated potassium (Kv) channels open upon membrane depolarization and proceed to spontaneously inactivate. Inactivation controls neuronal firing rates and serves as a form of short-term memory, and is implicated in various human neurological disorders. Here, we use high-resolution cryo-electron microscopy and computer simulations to determine one of the molecular mechanisms underlying this physiologically crucial process. Structures of the activated Shaker Kv channel and of its W434F mutant in lipid bilayers demonstrate that C-type inactivation entails the dilation of the ion selectivity filter, and the repositioning of neighboring residues known to be functionally critical. Microsecond-scale molecular dynamics trajectories confirm these changes inhibit rapid ion permeation through the channel. This long-sought breakthrough establishes how eukaryotic K+ channels self-regulate their functional state through the plasticity of their selectivity filters.

One-Sentence SummaryStructures of the Shaker Kv channel reveal the mechanism of slow C-type inactivation involves dilation of the selectivity filter.

sentences: Doxorubicin Induces Senescence in Intestinal Epithelial Cells Doxorubicin treatment induces DNA damage and apoptosis in rapidly dividing cell types like intestinal epithelial cells. This has been demonstrated both in vivo and in vitro. In certain cell types some cells do not undergo DNA damage-induced apoptosis in response to doxorubicin but instead become senescent. Induction of senescence in these cells can lead to dysfunction and chronic inflammation, which can lead to more damage. We questioned whether a single dose of doxorubicin would be able to induce apoptosis and senescence in intestinal epithelial cells in vitro. For these studies, we exposed IEC-6 small intestinal epithelial cells to doxorubicin to evaluate whether senescence is induced in a relatively homogeneous population of intestinal epithelial cells. Although some cells underwent apoptosis, those that did not showed traits of senescence. Our studies showed that doxorubicin treatment increased cell size and increased expression of senescence-associated {beta}-galactosidase. Concomitantly, we observed increased mRNA expression of several genes associated with a senescence-associated secretory phenotype including IL-6, Ptges, Faim2, and Cdkn1a and decreased expression of Sirt1. We also observed release of HMGB1, a cellular alarmin, from treated cells. Together, these data suggest that doxorubicin induces senescence in intestinal epithelial cells. Furthermore, our data indicate that cellular responses to a DNA damaging agent, such as doxorubicin, can differ within a population of cells suggesting differing levels of sensitivity within a relatively homogenous cell population. Further studies are needed to delineate the mechanisms that determine whether a cell moves down an apoptotic or senescent pathway following DNA damage.

sentences: Disruption of miR-18a alters proliferation, photoreceptor replacement kinetics, inflammatory signaling and microglia/macrophage numbers during retinal regeneration in zebrafish In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Muller glia to function as stem cells, producing progenitors that regenerate photoreceptors. MicroRNAs (miRNAs) regulate CNS neurogenesis, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miR-18a regulates photoreceptor differentiation. The purpose of the current study was to determine, in zebrafish, the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in-situ hybridization and immunohistochemistry showed that miR-18a expression increases throughout the retina between 1 and 5 days post-injury (dpi). To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess dividing progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 5-ethynyl-2'-deoxyuridine (EdU) and BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that in the injured zebrafish retina, disruption of miR-18a alters proliferation, inflammation, the microglia/macrophage response, and the timing of photoreceptor regeneration.

sentences: Parallel systems for social and spatial reasoning within the cortical apex What is the cognitive and neural architecture for high-level reasoning? We hypothesize that systems for understanding people and places remain separate throughout the brain, but share a parallel organization. We test this hypothesis using deep neuroimaging of individual human brains on diverse tasks involving reasoning and memory about familiar people, places, and objects. We find that thinking about people and places elicits responses in distinct areas of high-level association cortex, spanning the frontal, parietal, and temporal lobes. Person- and place-preferring brain regions are systematically yoked across cortical zones. These areas have strongly domain-specific response profiles across visual, semantic, and episodic tasks, and are specifically functionally connected to other parts of association cortex with like category preference. Social and spatial networks are anatomically separated even at the top of the cortical hierarchy, and include parts of cortex with anatomical connections to the hippocampal formation. These results demonstrate parallel, domain-specific networks within the cortical apex. They suggest that domain-specific systems for reasoning constitute components of a broader cortico-hippocampal system for long-term memory.

sentences: Modeling miRNA-driven post-transcriptional regulation by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses.

sentences: Computational and neural mechanisms of affected beliefs The feedback people receive on their behavior shapes the process of belief formation and self-efficacy in mastering a given task. However, the neural and computational mechanisms of how the subjective value of these beliefs, and the corresponding affect, bias the learning process remain unclear. We investigated these mechanisms during the learning of self-efficacy beliefs using fMRI, pupillometry, and computational modeling, and by analyzing individual differences in affective experience. Biases in the formation of self-efficacy beliefs were associated with affect, pupil dilation, and neural activity within the anterior insula, amygdala, ventral tegmental area/ substantia nigra, and mPFC. Specifically, neural and pupil responses map the valence of the prediction errors in correspondence with individuals experienced affect and learning bias during belief formation. Together with the functional connectivity dynamics of the anterior insula within this network, our results hint at neural and computational mechanisms that integrate affect into the process of belief formation.

sentences: Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

sentences: Infant gut strain persistence is associated with maternal origin, phylogeny, and functional potential including surface adhesion and iron acquisition Gut microbiome succession impacts infant development. However, it remains unclear what factors promote persistence of initial bacterial colonists in the developing gut. Here, we performed strain-resolved metagenomic analyses to compare gut colonization of preterm and full-term infants throughout the first year of life and evaluated links between strain persistence and strain origin as well as genetic potential. Analysis of 206 fecal metagenomes collected from full-term and preterm infants and their mothers revealed that infants initially distinct microbial communities converged by age one. Approximately 11% of early colonists, primarily Bacteroides and Bifidobacterium, persisted during the first year of life, and these were more prevalent in full-term compared to preterm infants. Gut-associated strains from mothers were significantly more likely to persist in the infant gut than other strains. Enrichment in genes for surface adhesion, iron acquisition and carbohydrate degradation may explain persistence of some strains through the first year of life.

sentences: Neuroprotective, antioxidant and antiproliferative activity of grapefruit IntegroPectin on SH-SY5Y cells Tested in vitro on SH-SY5Y neuroblastoma cells, grapefruit IntegroPectin is a powerful neuroprotective, antioxidant and antiproliferative agent. The strong antioxidant properties of grapefruit IntegroPectin, and its ability to preserve mitochondrial membrane potential and morphology, severely impaired in neurodegenerative disorders, make this new biopolymer highly soluble in water an attractive therapeutic agent for oxidative stress-associated brain disorders. Similarly, the ability of this new citrus pectin rich in naringin, linalool, linalool oxide and limonene adsorbed at the outer surface to inhibit cell proliferation or even kill, at high doses, neoplastic cells, coupled to its excellent health and safety profile, opens up new therapeutic strategies in cancer research. In order to take full advantage of its vast therapeutic and preventive potential, detailed studies of molecular mechanism involved in the antiproliferative and neuroprotective of IntegroPectin are urgently needed.

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Sparcle: assigning transcripts to cells in multiplexed images BackgroundImaging-based spatial transcriptomics has the power to reveal patterns of single-cell gene expression by detecting mRNA transcripts as individually resolved spots in multiplexed images. However, molecular quantification has been severely limited by the computational challenges of segmenting poorly outlined, overlapping cells, and of overcoming technical noise; the majority of transcripts are routinely discarded because they fall outside the segmentation boundaries. This lost information leads to less accurate gene count matrices and weakens downstream analyses, such as cell type or gene program identification.

ResultsHere, we present Sparcle, a probabilistic model that reassigns transcripts to cells based on gene covariation patterns and incorporates spatial features such as distance to nucleus. We demonstrate its utility on both multiplexed error-robust fluorescence in situ hybridization (MERFISH) and single-molecule FISH (smFISH) data.

ConclusionsSparcle improves transcript assignment, providing more realistic per-cell quantification of each gene, better delineation of cell boundaries, and improved cluster assignments. Critically, our approach does not require an accurate segmentation and is agnostic to technological platform.

sentences: Integrative transcriptomic analysis of tissue-specific metabolic crosstalk after myocardial infarction Abstract/SummaryMyocardial infarction (MI) promotes a range of systemic effects, many of which are unknown. Here, we investigated the alterations associated with MI progression in heart and other metabolically active tissues (liver, skeletal muscle, and adipose) in a mouse model of MI (induced by ligating the left ascending coronary artery) and sham-operated mice. We performed a genome-wide transcriptomic analysis on tissue samples obtained 6- and 24-hours post MI or sham operation. By generating tissue-specific biological networks, we observed: (1) dysregulation in multiple biological processes (including immune system, mitochondrial dysfunction, fatty-acid beta-oxidation, and RNA and protein processing) across multiple tissues post MI; and (2) tissue-specific dysregulation in biological processes in liver and heart post MI. Finally, we validated our findings in two independent MI cohorts. Overall, our integrative analysis highlighted both common and specific biological responses to MI across a range of metabolically active tissues.

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TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations BackgroundTopologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs was shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing led to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains -- highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies. Recent computational modeling studies suggest that epigenetic interactions may underlie the formation of the single-cell domains.

ResultsHere we use chromatin tracing to visualize in female human cells the fine-scale chromatin folding of inactive and active X chromosomes, which are known to have distinct global epigenetic landscapes and distinct population-averaged TAD profiles, with inactive X chromosomes largely devoid of TADs and cohesin. We show that both inactive and active X chromosomes possess highly variant single-cell domains across the same genomic region despite the fact that only active X chromosomes show clear TAD structures at the population level. These X chromosome single-cell domains exist in distinct cell lines. Perturbations of major epigenetic components did not significantly affect the frequency or strength of the single-cell domains. Increased chromatin compaction of inactive X chromosomes occurs at a length scale above that of the single-cell domains.

ConclusionsIn sum, this study suggests that single-cell domains are genome architecture building blocks independent of the tested major epigenetic components.

sentences: CXCL17 is an endogenous inhibitor of CXCR4 via a novel mechanism of action CXCL17 is the most recently described chemokine. It is principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, as well as being highly upregulated during viral infections of the lung. However, the exact role of CXCL17 in health and disease is largely unknown, mainly due to a lack of known molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET) based assays, here we demonstrate that CXCL17 inhibits CXCR4-mediated signalling and ligand binding. Moreover, CXCL17 interacts with neuropillin-1, a VEGFR2 co-receptor. Additionally, we find CXCL17 only inhibits CXCR4 ligand binding in intact cells and demonstrate that this effect is mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. This indicates that CXCL17 inhibits CXCR4 by a unique mechanism of action that potentially requires the presence of a glycosaminoglycan containing accessory protein. Altogether, our results reveal that CXCL17 is an endogenous inhibitor of CXCR4 and represents an important discovery in our understanding of the (patho) physiological functions of CXCL17 and regulation of CXCR4 signalling.

sentences: Nitrate enhances anaerobic oxidation of methane in boreal lake sediments The identity of electron acceptors in promoting anaerobic oxidation of methane (AOM) in the sediments of boreal lakes is currently unknown. Here, we studied the AOM rate of sediment slurries collected from three profundal stations of a nitrate-rich, oligo-mesotrophic, boreal lake (Lake Paajarvi, Finland), under varying nitrate concentrations using 13C-labelling. Furthermore, vertical profiles of the sediment and porewater geochemistry, and the microbial communities (16S rRNA gene and shotgun metagenomic sequencing) were analyzed. Despite geochemical data indicating that simultaneous consumption of nitrate and methane took place at the sediment layers chosen for incubations, AOM rate was not enhanced by nitrate amendments at either of the stations. AOM rate was much higher at the shallow Station 1 (0.9-6.8 nmol C cm-3 d-1) with high contents of labile phytoplankton-derived organic matter, than at the deeper stations, Station 2 (0-0.3 nmol C cm-3 d-1) and 3 (0-0.2 nmol C cm-3 d-1). Accordingly, a higher relative abundance of methanotrophic archaea (Candidatus Methanoperedens) and bacteria (Methylococcales) were observed in the layers chosen for incubations at Station 1 than at the other stations. Besides nitrate, the geochemical profiles indicated that AOM was potentially coupled with iron or sulfate reduction at all stations. Furthermore, putative nitrite-reducing methanotrophs (Ca. Methylomirabilis) were the most abundant methanotrophs above the incubation layer at Station 2 and 3, which suggests that nitrite reduction also plays a role in driving AOM in the study lake. We conclude that AOM is not uniquely coupled to nitrate reduction in sediments of nitrate-rich, oligo-mesotrophic, boreal lakes.

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Probing the association between resting state brain network dynamics and psychological resilience This study aimed at replicating a previously reported negative correlation between node flexibility and psychological resilience, i.e., the ability to retain mental health in the face of stress and adversity. To this end, we used multiband resting-state BOLD fMRI (TR = .675 sec) from 52 participants who had filled out three psychological questionnaires assessing resilience. Time-resolved functional connectivity was calculated by performing a sliding window approach on averaged time series parcellated according to different established atlases. Multilayer modularity detection was performed to track network reconfigurations over time and node flexibility was calculated as the number of times a node changes community assignment. In addition, node promiscuity (the fraction of communities a node participates in) and node degree (as proxy for time-varying connectivity) were calculated to extend previous work. We found no substantial correlations between resilience and node flexibility. We observed a small number of correlations between the two other brain measures and resilience scores, that were however very inconsistently distributed across brain measures, differences in temporal sampling, and parcellation schemes. This heterogeneity calls into question the existence of previously postulated associations between resilience and brain network flexibility and highlights how results may be influenced by specific analysis choices.

Author SummaryWe tested the replicability and generalizability of a previously proposed negative association between dynamic brain network reconfigurations derived from multilayer modularity detection (node flexibility) and psychological resilience. Using multiband resting-state BOLD fMRI data and exploring several parcellation schemes, sliding window approaches, and temporal resolutions of the data, we could not replicate previously reported findings regarding the association between node flexibility and resilience. By extending this work to other measures of brain dynamics (node promiscuity, degree) we observe a rather inconsistent pattern of correlations with resilience, that strongly varies across analysis choices. We conclude that further research is needed to understand the network neuroscience basis of mental health and discuss several reasons that may account for the variability in results.

sentences: Optogenetic activators of apoptosis, necroptosis and pyroptosis for probing cell death dynamics and bystander cell responses Targeted and specific induction of cell death in individual or groups of cells holds the potential for new insights into the response of tissues or organisms to different forms of death. Here we report the development of optogenetically-controlled cell death effectors (optoCDEs), a novel class of optogenetic tools that enables light-mediated induction of three types of programmed cell death (PCD) - apoptosis, pyroptosis and necroptosis - using Arabidopsis thaliana photosensitive protein Cryptochrome2. OptoCDEs enable rapid and highly specific induction of PCD in human, mouse and zebrafish cells and are suitable for a wide range of applications, such as sub-lethal cell death induction or precise elimination of single cells or cell populations in vitro and in vivo. As the proof-of-concept, we utilize optoCDEs to assess the differences in the neighboring cell response to apoptotic or necrotic PCD, revealing a new role for shingosine-1-phosphate signaling in regulating the efferocytosis of apoptotic cell by epithelia.

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Inhibition of CDK4/6 Overcomes Primary Resistance to PD-1 Blockade in Malignant Mesothelioma BackgroundDespite the profound number of malignant pleural mesothelioma (MPM) patients now treated with PD-1 blockade, insight into the underpinnings of rational therapeutic strategies to treat resistance to checkpoint immunotherapy remain unrealized. Our objective was to develop a novel therapeutic approach to overcome primary resistance to PD-1 blockade in MPM.

MethodsWe generated a transcriptome signature of resistance to PD-1 blockade in MPM patients treated with nivolumab (4 responders and 4 non-responders). We used the TCGA MPM cohort (N=73) to determine what genomic alterations were associated with the resistance signature. We tested whether regulation of identified molecules could overcome resistance to PD-1 blockade in an immunocompetent mouse malignant mesothelioma model.

ResultsImmunogenomic analysis by applying our anti-PD-1 resistance signature to the TCGA cohort revealed that deletion of CDKN2A was highly associated with primary resistance to PD-1 blockade. Under the hypothesis that resistance to PD-1 blockade can be overcome by CDK4/6 inhibition, we tested whether CDK4/6 inhibitors could overcome resistance to PD-1 blockade in subcutaneous tumors derived from Cdkn2a(-/-) AB1 malignant mesothelioma cells, which were resistant to PD-1 blockade. The combination of daily oral administration of CDK4/6 inhibitors (abemaciclib or palbociclib) and intraperitoneal anti-PD-1 treatment markedly suppressed tumor growth, compared with anti-PD-1 or CDK4/6 inhibitor alone.

ConclusionsWe identified a novel therapeutic target, CDK4/6, to overcome primary resistance to PD-1 blockade through comprehensive immunogenomic approaches. These data provide a rationale for undertaking clinical trials of CDK4/6 inhibitors in the more than 40% of patients with MPM who demonstrate loss of CDKN2A.

sentences: Distinct sensitivities to SARS-CoV-2 variants in vaccinated humans and mice The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOC) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOC. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses against VOC, as compared to non-human primates or humans, suggesting caution should be exercised when interpreting data for this animal model.

sentences: Prior selfing occur in larger flowers in a mixed mating species with delayed selfing Theory predicts that prior self-pollination (prior selfing) should not evolve in mixed mating species that enable delayed selfing. In this study, we test the hypotheais that prior selfing has evolved under severe pollinator limitation in the mixed mating species Commelina communis which can reproduce via delayed selfing. The hypothesis predicts that prior selfing occurs more frequently in populations with very low pollinator availability and/or in smaller flowers which receive infrequent visitations. We tested the predictions by comparing the degree of prior selfing among ten populations experiencing various levels of pollinator limitation and by examining a relationship between individual flower size and the occurrence of prior selfing. Populations with higher pollinator availability had higher prior selfing rate. Moreover, prior selfing occurs more frequently in larger flowers. These findings were totally opposite patterns of the predictions and the previous findings. We proposed new hypotheses that prior selfing has been maintained by the presence of reproductive interference from the congener and/or propotency in C. communis to explain our unexpected findings. We should verify potential effects of reproductive interference and propotency in future to elucidate the mystery of prior selfing in this mixed mating species with delayed selfing.

sentences: Role of Anthropogenic drivers in altering the forest community structure in a prime tiger habitat in central India Tropical forests are complex systems with heterogenous community assemblages often threatened under conservation conflicts. Herbivory and disturbances affect the diversity and species assemblages within forest patches having different disturbance regimes. We studied the change in plant community composition and structure under a disturbance gradient in the tropical dry deciduous forest of the corridor area between Nagzira-Navegaon Tiger reserve of central India. We tested the hypothesis that the plant community will change along the proximity gradient from the human settlement depending on the anthropogenic stress. We sampled 183 nested quadrat plots to collect data on species abundance and various disturbance parameters. Density, diversity, and Importance Value Index were calculated from the collected data on species abundance and girth at breast height (GBH) of individual tree species. We did multivariate analysis to assess the changes in species assemblage along the disturbance gradients. We found 76% dissimilarity between the plant communities in the three disturbance gradients from near to far from the villages perpetrated by the difference in mean abundance of species like Tectona grandis, Terminalia sp, and Largerstroemia parviflora. The anthropogenic factors significantly influence the density and diversity of tree species and regeneration classes. We found the abundance of regeneration class increased along the distance from the villages. The study intensifies the need for proper management and conservative approach to preserve the minimum diversity of the forest patches for its structural and functional contiguity as a corridor in the central Indias highly susceptible and intricate corridor framework.

sentences: A repurposed drug screen identifies compounds that inhibit the binding of the COVID-19 spike protein to ACE2 Repurposed drugs that block the interaction between the SARS-CoV-2 spike protein and its receptor ACE2 could offer a rapid route to novel COVID-19 treatments or prophylactics. Here, we screened 2701 compounds from a commercial library of drugs approved by international regulatory agencies for their ability to inhibit the binding of recombinant, trimeric SARS-CoV-2 spike protein to recombinant human ACE2. We identified 56 compounds that inhibited binding by <90%, measured the EC50 of binding inhibition, and computationally modeled the docking of the best inhibitors to both Spike and ACE2. These results highlight an effective screening approach to identify compounds capable of disrupting the Spike-ACE2 interaction as well as identifying several potential inhibitors that could serve as templates for future drug discovery efforts.

sentences: Early functional and cognitive declines measured by auditory evoked cortical potentials in Alzheimer's disease mice Alzheimers disease (AD) is characterized with a progressive loss of memory and cognitive decline. However, assessment of AD-associated functional and cognitive changes is still a big challenge. Auditory evoked cortical potential (AECP) is an event-related potential reflecting not only neural activation in the auditory cortex but also cognitive activity in the brain. In this study, we used the subdermal needle electrodes with the same electrode setting as the auditory brainstem response (ABR) recording and recorded AECP in normal aging CBA/CaJ mice and APP/PS1 AD mice. AECP in mice usually appeared three positive peaks, i.e., P1, P2, and P3, and three corresponding negative peaks N1, N2, and N3. In normal aging CBA mice, the early sensory peaks P1, N1, and P2 were reduced as age increased, whereas the later cognitive peaks N2, P3, and N3 were increased or had no changes with aging. Moreover, the latency of P1 peak was increased as age increased, although the latencies of later peaks had significant reduction with aging. In AD mice, peak P1 was significantly reduced in comparison with wild-type (WT) littermates at young ages, proceeding AD phenotype presentation. In particular, the later cognitive peak P3 was diminished after 3 months old, different from normal aging effect. However, the latencies of AECP peaks in AD mice generally had no significant delay or changes with aging. Finally, consistent with AECP changes, the accumulation of amyloid precursor protein (APP) at the auditory cortex was visible in AD mice as early as 2 months old. These data suggest that AECP could serve as an early, noninvasive, and objective biomarker for detecting AD and AD-related dementia.

sentences: Allopeptimicins: unique antibacterial metabolites generated by hybrid PKS-NRPS, with original self-defense mechanism in Actinoallomurus In the search for structurally novel metabolites with antibacterial activity, innovative approaches must be implemented to increase the probability of discovering novel chemistry from microbial sources. Here we report on the application of metabolomic tools to the genus Actinoallomurus, a poorly explored member of the Actinobacteria. From examining extracts derived from 88 isolates belonging to this genus, we identified a family of cyclodepsipeptides acylated with a C20 polyketide chain, which we named allopeptimicins. These molecules possess unusual structural features, including several double bonds in the amino-polyketide chain and four non-proteinogenic amino acids in the octapeptide. Remarkably, allopeptimicins are produced as a complex of active and inactive congeners, the latter carrying a sulfate group on the polyketide amine. This modification is also a mechanism of self-protection in the producer strain. The structural uniqueness of allopeptimicins is reflected in a biosynthetic gene cluster showing a mosaic structure, with dedicated gene cassettes devoted to formation of specialized precursors and modular assembly lines related to those from different pathways.

sentences: Thymic macrophages consist of two populations with distinct localization and origin Tissue-resident macrophages are essential for protection from pathogen invasion and maintenance of organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and found out that they express typical tissue-resident macrophage genes yet also exhibit organ-specific features. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor SpiC with these cells. Using shield chimeras, transplantation of embryonic thymuses, and fate mapping, we found that three distinct waves of precursors generate thymic macrophages. Moreover, some of them proliferated in situ. Single-cell RNA sequencing showed that the macrophages in the adult thymus are composed of two populations with distinct localization and origin. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.

sentences: Functional Specialization of Specific DC Subsets in the Synovial Fluid of JIA Patients Dendritic cells (DC) are crucial for initiating and shaping immune responses. So far, little is known about heterogeneity and functional specialization of human DC subsets in (local) inflammatory conditions. We profiled conventional (c)DC1, cDC2 and monocytes based on phenotype, transcriptome and function from a local inflammatory site, namely synovial fluid (SF) from patients suffering from a chronic inflammatory condition, Juvenile Idiopathic Arthritis (JIA). cDC1, a relatively small dendritic cell subset in blood, are strongly enriched in SF. SF cDC1 showed a quiescent immune signature without a clear inflammatory profile low expression of PRR, chemokine, and cytokine receptors, and poor induction of T cell proliferation and cytokine production, but selective production of IFN{lambda} upon polyinosinic:polycytidylic acid exposure. In stark contrast, cDC2 and monocytes from the same environment, showed a pro-inflammatory transcriptional profile, high levels of (spontaneous) pro-inflammatory cytokine production, and strong induction of T cell proliferation and cytokine production, including IL-17. Although the cDC2 and monocytes showed an overlapping transcriptional core profile, there were clear differences in the transcriptional landscape and functional features, indicating that these cell types retain their lineage identity in chronic inflammatory conditions. In conclusion, our findings suggest that at the site of inflammation, there is specific functional programming of human DC subsets, especially cDC2. In contrast, the enriched subset of cDC1 remains relatively quiescent and seemingly unchanged under inflammatory conditions, pointing to a more regulatory role.

sentences: Electroencephalographic dynamics of rhythmicbreath-based meditation Meditation has been practised for millennia but the neuroscientific understanding of the dynamics is still lacking. Sudarshan Kriya Yoga (SKY) is an evidence based breathing based meditation technique that utilizes rhythmic breathing to induce a deep state of relaxation and calm. Multiple studies have found benefits of the SKY technique from genetic, physiological, psychological to behaviour levels. We collected Electroencephalographic (EEG) data in 43 subjects who underwent the SKY technique and analysed the brain rhythms at different stages of the technique namely preparatory breathing (Pranayama), rhythmic breathing (Kriya) and meditation (Yoga Nidra) using newly developed methods to analyse periodic and aperiodic components. Alpha waves amplitude in the parieto-occipital region decreased as the rhythmic breathing progressed and dropped sharply during the meditation period. Theta amplitudes and peak frequency increased in the centro-frontal region during the rhythmic breathing period but were marked by sustained low theta waves during the meditation period. The delta wave amplitude was not affected by breathing but both delta band power and peak frequency increased during the meditation period in the centro-frontal region. We also saw a decrease in the 1/f aperiodic signal across the brain during the meditation period suggesting a modification of excitation-inhibition balance. We see an overall slowing down of brain oscillations from alpha to theta to delta as the meditation progressed. The paper studies in depth the transitional dynamics of the SKY technique analysing the alpha, theta, delta waves and aperiodic signals and demonstrates that each phase in a breathing based meditation has a unique electrophysiological signature.

sentences: Alterations in human gut microbiome composition and metabolism after exposure to glyphosate and Roundup and/or a spore-based formulation using the SHIME(R) technology Despite extensive research into the toxicology of the herbicide glyphosate, there are still major unknowns regarding its effects on the human gut microbiome. As a step in addressing this knowledge gap, we describe for the first time the effects of glyphosate and a Roundup glyphosate-based herbicide on infant gut microbiota using SHIME technology, which mimics the entire gastrointestinal tract. SHIME(R) microbiota culture was undertaken in the presence of a concentration of 100 mg/L (corresponding to a dose of 1.6 mg/kg/day) glyphosate and the same glyphosate equivalent concentration of Roundup, which is in the range of the US chronic reference dose, and subjected to molecular profiling techniques to assess outcomes. Roundup and to a lesser extent glyphosate caused an increase in fermentation activity, resulting in acidification of the microbial environment. This was also reflected by an increase in lactate and acetate production concomitant to a decrease in the levels of propionate, valerate, caproate and butyrate. Ammonium production reflecting proteolytic activities was increased by Roundup exposure. Global metabolomics revealed large scale disturbances in metabolite profiles, including an increased abundance of long chain polyunsaturated fatty acids (n3 and n6). Although changes in bacterial composition measured by qPCR and 16S rRNA sequencing were less clear, our results suggested that lactobacilli had their growth stimulated as a result of microenvironment acidification. Co-treatment with the spore-based probiotic formulation MegaSporeBiotic reverted some of the changes in short-chain fatty acid levels. Altogether, our results suggest that glyphosate can exert effects on human gut microbiota at permitted regulatory levels of exposure, highlighting the need for epidemiological studies aimed at evaluating the effects of glyphosate herbicides on human gut microbiome function.

sentences: Longitudinal dynamics of clonal hematopoiesis identifies gene-specific fitness effects The prevalence of clonal haematopoiesis of indeterminate potential (CHIP) in healthy individuals increases rapidly from age 60 onwards and has been associated with increased risk for malignancy, heart disease and ischemic stroke. CHIP is driven by somatic mutations in stem cells that are also drivers of myeloid malignancies. Since mutations in stem cells often drive leukaemia, we hypothesised that stem cell fitness substantially contributes to transformation from CHIP to leukaemia. Stem cell fitness is defined as the proliferative advantage over cells carrying no or only neutral mutations. It is currently unknown whether mutations in different CHIP genes lead to distinct fitness advantages that could form the basis for patient stratification. We set out to quantify the fitness effects of CHIP drivers over a 12 year timespan in older age, using longitudinal error-corrected sequencing data. We developed a new method based on drift-induced fluctuation (DIF) filtering to extract fitness effects from longitudinal data, and thus quantify the growth potential of variants within each individual. Our approach discriminates naturally drifting populations of cells and faster growing clones, while taking into account individual mutational context. We show that gene-specific fitness differences can outweigh inter-individual variation and therefore could form the basis for personalised clinical management.

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Distinct bile salt hydrolase substrate preferences dictate C. difficile pathogenesis Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence intestinal diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate preferences and impact on C. difficile remain elusive. Here, we survey the diversity of Lactobacillus BSHs and unravel the structural basis of their substrate preference. We show that leveraging BSH activity and specificity is an effective strategy to prevent C. difficile growth in clinically relevant CDI models. A range of non-canonical conjugated BAs is also identified, comprising unique BSH substrates that also inhibit C. difficile spore germination. These findings establish BSHs as intestinal enzymes essential to BA homeostasis and colonization resistance against C. difficile.

One sentence summaryBile salt hydrolase activity inhibits C. difficile by shaping the conventional and non-canonical conjugated bile acid pools

sentences: Improved antibody structure prediction by deep learning of side chain conformations Antibody engineering is becoming increasingly popular in medicine for the development of diagnostics and immunotherapies. Antibody function relies largely on the recognition and binding of antigenic epitopes via the loops in the complementarity determining regions. Hence, accurate high-resolution modeling of these loops is essential for effective antibody engineering and design. Deep learning methods have previously been shown to effectively predict antibody backbone structures described as a set of inter-residue distances and orientations. However, antigen binding is also dependent on the specific conformations of surface side chains. To address this shortcoming, we created DeepSCAb: a deep learning method that predicts inter-residue geometries as well as side chain dihedrals of the antibody variable fragment. The network requires only sequence as input, rendering it particularly useful for antibodies without any known backbone conformations. Rotamer predictions use an interpretable self-attention layer, which learns to identify structurally conserved anchor positions across several species. We evaluate the performance of our model for discriminating near-native structures from sets of decoys and find that DeepSCAb outperforms similar methods lacking side chain context. When compared to alternative rotamer repacking methods, which require an input backbone structure, DeepSCAb predicts side chain conformations competitively. Our findings suggest that DeepSCAb improves antibody structure prediction with accurate side chain modeling and is adaptable to applications in docking of antibody-antigen complexes and design of new therapeutic antibody sequences.

sentences: In silico analyses of neuropeptide-like protein (NLP) profiles in parasitic nematodes Nematode parasite infections cause disease in humans and animals and threaten global food security by reducing productivity in livestock and crop farming. The escalation of anthelmintic resistance in economically important nematode parasites underscores the need for the identification of novel drug targets in these worms. Nematode neuropeptide signalling is an attractive system for potential chemotherapeutic exploitation, with neuropeptide G-protein coupled receptors (NP-GPCRs) representing the leading target candidates therein. In order to successfully validate NP-GPCRs as targets for parasite control it is necessary to characterise their function and importance to nematode biology. This can be aided through identifying receptor activating ligand(s) in a process known as receptor deorphanisation. Such efforts first require the identification of all neuropeptide ligands within parasites. Here we comb the genomes of nine therapeutically relevant pathogenic nematode species to comprehensively characterise the nematode parasite neuropeptide-like protein (NLP) complements, and details the discovery of several previously unreported, yet conserved, neuropeptides and neuropeptide-encoding genes. We identify the neuropeptides that are most highly conserved in all parasites examined, and characterise their physiological activity on the reproductive musculature of the parasite, Ascaris suum. These data suggest conserved neuropeptide functions in both free living and parasitic nematodes, and support the potential for exploitation of the neuropeptide signalling system as an anthelmintic target.

sentences: Masculinizer-induced dosage compensation is achieved by transcriptional downregulation of both copies of Z-linked genes in the silkworm, Bombyx mori Dosage compensation balances the expression of sex-chromosome-linked genes with autosome-linked genes in the heterogametic sex. The silkworm (Bombyx mori), a lepidopteran model insect, uses the female heterogametic WZ sex determination system. In B. mori, a Z-linked gene, Masculinizer (Masc), is the primary determinant of maleness and dosage compensation. However, it remains unknown whether one of the two Z chromosomes is inactivated or both Z chromosomes are suppressed in B. mori males. Hence, we performed transcriptome analysis using hybrids between two B. mori strains and analyzed the allele-specific expression (ASE) to solve the problem. The ASE analysis revealed that the genes located on the maternal and paternal Z chromosomes are transcriptionally upregulated in Masc knocked down males. Also, our results revealed that two Z chromosomes are transcriptionally downregulated in B. mori males as observed in Caenorhabditis elegans.

sentences: Potential transmission chains of variant B.1.1.7 and co-mutations of SARS-CoV-2 The presence of SARS-CoV-2 mutants, including the emerging variant B.1.1.7, has raised great concerns in terms of pathogenesis, transmission, and immune escape. Characterizing SARS-CoV-2 mutations, evolution, and effects on infectivity and pathogenicity is crucial to the design of antibody therapies and surveillance strategies. Here we analyzed 454,443 SARS-CoV-2 spike genes/proteins and 14,427 whole-genome sequences. We demonstrated that the early variant B.1.1.7 may not have evolved spontaneously in the United Kingdom or within human populations. Our extensive analyses suggested that Canidae, Mustelidae or Felidae, especially the Canidae family (for example, dog) could be a possible host of the direct progenitor of variant B.1.1.7. An alternative hypothesis is that the variant was simply yet to be sampled. Notably, the SARS-CoV-2 whole genome represents a large number of potential co-mutations with very strong statistical significances (p value<E- 44). In addition, we used an experimental SARS-CoV-2 reporter replicon system to introduce the dominant co-mutations NSP12_c14408t, 5UTR_c241t, and NSP3_c3037t into the viral genome, and to monitor the effect of the mutations on viral replication. Our experimental results demonstrated that the co-mutations significantly attenuated the viral replication. The study provides valuable clues for discovering the transmission chains of variant B.1.1.7 and understanding the evolutionary process of SARS-CoV-2.

sentences: Prostaglandin EP3 receptor-expressing preoptic neurons bidirectionally control body temperature via tonic GABAergic signaling The circuit mechanism of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here we show prostaglandin EP3 receptor-expressing POA neurons (POAEP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POAEP3R neurons are activated in response to elevated ambient temperature, but inhibited by prostaglandin E2, a pyrogenic mediator. Chemogenetic stimulation of POAEP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POAEP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic inhibitory signaling. Although many POAEP3R neuronal cell bodies express a glutamatergic mRNA marker, paradoxically, their axons in the DMH predominantly contain terminals with GABAergic presynaptic proteins, which are increased by chronic heat exposure. These findings indicate that tonic GABAergic inhibitory signaling from POAEP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.

sentences: What is the evidence available to support our knowledge about threats to the conservation of Galliformes in the Greater Himalaya? 1.Biodiversity is at a heightened risk of extinction and we are losing species faster than any other time. It is important to understand the threats that drive a species towards extinction in order to address those drivers. In this paper, we assess our knowledge of the threats faced by 24 Himalayan Galliformes species by undertaking a review to identify threats reported in the published literature and the supporting evidence that the threat is having an impact on the species population. Only 24 papers were deemed suitable to be included in the study. We found that biological resource use, agriculture and aquaculture are the predominant threats to the Galliformes in the Greater Himalaya but the evidence available in the studies is quite poor as only one paper quantified the impact on species. This study shows that major gaps exist in our understanding of threats to species and it is imperative to fill those gaps if we want to prevent species from going extinct.

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Visual recognition is heralded by shifts in local field potential oscillations and inhibitory networks in primary visual cortex Learning to recognize and filter familiar, irrelevant sensory stimuli eases the computational burden on the cerebral cortex. Inhibition is a candidate mechanism in this filtration process, and oscillations in the cortical local field potential (LFP) serve as markers of the engagement of different inhibitory neurons. We show here that LFP oscillatory activity in visual cortex is profoundly altered as male and female mice learn to recognize an oriented grating stimulus--low frequency ([~]15 Hz peak) power sharply increases while high frequency ([~]65 Hz peak) power decreases. These changes report recognition of the familiar pattern, as they disappear when the stimulus is rotated to a novel orientation. Two-photon imaging of neuronal activity reveals that parvalbumin-expressing inhibitory neurons disengage with familiar stimuli and reactivate to novelty, whereas somatostatin-expressing inhibitory neurons show opposing activity patterns. We propose a model in which the balance of two interacting interneuron circuits shifts as novel stimuli become familiar.

Significance StatementHabituation, familiarity and novelty detection are fundamental cognitive processes that enable organisms to adaptively filter meaningless stimuli and focus attention on potentially important elements of their environment. We have shown that this process can be studied fruitfully in the mouse primary visual cortex by using simple grating stimuli for which novelty and familiarity are defined by orientation, and by measuring stimulus-evoked and continuous local field potentials. Altered event-related and spontaneous potentials, and deficient habituation, are well-documented features of several neurodevelopmental psychiatric disorders. The paradigm described here will be valuable to interrogate the origins of these signals and the meaning of their disruption more deeply.

sentences: SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-{beta} signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-{beta} signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

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Experimental evaluation of thermodynamic speed limit in living cells via information geometry Chemical reactions are responsible for information processing in living cells, and thermodynamic trade-off relations can explain their accuracy and speed. Its experimental test in living cells had not existed despite its importance because it is hard to justify sample size sufficiency. This paper reports the first experimental test of the thermodynamic trade-off relation, namely the thermodynamic speed limit, in living systems at the single-cell level where the sample size is relatively small. Due to the information-geometric approach, we can demonstrate the thermodynamic speed limit for the extracellular signal-regulated kinase phosphorylation using time-series fluorescence imaging data. Our approach quantifies the intrinsic speed of cell proliferation and can potentially apply other signal transduction pathways to detect their information processing speed.

One-Sentence SummaryExperimental measurement of information thermodynamic speed by fluorescence imaging in living cells

sentences: SPATIAL ORGANIZATION OF TRANSCRIBED EUKARYOTIC GENES Despite the well-established role of nuclear organization in gene expression regulation, little is known about the reverse: how transcription shapes the spatial organization of the genome. Owing to small sizes of most previously studied genes and the limited resolution of microscopy, the structure and spatial arrangement of a single transcribed gene are still poorly understood. Here, we make use of several long highly expressed genes and demonstrate that they form transcription loops with polymerases moving along the loops and carrying nascent RNAs. Transcription loops can span across microns resembling lampbrush loops and polytene puffs. Extension and shape of transcription loops suggest their intrinsic stiffness, which we attribute to decoration with multiple voluminous nascent RNPs. Our data contradict the model of transcription factories and indicate that although microscopically resolvable transcription loops are specific for long highly expressed genes, the mechanisms underlying their formation can represent a general aspect of eukaryotic transcription.

sentences: High Level of Interaction between Phages and Bacteria in an Artisanal Raw Milk Cheese Microbial Community Endogenous starter cultures are used in the production of several cheeses around the world, such as Parmigiano-Reggiano, in Italy, Epoisses, in France, and Canastra, in Brazil. These microbial communities are responsible for many of the intrinsic characteristics of each of these cheeses. Bacteriophages are ubiquitous around the world, well known to be involved in the modulation of complex microbiological processes. However, little is known about phage-bacteria growth dynamics in cheese production systems, where phages are normally treated as problems, as the viral infections can negatively affect or even eliminate the starter culture during production. Furthermore, a recent metagenomic based meta-analysis has reported that cheeses contain a high abundance of phage-associated sequences. Here, we analyse the viral and bacterial metagenomes of Canastra cheese, a tradition artisanal cheese produced using an endogenous starter culture. We observe a very high phage diversity level, mostly composed of novel sequences. We detect several metagenomic assembled bacterial genomes at strain level resolution, and several putative phage-bacteria interactions, evidenced by the recovered viral and bacterial genomic signatures. We postulate that at least one bacterial strain detected could be endogenous to the Canastra region, in Brazil, and that its growth seems to be modulated by native phages present in this artisanal production system. This relationship is likely to influence the fermentation dynamics and ultimately the sensorial profile of these cheeses, with implication for all cheeses that employ similar production processes around the world.

sentences: Sex without crossing over in the yeast Saccharomycodes ludwigii Meiotic recombination is a ubiquitous function of sexual reproduction throughout eukaryotes. Here we report that recombination is extremely suppressed during meiosis in the yeast species Saccharomycodes ludwigii. DNA double-strand break formation, processing and repair are required for normal meiosis but do not lead to crossing over. Although the species has retained an intact meiotic gene repertoire, genetic and population analyses suggest the exceptionally rare occurrence of meiotic crossovers. We propose that Sd. ludwigii has followed a unique evolutionary trajectory that possibly derives fitness benefits from the combination of frequent fertilization within the meiotic tetrad with the absence of meiotic recombination.

sentences: Snowball Earths, population bottlenecks, and the evolution of marine photosynthetic bacteria Prochlorococcus are the most abundant photosynthetic organisms in the modern ocean. A massive DNA loss event occurred in their early evolutionary history, leading to highly reduced genomes in nearly all lineages, as well as enhanced efficiency in both nutrient uptake and light absorption. The environmental landscape that shaped this ancient genome reduction, however, remained unknown. Through careful molecular clock analyses, we established that this Prochlorococcus genome reduction occurred during the Neoproterozoic Snowball Earth climate catastrophe. The lethally low temperature and exceedingly dim light during the Snowball Earth event would have inhibited Prochlorococcus growth and proliferation and caused severe population bottlenecks. These bottlenecks are recorded as an excess of deleterious mutations that accumulated across genomic regions in the descendant lineages. Prochlorococcus adaptation to extreme environmental conditions during Snowball Earth intervals can be inferred by tracing the evolutionary paths of genes that encode key metabolic potential. This metabolic potential includes modified lipopolysaccharide structure, strengthened peptidoglycan biosynthesis, the replacement of a sophisticated circadian clock with an hourglass-like mechanism that resets daily for dim light adaption, and the adoption of ammonia diffusion as an efficient membrane transporter-independent mode of nitrogen acquisition. In this way, the Neoproterozoic Snowball Earth event altered the physiological characters of Prochlorococcus, shaping their ecologically vital role as the most abundant primary producers in the modern oceans.

sentences: Using antibody synergy to engineer a high potency biologic cocktail against C. difficile We applied a mathematical framework originally used to model the effects of multiple inhibitors on enzyme activity to guide the development a therapeutic antibody cocktail, LMN-201, to prevent and treat C. difficile infection (CDI). CDI causes hundreds of thousands of cases of severe, often recurrent diarrhea and colitis in the United States annually and is associated with significant morbidity and mortality worldwide. Current therapies for preventing recurrent CDI are only partially successful, and there are no options available to prevent initial bouts of CDI in at-risk populations. Almost all antibody therapies have been developed and administered as monotherapies. Antibody cocktails are relatively rare even though they have the potential to greatly increase efficacy. One reason for this is our limited understanding of how antibody interactions can enhance potency, which makes it difficult to identify and develop antibodies that can be assembled into optimally effective cocktails. In contrast to the view that antibody synergies depend on unusual instances of cooperativity or allostery, we show that synergistic efficacy requires nothing more than that the antibodies bind independently to distinct epitopes on a common target. Therefore, synergy may be achieved much more readily than is generally appreciated. Due to synergy the LMN-201 antibody cocktail, which targets the C. difficile exotoxin B (TcdB), is 300- to 3000-fold more potent at neutralizing the most clinically prevalent TcdB toxin types than bezlotoxumab, the only monoclonal antibody currently approved for treatment or prevention of CDI. The efficacy of LMN-201 is further enhanced by inclusion of a phage-derived endolysin that destroys the C. difficile bacterium, and which therefore has a complementary mechanism of action to the antibody cocktail. These observations may serve as a paradigm for the development of high potency biologic cocktails against targets that have proven challenging for single-agent therapies.

sentences: Bacterial small RNAs and host epigenetic effectors of a transgenerational memory of pathogens in C. elegans. The inheritance of memories is adaptive for survival. Microbes interact with all organisms challenging their immunity and triggering behavioral adaptations. Some of these behaviors induced by bacteria can be inherited although the mechanisms of action are largely unexplored. In this work, we use C. elegans and its bacteria to study the transgenerational RNA dynamics of an interspecies crosstalk leading to a heritable behavior. Heritable responses to bacterial pathogens in the nematode include avoidance and pathogen-induced diapause (PIDF), a state of suspended animation to evade the pathogen threat. We identify a small RNA RsmY, involved in quorum sensing from P. aeruginosa as required for initiation of PIDF. Histone methyltransferase SET-18/SMYD3 is also needed for PIDF initiation in C. elegans. In contrast, SET-25/EHMT2 is necessary for the maintenance of the memory of pathogen exposure in the transgenerational lineage. This work can be a starting point to understanding microbiome-induced inheritance of acquired traits.

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Bioactive constituents of Verbena officinalis regulate killing efficiency of primary human natural killer cells by accelerating killing processes BackgroundNatural killer (NK) cells play a key role in eliminating tumorigenic and pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine, exhibiting anti-tumor and anti-inflammation activity, but its role in NK cells remains unclear.

PurposeThe impact of bioactive constituents of V. officinalis on immune responses remains largely elusive. In this work we investigated the potential targets of V. officinalis and focused on killing efficiency and related functions of NK cells regulated by bioactive constituents of V. officinalis.

Study design/methodsWe used primary human NK cells from peripheral blood mononuclear cells. Potential regulatory roles of selected compounds in NK killing function-related genes were first analyzed by network pharmacology approaches. Potential engagement between compounds and NK receptors was examined by molecular docking assay. Killing efficiency of NK cells was determined with real-time killing assay and live-cell imaging. Proliferation was examined by CFSE staining. Expression of cytotoxic proteins was analyzed using flow cytometry. Lytic granule release was quantified by CD107a degranulation assay. Contact time required for killing and determination of serial killers were analyzed using live cell imaging results.

ResultsResults from network pharmacology approaches and molecular docking analyses predicted that the five bioactive compounds (acteoside, apigenin, kaempferol, verbenalin and hastatoside) from V. officinalis can regulated various key genes essential for NK effector functions and can bind to NK inhibitory receptors as well as activating receptors, indicating their potential role in regulating NK killing capacity. Analyses of NK killing efficiency revealed that out of these five compounds verbenalin exhibited an encouraging potency to enhance NK killing efficiency. Further investigation showed that verbenalin did not affect proliferation, expression of cytotoxic proteins, or lytic granule degranulation, but rather reduced contact time required for killing, enhanced total killing events per NK cell, and elevated the fraction of killing competent NK cells.

ConclusionsIn this work, we have identified verbenalin as the most potent bioactive constituent from V. officinalis to enhance NK cell killing efficiency via accelerating killing processes and converting non-killers to killing competent NK cells without affecting the lytic granule pathway.

sentences: Comparison of Genomic Selection Models for Exploring Predictive Ability of Complex Traits in Breeding Programs Traits with a complex unknown genetic architecture are common in breeding programs. However, they pose a challenge for selection due to a combination of complex environmental and pleiotropic effects that impede the ability to create mapping populations to characterize the traits genetic basis. One such trait, seedling emergence of wheat (Triticum aestivum L.) from deep planting, presents a unique opportunity to explore the best method to use and implement GS models to predict a complex trait. 17 GS models were compared using two training populations, consisting of 473 genotypes from a diverse association mapping panel (DP) phenotyped from 2015-2019 and the other training population consisting of 643 breeding lines phenotyped in 2015 and 2020 in Lind, WA with 40,368 markers. There were only a few significant differences between GS models, with support vector machines reaching the highest accuracy of 0.56 in a single breeding line trial using cross-validations. However, the consistent moderate accuracy of cBLUP and other parametric models indicates no need to implement computationally demanding non-parametric models for complex traits. There was an increase in accuracy using cross-validations from 0.40 to 0.41 and independent validations from 0.10 to 0.17 using diversity panels lines to breeding lines. The environmental effects of complex traits can be overcome by combining years of the same populations. Overall, our study showed that breeders can accurately predict and implement GS for a complex trait by using parametric models within their own breeding programs with increased accuracy as they combine training populations over the years.

sentences: Factors Associated with Emerging and Re-emerging of SARS-CoV-2 Variants Global spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has triggered unprecedented scientific efforts, as well as containment and treatment measures. Despite these efforts, SARS-CoV-2 infections remain unmanageable in some parts of the world. Due to inherent mutability of RNA viruses, it is not surprising that the SARS-CoV-2 genome has been continuously evolving since its emergence. Recently, four functionally distinct variants, B.1.1.7, B.1.351, P.1 and CAL.20C, have been identified, and they appear to more infectious and transmissible than the original (Wuhan-Hu-1) virus. Here we provide evidence based upon a combination of bioinformatics and structural approaches that can explain the higher infectivity of the new variants. Our results show that the greater infectivity of SARS-CoV-2 than SARS-CoV can be attributed to a combination of several factors, including alternate receptors. Additionally, we show that new SARS-CoV-2 variants emerged in the background of D614G in Spike protein and P323L in RNA polymerase. The correlation analyses showed that all mutations in specific variants did not evolve simultaneously. Instead, some mutations evolved most likely to compensate for the viral fitness.

sentences: The mitochondrial calcium uniporter regulates mitochondrial mass and dendritic localization in distinct hippocampal circuits CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2, however the functional significance of this enrichment remains unclear. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Further, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites. Genetic knockdown of MCU in CA2 resulted in smaller mitochondria in CA2 distal dendrites, indicating that MCU expression plays a role in regulating mitochondrial mass in CA2. MCU overexpression in neighboring CA1 led to larger mitochondria preferentially in proximal dendrites compared to distal dendrites and GFP controls. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and that MCU expression cell-autonomously regulates mitochondrial mass, but layer-specific dendritic localization depends on cell type. Our data support the idea that CA2 mitochondria are functionally distinct from CA1 mitochondria, which may confer unique synaptic and circuit properties underlying CA2 function in social memory.

sentences: Use of Induced Pluripotent Stem Cells to Build Isogenic Systems and Investigate Type 1 Diabetes Type 1 diabetes is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of {beta}-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individuals collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and {beta}-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.

sentences: Engineering the AEG1 promoter from cotton to develop male sterile lines This work reports on modifying the Upstream Regulatory Module (URM, 1.5 Kb region upstream of the open reading frame) of Anther Expressing Gene 1 (AEG1) from cotton to achieve anther specific activity. AEG1 was identified in a previous study aimed to isolate a promoter with tapetum specific activity. Such a promoter could then be used to express barnase and barstar genes for developing male sterile and restorer lines for hybrid seed production in cotton. The AEG1 URM was observed to be active in tapetum as well as in roots making it unusable to drive the expression of barnase gene. Analysis of the URM showed the presence of several root specific motifs. Two modified AEG1 URMs were developed, by removing or mutating these motifs and its activity checked in tobacco. The activity of one of the modified URMs, AEG1({Delta}Bmut) was restricted to the anther tissue as observed using the reporter gene {beta}-glucuronidase. The study also demonstrates that male sterile lines could be developed in tobacco using the AEG1({Delta}Bmut) URM to express the barnase gene. This work thus shows the possibility of engineering promoters to achieve tissue specificity and to develop male sterile lines in cotton.

sentences: Single-sample proteome enrichment enables missing protein recovery and phenotype association Proteomic studies characterize the protein composition of complex biological samples. Despite recent developments in mass spectrometry instrumentation and computational tools, low proteome coverage remains a challenge. To address this, we present Proteome Support Vector Enrichment (PROSE), a fast, scalable, and effective pipeline for scoring protein identifications based on gene co-expression matrices. Using a simple set of observed proteins as input, PROSE gauges the relative importance of proteins in the phenotype. The resultant enrichment scores are interpretable and stable, corresponding well to the source phenotype, thus enabling reproducible recovery of missing proteins. We further demonstrate its utility via reanalysis of the Cancer Cell Line Encyclopedia (CCLE) proteomic data, with prediction of oncogenic dependencies and identification of well-defined regulatory modules. PROSE is available as a user-friendly Python module from https://github.com/bwbio/PROSE.

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Inhibition of Metastatic Uveal Melanoma Cell Proliferation by the Histone Deacetylase 6 Inhibitor, Ricolinostat, is Linked to Altered Microphthalmia-associated Transcription Factor and Phospho-ERK Expression Metastatic uveal melanoma (MUM) is characterized by poor patient survival. Unfortunately, current treatment options demonstrate limited benefits. In this study, we evaluate the efficacy of ACY-1215, a histone deacetylase 6 inhibitor (HDAC6i), to attenuate MUM cell growth in vitro and in vivo, and elucidate the underlying molecular mechanisms. Treatment of OMM2.5 MUM cells with ACY-1215 resulted in a significant (p = 0.0001), dose-dependent reduction in cell survival and proliferation in vitro, and in vivo regression of primary OMM2.5 xenografts in zebrafish larvae. Furthermore, flow cytometry analysis revealed that ACY-1215 significantly arrested the OMM2.5 cell cycle in S phase (p = 0.0006) following 24 hours of treatment and significant apoptosis was triggered in a time- and dose-dependent manner (p = <0.0001). Additionally, ACY-1215 treatment resulted in a significant reduction in OMM2.5 p-ERK expression levels. Through proteome-profiling, attenuation of the microphthalmia-associated transcription factor (MITF) signaling pathway was linked to the observed anti-cancer effects of ACY-1215. In agreement, pharmacological inhibition of MITF signaling with ML329, significantly reduced OMM2.5 cell survival and viability in vitro (p = 0.0001) and in vivo (p = 0.0006). Our findings provide evidence that ACY-1215 and ML329 are efficacious against growth and survival of MUM cells and are potential therapeutic options for MUM.

Simple SummaryUveal melanoma (UM) is the most common adult eye cancer. UM originates in the iris, ciliary body, or choroid (collectively known as the uvea), in the middle layer of the eye. This first or primary UM is treated by targeting the cancer cells using ocular radiation implants or by surgical removal of the eye. However, when UM spreads to the liver and other parts of the body, patients have a poor survival prognosis. Unfortunately, there are no effective treatment options for UM that has spread. Our aim is to help identify effective treatments for UM cancer that has spread. In our study, we identified that the drug ACY-1215 prevents the growth of UM cells from the liver. Our study has found a promising treatment approach for advanced UM.

sentences: A microbial mutualist within host individuals increases parasite transmission between host individuals: Evidence from a field mesocosm experiment The interactions among host-associated microbes and parasites can have clear consequences for disease susceptibility and progression within host individuals. Yet, empirical evidence for how these interactions impact parasite transmission between host individuals remains scarce. We address this scarcity by using a field mesocosm experiment to investigate the interaction between a systemic fungal endophyte, Epichloe coenophiala, and a fungal parasite, Rhizoctonia solani, in leaves of a grass host, tall fescue. Specifically, we investigated how this interaction impacted parasite transmission under field conditions in replicated experimental host populations. Epichloe-inoculated populations tended to have greater disease prevalence over time, though this difference had weak statistical support. More clearly, Epichloe-inoculated populations experienced higher peak parasite prevalences than Epichloe-free populations. Epichloe conferred a benefit in growth; Epichloe-inoculated populations had greater aboveground biomass than Epichloe-free populations. Using biomass as a proxy, host density was correlated with peak parasite prevalence, but Epichloe still increased peak parasite prevalence after controlling for the effect of biomass. Together, these results suggest that within-host microbial interactions can impact disease at the population level. Further, while Epichloe is clearly a mutualist of tall fescue, it may not be a defensive mutualist in relation to R. solani.

sentences: Coupled protein quality control during nonsense mediated mRNA decay Translation of mRNAs containing premature termination codons (PTCs) can result in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance path-way responsible for detecting and degrading PTC containing transcripts. While the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational, and dependent on an intact ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors, and suggested a lack of dependence on the canonical ribosome-quality control (RQC) pathway. Finally, one of the strongest hits in our screens was the E3 ubiquitin ligase CNOT4, a member of the CCR4-NOT complex, which is involved in initiating mRNA degradation. We show that CNOT4 is involved in NMD coupled protein degradation, and its role depends on a functional RING ubiquitin ligase domain. Our results demonstrate the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a framework for identifying and characterizing factors involved in this process.

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The cellular characterisation of SARS-CoV-2 spike protein in virus-infected cells using Receptor Binding Domain-binding specific human monoclonal antibodies. A human monoclonal antibody panel (PD4, PD5, PD7, SC23 and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2- infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 Spike (S) protein, only PD5, PD7, and SC23 were able to bind to the Receptor Binding Domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localised within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognised the uncleaved S protein indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining was demonstrated for each isolate, the SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders, and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection.

ImportanceThe SARS CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID19 patients to monitor the RBD in cells infected with SARS CoV-2 clinical isolates. These immunological reagents specifically recognise the correctly folded RBD and were used to monitor the appearance of the RBD in SARS CoV-2-infected cells and identified the site where the RDB first appears.

sentences: Manifold learning analysis suggests novel strategies for aligning single-cell multi-modalities and revealing functional genomics for neuronal electrophysiology Recent single-cell multi-modal data reveal multi-scale characteristics of single cells, such as transcriptomics, morphology, and electrophysiology. However, our understanding of functional genomics and gene regulation leading to various cellular characteristics remains elusive. To address this, we applied multiple machine learning methods to align gene expression and electrophysiological data of single neuronal cells in the mouse brain. We found that nonlinear manifold learning outperforms other methods. After manifold alignment, the cell clusters highly correspond to transcriptomic and morphological cell-types, suggesting a strong nonlinear relationship between gene expression and electrophysiology at the cell-type level. The aligned cells form developmental trajectories and show continuous changes of electrophysiological features, implying the underlying developmental process. We also found that the manifold-aligned cell clusters differentially expressed genes can predict many electrophysiological features. Functional enrichment and gene regulatory network analyses for those cell clusters revealed potential genome functions and molecular mechanisms from gene expression to neuronal electrophysiology.

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Precocious hippocampal structural and functional changes in gestational protein-restricted male elderly offspring: an Alzheimer-simile disease model? BackgroundMaternal undernutrition has been associated with psychiatric and neurological disorders characterized by learning and memory impairment. Considering the lack of evidence for this, we aimed to analyze the effects of gestational protein restriction on learning and memory function later in life. This research associates behavioral findings with hippocampal cell numbers and protein content related to neurodegenerative brain disease.

MethodsExperiments were conducted in animals subjected to a low-protein (LP, 6% casein) or regular-protein (NP, 17% casein) diet throughout their pregnancy. Behavioral tests, isolated hippocampal isotropic fractionator cell studies, immunoblotting, and survival lifetime tests were performed. The results confirmed that the birthweight of LP male pups significantly reduced relative to NP male pups and that hippocampal mass increased in 88-week-old LP compared to age-matched NP offspring. We used the Morris water maze proximity measure, which is the sum of 10 distances each second between rat position and location of a hidden platform target, as a suitable test for assessing age-related learning or memory impairment in aged offspring.

ResultsThe results showed an increased proximity measure in 87-week-old LP rats (52.6 x 104 {+/-} 10.3 x 104 mm) as compared to NP rats (47.0 x 104 {+/-} 10.6 x 103 mm, p = 0.0007). In addition, LP rats exhibited anxiety-like behaviors compared to NP rats at 48 and 86 weeks of life.

Additionally, the estimated neuron number was unaltered in LP rats; however, glial and other cell numbers increased in LP compared to NP rats. Here, we showed unprecedented hippocampal deposition of brain-derived neurotrophic factor, {beta}-amyloid peptide (A{beta}), and tau protein in 88-week-old LP compared to age-matched NP offspring. To date, no predicted studies showed changes in hippocampal neuron and glial cell numbers in maternal protein-restricted elderly offspring. The current data suggest that maternal protein restriction has a high impact on lifespan and brain structure, and function.

Conclusionthe gestational protein restriction may accelerate hippocampal function loss, impacting learning/memory performance, and supposedly developing diseases similar to Alzheimers disease (AD) in elderly offspring. Thus, we propose that maternal protein restriction could be a probable, elegant, and novel method for constructing an AD-like model in adult male offspring.

sentences: The Systematic Optimization of Square Wave Electroporation for Six Commonly Used Human Cell Lines During cellular electroporation, the formation of transient pores allow for the diffusion of innately impermeable molecules. The diversity of cell and membrane structure results in unique properties with respect to sensitivity to electric fields. The growing use of human cell lines in biomedical research and technology has led to a demand for protocols that can effectively and economically perform electroporation. We electroporated six human cell lines using a fluorescent reporter to investigate the effects of pulse electric field strength, pulse duration, and DNA concentration during electroporation. It was found that the cell lines all responded to electric field strengths within 400-950V/cm with viability decreasing with increasing voltage. It was also observed that the concentration of DNA used directly impacts transfection efficiency and cell viability as well. To better characterize square wave electroporation, we adopted a model where the pulse is described by its energy density (J/L) with respect to the sample buffer volume. It was determined that the key electrical characteristics of electroporation can be generalized with this value to provide a simplified measure of pulse intensity. The resulting analysis was consistent with other models, indicating cell type specific optimal electrical and DNA concentrations.

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Genetic diversity of wild and cultivated Coffea canephora in northeastern DR Congo and the implications for conservation. PremiseMany cultivated coffee varieties descend from Coffea canephora, commonly known as Robusta coffee. The Congo Basin has a century long history of Robusta coffee cultivation and breeding, and is hypothesized to be the region of origin of many of the cultivated Robusta varieties. Since little is known about the genetic composition of C. canephora in this region, we assessed the genetic diversity of wild and cultivated C. canephora shrubs in the Democratic Republic of the Congo.

MethodsUsing 18 microsatellite markers, we studied the genetic composition of wild and backyard-grown C. canephora shrubs in the Tshopo and Ituri provinces, and from the INERA Yangambi Coffee Collection. We assessed genetic clustering patterns, genetic diversity, and genetic differentiation between populations.

Key resultsGenetic differentiation was relatively strong between wild and cultivated C. canephora shrubs, and both gene pools harbored multiple unique alleles. Strong genetic differentiation was also observed between wild populations. The level of genetic diversity in wild populations was similar to that of the INERA Yangambi Coffee Collection, but local wild genotypes were mostly missing from that collection. Shrubs grown in the backyards were genetically similar to the breeding material from INERA Yangambi.

ConclusionsMost C. canephora that is grown in local backyards originated from INERA breeding programs, while a few shrubs were obtained directly from surrounding forests. The INERA Yangambi Coffee Collection could benefit from an enrichment with local wild genotypes, to increase the genetic resources available for breeding purposes, as well as to support ex situ conservation.

sentences: Mismatch between bird species sensitivity and the protection of intact habitats across the Americas Protected areas, the most prevalent international policy mechanism for biodiversity conservation, are highly heterogeneous in their effectiveness at buffering ecosystems and species habitats from human pressure. Protected areas with intense human pressure cannot protect species that are highly sensitive to human activities. Here, we use 60 million bird observations from the eBird citizen science platform to estimate the sensitivity to human pressure of each bird species breeding in the Americas (Nearctic and Neotropical regions). We find that high-sensitivity species, while found in all ecoregions, are concentrated in the tropical biomes. Ecoregions with large proportions of high-sensitivity species do not have more intact protected habitat, resulting in a low coverage of intact protected habitat for many high-sensitivity species. What is more, 139 high-sensitivity species have little or no intact protected habitat within their distributions while being threatened with extinction. Finally, we show that protected area intactness is decreasing faster in ecoregions with many high-sensitivity species. Our results highlight a major mismatch between species conservation needs and the coverage of intact protected habitats, and will likely hamper the long-term effectiveness of protected areas at retaining species. We highlight ecoregions where the protection and management of intact habitats, complemented by the restoration of degraded ones, is urgently needed to avoid extinctions.

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SIMPATI: patient classifier identifies signature pathways as patient similarity networks for the disease prediction BACKGROUNDPathway-based patient classification is a supervised learning task which implies a model learning pathways as features to predict the classes of patients. The counterpart of enrichment tools for the pathway analysis are fundamental methods for clinicians and biomedical scientists. They allow to find signature cellular functions which help to define and annotate a disease phenotype. They provide results which lead human experts to manually classify patients. It is a paradox that pathwaybased classifiers which natively resolve this objective are not strongly developed. They could simulate the human way of thinking, decipher hidden multivariate relationships between the deregulated pathways and the disease phenotype, and provide more information than a probability value. Instead, there are currently only two classifiers of such kind, they require a nontrivial hyperparameter tuning, are difficult to interpret and lack in providing new insights. There is the need of new classifiers which can provide novel perspectives about pathways, be easy to apply with different biological omics and produce new data enabling a further analysis of the patients.

RESULTSWe propose Simpati, an innovative and interpretable patient classifier based on pathway-specific patient similarity networks. The first classifier to adopt ad-hoc novel algorithms for such graph type. It standardizes the biological high-throughput dataset of patients profiles with a propagation algorithm that considers the interconnected nature of the cells molecules for inferring a new activity score. This allows Simpati to classify with dense, sparse, and non-homogenous omic data. Simpati organizes patients molecules in pathways represented by patient similarity networks for being interpretable, handling missing data and preserving the patient privacy. A network represents patients as nodes and a novel similarity measure determines how much every pair act co-ordinately in a pathway. Simpati detects signature biological processes based on how much the topological properties of the related networks separate the patient classes. In this step, it includes a new cohesive subgroup detection algorithm to handle patients not showing the same pathway activity as the other class members. An unknown patient is then classified by a unique recommender system which considers how much is similar to known patients and distant from being an outlier. Simpati outperforms previously published classifiers on five cancer datasets described with two biological omics, classifies well with sparse data, identifies more relevant pathways associated to the patients disease than the competitors and has the lowest computational requirements.

CONCLUSIONSimpati can serve as generic-purpose pathway-based classifier of patient classes. It provides signature pathways to unveil the altered biological mechanisms of a disease phenotype and to classify patients according to the learnt pathway-specific similarities. The signature condition and patient prediction can be deciphered considering the patient similarity networks which must reveal the members of a patient class more cohesive and similar than the non-members. Simpati divides the pathways in up and downinvolved. Upinvolved when the signaling cascades generated by the altered molecules of the disease patients impact stronger the pathway than the ones of the control class. We provide an R implementation, a graphical user interface and a visualization function for the patient similarity networks. The software is available at: https://github.com/LucaGiudice/Simpati

sentences: Autoantibody and hormone activation of the thyrotropin G protein-coupled receptor Thyroid hormones are vital to growth and metabolism. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR). Autoantibodies that activate the TSHR pathologically increase thyroid hormones in Graves disease. How autoantibodies mimic TSH function remains unclear. We determined cryogenic-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. TSH selects an upright conformation of the extracellular domain due to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody selects a similar upright conformation of the extracellular domain. Conformational changes in the extracellular domain are transduced to the seven transmembrane domain via a conserved hinge domain, a tethered peptide agonist, and a phospholipid that binds within the seven transmembrane domain. Rotation of the TSHR ECD relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to G protein-coupled receptors with large extracellular domains.

sentences: New insights into large tropical tree mass and structure from direct harvest and terrestrial lidar A large portion of the terrestrial vegetation carbon stock is stored in the above-ground biomass (AGB) of tropical forests, but the exact amount remains uncertain, partly due to the lack of measurements. To date, accessible peer-reviewed data are available for just 10 large tropical trees in the Amazon that have been harvested and directly measured entirely via weighing. Here, we harvested four large tropical rainforest trees (stem diameter: 0.6-1.2m, height: 30-46m, AGB: 3960-18 584 kg) in intact old-growth forest in East Amazonia, and measured above-ground green mass, moisture content and woody tissue density. We first present rare ecological insights provided by these data, including unsystematic intra-tree variations in density, with both height and radius. We also found the majority of AGB was usually found in the crown, but varied from 42-62%. We then compare non-destructive approaches for estimating the AGB of these trees, using both classical allometry, and new lidar-based methods. Terrestrial lidar point clouds were collected pre-harvest, on which we fitted cylinders to model woody structure, enabling retrieval of volume-derived AGB. Estimates from this approach were more accurate than allometric counterparts (mean tree-scale relative error: 3% vs. 15%), and error decreased when up-scaling to the cumulative AGB of the four trees (1% vs. 15%). Further, whilst allometric error increased four-fold with tree size over the diameter range, lidar error remained constant. This suggests error in these lidar-derived estimates is random and additive. Were these results transferable across forest scenes, terrestrial lidar methods would reduce uncertainty in stand-scale AGB estimates, and therefore advance our understanding of the role of tropical forests in the global carbon cycle.

sentences: Northward Expansion of Amblyomma americanum (Acari: Ixodidae) into Southern Michigan Amblyomma americanum (Linnaeus) (Acari: Ixodidae) (lone star tick) is an aggressive, generalist parasite that vectors numerous important human and animal pathogens. In recent decades its geographic range has expanded northwards from endemic regions in the southeastern and southcentral US. In 2019 five questing A. americanum comprising two life stages were detected at one site in Berrien County, in southwestern Michigan, satisfying one CDC criterium for an established population for the first time in the state. To better characterize the northern extent of emerging A. americanum, we conducted active surveillance (i.e., drag sampling) in summer 2020 throughout Michigans southern counties and detected one adult A. americanum from each of six widespread sites, including where they had been detected in 2019. A larger established population was identified at another site in Berrien County, which yielded 691 A. americanum comprising three life stages. Questing tick phenologies at this site were similar to that reported for other regions. Statewide surveillance in 2021 revealed no A. americanum outside of Berrien County, but establishment criteria were met again at the two sites where established populations were first detected respectively in 2019 and 2020. These observations may represent the initial successful invasion of A. americanum into Michigan. Data from passive (1999-2020) and active surveillance (2004-2021) efforts, including a domestic animal sentinel program (2015-2018), are reported to provide context for this nascent invasion. Continued active surveillance is needed to help inform the public, medical professionals, and public health officials of the health risks associated with this vector.

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The division defect of a Bacillus subtilis minD noc double mutant can be suppressed by Spx-dependent and Spx-independent mechanisms During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, an intense ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here, we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a minD noc double mutant indicated that FtsZ formed proto-Z-rings at periodic inter-chromosome locations but that the rings failed to mature and become functional. Extragenic suppressor analysis indicated that a variety of mutations restored high temperature growth to the minD noc double mutant, and while many were likely pleiotropic, others implicated the proteolysis of the transcription factor Spx. Further analysis indicated that a Spx-dependent pathway activated the expression of ZapA, a protein that primarily compensates for the absence of Noc. Additionally, an Spx-independent pathway increased the activity of the divisome to reduce the length of the cytokinetic period. Finally, we provide evidence of an as-yet-unidentified protein that is activated by Spx and governs the frequency of polar division and minicell formation.

IMPORTANCEBacteria must properly position the location of the cell division machinery in order to grow, divide, and ensure each daughter cell receives one copy of the chromosome. In B. subtilis, cell division site selection is thought to depend on two systems called Min and Noc, and while neither is individually essential, cells fail to grow at high temperature when both are mutated. Here, we show that cell division fails in the absence of Min and Noc, not due to a defect in FtsZ localization, but rather a failure in the maturation of the cell division machinery. To understand what happens when the division machinery fails to mature, suppressor mutations that bypass the need for Min, Noc, or both were selected. Some of the mutants activated the Spx stress response pathway while others appeared to directly enhance divisome activity.

sentences: Biophysical basis of cellular multi-specificity encoded in a model molecular switch Molecular switches are central to signal transduction in protein interaction networks. One switch protein can independently regulate distinct cellular processes, but the molecular mechanisms enabling this functional multi-specificity remain unclear. Here we integrate system-scale cellular and biophysical measurements to study how a paradigm switch, the small GTPase Ran/Gsp1, achieves its functional multi-specificity. We make 55 targeted point mutations to individual interactions of Ran/Gsp1 and show through quantitative, systematic genetic and physical interaction mapping that Ran/Gsp1 interface perturbations have widespread cellular consequences that cluster by biological processes but, unexpectedly, not by the targeted interactions. Instead, the cellular consequences of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle, and cycle kinetics are allosterically tuned by distal interface mutations. We propose that the functional multi-specificity of Ran/Gsp1 is encoded by a differential sensitivity of biological processes to different kinetic parameters of the Gsp1 switch cycle, and that Gsp1 partners binding to the sites of distal mutations act as allosteric regulators of the switch. Similar mechanisms may underlie biological regulation by other GTPases and biological switches. Finally, our integrative platform to determine the quantitative consequences of cellular perturbations may help explain the effects of disease mutations targeting central switches.

sentences: Induction of Bdnf from promoter I following electroconvulsive seizures contributes to structural plasticity in neurons of the piriform cortex The efficacy of electroconvulsive therapy (ECT) as a treatment for psychiatric disorders, including major depressive disorder (MDD) is hypothesized to depend on induction of molecular and cellular events that trigger structural plasticity in neurons. Electroconvulsive seizures (ECS) in animal models can help to inform our understanding of how electroconvulsive therapy (ECT) impacts the brain. ECS induces structural plasticity in neuronal dendrites in many brain regions, including the piriform cortex, a highly epileptogenic region that has also been implicated in depression. ECS-induced structural plasticity is associated with differential expression of unique isoforms encoding the neurotrophin, brain-derived neurotrophic factor (BDNF), but the functional significance of these transcripts in dendritic plasticity is not clear. Here, we demonstrate that different Bdnf isoforms are expressed non-stochastically across neurons of the piriform cortex following ECS. Specifically, cells expressing Bdnf exon 1-containing transcripts show a unique spatial recruitment pattern in response to ECS. We further demonstrate that Bdnf Ex1 expression in these cells is necessary for ECS-induced dendritic spine plasticity.

sentences: Resetting H3K4me3, H3K27ac, H3K9me3 and H3K27me3 during the maternal-to-zygotic transition and blastocyst lineage specification in bovine embryos It remains poorly understood how histone modifications regulate changes in gene expression during preimplantation development. Using a bovine model, we profiled changes in two activating (H3K4me3 and H3K27ac) and two repressive (H3K9me3 and H3K27me3) marks in oocytes, 2-, 4- and 8-cell embryos (that developed in the presence or absence of the transcription inhibitor a-amanitin), morula, blastocysts, inner cell mass cells and trophectoderm. In oocytes, we find that broad bivalent domains of H3K4me3 and H3K27me3 mark developmental genes, and that prior to genome activation, H3K9me3 and H3K27me3 co-occupy gene bodies. During genome activation, chromatin accessibility is established before canonical H3K4me3 and H3K27ac, and although embryonic transcription is required for this active remodeling, it is dispensable for maintenance of pre-established histone marks. Finally, blastocyst lineages are defined by differential Polycomb repression and transcription factor activity. Overall, these results further support the use of bovine as a more appropriate model system than the mouse to study genome activation and cell lineage specification during human preimplantation development.

sentences: Pericytes and Astrocytes Instruct Glioblastoma Invasion, Proliferation, and Therapeutic Response within an Engineered Brain Perivascular Niche Model Glioblastoma (GBM) tumor cells are found in the perivascular niche microenvironment and are believed to associate closely with the brain microvasculature. However, it is largely unknown how the resident cells of the perivascular niche, such as endothelial cells, pericytes, and astrocytes, influence GBM tumor cell behavior and disease progression. We describe a three-dimensional in vitro model of the brain perivascular niche developed by encapsulating brain-derived endothelial cells, pericytes, and astrocytes in a gelatin hydrogel. We show that pericytes and astrocytes explicitly contribute to vascular architecture and maturation. We use co-cultures of patient-derived GBM tumor cells with brain microvascular cells to identify a role for pericytes and astrocytes in establishing a perivascular niche environment that modulates GBM cell invasion, proliferation, and therapeutic response. Engineered models provides unique insight regarding the spatial patterning of GBM cell phenotypes in response to a multicellular model of the perivascular niche. Critically, we show that engineered perivascular models provide an important resource to evaluate mechanisms by which inter- cellular interactions modulate GBM tumor cell behavior, drug response, and provide a framework to consider patient-specific disease phenotypes.

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Identification of hub genes and pathways in nonalcoholic steatohepatitis by integrated bioinformatics analysis BackgroundCurrently, non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in the world. Forecasting the short-term, up to 2025, NASH due to fibrosis is one of the leading causes of liver transplantation. Cohort studies revealed that non-alcoholic steatohepatitis (NASH) has a higher risk of fibrosis progression among NAFLD patients. Identifying differentially expressed genes helps to determine NASH pathogenic pathways, make more accurate diagnoses, and prescribe appropriate treatment.

Methods and ResultsIn this study, we found 11 NASH datasets by searching in the Gene Expression Omnibus (GEO) database. Subsequently, NASH datasets with low-quality control scores were excluded. Four datasets were analyzed with packages of R/Bioconductor. Then, all integrated genes were Imported into Cytoscape to illustrate the protein-protein interactions network. All hubs and nodes degree has been calculated to determine the hub genes with critical roles in networks.Possible correlations between expression profiles of mutual DEGs were identified employing Principal Component Analysis (PCA). Primary analyzed data were filtered based on gene expression (logFC > 1, logFC < -1) and adj-P-value (<0.05). Ultimately, among 379 DEGs, we selected the top 10 genes (MYC, JUN, EGR1, FOS, CCL2, IL1B, CXCL8, PTGS2, IL6, SERPINE1) as candidates among up and down regulated genes, and critical pathways such as IL-6, IL-17, TGF {beta}, and TNF were identified.

ConclusionThe present study suggests an important DEGs, biological processes, and critical pathways involved in the pathogenesis of NASH disease. Further investigations are needed to clarify the exact mechanisms underlying the development and progression of NASH disease.

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Towards reliable quantification of cell state velocities A few years ago, it was proposed to use the simultaneous quantification of unspliced and spliced messenger RNA (mRNA) to add a temporal dimension to high-throughput snapshots of single cell RNA sequencing data. This concept can yield additional insight into the transcriptional dynamics of the biological systems under study. However, current methods for inferring cell state velocities from such data (known as RNA velocities) are afflicted by several theoretical and computational problems, hindering realistic and reliable velocity estimation. We discuss these issues and propose new solutions for addressing some of the current challenges in consistency of data processing, velocity inference and visualisation. We translate our computational conclusion in two velocity analysis tools: one detailed method{kappa} -velo and one heuristic method eco-velo.

Author summarySingle cell transcriptomics has been used to study dynamical biological processes such as cell differentiation or disease progression. An ideal study of these systems would track individual cells in time but this is not directly feasible since cells are destroyed as part of the sequencing protocol. Because of asynchronous progression of cells, single cell snapshot datasets often capture cells at different stages of progression. The challenge is to infer both the overall direction of progression (pseudotime) as well as single cell specific variations in the progression. Computational methods development for inference of the overall direction are well advanced but attempts to address the single cell level variations of the dynamics are newer. La Manno et al. [1] proposed that simultaneous measurement of abundances of new (unspliced) and older (spliced) mRNA in the same single cell adds a temporal dimension to the data which can be used to infer the time derivative of single cells progression through the dynamical process. State-of-the-art methods for inference of cell state velocities from RNA-seq data (also known as RNA velocity) have multiple unaddressed issues. In this manuscript, we discuss these issues and propose new solutions. In previous works, agreement of RNA velocity estimations with pseudotime has been used as validation. We show that this in itself is not proof that the method works reliably and the overall direction of progression has to be distinguished from individual cells behaviour. We propose two new methods (one detailed and one cost efficient heuristic) for estimation and visualisation of RNA velocities and show that our methods faithfully capture the single-cell variances and overall trend on simulation. We further apply the methods to a dataset of developing mouse pancreas and show how the method can help us gain biological insight from real data.

sentences: Latitudinal patterns in intertidal ecosystem structure in West Greenland suggest resilience to climate change Climate change has ecosystem-wide cascading effects. Little is known, however, about the resilience of Arctic marine ecosystems to environmental change. Here we quantify and compare large-scale patterns in rocky intertidal biomass, coverage and zonation in six regions along a north-south gradient of temperature and ice conditions in West Greenland (60-72{degrees}N). We related the level and variation in assemblage composition, biomass and coverage to latitudinal-scale environmental drivers. Across all latitudes, the intertidal assemblage was dominated by a core of stress-tolerant foundation species that constituted >95% of the biomass. Hence, canopy-forming macroalgae, represented by Fucus distichus subsp. evanescens and F. vesiculosus and, up to 69 {degrees}N, also Ascophyllum nodosum, together with Semibalanus balanoides, occupied >70% of the vertical tidal range in all regions. Thus, a similar functional assemblage composition occurred across regions, and no latitudinal depression was observed. The most conspicuous difference in species composition from south to north was that three common species (the macroalgae Ascophyllum nodosum, the amphipod Gammarus setosus and the gastropod Littorina obtusata) disappeared from the mid-intertidal, although at different latitudes. There were no significant relationships between assemblage metrics and air temperature or sea ice coverage as obtained from weather stations and satellites, respectively. Although the mean biomass decreased >50% from south to north, local biomass in excess of 10 000 g ww m-2 was found even at the northernmost site, demonstrating the patchiness of this habitat and the effect of small-scale variation in environmental characteristics. Hence, using the latitudinal gradient in a space-for-time substitution, our results suggest that while climate modification may lead to an overall increase in the intertidal biomass in north Greenland, it is unlikely to drive dramatic functional changes in ecosystem structure in the near future. Our dataset provides an important baseline for future studies to verify these predictions for Greenlands intertidal zone.

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Genomic analyses of the extinct Sardinian dhole (Cynotherium sardous) reaveal its evolutionary history The Sardinian dhole (Cynotherium sardous)1 was an iconic and unique canid species of canid that was endemic of Sardinia and Corsica until it became extinct at the end of the Late Pleistocene2-5. Given its peculiar dental morphology, small body size and high level of endemism, several canids have been proposed as possible ancestors of the Sardinian dhole, including the Asian dhole and African hunting dog ancestor 3,6-9. Morphometric analyses3,6,8-12 have failed to clarify the evolutionary relationship with other canids.

We sequenced the genome of a ca 21,100 year old Sardinian dhole in order to understand its genomic history and clarify its phylogenetic position. We found it represents a separate taxon from all other living canids from Eurasia, Africa and North America, and that the Sardinian and Asian dhole lineages diverged ca 885 ka. We additionally detected historical gene flow between the Sardinian and Asian dhole lineages, that ended approximately 500-300 ka, when the landbridge between Sardinia and mainland Italy was broken, severing their population connectivity. Our sample showed low genome-wide diversity compared to other extant canids - probably a result of the long-term isolation - that could have contributed to the subsequent extinction of the Sardinian dhole.

sentences: A novel approach to comparative RNA-Seq does not support a conserved set of genes underlying animal regeneration Molecular studies of animal regeneration typically focus on conserved genes and signaling pathways that underlie morphogenesis. To date, a holistic analysis of gene expression across animals has not been attempted, as it presents a suite of problems related to differences in experimental design and gene homology. By combining orthology analysis with a novel statistical method for testing gene enrichment across large datasets, we are able to test whether biological processes across organisms share transcriptional regulation. We applied this method to six publicly available RNA-seq datasets from diverse examples of animal regeneration. We recovered 160 conserved orthologous gene clusters, which are enriched in structural genes as opposed to those regulating morphogenesis. A breakdown of gene presence/absence provides only limited support for the conservation of pathways typically implicated in regeneration, such as Wnt signaling and cell pluripotency. Specifically, these pathways are only conserved if we allow gene paralogs to be interchangeable through evolution. Overall, our analysis does not support the hypothesis that a shared set of ancestral genes underlie regeneration mechanisms in animals. The methods described in this paper will be broadly applicable for studying the genetic underpinnings of traits across distantly related organisms.

sentences: Bees can be trained to identify SARS-CoV-2 infected samples. The COVID-19 pandemic has illustrated the need for the development of fast and reliable testing methods for novel, zoonotic, viral diseases in both humans and animals. Pathologies lead to detectable changes in the Volatile Organic Compound (VOC) profile of animals, which can be monitored, thus allowing the development of a rapid VOC-based test. In the current study, we successfully trained honeybees (Apis mellifera) to identify SARS-CoV-2 infected minks (Neovison vison) thanks to Pavlovian conditioning protocols. The bees can be quickly conditioned to respond specifically to infected minks odours and could therefore be part of a wider SARS-CoV-2 diagnostic system. We tested two different training protocols to evaluate their performance in terms of learning rate, accuracy and memory retention. We designed a non-invasive rapid test in which multiple bees are tested in parallel on the same samples. This provided reliable results regarding a subjects health status. Using the data from the training experiments, we simulated a diagnostic evaluation trial to predict the potential efficacy of our diagnostic test, which yielded a diagnostic sensitivity of 92% and specificity of 86%. We suggest that a honeybee-based diagnostics can offer a reliable and rapid test that provides a readily available, low-input addition to the currently available testing methods. A honeybee-based diagnostic test might be particularly relevant for remote and developing communities that lack the resources and infrastructure required for mainstream testing methods.

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The effect of indoor daylight spectrum and intensity on viability of indoor pathogens on different surface materials Built environments play a key role in the transmission of infectious diseases. Ventilation rates, air temperature and humidity affect airborne transmission while cleaning protocols, material properties and light exposure can influence viability of pathogens on surfaces. We investigated how indoor daylight intensity and spectrum through electrochromic (EC) windows can impact the growth rate and viability of indoor pathogens on different surface materials (polyvinyl chloride (PVC) fabric, polystyrene (PS), and glass) compared to traditional blinds. Our results showed that tinted EC windows let in higher energy, shorter wavelength daylight than those with clear window and blind. The growth rates of pathogenic bacteria and fungi were significantly lower in spaces with EC windows compared to blinds: nearly 100% growth rate reduction was observed when EC windows were in their clear state followed by 41-100% reduction in bacterial growth rate and 26-42% reduction in fungal growth rate when EC windows were in their darkest tint. Moreover, bacterial viabilities were significantly lower on PVC fabric when they were exposed to indoor light at EC-tinted window. These findings are deemed fundamental to the design of healthy modern buildings, especially those that encompass sick and vulnerable individuals.

PO_SCPLOWRACTICALC_SCPLOW IO_SCPLOWMPLICATIONSC_SCPLOWO_LILight is an important factor that influences occupant health.
C_LIO_LIHealthcare Associated Infections (HAI) bring substantial costs on the healthcare systems hence new disinfection methods are always needed to minimize fomites especially with the increasing antibiotic resistance.
C_LIO_LIWe found that indoor light modulated by the EC smart windows can significantly reduce the growth rate and viability of pathogenic bacteria and fungi, which is mainly due to the high energy blue light spectrum at wavelength of 400-500nm.
C_LIO_LIPathogenic fungi are found to be more affected by the indoor light intensity, while indoor bacteria on surfaces are more susceptible to the light spectrums.
C_LIO_LIThese results also demonstrate the promising potential of indoor daylight exposure as an alternative for fomite disinfection strategy and expand the benefits of EC window as part of healthy building design in the future.
C_LI

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Known mechanisms account for less than half of antimicrobial resistance in a diverse collection of non-aureus staphylococci. IntroductionNon-aureus staphylococci (NAS) are implicated in many healthcare-acquired infections and an understanding of the genetics of antimicrobial resistance in NAS is important in relation to both clinical intervention and the role of NAS as a reservoir of resistance genes.

Gap statementThe burden of antimicrobial resistance in NAS, particularly to clinically relevant antimicrobials, is understudied.

MethodologyWe sourced 394 NAS isolates from clinical samples, healthy human volunteers, animals and type cultures and subjected them to agar dilution susceptibility testing against eight antimicrobials. We performed whole genome sequencing on 316 isolates and analysed these genotypically for the presence of genetic mechanisms responsible for the phenotypic levels of reduced antimicrobial susceptibility.

ResultsCefoxitin is used to screen for methicillin resistance in S. aureus, as it stimulates expression of mecA. We observed 174 isolates with an MIC of at least 4 g/ml to cefoxitin, of which sequencing revealed 47.6% (80/168) did not harbour a known mec homologue. Seven clinical NAS isolates displayed high daptomycin minimum inhibitory concentrations (MICs) (>4 g/ml), with no known mechanism identified. Differences in MICs against erythromycin were attributable to the presence of different resistance genes (msrA and ermC). In total, 49% (187/394) of isolates displayed reduced susceptibility to three or more of the antimicrobials tested.

ConclusionsThe widespread presence of reduced antimicrobial susceptibility in NAS is a concern, with an increased likelihood of (1) harder to treat infections caused directly by NAS, and (2) resistance genes being passed on to other bacteria via horizontal gene transfer, both of which have clinical implications for treatment and management of patients.

sentences: Sources of information waste in neuroimaging:mishandling structures, thinking dichotomously, and over-reducing data Neuroimaging relies on separate statistical inferences at tens of thousands of spatial locations. Such massively univariate analysis typically requires an adjustment for multiple testing in an attempt to maintain the family-wise error rate at a nominal level of 5%. First, we examine three sources of substantial information loss that are associated with the common practice under the massively univariate framework: (a) the hierarchical data structures (spatial units and trials) are not well maintained in the modeling process; (b) the adjustment for multiple testing leads to an artificial step of strict thresholding; (c) information is excessively reduced during both modeling and result reporting. These sources of information loss have far-reaching impacts on result interpretability as well as reproducibility in neuroimaging. Second, to improve inference efficiency, predictive accuracy, and generalizability, we propose a Bayesian multilevel modeling framework that closely characterizes the data hierarchies across spatial units and experimental trials. Rather than analyzing the data in a way that first creates multiplicity and then resorts to a post hoc solution to address them, we suggest directly incorporating the cross-space information into one single model under the Bayesian framework (so there is no multiplicity issue). Third, regardless of the modeling framework one adopts, we make four actionable suggestions to alleviate information waste and to improve reproducibility: 1) abandon strict dichotomization, 2) report full results, 3) quantify effects, and 4) model data hierarchies. We provide examples for all of these points using both demo and real studies, including the recent NARPS investigation.

sentences: Prolactin action is necessary for parental behavior in male mice Parental care is critical for successful reproduction in mammals. In comparison to maternal care, the neuroendocrine mechanisms supporting paternal care are less well-studied. Laboratory mice show a mating-induced suppression of infanticide (normally observed in virgins) and onset of paternal behavior. Using this model, we sought to investigate whether the hormone prolactin plays a role in paternal behavior, as it does for maternal behavior. First, using c-fos immunoreactivity in Prlr-IRES-Cre-tdtomato reporter mouse sires, we show that the circuitry activated during paternal interactions contains prolactin-responsive neurons, including the medial preoptic area, bed nucleus of the stria terminalis, and medial amygdala. To evaluate whether prolactin action is required for the establishment and display of paternal behavior, we conditionally deleted the prolactin receptor (Prlr) from 3 distinct cell types: glutamatergic, GABAergic, and CaMKII-expressing forebrain neurons. Prlr-deletion from CaMKII-expressing forebrain neurons, but not from glutamatergic or GABAergic cells, resulted in a profound effect on paternal behavior, as none of these males completed the pup retrieval task. Finally, although sires do not show an acute increase in circulating prolactin levels in response to pups, pharmacological blockade of prolactin-release at the time of pup exposure resulted in failure to retrieve pups, similar to when the Prlr was deleted from CaMKII neurons, with prolactin administration rescuing this behavior. Taken together, our data show that paternal behavior in sires is dependent on basal levels of circulating prolactin acting at the Prlr on CaMKII-expressing neurons. These new data in male mice demonstrate that prolactin has a similar action in both sexes to promote parental care.

sentences: Recut: a Concurrent Framework for Sparse Reconstruction of Neuronal Morphology Advancement in modern neuroscience is bottlenecked by neural reconstruction, a process that extracts 3D neuron morphology (typically in tree structures) from image volumes at the scale of hundreds of GBs. We introduce Recut, an automated and accelerated neural reconstruction pipeline, which provides a unified, and domain specific sparse data representation with 79x reduction in the memory footprint. Recuts reconstruction can process 111 Kneurons/day or 79 TB/day on a 24-core workstation, placing the throughput bottleneck back on microscopic imaging time. Recut allows the full brain of a mouse to be processed in memory on a single server, at 89.5x higher throughput over existing I/O-bounded methods. Recut is also the first fully parallelized end-to-end automated reconstruction pipeline for light microscopy, yielding tree morphologies closer to ground truth than the state-of-the-art while removing involved manual steps and disk I/O overheads. We also optimized pipeline stages to linear algorithmic complexity for scalability in dense settings and allow the most timing-critical stages to optionally run on accelerated hardware.

sentences: Network reconstruction and modelling made reproducible with moped Mathematical modeling of metabolic networks is a powerful approach to investigate the underlying principles of metabolism and growth. Such approaches include, amongst others, differential equation based modeling of metabolic systems, constraint based modeling and topological analysis of metabolic networks. Most of these methods are well established and are implemented in numerous software packages, but these are scattered between different programming languages, packages and syntaxes. This complicates establishing straight forward pipelines integrating model construction and simulation. We present the Python package moped which serves as an integrative hub for reproducible construction, modification, curation and analysis of metabolic models. moped supports draft reconstruction of models directly from genome/proteome sequences and path-way/genome databases utilizing GPR annotations, providing a completely reproducible model construction and curation process within executable Python scripts. Alternatively, existing models published in SBML format can be easily imported. Models are represented as Python objects, for which a wide spectrum of easy-to-use modification and analysis methods exist. The model structure can be manually altered by adding, removing or modifying reactions, and gap filling reactions can be found and inspected. This greatly supports the development of draft models, as well as the curation and testing of models. Moreover, moped provides several analysis methods, in particular including the calculation of biosynthetic capacities using metabolic network expansion. The integration with other Python based tools is facilitated through various model export options. For example, a model can be directly converted into a CobraPy object for constraint-based analyses. moped is a fully documented and expandable Python package. We demonstrate the capability to serve as a hub for integrating reproducible model construction and curation, database import, topological analysis and export for constraint-based analyses.

sentences: Vocal tract allometry in a mammalian vocal learner Acoustic allometry occurs when features of animal vocalisations can be predicted from body size measurements. Despite this being considered the norm, allometry sometimes breaks, resulting in species sounding smaller or larger than expected for their size. A recent hypothesis suggests that allometry-breaking mammals cluster into two groups: those with anatomical adaptations to their vocal tracts and those capable of learning new sounds (vocal learners). Here we test which mechanism is used to escape from acoustic allometry by probing vocal tract allometry in a proven mammalian vocal learner, the harbour seal (Phoca vitulina). We test whether vocal tract structures and body size scale allometrically in 68 young individuals. We find that both body length and body mass accurately predict vocal tract length and one tracheal dimension. Independently, body length predicts vocal fold length while body mass predicts a second tracheal dimension. All vocal tract measures are larger in weaners than in pups and some structures are sexually dimorphic within age classes. We conclude that harbour seals do comply with anatomical allometric constraints. However, allometry between body size and vocal fold length seems to emerge after puppyhood, suggesting that ontogeny may modulate the anatomy-learning distinction previously hypothesised as clear-cut. We suggest that seals, like other species producing signals that deviate from those expected from their vocal tract dimensions, may break allometry without morphological adaptations. In seals, and potentially other vocal learning mammals, advanced neural control over vocal organs may be the main mechanism for breaking acoustic allometry.

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Nucleus accumbens neurons encode initiation and vigor of reward approach behavior The nucleus accumbens (NAc) is considered an interface between motivation and action, with NAc neurons playing an important role in promoting reward approach. However, the encoding by NAc neurons that contribute to this role remains unknown. Here, we trained male rats to find rewards in an 8-arm radial maze. The activity of 62 neurons, mostly in the shell of the NAc, were recorded while rats ran towards each reward place. General linear model (GLM) analysis showed that variables related to the vigor of the locomotor approach, like speed and acceleration, and the fraction of the approach run completed were the best predictors of the firing rate for most NAc neurons. Nearly 23% of the recorded neurons, here named locomotion-off cells, were inhibited during the entire approach run, suggesting that reduction in firing of these neurons promotes initiation of locomotor approach. Another 24% of the neurons presented a peak of activity during acceleration followed by a valley during deceleration (peak-valley cells). Together, these neurons accounted for most of the speed and acceleration encoding identified in the GLM analysis. Cross-correlations between firing and speed indicated that the spikes of peak-valley cells were followed by increases in speed, suggesting that the activity of these neurons drives acceleration. In contrast, a further 19% of neurons presented a valley during acceleration followed by a peak just prior to or after reaching reward (valley-peak cells). These findings suggest that these three classes of NAc neurons control the initiation and vigor of the locomotor approach to reward.

Significance StatementDeciphering the mechanisms by which the NAc controls the vigor of motivated behavior is critical to better understand and treat psychiatric conditions in which motivation is dysregulated. Manipulations of the NAc profoundly impair subjects ability to spontaneously approach reward-associated locations, preventing them from exerting effort to obtain reward. Here, we identify for the first time specific activity of NAc neurons in relation to spontaneous approach behavior. We discover three classes of neurons that could control initiation of movement and the speed vs. time trajectory during locomotor approach. These results suggest a prominent but heretofore unknown role for the NAc in regulating the kinematics of reward approach locomotion.

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The C-terminal PARP domain of the long ZAP isoform contributes essential effector functions for CpG-directed antiviral activity The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNA and, through the recruitment of TRIM25, KHNYN and other cellular RNA degradation machinery, target them for degradation or prevent their translation. ZAPs activity requires the N-terminal RNA binding domain that selectively binds CpG-containing RNA. However, much less is known about the functional contribution of the remaining domains. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain of the long ZAP isoform (ZAP-L) is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the inactive catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks CpG-dependent antiviral activity despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-Ls CpG-directed antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in the ZAP-L-mediated antiviral activity.

Author summaryCell-intrinsic antiviral factors, such as the zinc-finger antiviral protein (ZAP), provide a first line of defence against viral pathogens. ZAP acts by selectively binding CpG dinucleotide-rich RNAs, which are more common in some viruses than their vertebrate hosts, leading to their degradation. Here, we show that the ability to target these foreign elements is not only dependent on ZAPs N-terminal RNA-binding domain, but additional determinants in the central and C-terminal regions also regulate this process. The PARP domain and its associated CaaX box, are crucial for ZAPs CpG-specific activity and required for optimal binding to cofactors TRIM25 and KHNYN. Furthermore, a CaaX box, known to mediate post-translational modification by a hydrophobic S-farnesyl group, caused re-localization of ZAP from the cytoplasm and increased its association with intracellular membranes. This change in ZAPs distribution was essential for inhibition of both a ZAP-sensitized HIV-1 and SARS-CoV-2. Our work unveils how the determinants outside the CpG RNA-binding domain assist ZAPs antiviral activity and highlights the role of S-farnesylation and membrane association in this process.

sentences: A reference genome sequence resource for the sugar beet root rot pathogen Aphanomyces cochlioides Aphanomyces cochlioides, the causal agent of damping-off and root rot of sugar beet (Beta vulgaris L.), is a soil-dwelling oomycete responsible for yield losses in all major sugar beet growing regions. Currently, genomic resources for A. cochlioides are limited. Here we report a de novo genome assembly using a combination of long-read MinION (Oxford Nanopore Technologies) and short-read Illumina sequence data for A. cochlioides isolate 103-1, from Breckenridge, MN. The assembled genome was 76.3 Mb, with a contig N50 of 2.6 Mb. The reference assembly was annotated and was composed of 32.1% repetitive elements and 20,274 gene models. This high-quality genome assembly of A. cochlioides will be a valuable resource for understanding genetic variation, virulence factors, and comparative genomics of this important sugar beet pathogen.

sentences: Genomic Surveillance of COVID-19 Variants with Language Models and Machine Learning The global efforts to control COVID-19 are threatened by the rapid emergence of novel SARS-CoV-2 variants that may display undesirable characteristics such as immune escape, increased transmissibility or pathogenicity. Early prediction for emergence of new strains with these features is critical for pandemic preparedness. We present Strainflow, a supervised and causally predictive model using unsupervised latent space features of SARS-CoV-2 genome sequences. Strainflow was trained and validated on 0.9 million sequences for the period December, 2019 to June, 2021 and the frozen model was prospectively validated from July, 2021 to December, 2021. Strainflow captured the rise in cases two months ahead of the Delta and Omicron surges in most countries including the prediction of a surge in India as early as beginning of November, 2021. Entropy analysis of Strainflow unsupervised embeddings clearly reveals the explore-exploit cycles in genomic feature-space, thus adding interpretability to the deep learning based model. We also conducted codon-level analysis of our model for interpretability and biological validity of our unsupervised features. Strainflow application is openly available as an interactive web-application for prospective genomic surveillance of COVID-19 across the globe.

sentences: Adolescent Binge Drinking is Associated with Accelerated Decline of Gray Matter Volume The age- and time-dependent effects of binge-drinking on adolescent brain development have not been well characterized even though binge drinking is a health crisis among adolescents. The impact of binge drinking on gray matter volume development was examined using longitudinal data from the National Consortium on Alcohol and NeuroDevelopment in Adolescence (NCANDA). Non-binge drinkers (n=177) were matched to binge drinkers (n=164) on potential confounders. Number of binge drinking episodes in the past year was linked to decreased volumes for total gray matter, frontal, parietal, temporal, and occipital lobes (ps<.001). Interactions of binge drinking episodes and age demonstrated stronger effects in younger subjects for total gray matter, frontal, temporal, and occipital lobes (ps<.001). Subsequent models included binge drinking coded in multiple ways. Models sensitive to number of episodes and temporal proximity to outcomes provided the best fits. Declines in gray matter volume association with binge drinking are potentially related to changes in cognition frequently reported among binge drinking adolescents. Results underscore the potential importance of delaying initiation of binge drinking and provide evidence for a dose-response relationship of binge drinking to gray matter decline. Temporally proximal binge drinking was associated more strongly with gray matter decline, suggesting the potential for recovery.

sentences: Zetaproteobacteria pan-genome reveals candidate gene cluster for twisted stalk biosynthesis and export Twisted stalks are morphologically unique bacterial extracellular organo-metallic structures containing Fe(III) oxyhydroxides that are produced by microaerophilic Fe(II)-oxidizers belonging to the Betaproteobacteria and Zetaproteobacteria. Understanding the underlying genetic and physiological mechanisms of stalk formation is of great interest based on their potential as novel biogenic nanomaterials and their relevance as putative biomarkers for microbial Fe(II) oxidation on ancient Earth. Despite the recognition of these special biominerals for over 150 years, the genetic foundation for the stalk phenotype has remained unresolved. Here we present a candidate gene cluster for the biosynthesis and secretion of the stalk organic matrix that we identified with a trait-based analyses of a pan-genome comprising 16 Zetaproteobacteria isolate genomes. The "stalk formation in Zetaproteobacteria" (sfz) cluster comprises six genes (sfz1-sfz6), of which sfz1 and sfz2 were predicted with functions in exopolysaccharide synthesis, regulation, and export, sfz4 and sfz6 with functions in cell wall synthesis manipulation and carbohydrate hydrolysis, and sfz3 and sfz5 with unknown functions. The stalk-forming Betaproteobacteria Ferriphaselus R-1 and OYT-1, as well as dread-forming Zetaproteobacteria Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8 contain distant sfz gene homologues, whereas stalk-less Zetaproteobacteria and Betaproteobacteria lack the entire gene cluster. Our pan-genome analysis further revealed a significant enrichment of clusters of orthologous groups (COGs) across all Zetaproteobacteria isolate genomes that are associated with the regulation of a switch between sessile and motile growth controlled by the intracellular signaling molecule c-di-GMP. Potential interactions between stalk-former unique transcription factor genes, sfz genes, and c-di-GMP point towards a c-di-GMP regulated surface attachment function of stalks during sessile growth.

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RCytoGPS: An R Package for Reading and Visualizing Cytogenetics Data SummaryCytogenetics data, or karyotypes, are among the most common clinically used forms of genetic data. Karyotypes are stored as standardized text strings using the International System for Human Cytogenomic Nomenclature (ISCN). Historically, these data have not been used in large-scale computational analyses due to limitations in the ISCN text format and structure. Recently developed computational tools such as CytoGPS have enabled large-scale computational analyses of karyotypes. To further enable such analyses, we have now developed RCytoGPS, an R package that takes JSON files generated from CytoGPS.org and converts them into objects in R. This conversion facilitates the analysis and visualizations of karyotype data. In effect this tool streamlines the process of performing large-scale karyotype analyses, thus advancing the field of computational cytogenetic pathology.

Availability and ImplementationFreely available at https://CRAN.R-project.org/package=RCytoGPS

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Spatial dynamics and vaccine-induced fitness changes of Bordetella pertussis Competitive interactions between pathogen strains drive infection risk. Vaccines are thought to perturb strain diversity through shifts in immune pressures, however, this has rarely been measured due to inadequate data and analytical tools. Bordetella pertussis (B. pertussis), responsible for 160,000 deaths annually1, provides a rare natural experiment as many countries have switched from whole cell vaccines to acellular vaccines, which have very different immunogenic properties2,3. Here we use 3,344 sequences from 23 countries and build phylogenetic models to reveal that B. pertussis has substantial diversity within communities, with the relative fitness of local genotypes changing in response to switches in vaccine policy. We demonstrate that the number of transmission chains circulating within subnational regions is strongly associated with host population size. It takes 5-10 years for individual lineages to be homogeneously distributed throughout Europe or the United States. Increased fitness of pertactin-deficient strains following implementation of acellular vaccines, but reduced fitness otherwise, can explain long-term genotype dynamics. These findings highlight the role of national vaccine policies in shifting local diversity of a pathogen that still poses a large burden on global public health.

sentences: Authentication of In Situ Measurements for Thoracic Aortic Aneurysms in Mice Aortic diameter is a standard parameter for defining disease severity of thoracic aortic aneurysms. In mouse studies, aortic diameters can be measured in situ directly, but this approach has a potential confounder of underestimation due to the absence of physiological arterial blood pressure. In the present study, we developed an in situ approach for authentic aortic measurements. Thoracic aortic aneurysms were induced by beta-aminopropionitrile (BAPN, 0.5% wt/vol) administration in 4-week-old male C57BL/6J mice. Ultrasonography was performed to examine aortic dimensions, and mice with thoracic aortic dilatations were terminated subsequently. After saline perfusion through the left ventricle, periaortic tissues were removed to expose thoracic aortas. Optimal cutting temperature (OCT) compound was injected via the left ventricle to maintain aortic patency. In situ aortic images were captured pre- and post-OCT injection. In mice with severe thoracic aortic aneurysms, smaller aortic diameters were observed prior to OCT injection compared to ultrasound measurements, while aortic diameters in situ after OCT were comparable to diameters measured using ultrasound. A telemetry system demonstrated that maximal luminal pressures during 150 ul of OCT injection were 90 mmHg. Immunostaining for CD31 revealed that endothelial cells were preserved in the intima after OCT injection. These results indicate that OCT injection does not cause aortic damages due to excess pressures. In conclusion, in situ imaging with OCT injection provides authentic aortic measurements without overt aortic damage in mice with thoracic aortic aneurysms.

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Climate, latitude, and land cover predict flying insect biomass across a German malaise trap network O_LIAmong the many concerns for biodiversity in the Anthropocene, recent reports of flying insect loss are particularly alarming, given their importance as pollinators and as a food source for many predators. Few insect monitoring programs cover large spatial scales required to provide more generalizable estimates of insect responses to global change drivers.
C_LIO_LIWe ask how climate and surrounding habitat affect flying insect biomass and day of peak biomass using data from the first year of a new standardized distributed monitoring network at 84 locations across Germany comprising spatial gradient of land-cover types from protected to urban areas.
C_LIO_LIFlying insect biomass increased linearly with monthly temperature across Germany. However, the effect of temperature on flying insect biomass flipped to negative in the hot months of June and July when local temperatures most exceeded long-term averages.
C_LIO_LILand-cover explained little variation in insect biomass, but biomass was lowest in forested sites. Grasslands, pastures and orchards harbored the highest insect biomass. The date of peak biomass was primarily driven by surrounding land-cover type, with grasslands especially having earlier insect biomass phenologies.
C_LIO_LIStandardized, large-scale monitoring is pivotal to uncover underlying processes of insect decline and to develop climate-adapted strategies to promote insect diversity. In a temperate climate region, we find that the benefits of temperature on flying insect biomass diminish in a German summer at locations where temperatures most exceeded long-term averages. These results highlighting the importance of local adaptation in climate change-driven impacts on insect communities.
C_LI

sentences: A systematic benchmark of Nanopore long read RNA sequencing for transcript level analysis in human cell lines The human genome contains more than 200,000 gene isoforms. However, different isoforms can be highly similar, and with an average length of 1.5kb remain difficult to study with short read sequencing. To systematically evaluate the ability to study the transcriptome at a resolution of individual isoforms we profiled 5 human cell lines with short read cDNA sequencing and Nanopore long read direct RNA, amplification-free direct cDNA, PCR-cDNA sequencing. The long read protocols showed a high level of consistency, with amplification-free RNA and cDNA sequencing being most similar. While short and long reads generated comparable gene expression estimates, they differed substantially for individual isoforms. We find that increased read length improves read-to-transcript assignment, identifies interactions between alternative promoters and splicing, enables the discovery of novel transcripts from repetitive regions, facilitates the quantification of full-length fusion isoforms and enables the simultaneous profiling of m6A RNA modifications when RNA is sequenced directly. Our study demonstrates the advantage of long read RNA sequencing and provides a comprehensive resource that will enable the development and benchmarking of computational methods for profiling complex transcriptional events at isoform-level resolution.

sentences: The polyglutamine amyloid nucleus in living cells is a monomer with competing dimensions of order A long-standing goal of the study of amyloids has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of that event has made it inaccessible to classical biochemistry, structural biology, and computational approaches. Here, we addressed that limitation by measuring the dependence of amyloid formation on concentration and conformational templates in living cells, whose volumes are sufficiently small to resolve the outcomes of independent nucleation events. We characterized numerous rationally designed sequence variants of polyglutamine (polyQ), a polypeptide that precipitates Huntingtons and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. This effort uncovered a pattern of approximately twelve Qs, only for polypeptides exceeding the clinical length threshold, that allow for amyloid nucleation to occur spontaneously within single polypeptides. Nucleation was inhibited by intermolecular phase separation. Using atomistic molecular dynamics simulations, we found that the pattern encodes a minimal steric zipper of interdigitated side chains. Lateral growth of the steric zipper competed with axial growth to produce "pre-amyloid" oligomers. By illuminating the structural mechanism of polyQ amyloid formation in cells, our findings reveal a potential molecular etiology for polyQ diseases, and may provide a roadmap for the design of new therapies.

sentences: Interacting phenotypes and the coevolutionary process: Interspecific indirect genetic effects alter coevolutionary dynamics Coevolution occurs when species interact to influence one anothers fitness, resulting in reciprocal evolutionary change. In many coevolving lineages, trait expression in one species is modified by the genotypes and phenotypes of the other, forming feedback loops reminiscent of models of intraspecific social evolution. Here, we adapt the theory of within-species social evolution, characterized by indirect genetic effects and social selection imposed by interacting individuals, to the case of interspecific interactions. In a trait-based model, we derive general expressions for multivariate evolutionary change in two species and the expected between-species covariance in evolutionary change when selection varies across space. We show that reciprocal interspecific indirect genetic effects can dominate the coevolutionary process and drive patterns of correlated evolution beyond what is expected from direct selection alone. In extreme cases, interspecific indirect genetic effects can lead to coevolution when selection does not covary between species or even when one species lacks genetic variance. Moreover, our model indicates that interspecific indirect genetic effects may interact in complex ways with cross-species selection to determine the course of coevolution. Importantly, our model makes empirically testable predictions for how different forms of reciprocal interactions contribute to the coevolutionary process.

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Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in PSC. ObjectiveConflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. Goal: Define complex interactions between resident microbes and their association in PSC patients by studying antibiotic-treated specific pathogen-free (SPF) and germ-free (GF) multi-drug-resistant 2 deficient (mdr2-/-) mice.

DesignWe measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, fecal 16s rRNA gene profiling, and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2-/- mice and targeted metagenomic analysis in PSC patients.

ResultsGF mdr2-/- mice had exaggerated hepatic inflammation and fibrosis with 100% mortality by 8 weeks; early SPF autologous stool transplantation rescued liver-related mortality. Broad-spectrum antibiotics and vancomycin alone accelerated disease in weanling SPF mdr2-/- mice, indicating that vancomycin-sensitive resident microbiota protect against hepatobiliary disease. Vancomycin treatment selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased cytolysin-expressing E. faecalis and E. coli liver translocation; colonization of gnotobiotic mdr2-/- mice with translocated E. faecalis and E. coli strains accelerated liver inflammation and mortality. Lachnospiraceae colonization of antibiotic pre-treated mdr2-/- mice reduced liver fibrosis, inflammation and translocation of pathobionts, while Lachnospiraceae-produced SCFA decreased fibrosis. Fecal E. faecalis/ Enterobacteriaceae was positively and Lachnospiraceae was negatively associated with PSC patients clinical severity Mayo risk scores.

ConclusionsWe identified specific functionally protective and detrimental resident bacterial species in mdr2-/- mice and PSC patients with associated clinical outcomes. These insights may guide personalized targeted therapeutic interventions in PSC patients.

sentences: Association of a common genetic variant with Parkinson's disease is propagated through microglia Studies of the genetic basis of Parkinsons disease (PD) have identified many disease-associated genetic variants, but the mechanisms linking variants to pathogenicity are largely unknown. PD risk is attributed to both coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene and to common non-coding variation upstream of the LRRK2 locus. Here we show that the influence of genotype at non-coding variant rs76904798 on LRRK2 expression is propagated specifically through microglia, in contrast to evaluations based on general rather than genotype-dependent expression. We find evidence of microglia-specific regulatory regions that may modulate LRRK2 expression using single nuclei sequencing analyses of human frontal cortex and confirm these results in a human induced pluripotent stem cell-derived microglia model. Our study demonstrates that cell type is an important consideration in interrogation of the role of non-coding variation in disease pathogenesis.

sentences: High temperature molecular motions within a model protomembrane architecture Modern phospholipid membranes are known to be in a functional, physiological state, corresponding to the liquid crystalline phase, only under very precise external conditions. The phase is characterised by specific lipid motions, which seem mandatory to permit sufficient flexibility and stability for the membrane. It can be assumed that similar principles hold for proto-membranes at the origin of life although they were likely composed of simpler, single chain fatty acids and alcohols. In the present study we investigated molecular motions of four types of model membranes to shed light on the variations of dynamics and structure as a function of temperature as protocells might have existed close to hot vents. We find a clear hierarchy among the flexibilities of the samples, where some structural parameters seem to depend on the lipids used while others do not.

sentences: Increased frequencies of IgH locus suicide recombination points on Chronic Lymphocytic Leukemia with low rate of AID related somatic mutations, cMYC overexpression and short telomeres In normal activated B-cells, Activation Induced-cytidine deaminase (AID) is absolutely required for immunoglobulin (Ig) class switch recombination (CSR) and IGHV somatic hypermutation (SHM). AID is also implicated in the Locus Suicide Recombination (LSR) of the Ig heavy (IgH) locus, resulting in the deletion of the IgH constant part. Chronic lymphocytic leukemia (CLL) is an indolent non-Hodgkin B-cell lymphoma characterized by tumor CLL B-cells that weakly express a B cell receptor (BCR) on their surface. The great majority of CLL tumor B-cells are non class-switched. Searching for abnormalities of IgH locus recombination in CLL, we investigated CSR and LSR in samples from CLL patients (N=47) with high blood tumor cell infiltration (>98%) and in healthy volunteers (HV) as controls (N=9). LSR was detectable at comparable levels in both HV and CLL groups. CSR counts were decreased in CLL samples as expected. As distribution of LSR counts was bimodal, we separated CLL patients in two groups so called LSRHigh and LSRLow. LSRHigh CLLs exhibited very weak AID expression and low mutation rate of IgHV region and of the AID off-target PIM1 gene. LSR junction diversity, evaluated using the Shannon index, was increased in LSRHigh CLLs suggesting that LSR was on-going in these cells. Also, shorter telomeres were observed in LSRHigh CLLs suggesting an increased number of past mitosis. Consistently, increased levels of cMYC expression were detected in LSRHigh CLLs and treatment free survival of these cases was markedly decreased. We hypothesized that LSR in LSRHigh CLLs is AID independent and could be due to DNA lesion and inaccurate DSB repair within the IgH locus, which could be accessible to recombination machinery due to increased IgH locus transcription. Altogether, our results indicate the accessibility of IgH locus and the proliferation increase LSR rate in LSRHigh CLLs could be related to cMYC resulting in shorter treatment free survival of patients and point on an AID independent mechanism of IgH recombination.

sentences: Possible link between higher transmissibility of B.1.617 and B.1.1.7 variants of SARS-CoV-2 and increased structural stability of its spike protein and hACE2 affinity The Severe Acute syndrome corona Virus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has caused alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests, which also might lead to inefficacy of the available drugs. It has also increased the chances of reinfection and immune escape. We have performed Molecular Dynamic simulations of three different Spike-ACE2 complexes, namely Wildtype (WT), B.1.1.7 variant (N501Y Spike mutant) and B.1.617 variant (L452R, E484Q Spike mutant) and compared their dynamics, binding energy and molecular interactions. Our result shows that mutation has caused the increase in the binding energy between the Spike and hACE2. In the case of B.1.617 variant, the mutations at L452R and E484Q increased the stability and intra-chain interactions in the Spike protein, which may change the interaction ability of human antibodies to this Spike variant. Further, we found that the B.1.1.7 variant had increased hydrogen interaction with LYS353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.

sentences: Coronavirus associated molecular mimicry common to SARS-CoV-2 peptide This study aims to predict autoimmunity-related pathological mechanisms that possess risk for individuals with specific human leukocyte antigen (HLA) serotypes and shared by certain coronaviruses including SARS-CoV-2, based on homology to a SARS-CoV-2 peptide. With the given aim, 1-) coronavirus-associated sequences, which are homologous to the 15mer SARS-CoV-2 peptide CFLGYFCTCYFGLFC, are obtained. 2-) Human peptides that have at least 7 residue matches with those coronavirus sequences, and the SARS-CoV-2 15mer, are found. 3-) Epitope pairs, which are sourced by those aligned coronavirus and human sequences are identified. 4-) Epitope pairs that are predicted to bind strongly not only to the same HLA allele with each other but also to the same HLA allele as those of the respective alignment of the SARS-CoV-2 peptide are selected. Following are the identified proteins or peptides (with HLA-A*02:01 or HLA-A*24:02 epitopes), as described in 1-to-4: Immunoglobulin heavy chain junction regions, CRB1 isoform I precursor, slit homolog 2 protein, hCG1995581, hCG2028737, phospholipid phosphatase-related protein type 2. Among those, CRB1 isoform I precursor sequence with the predicted HLA-A*24:02 epitope aligns with the highest number of different sequences. Results imply autoimmunity risk in COVID-19 patients with HLA-A*02:01 and HLA-A*24:02 serotypes, through molecular mimicry, as a shared pathogenicity risk that can be prevalent upon getting infected with certain coronaviruses. These can pave way to improved risk groups assessment and autoimmunity treatment options, for COVID-19 and its associated diseases. Also, the approach in this study can be used to predict prospective pathologies of the transmissible variants in susceptible humans.

sentences: Nitric oxide negatively regulates gibberellin signaling to coordinate growth and salt tolerance in Arabidopsis In response to dynamically altered environments, plants must finely coordinate the balance between growth and stress responses for their survival. However, the underpinning regulatory mechanisms remain largely elusive. The phytohormone gibberellin promotes growth via a derepression mechanism by proteasomal degradation of the DELLA transcription repressors. Conversely, the stress-induced burst of nitric oxide (NO) enhances stress tolerance, largely relaying on NO-mediated S-nitrosylation, a redox-based posttranslational modification. Here, we show that S-nitrosylation of Cys-374 in the Arabidopsis RGA protein, a key member of DELLAs, inhibits its interaction with the F-box protein SLY1, thereby preventing its proteasomal degradation under salinity condition. The accumulation of RGA consequently retards growth but enhances salt tolerance. We propose that NO negatively regulates gibberellin signaling via S-nitrosylation of RGA to coordinate the balance of growth and stress responses when challenged by adverse environments.

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1,25(OH)2D3 attenuates   hepatic insulin resistance  in rats with streptozotocin -induced diabetes via antioxidation AimThis study was conducted to explore the mechanism by which 1,25(OH)2D3 ameliorates hepatic insulin resistance in rats with streptozotocin (STZ)-induced type 2 diabetes.

MethodsSprague Dawley (SD) rats were randomly divided into five groups: normal, control, and 1,25(OH)2D3 administered at 0.075, 0.15, and 0.3 g/kg/d. Tissue and blood samples were obtained from all five groups after 4 and 12 weeks of treatment. Morphological changes in the livers were observed with HE staining. The levels of fasting blood glucose (FBG), insulin, and lipids were determined using an automatic biochemistry analyzer. The levels of hepatic glycogen, superoxide dismutase (SOD), malondialdehyde (MDA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were analyzed by spectrophotometry. Protein expression of AKT and GLUT2 was examined by western blotting.

Results1,25(OH)2D3 treatment reduced the FBG content as well as hepatic MDA, AST, and ALT levels, improved the activity of SOD, and enhanced the expression of GLUT2 and AKT in diabetic rats. These findings suggest that 1,25(OH)2D3, exerts antioxidant effects and ameliorates insulin resistance in the liver.

Conclusion1,25(OH)2D3 is a potential agent that could be used in the treatment of insulin resistance in the liver.

sentences: Spontaneous myocarditis in mice predisposed to autoimmune disease: Including Vaccination-induced onset Non-obese diabetic (NOD)/ShiLtJ mice, like biobreeding rats, are used as an animal model for type 1 diabetes. Diabetes develops in NOD mice as a result of insulitis, a leukocytic infiltrate of the pancreatic islets. The onset of diabetes is associated with a moderate glycosuria and a non-fasting hyperglycemia. Previously, in non-obese diabetic (NOD)/ShiLtJ mice spontaneously developing type 1 diabetes, the possible involvement of decreased expression of LMP2/{beta}1i, an immunoproteasome {beta} subunit, and associated decreased expression of NF-{kappa}B1 (also called as p50) in the development of type 1 diabetes was argued between our research team and other research groups. In response to these arguments, we created NOD mice in which NF-{kappa}B1 expression is not consistently observed. Unexpectedly, most NOD Nf{kappa}b1 homozygote mice were found to die by the 8th week of age due to the development of severe myocarditis. Furthermore, in all NOD Nf{kappa}b1 heterozygote mice, the onset of Insulitis was observed from 4 months of age. In addition, in NOD Nf{kappa}b1 heterozygote mice, an increase in cTnT due to vaccination with influenza or HBV vaccine was observed without gender difference. Now, we found a direct involvement of decreased expression of NF-{kappa}B1 in the development of autoimmune diseases in NOD/ShiLtJ mice. Therefore, we would like to introduce new research results on autoimmune diseases, including findings on important risk factors for the development of myocarditis observed after vaccination with mRNA-based COVID-19 vaccine.

sentences: Testing for genetic assimilation with phylogenetic comparative analysis: Conceptual, methodological, and statistical considerations Genetic assimilation is a process that leads to reduced phenotypic plasticity during adaptation to novel conditions, a potentially important phenomenon under global environmental change. Null expectations when testing for genetic assimilation, however, are not always clear. For instance, the statistical artifact of regression to the mean could bias us towards detecting genetic assimilation when it has not occurred. Likewise, the specific mechanism underlying plasticity expression may affect null expectations under neutral evolution. We used macroevolutionary numerical simulations to examine both of these important issues and their interaction, varying whether or not plasticity evolves, the evolutionary mechanism, trait measurement error, and experimental design. We also modified an existing reaction norm correction method to account for phylogenetic non-independence. We found: 1) regression to the mean is pervasive and can generate spurious support for genetic assimilation; 2) experimental design and post-hoc correction can minimize this spurious effect; and 3) neutral evolution can produce patterns consistent with genetic assimilation without constraint or selection, depending on the mechanism of plasticity expression. Additionally, we re-analyzed published macroevolutionary data supporting genetic assimilation, and found that support was lost after proper correction. Considerable caution is thus required whenever investigating genetic assimilation and reaction norm evolution at macroevolutionary scales.

sentences: Virgin Birth: A genetic basis for facultative parthenogenesis Sexual reproduction evolved 1-2 billion years ago and underlies the biodiversity of our planet. Nevertheless, devolution of sexual into asexual reproduction can occur across all phyla of the animal kingdom. The genetic basis for how parthenogenesis can arise is completely unknown. To understand the mechanism and benefits of parthenogenesis, we have sequenced the genome of the facultative parthenogen, Drosophila mercatorum, and compared its organisation and expression pattern during parthenogenetic or sexual reproduction. We identified three genes, desat2, Myc, and polo in parthenogenetic D. mercatorum that when mis-regulated in a non-parthenogenetic species, D. melanogaster, enable facultative parthenogenetic reproduction. This simple genetic switch leads us to propose that sporadic facultative parthenogenesis could evolve as an  escape route preserving the genetic lineage in the face of sexual isolation.

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Comparative metaproteomics demonstrates different views on the complex granular sludge microbiome The tremendous progress in sequencing technologies has made 16S amplicon and whole metagenome sequencing routine in microbiome studies. Furthermore, advances in mass spectrometric techniques has expanded conventional proteomics into the field of microbial ecology. Commonly referred to as metaproteomics, this approach measures the gene products (i.e., proteins) to subsequently identify the actively-expressed metabolic pathways and the protein-biomass composition of complete microbial communities.

However, more systematic studies on metaproteomic and genomic approaches are urgently needed, to determine the orthogonal character of these approaches. Here we describe a deep, comparative metaproteomic study on the complex aerobic granular sludge microbiome obtained from different wastewater treatment plants. Thereby, we demonstrate the different views that can be obtained on the central nutrient-removing organisms depending on the  omic approach and reference sequence databases. Furthermore, we demonstrate a  homogenized Genome Taxonomy Database (GTDB) that subsequently enables a more accurate interpretation of data from different omics approaches. Ultimately, our systematic study underscores the importance of metaproteomics in the characterization of complex microbiomes; and the necessity of accurate reference sequence databases to improve the comparison between approaches and accuracy in scientific reporting.

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Increased mitochondrial activity upon CatSper channel activation is required for sperm capacitation. To fertilize an oocyte, sperm must become hyperactive. However, whether they obtain ATP for hyperactivated motility via glycolysis or mitochondrial respiration is unclear. Here, high-resolution respirometry, flow cytometry, and confocal microscopy experiments revealed that mitochondrial respiration and membrane potential increased during mouse sperm capacitation. Treatment with inhibitors of mitochondrial respiration prevented sperm from hyperactivating and fertilizing an oocyte. Mitochondrial respiration was impaired in sperm from mice lacking the calcium channel CatSper. We developed a method to image mitochondrial calcium in sperm and found that CatSper activation led to increased mitochondrial calcium concentration. Finally, treating sperm with an inhibitor of mitochondrial calcium import impaired mitochondrial function and sperm hyperactivation. Together, our results uncover a new role of sperm mitochondria and reveal a new pathway connecting calcium influx through CatSper to mitochondrial activity and the sperm hyperactivation required to fertilize an oocyte.

SummaryThe source of ATP for sperm hyperactivation is unclear. Ferreira et al. show that mitochondrial activity increases during, and is required for, hyperactivation and fertilization ability. Increased mitochondrial activity depends on calcium influx through the channel CatSper.

sentences: Automated Analysis of Bacterial Flow Cytometry Data with FlowGateNIST Flow cytometry is commonly used to evaluate the performance of engineered bacteria. With increasing use of high-throughput experimental methods, there is a need for automated analysis methods for flow cytometry data. Here, we describe FlowGateNIST, a Python package for automated analysis of bacterial flow cytometry data. The main components of FlowGateNIST perform automatic gating to differentiate between cells and background events and then between singlet and multiplet events. FlowGateNIST also includes a method for automatic calibration of fluorescence signals using fluorescence calibration beads. FlowGateNIST is open source and freely available with tutorials and example data to facilitate adoption by users with minimal programming experience.

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Genome wide association studies define minimal set of loci for prediction of penicillin and tetracycline susceptibility in Neisseria gonorrhoeae BackgroundWhile Neisseria gonorrhoeae poses an urgent public health threat because of increasing antimicrobial resistance, much of the circulating population remains susceptible to historical treatment regimens. Point-of-care diagnostics that report susceptibility could allow for reintroduction of these regimens, but development of such diagnostics has been limited to ciprofloxacin, for which susceptibility can be predicted from a single locus.

MethodsWe assembled a dataset of 12,045 N. gonorrhoeae genomes with phenotypic resistance data for tetracycline (n = 3,611) and penicillin (n = 6,935). Using conditional genome wide association studies (GWAS), we sought to define genetic variants associated with susceptibility to penicillin and tetracycline. We evaluated the sensitivity and specificity of these variants for predicting susceptibility and non-resistance in our collection of gonococcal genomes.

FindingsIn our conditional penicillin GWAS, the presence of a genetic variant defined by a non-mosaic penA allele without an insertion at codon 345 was significantly associated with penicillin susceptibility and had the highest negative effect size of significant variants (p = 5.0 x 10-14, {beta} = -2.5). In combination with the absence of blaTEM, this variant predicted penicillin susceptibility with high specificity (99.8%) and modest sensitivity (36.7%). For tetracycline, the wild type allele at rpsJ codon 57, encoding valine, was significantly associated with tetracycline susceptibility (p = 5.6 x 10-16, {beta} = -1.6) after conditioning on the presence of tetM. The combination of rpsJ codon 57 allele and tetM absence predicted tetracycline susceptibility with high specificity (97.2%) and sensitivity (88.7%).

InterpretationAs few as two genetic loci can predict susceptibility to penicillin and tetracycline in N. gonorrhoeae with high specificity. Molecular point-of-care diagnostics targeting these loci have the potential to increase available treatments for gonorrhea.

FundingNational Institute of Allergy and Infectious Diseases, the National Science Foundation, and the Smith Family Foundation

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with the terms "Neisseria gonorrhoeae" and "diagnostic" or "assay" plus "penicillin" or "tetracycline" for reports in any language published up to July 1, 2021. We additionally searched for "Neisseria gonorrhoeae" and "genome wide association study". We found that previously proposed molecular diagnostics for penicillin and tetracycline susceptibility either exclusively focused on plasmid-mediated resistance (i.e., targeting blaTEM or tetM) or did not include variants in genes encoding antibiotic targets (e.g., did not include penA or rpsJ). Targets for molecular surveillance have focused on resistance-associated alleles rather than susceptibility-associated alleles. We did not find any previous penicillin or tetracycline conditional genome wide association studies (GWAS) in N. gonorrhoeae.

Added value of this studyTo identify targets for molecular diagnostics that predict penicillin and tetracycline susceptibility, we conducted GWAS conditioning on the presence of plasmid-mediated resistance determinants to detect chromosomal loci with the highest association with susceptibility. We discovered a sequence (penA_01) that differentiates susceptible isolates from those with a resistance-associated insertion at codon 345 and from those with mosaic penA alleles, which is associated with penicillin susceptibility. We also found that rpsJ codon 57 was the chromosomal locus contributing the most to tetracycline susceptibility. The combination of these chromosomal loci and the absence of plasmid encoded determinants predicts penicillin and tetracycline susceptibility with high specificity in both a large global collection of N. gonorrhoeae and a validation dataset consisting of recently published genomes from CDCs Gonococcal Isolate Surveillance Program (GISP) surveillance collected in 2018.

Implications of all the available evidenceThe chromosomal loci penA_01 and rpsJ codon 57 in combination with plasmid loci blaTEM and tetM are candidates for the development of point-of-care molecular diagnostics for penicillin and tetracycline susceptibility. The loci may be combined with the currently available ciprofloxacin susceptibility diagnostics to predict susceptibility to multiple antibiotics. Additionally, our study suggests that conditional GWAS focused on variants associated with susceptibility may be a promising approach to identify minimal sets of loci for molecular diagnostics and surveillance.

sentences: SNP- Based Assessment of Genetic Purity and Diversity in Maize Hybrid Breeding Assessment of genetic purity of inbred lines and their resultant F1 hybrids is an essential quality control check in maize hybrid breeding, variety release and seed production. In this study, genetic purity, parent-offspring relationship and diversity among the inbred lines were assessed using 92 single-nucleotide polymorphism (SNP) markers. A total of 188 maize genotypes, comprising of 26 inbred lines, four doubled haploid (DH) lines and 158 single-cross maize hybrids were investigated in this study using Kompetitive Allele Specific Polymerase Chain Reaction (KASP) genotyping assays. The bi-allelic data was analyzed for genetic purity and diversity parameters using GenAlex software. The SNP markers were highly polymorphic and 90% had polymorphic information content (PIC) values of > 0.3. Pairwise genetic distances among the lines ranged from 0.05 to 0.56, indicating a high level of dissimilarity among the inbred lines. Maximum genetic distance of (0.56) was observed for CKDHL0089, CML443 and CB323, while the lowest (0.05) was between I-42 and I-40. The majority (67%) of the inbred lines studied were genetically pure with residual heterozygosity of <5%, while only 33% were had heterozygosity levels of >5%. Inbred lines, which were not pure, require purification through further inbreeding. Cluster analysis partitioned the lines into three distinct genetic clusters with the potential to contribute new beneficial alleles to the maize breeding program. Out of the 68 hybrids (43%) that passed the parent-offspring test, seven hybrids namely; SCHP29, SCHP95, SCHP94, SCHP134, SCHP44, SCHP114 and SCHP126, were selected as potential candidates for further evaluation and release due to their outstanding yield performance.

sentences: Shrimp parvovirus circular DNA fragments arise from both endogenous viral elements (EVE) and the infecting virus Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp (Penaeus monodon) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp (Penaeus vannamei) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs.

sentences: Sea Surface Temperatures Drive Historical Demography of Deep-Sea Fishes Demographic histories are largely understood to be a product of their environment, as populations expand or contract in response to major environmental changes. Deep-pelagic fishes inhabit one of the most temporally and spatially stable habitats on the planet, so they may be resistant to the demographic instability commonly reported in other marine habitats, but their demographic histories are poorly understood. We reconstructed the demographic histories of thirteen species of deep-pelagic fishes using mitochondrial and nuclear DNA sequence data. We uncovered widespread evidence of demographic expansion in our study species, a counterintuitive result bases on the nature of the deep-pelagic. The frequency-based methods detected potential demographic changes in eleven species, while the Extended Bayesian Skyline Plots were more conservative and identified population expansion in five species. The dates of expansion largely coincide with periods of warm sea-surface temperature at the northern and southern boundaries for the ranges these species inhabit. We suggest that this is the result of the pelagic larval phase shared by most deep-pelagic fishes, where the larvae inhabit the upper 200 meters. Changes in sea surface conditions likely alter the suitability of the habitat in a given region for the larval phase, affecting the species range and in turn population size. These results are critical to our understanding of how the deep-pelagic fish community will respond to future climatic changes.

sentences: The RIFF: an automated environment for studying the neural basis of auditory-guided complex behavior Behavior consists of the interaction between an organism and its environment, and is controlled by the brain. Brain activity varies at sub-second time scales, but behavioral measures are usually coarse (often consisting of only binary trial outcomes). To overcome this mismatch, we developed the RIFF: a programmable interactive arena for freely-moving rats with multiple feeding areas, multiple sound sources, high-resolution behavioral tracking, and simultaneous electrophysiological recordings. We describe two complex tasks implemented in the RIFF. Rats quickly learned these tasks and developed anticipatory behavior. Neurons in auditory cortex and posterior insula showed sensitivity to non-auditory parameters such as location and pose. Our combination of wireless electrophysiology and detailed behavioral documentation in a controlled environment produces insights into the cognitive capabilities and learning mechanisms of rats and opens the way to a better understanding of how brains control behavior.

sentences: Molecular epidemiology of third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia SynopsisThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using rapid ([~]40 min) real-time PCR assays targeting the most common extended spectrum {beta}-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). AmpC CMY {beta}-lactamase prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one {beta}-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. A subset of isolates (n=98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants, and to verify genotyping accuracy. WGS of {beta}-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

sentences: Neuroinvasion and neurotropism by SARS-CoV-2 variants in the K18-hACE2 mouse Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) not only affects the respiratory tract but also causes neurological symptoms such as loss of smell and taste, headache, fatigue or severe cerebrovascular complications. Using transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) we investigated the spatiotemporal distribution and pathomorphological features in the CNS following intranasal infection with SARS-CoV-2 variants, also after prior influenza A virus infection. Apart from Omicron, we found all variants to frequently spread to and within the CNS. Infection was restricted to neurons and appeared to spread from the olfactory bulb mainly in basally orientated regions in the brain and into the spinal cord, independent of ACE2 expression and without evidence of neuronal cell death, axonal damage or demyelination. However, microglial activation, microgliosis and a mild macrophage and T cell dominated inflammatory response was consistently observed, accompanied by apoptotic death of endothelial, microglial and immune cells, without their apparent infection. Microgliosis and immune cell apoptosis indicate a potential role of microglia for pathogenesis and viral effect in COVID-19 and possible impairment of neurological functions, especially in long COVID. These data may also be informative for the selection of therapeutic candidates, and broadly support investigation of agents with adequate penetration into relevant regions of the CNS.

sentences: Occupancy-collection models: Towards bias-corrected modeling of species' distributions using unstructured occurrence data from museums and herbaria The digitization of museum collections as well as an explosion in citizen science initiatives has resulted in a wealth of data that can be useful for understanding the global distribution of biodiversity, provided that the well-documented biases inherent in unstructured opportunistic data are accounted for. While traditionally used to model imperfect detection using structured data from systematic surveys of wildlife, occupancy-detection models provide a framework for modelling the imperfect collection process that results in digital specimen data. In this study, we explore methods for adapting occupancy-detection models for use with biased opportunistic occurrence data from museum specimens and citizen science platforms using 7 species of Anacardiaceae in Florida as a case study. We explored two methods of incorporating information about collection effort to inform our uncertainty around species presence: (1) filtering the data to exclude collectors unlikely to collect the focal species and (2) incorporating collection covariates (collection type and history of previous detections) into a model of collection probability. We found that the best models incorporated both the background data filtration step as well as the incorporation of collector covariates associated with the probability of collection. We found that month, method of collection and whether a collector had previously collected the focal species were important predictors of collection probability. Efforts to standardize meta-data associated with data collection will improve efforts for modeling the spatial distribution of a variety of species.

sentences: Common resistance mechanisms are deployed by plants against sap-feeding herbivorous insects: insights from a meta-analysis and systematic review Sap-feeding insects cause significant yield losses to the worlds crops, these insects feed using syringe-like mouthparts and electrophysiology can be used to compare feeding behaviour on susceptible and resistant plants to identify the mechanistic processes behind resistant phenotypes. Data extracted from 129 studies, comprising 41 insect species across eight insect taxa and 12 host-plant families representing over 30 species, demonstrates that mechanisms deployed by resistant plants have common consequences on the feeding behaviour of diverse insect groups. We show that insects feeding on resistant plants take longer to establish a feeding site and have their feeding duration suppressed two-fold compared with insects feeding on susceptible plants. Our results reveal that the underlying traits contributing towards resistant phenotypes are conserved across plant families, deployed against taxonomically diverse insect groups, and that the underlying resistance mechanisms are conserved. These findings provide new insight that will be beneficial when developing future crop varieties.

sentences: Production of Offspring from Azoospermic Mice with Meiotic Failure: Precise Biparental Meiosis within Halved Oocytes While the large volume of mammalian oocytes is necessary for embryo development, it can lead to error-prone chromosomal segregation during meiosis. Conversely, we hypothesized that smaller oocytes would have a great unidentified potential to stabilize unstable meiosis and improve the development of the resultant embryos. Here, we show that reducing ooplasmic volume can rescue highly error-prone fertilization using primary spermatocytes by preventing segregation errors of chromosomes during biparental meiosis. High-resolution live-imaging analysis revealed that erroneous chromosome segregation occurred in most (90%) spermatocyte-injected oocytes of normal size, but could be ameliorated to 40% in halved oocytes. The birth rate improved remarkably from 1% to 19% (P < 0.0001). Importantly, this technique enabled the production of offspring from azoospermic mice with spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans. Thus, contrary to popular opinion, oocytes inherently have a strong potential for precise meiotic divisions, which can be evoked by reduction of the ooplasmic volume. Their potential might help rescue cases of untreatable human azoospermia with spermatocyte arrest.

sentences: The secreted neuronal signal Spock1 regulates the blood-brain barrier The blood-brain barrier (BBB) is comprised of a single layer of endothelial cells with uniquely restrictive properties required for maintaining a tightly controlled homeostatic environment in the brain. Classic quail-chick grafting experiments showed that BBB properties are not intrinsic to brain endothelial cells, but instead are induced by signals from the embryonic brain microenvironment. Here we have identified a neuronally produced signal, Spock1, that specifically regulates BBB functional development in both zebrafish and mice without affecting angiogenesis. Using a combination of mosaic genetic analysis, tracer leakage assays and live imaging we show that Spock1 from neurons can regulate brain vasculature permeability non-cell autonomously. Electron microscopy analyses of spock1 mutants revealed that the leakage arises predominantly through increased endothelial transcytosis of both clathrin-independent small and large vesicles due to altered pericyte-endothelial interactions. Single-cell RNA sequencing analyses revealed a reduction in vascular expression of the cell adhesion molecule mcamb in the spock1 mutants, and this down-regulation of mcamb occurred specifically in regions with increased BBB leakage. These analyses indicate that the neuronal signal Spock1 regulates BBB properties by altering vascular gene expression and cellular interactions.

sentences: Defining the KRAS-regulated kinome in KRAS-mutant pancreatic cancer Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines, and then applied Multiplexed Inhibitor Bead/Mass Spectrometry (MIB/MS) chemical proteomics to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transforming activities, and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGFBR1, ILK), and pharmacologic inhibition of the upregulated kinase, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacologic inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death than WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.

sentences: CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever Dengue virus infection can cause dengue hemorrhagic fever (DHF). Dengue NS1 is multifunctional: the intracellular dimeric NS1 (iNS1) forms part of the viral replication complex, the extracellular multi-oligomeric secreted NS1 (sNS1) is a major factor contributing to DHF. The structure of the iNS1 is well studied but not sNS1. Here we show the tetrameric (stable and loose conformation) and hexameric structures of sNS1. Stability of the stable and loose tetramers is determined by the conformation of their N-terminal domain - elongated {beta}-sheet or {beta}-roll. Binding of an anti-NS1 Fab breaks the loose tetrameric and hexameric sNS1 into dimers, whereas the stable tetramer remains largely unbound. Our results show detailed quaternary organization of different oligomeric states of sNS1 and will contribute towards the design of dengue therapeutics.

sentences: TatA and TatB generate a hydrophobic mismatch that is important for function and assembly of the Tat translocon in Escherichia coli The Tat system has the unique purpose to translocate folded proteins across energy-transducing membranes. It occurs in bacteria and archaea, as well as in eukaryotic organelles of bacterial origin. In the bacterial model system Escherichia coli, the three components TatA, TatB, and TatC assemble to functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) that is followed by an amphipathic helix (APH). The TMHs of TatA and TatB generate a hydrophobic mismatch with only 12 consecutive hydrophobic residues that span the membrane. We shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both, and analyzed effects on transport functionality and translocon assembly. The wild type length functioned best but was not an absolute requirement, as some variation was clearly tolerated. Defects of shortenings or extensions were enhanced by simultaneous mutations in TatA and TatB, indicating partial compensations of mutations in TatA by wild type TatB or vice versa. Length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residues-length is important for Tat component interactions and translocon assembly. To also address potential effects on TatA associations, we characterized these by metal tagging transmission electron microscopy and carried out molecular dynamics simulations. In these simulations, interacting short TMHs of larger TatA assemblies were thinning the membrane together with laterally aligned tilted APHs that generated a deep V-shaped groove. The conserved length of 12 hydrophobic residues may thus not only be important for translocon interactions, but also for a membrane destabilization during Tat transport. If this is the case, the specific short length could be a compromise between functionality and proton leakage minimization.

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Calcium-induced environmental adaptability of the blood protein vitronectin The adaptability of proteins to their work environments is fundamental for cellular life. Here we describe how the hemopexin-like (HX) domain of the multifunctional blood glycoprotein vitronectin (Vn) binds Ca2+ to adapt to excursions of temperature and shear stress. Using X-ray crystallography and molecular dynamics (MD) simulations, nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSF), we describe how Ca2+ and its flexible hydration shell enable the protein to perform conformational changes, that relay beyond the Ca2+ binding site to alter the number of polar contacts and confer conformational stability. By means of mutagenesis, we identify key residues that cooperate with Ca2+ to promote protein stability, and we show that Ca2+ association confers protection against shear stress, a property that provides conformational advantage for proteins that circulate in the vasculature like Vn. The data reveal a mechanism of adaptation.

Significance StatementThe protein vitronectin (Vn) plays important roles in cell adhesion and migration, bone remodeling and immunity. It circulates in blood, but it is also found in the extracellular matrix, and it accumulates with plaques associated with age-related macular degeneration, Alzheimers disease, atherosclerosis and other degenerative disorders. Vn is a calcium-binding protein, and here, we show that calcium helps Vn alter its structure in response to diverse environmental conditions. The results shed light on the way in which Vn adapts to its surroundings. This structural knowledge is important for the development of diagnostic, preventive or therapeutic approaches.

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Plotgardener: Cultivating precise multi-panel figures in R The R programming language is one of the most widely used programming languages for transforming raw genomic data sets into meaningful biological conclusions through analysis and visualization, which has been largely facilitated by infrastructure and tools developed by the Bioconductor project. However, existing plotting packages rely on relative positioning and sizing of plots, which is often sufficient for exploratory analysis but is poorly suited for the creation of publication-quality multi-panel images inherent to scientific manuscript preparation. We present plotgardener, a coordinate-based genomic data visualization package that offers a new paradigm for multi-plot figure generation in R. Plotgardener allows precise, programmatic control over the placement, aesthetics, and arrangements of plots while maximizing user experience through fast and memory-efficient data access, support for a wide variety of data and file types, and tight integration with the Bioconductor environment. Plotgardener also allows precise placement and sizing of ggplot2 plots, making it an invaluable tool for R users and data scientists from virtually any discipline.

AvailabilityPackage: https://bioconductor.org/packages/plotgardener

Code: https://github.com/PhanstielLab/plotgardener

Documentation: https://phanstiellab.github.io/plotgardener/

sentences: Cerebral laterality for writing in right- and left-handers: A functional transcranial Doppler ultrasound study The cerebral lateralization of written language has received very limited research attention in comparison to the wealth of studies on the cerebral lateralization of oral language. The purpose of the present study was to further our understanding of written language lateralization, by elucidating the relative contribution of language and motor functions. We compared written word generation with a task that has equivalent visuomotor demands but does not include language: the repeated drawing of symbols. We assessed cerebral laterality using functional transcranial Doppler ultrasound (fTCD), a non-invasive, perfusion-sensitive neuroimaging technique in 23 left- and 31 right-handed participants. Findings suggest that the linguistic aspect of written word generation recruited more left-hemispheric areas during writing, in right-handers compared to left-handers. This difference could be explained by greater variability in cerebral laterality patterns within left-handers or the possibility that the areas subserving language in left-handers are broader than in right-handers. Another explanation is that the attentional demands of the more novel symbol copying task (compared to writing) contributed more right-hemispheric activation in right-handers, but this could not be captured in left-handers due to ceiling effects. Future work could investigate such attentional demands using both simple and complex stimuli in the copying condition.

sentences: On the Apportionment of Archaic Human Diversity The apportionment of human genetic diversity within and between populations has been measured to understand human relatedness and demographic history. Likewise, the distribution of archaic ancestry in modern populations can be leveraged to better understand the interaction between our species and its archaic relatives, and the impact of natural selection on archaic segments of the human genome. Resolving these interactions can be difficult, as archaic variants in modern populations have also been shaped by genetic drift, bottlenecks, and gene flow. Here, we investigate the apportionment of archaic variation in Eurasian populations. We find that archaic genome coverage at the individual- and population-level present unique patterns in modern human population: South Asians have an elevated count of population-unique archaic SNPs, and Europeans and East Asians have a higher degree of archaic SNP sharing, indicating that population demography and archaic admixture events had distinct effects in these populations. We confirm previous observations that East Asians have more Neanderthal ancestry than Europeans at an individual level, but surprisingly Europeans have more Neandertal ancestry at a population level. In comparing these results to our simulated models, we conclude that these patterns likely reflect a complex series of interactions between modern humans and archaic populations.

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The dorsal visual pathway represents object-centered spatial relations for object recognition Although there is mounting evidence that input from the dorsal visual pathway is crucial for object processes in the ventral pathway, the specific functional contributions of dorsal cortex to these processes remain poorly understood. Here, we hypothesized that dorsal cortex computes the spatial relations among an objects parts - a processes crucial for forming global shape percepts - and transmits this information to the ventral pathway to support object categorization. Using fMRI with human participants (females and males), we discovered regions in the intraparietal sulcus (IPS) that were selectively involved in computing object-centered part relations. These regions exhibited task-dependent functional and effective connectivity with ventral cortex, and were distinct from other dorsal regions, such as those representing allocentric relations, 3D shape, and tools. In a subsequent experiment, we found that the multivariate response of posterior IPS, defined on the basis of part-relations, could be used to decode object category at levels comparable to ventral object regions. Moreover, mediation and multivariate effective connectivity analyses further suggested that IPS may account for representations of part relations in the ventral pathway. Together, our results highlight specific contributions of the dorsal visual pathway to object recognition. We suggest that dorsal cortex is a crucial source of input to the ventral pathway and may support the ability to categorize objects on the basis of global shape.

Significance StatementHumans categorize novel objects rapidly and effortlessly. Such categorization is achieved by representing an objects global shape structure, that is, the relations among object parts. Yet, despite their importance, it is unclear how part relations are represented neurally. Here, we hypothesized that object-centered part relations may be computed by the dorsal visual pathway, which is typically implicated in visuospatial processing. Using fMRI, we identified regions selective for the part relations in dorsal cortex. We found that these regions can support object categorization, and even mediate representations of part relations in the ventral pathway, the region typically thought to support object categorization. Together, these findings shed light on the broader network of brain regions that support object categorization.

sentences: Pyruvate kinase M1 suppresses development and progression of prostate adenocarcinoma Most cancers, including prostate cancers, express the M2 splice isoform of pyruvate kinase (Pkm2). This isoform can promote anabolic metabolism to support cell proliferation; however, Pkm2 expression is dispensable for many cancers in vivo. Pyruvate kinase M1 (Pkm1) isoform expression is restricted to relatively few tissues and has been reported to promote growth of select tumors, but the role of PKM1 in cancer has been less studied. Pkm1 is expressed in normal prostate tissue; thus, to test how differential pyruvate kinase isoform expression affects cancer initiation and progression we generated mice harboring a conditional allele of Pkm1 and crossed this allele, as well as a Pkm2 conditional allele, to a Pten loss-driven prostate cancer model. We found that Pkm1 loss leads to Pkm2 expression and accelerates prostate cancer, while deletion of Pkm2 leads to increased Pkm1 expression and suppresses cancer. Consistent with these data, a small molecule pyruvate kinase activator that mimics a PKM1-like state suppresses progression of established prostate tumors. PKM2 expression is retained in most human prostate cancers, arguing that pharmacological PKM2 activation may be beneficial for some prostate cancer patients.

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Timing of meristem initiation and maintenance determines the morphology of fern gametophytes The alternation of generations in land plants occurs between the sporophyte phase and the gametophyte phase. The sporophytes of seed plants develop self-maintained, multicellular meristems, and these meristems determine plant architecture. The gametophytes of seed plants lack meristems and are heterotrophic. In contrast, the gametophytes of seed-free vascular plants, including ferns, are autotrophic and free-living, developing meristems to sustain their independent growth and proliferation. Compared to meristems in the sporophytes of seed plants, the cellular mechanisms underlying meristem development in fern gametophytes remain largely unknown. Here, using confocal time-lapse live imaging and computational segmentation and quantification, we determined different patterns of cell divisions associated with the initiation and proliferation of two distinct types of meristems in fern gametophytes. Our results reveal how the simple timing of a switch between two meristems has considerable consequences for the divergent gametophyte morphologies of two closely related ferns from Pteridaceae (Pteris and Ceratopteris). Our result provides evolutionary insight into the function and regulation of gametophyte meristems in seed-free vascular plants.

HighlightLive-imaging of cell growth and division in apical initials and lateral meristems reveals that the timing of a switch between the two meristem identities drives morphology variation in fern gametophytes.

sentences: Inferring the timing and strength of natural selection and gene migration in the evolution of chicken from ancient DNA data With the rapid growth of the number of sequenced ancient genomes, there has been increasing interest in using this new information to study past and present adaptation. Such an additional temporal component has the promise of providing improved power for the estimation of natural selection. Over the last decade, statistical approaches for detection and quantification of natural selection from ancient DNA (aDNA) data have been developed. However, most of the existing methods do not allow us to estimate the timing of natural selection along with its strength, which is key to understanding the evolution and persistence of organismal diversity. Additionally, most methods ignore the fact that natural populations are almost always structured, which can result in overestimation of the effect of natural selection. To address these issues, we introduce a novel Bayesian framework for the inference of natural selection and gene migration from aDNA data with Markov chain Monte Carlo techniques, co-estimating both timing and strength of natural selection and gene migration. Such an advance enables us to infer drivers of natural selection and gene migration by correlating genetic evolution with potential causes such as the changes in the ecological context in which an organism has evolved. The performance of our procedure is evaluated through extensive simulations, with its utility shown with an application to ancient chicken samples.

sentences: Superior colliculus saccade motor bursts do not dictate movement kinematics The primate superior colliculus (SC) contains a topographic map of visual field locations, such that the anatomical location of any given active neuron defines a desired eye movement amplitude and direction. Complementing such a spatial code, SC neurons also exhibit saccade-related bursts that are tightly synchronized with movement onset. Current models suggest that such bursts, and their properties, constitute a temporal rate code that may dictate moment-to-moment movement evolution. However, a recent result demonstrated altered movement properties with minimal changes in SC motor burst strengths (Buonocore, Tian, Khademi, & Hafed, 2021). Here, I support such a dissociation between the SC temporal rate code and instantaneous movement evolution: SC burst strength varies depending on whether saccades are directed towards the upper or lower visual fields, but the movements themselves have similar kinematics. Thus, SC saccade-related motor bursts do not necessarily dictate movement kinematics, motivating investigating other possible functional roles for these bursts.

sentences: To draw or not to draw: understanding the temporal organization of drawing behaviour using fractal analyses Studies on drawing often focused on spatial aspects of the finished products. Here, the drawing behaviour was studied by analysing its intermittent process, between drawing (i.e. marking a surface) and interruption (i.e. a pause in the marking gesture). To assess how this intermittence develops with age, we collected finger-drawings on a touchscreen by 185 individuals (children and adults). We measured the temporal structure of each drawing sequence to determine its complexity. To do this, we applied temporal fractal estimators to each drawing time series before combining them in a Principal Component Analysis procedure. The youngest children (3 years-old) drew in a more stereotypical way with long-range dependence detected in their alternations between states. Among older children and adults, the complexity of drawing sequences increased showing a less predictable behaviour as their drawings become more detailed and figurative. This study improves our understanding of the temporal aspects of drawing behaviour, and contributes to an objective understanding of its ontogeny.

sentences: Arc rescues defective synaptic plasticity and cognitive dysfunction in sepsis-associated encephalopathy Sepsis-associated encephalopathy (SAE) is a frequent complication in patients with severe systemic infection resulting in acute brain dysfunction and incapacitating long-term sequelae. SAE includes delirium, premature death, post-traumatic stress disorder, and major long-term cognitive impairment. The underlying pathophysiology of SAE is largely unresolved and specific treatment options are missing. We induced the peritoneal contamination and infection (PCI) sepsis model in 769 mice and compared these with 259 control mice. We found that experimental sepsis causes synaptic pathology in the brain characterized by severely disordered synaptic plasticity with reduced long-term potentiation, changes in CA1 pyramidal neuron dendritic spines, and behavioral abnormalities indicating cognitive dysfunction. Using electrophysiology, we found reduced frequency of quantal and spontaneous excitatory postsynaptic currents whereas amplitudes of miniature, spontaneous, and evoked excitatory currents were increased, pointing towards a homeostatic synaptic scaling mechanism. Corresponding to dysfunctional excitatory synaptic function we discovered downregulation of genes related to neuronal and synaptic signaling in the brain, including the gene for activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), members of the transcription-regulatory EGR gene family, and the gene for dual-specificity phosphatase 6 (Dusp6). At the protein level, ARC expression and MAP kinase signaling in the brain were affected. For targeted rescue of dysfunctional synaptic signaling and plasticity, we overexpressed ARC in the hippocampus by microinjection of an adeno-associated virus containing a neuron-specific plasmid of the ARC transgene. Hereby we achieved recovery of defective synaptic plasticity in the hippocampal Schaffer collateral-CA1 pathway and improvement of memory dysfunction. Using a different rescue paradigm, PCI mice were subjected to enriched environment providing multiple activating stimuli. Enriched environment led to restoration of disordered long-term potentiation and memory, thus demonstrating the potential for activity-induced improvement. Together, we identified synaptic pathomechanisms of SAE after severe systemic infection and provide a conceptual approach to treat SAE-related disease mechanisms which may be applicable to patients afflicted with SAE.

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Hyperuricemia triggers Renal Tubular Epithelial Pyroptosis by using ROS to activate the NLRP3 inflammasome Renal injury resulting from hyperuricemia has gained a lot of interest. Pyroptosis refers to inflammatory cell death. The activated caspase-1 cleavage, and the pivotal protein - GSDMD could have an association with the hyperuricemic kidney lesion pathogenesis. ROS is a vital NLRP3 inflammasome antagonist in various cells. We investigated the mechanism through which ROS stimulates NLRP3 to modulate pyroptosis in renal tubular epithelial cells as well as hyperuricemic rat kidneys.

MethodsIn vitro cultured renal tubular epithelial cells (NRK-52E cells) were incubated with a gradient concentration of uric acid for 24 hr to investigate the pyroptosis through flow cytometry. Next, we used the inhibitors of ROS, mitochondrial ROS, NLRP3 and Caspase-1 respectively to intervene in uric acid treated cells to analyse pyproptosis and activation of ROS- NLRP3 inflammasome signal pathway. Finally, we evaluated the mechanism of hyperuricemia triggering renal tubular epithelial pyroptosis in rat kidney tissues.

ResultsThe levels of ROS and mitochondrial ROS, the mRNA and protein expression of pyroptosis-associated factors Caspase-1 (p45, p20/10), NLRP3, and GSDMD were upregulated in uric acid, the induced NRK-52E cells as well as hyperuricemic model kidneys. The inhibition of ROS, mitochondrial ROS, NLRP3, or caspase-1 in the uric acid-induced NRK-52E cells may help in controlling pyroptosis. The expression of mRNA and protein by the cytokines IL-18 and IL-1{beta} also increased.

ConclusionsGenerally, hyperuricemia triggered renal tubular epithelial pyroptosis via excessive ROS to modulate NLRP3 inflammasome activation in uric acid stimulated renal tubular epithelial cells as well as the oxonic acid potassium induced hyperuricemia.

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LY-CoV1404 potently neutralizes SARS-CoV-2 variants SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19.

In BriefLY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated.

HighlightsO_LILY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variants
C_LIO_LINo loss of potency against currently circulating variants
C_LIO_LIBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID database
C_LIO_LIBreadth of neutralizing activity and potency supports clinical development
C_LI

sentences: Combining deep learning and automated feature extraction to analyze minirhizotron images: development and validation of a new pipeline Root systems of crops play a significant role in agro-ecosystems. The root system is essential for water and nutrient uptake, plant stability, symbiosis with microbes and a good soil structure. Minirhizotrons, consisting of transparent tubes that create windows into the soil, have shown to be effective to non-invasively investigate the root system. Root traits, like root length observed around the tubes of minirhizotron, can therefore be obtained throughout the crop growing season. Analyzing datasets from minirhizotrons using common manual annotation methods, with conventional software tools, are time consuming and labor intensive. Therefore, an objective method for high throughput image analysis that provides data for field root-phenotyping is necessary. In this study we developed a pipeline combining state-of-the-art software tools, using deep neural networks and automated feature extraction. This pipeline consists of two major components and was applied to large root image datasets from minirhizotrons. First, a segmentation by a neural network model, trained with a small image sample is performed. Training and segmentation are done using "Root-Painter". Then, an automated feature extraction from the segments is carried out by "RhizoVision Explorer". To validate the results of our automated analysis pipeline, a comparison of root length between manually annotated and automatically processed data was realized with more than 58,000 images. Mainly the results show a high correlation (R=0.81) between manually and automatically determined root lengths. With respect to the processing time, our new pipeline outperforms manual annotation by 98.1 - 99.6 %. Our pipeline,combining state-of-the-art software tools, significantly reduces the processing time for minirhizotron images. Thus, image analysis is no longer the bottle-neck in high-throughput phenotyping approaches.

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Reduced cutaneous CD200:CD200R1 signalling in psoriasis enhances neutrophil recruitment to skin The skin immune system is tightly regulated to prevent inappropriate inflammation in response to harmless environmental substances. This regulation is actively maintained by mechanisms including cytokines and cell surface receptors and its loss results in inflammatory disease. In the case of psoriasis, inappropriate immune activation leads to IL-17-driven chronic inflammation, but molecular mechanisms underlying this loss of regulation are not well understood.

We reveal that immunoglobulin superfamily member CD200, and signalling via its receptor, CD200R1 are reduced in non-lesional psoriasis skin. To examine the consequences of this, CD200R1 was blocked in a mouse model of psoriasis demonstrating that the receptor limits psoriasis-like inflammation. Specifically, CD200R1 blockade enhances acanthosis, CCL20 production and neutrophil recruitment but surprisingly, macrophage function and IL-17 production were not affected, and neutrophil reactive oxygen species production was reduced.

Collectively, our data show that CD200R1 affects neutrophil function and limits inflammatory responses in healthy skin by restricting neutrophil recruitment. However, the CD200 pathway is reduced in psoriasis, resulting in a loss of immune control, and increased neutrophil recruitment in mouse models. In conclusion, we highlight a pathway that might be targeted to dampen inflammation in patients with psoriasis.

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A Novel Model for Encephalomyosynangiosis Surgery after Middle Cerebral Artery Occlusion-Induced Stroke in Mice Background and PurposeNo effective treatment is available for most patients who suffer ischemic stroke. Development of novel treatment options is imperative. The brain attempts to self-heal after ischemic stroke via various mechanism mediated by restored blood circulation in affected region of brain but this process is limited by inadequate angiogenesis or neoangiogenesis. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis with low morbidity in patients with moyamoya disease, reducing risk of stroke. However, EMS, surgery has never been studied as an therapeutic option after ischemic stroke. Here we described a novel procedure and feasibility data for EMS after ischemic stroke in mice.

MethodsA 60 mins of middle cerebral artery occlusion (MCAo) was used to induce ischemic stroke in mice. After 3-4 hours of MCAo onset/sham, EMS was performed. Mortality of EMS, MCAo and. MCAo+EMS mice was recorded up to 21 days after surgery. Graft tissue viability was measured using a nicotinamide adenine dinucleotide reduced tetrazolium reductase assay.

ResultsEMS surgery after ischemic stroke does not increase mortality compared to stroke alone. Graft muscle tissue remained viable 21 days after surgery.

ConclusionsThis novel protocol is effective and well-tolerated, may serve as novel platform for new angiogenesis and thus recovery after ischemic stroke. If successful in mice, EMS can a very feasible and novel treatment option for ischemic stroke in humans.

sentences: Interpretable and Generalizable Attention-Based Model for Predicting Drug-Target Interaction Using 3D Structure of Protein Binding Sites: SARS-CoV-2 Case Study and in-Lab Validation In this study, we introduce and implement an interpretable graph-based deep learning prediction model, which utilizes protein binding sites along with self-attention to learn which protein binding sites interact with a given ligand. Our proposed model enables interpretability by identifying the protein binding sites that contribute the most towards the Drug-Target Interaction. Results on three benchmark datasets show improved performance compared to previous graph-based models. More significantly, unlike previous studies our model performance remains close to the optimal performance when tested with new proteins (ie., high generalizablity). Through multidisciplinary collaboration, we further experimentally evaluate the practical potential of our proposed approach. To achieve this, we first computationally predict binding interaction of some candidate compounds with a target protein, then experimentally validate the binding interactions for these pairs in the laboratory. The high agreement between the computationally-predicted and experimentally-observed (measured) DTIs illustrates the potential of our method as an effective pre-screening tool in drug re-purposing applications.

sentences: SMILE: Mutual Information Learning for Integration of Single Cell Omics Data Deep learning approaches have empowered single-cell omics data analysis in many ways, generating new insights from complex cellular systems. As there is an increasing need for single cell omics data to be integrated across sources, types, and features of data, the challenges of integrating single-cell omics data are rising. Here, we present a deep clustering algorithm that learns discriminative representation for single-cell data via maximizing mutual information, SMILE (Single-cell Mutual Information Learning). Using a unique cell-pairing design, SMILE successfully integrates multi-source single-cell transcriptome data, removing batch effects and projecting similar cell types, even from different tissues, into the same representation space. SMILE can also integrate data from two or more modalities, such as joint profiling technologies using singlecell ATAC-seq, RNA-seq, DNA methylation, Hi-C, and ChIP data. SMILE works well even when feature types are unmatched, such as genes for RNA-seq and genome wide peaks for ATAC-seq.

sentences: Botulinum neurotoxin accurately separates tonic vs phasic transmission and reveals heterosynaptic plasticity rules in Drosophila In developing and mature nervous systems, diverse neuronal subtypes innervate common targets to establish and maintain functional neural circuits. A major challenge towards understanding the structural and functional architecture of neural circuits is to separate these inputs and determine their intrinsic and heterosynaptic relationships. The Drosophila larval neuromuscular junction is a powerful model system to study these questions, where two glutamatergic motor neurons, the strong phasic-like Is and weak tonic-like Ib, co-innervate individual muscle targets to coordinate locomotor behavior. However, complete neurotransmission from each input has never been electrophysiologically separated. We have developed a botulinum neurotoxin, BoNT-C, that eliminates both spontaneous and evoked neurotransmission without perturbing synaptic growth or structure, enabling the first approach that accurately isolates input-specific neurotransmission. Selective expression of BoNT-C in Is or Ib motor inputs disambiguates functional properties of each input. Importantly, the composite values of Is and Ib neurotransmission can be fully recapitulated by isolated physiology from each input. Finally, selective silencing by BoNT-C does not induce heterosynaptic structural or functional plasticity at the convergent input. Thus, BoNT-C establishes the first approach to cleanly separate neurotransmission between tonic vs phasic neurons and defines heterosynaptic plasticity rules in a powerful model glutamatergic circuit.

sentences: In silico screening and testing of FDA approved small molecules to block SARS-CoV-2 entry to the host cell by inhibiting Spike protein cleavage The COVID-19 pandemic began in 2019, but it is still active. The development of an effective vaccine reduced the number of deaths; however, a treatment is still needed. Here, we aimed to inhibit viral entry to the host cell by inhibiting Spike (S) protein cleavage by several proteases. We develop a computational pipeline to repurpose FDA-approved drugs to inhibit protease activity and thus prevent S protein cleavage. We tested some of our drug candidates and demonstrated a decrease in protease activity. We believe our pipeline will be beneficial in identifying a drug regimen for COVID-19 patients.

sentences: Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping and can be used for long-term cytological biobanking. We sought to determine whether it is possible to access microbial DNA from LBC specimens, and compared the performance of four different extraction protocols: (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens. LBC specimens from 20 patients (stored for 716 {+/-} 105 days) with CIN values of 2 or 3 were each aliquoted for each of the four kits. Loss of microbial diversity due to long-term LBC storage could not be assessed due to lack of fresh LBC samples. Comparisons with other types of cervical sampling were not performed. We observed that all DNA extraction kits provided equivalent accessibility to the cervical microbial DNA within stored LBC samples. Approximately 80% microbial genera were shared among all DNA extraction protocols. Potential kit contaminants were observed as well. Variation between individuals was a significantly greater influence on the observed microbial composition than was the method of DNA extraction. We also observed that HPV16 was significantly associated with community types that were not dominated by Lactobacillus iners.

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Fast and flexible processing of large FRET image stacks using the FRET-IBRA toolkit Ratiometric time-lapse FRET analysis requires a robust and accurate processing pipeline to eliminate bias in intensity measurements on fluorescent images before further quantitative analysis can be conducted. This level of robustness can only be achieved by supplementing automated tools with built-in flexibility for manual ad-hoc adjustments. FRET-IBRA is a modular and fully parallelized configuration file-based tool written in Python. It simplifies the FRET processing pipeline to achieve accurate, registered, and unified ratio image stacks. The flexibility of this tool to handle discontinuous image frame sequences with tailored configuration parameters further streamlines the processing of outliers and time-varying effects in the original microscopy images. FRET-IBRA offers cluster-based channel background subtraction, photobleaching correction, and ratio image construction in an all-in-one solution without the need for multiple applications, image format conversions, and/or plug-ins. The package accepts a variety of input formats and outputs TIFF image stacks along with performance measures to detect both the quality and failure of the background subtraction algorithm on a per frame basis. Furthermore, FRET-IBRA outputs images with superior signal-to-noise ratio and accuracy in comparison to existing background subtraction solutions, whilst maintaining a fast runtime. The FRET-IBRA package has been extensively used in quantifying the spatial distribution of calcium ions during pollen tube growth under mechanical constraints. Benchmarks against existing tools clearly demonstrate the need for FRET-IBRA in extracting reliable insights from FRET microscopy images of dynamic physiological processes at high spatial and temporal resolution. The source code for Linux and Mac operating systems is released under the BSD license and, along with installation instructions, test images, example configuration files, and a step-by-step tutorial, is freely available at github.com/gmunglani/fret-ibra.

Author SummaryFRET is a fundamental imaging technique used to generate fluorescence signals sensitive to molecular conformations and interactions. Despite its wide use and the large body of literature on the theoretical steps required to process images generated from this procedure, we were unable to locate a tool that contained the entire processing workflow, whilst allowing the user the flexibility to adjust parameters for maximum accuracy and runtime efficiency. FRET-IBRA was thus created to be an all-in-one, open-source, parallel solution to process FRET images, while eliminating complications arising from repeated image format conversions. Besides enhancing the background subtraction algorithm for FRET images, several additional options were implemented for the user to extract the cleanest signal possible for their specific use case. FRET-IBRA is primarily built for flexibility when handling large image stacks by supporting sequences of image frames to be treated independently, greatly reducing time spent on splitting and concatenating image stacks. In accuracy and speed benchmarks against more general background subtraction packages, FRET-IBRA was able to provide the cleanest results with a fast runtime, leading to reliable analysis without additional tuning.

sentences: A novel cholinergic neural pathway and its role in the drug relapse The lateral parabrachial nucleus (LPB) is critical hub implicated in the control of food intake, reward and aversion. Here, we identified a novel cholinergic projection from choline acetyltransferase (ChAT)-positive neurons in external portion of the lateral parabrachial nucleus (eLPBChAT) to {gamma}-aminobutyric acid (GABA) neurons in central nucleus of amygdala (CeAGABA), activation of which could block methamphetamine (METH)-primed conditioned place preference (CPP) in mice.

sentences: Minicells from Highly Genome Reduced Escherichia coli: Cytoplasmic and Surface Expression of Recombinant Proteins and Incorporation in the Minicells Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced (GR) bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, GR bacteria offer certain advantages. With GR bacteria, more recombinant genes can be placed into GR chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in GR E. coli. The minCminD mutant GR E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of GR bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from GR bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. As some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud, and because the surface-to-volume ratio of the small minicells is higher than bacteria, recombinant proteins expressed on surfaces of the GR bacteria are concentrated on the minicells. Minicells made from GR bacteria can enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

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Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria Cobalamin (vitamin B12), is a cofactor for crucial metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonise the algal phycosphere, forming stable communities that gain preferential access to exudates and in return provide compounds, such as B12. Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12-independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesise that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12-dependent algae.

Originality-Significance statementMicroalgae are fundamental to the global carbon cycle, and yet despite being photosynthetic they often rely on other organisms for micronutrients. One of these micronutrients is vitamin B12 (cobalamin), which they receive from bacteria. Many environmental studies support the widespread role of B12 in algal-bacterial mutualisms, so here we wished to investigate how these mutualisms may arise evolutionarily by using an experimentally evolved B12-dependent alga and various B12-producers. A B12-producing rhizobium, Mesorhizobium loti, could stably support the B12-dependent Chlamydomonas reinhardtii metE7 strain, and vice versa, but nutrient supplementation increased growth of both species further. metE7 could also be supported by E. coli strains engineered to produce B12, and engineering a strain to release a higher proportion of B12 led to better algal growth, which increased bacterial growth in turn. We suggest that as B12-based mutualisms develop, increased B12 release may be selected for and therefore lead to more productive symbioses.

sentences: Systems-level identification of key transcription factors in immune cell specification Transcription factors (TFs) are crucial for regulating cell differentiation during the development of the immune system. However, the key TFs for orchestrating the specification of distinct immune cells are not fully understood. Here, we integrated the transcriptomic and epigenomic measurements in 73 mouse and 61 human primary cell types, respectively, that span the immune cell differentiation pathways. We constructed the cell-type-specific transcriptional regulatory network and assessed the global importance of TFs based on the Taiji framework, which is a method we have previously developed that can infer the global impact of TFs using integrated transcriptomic and epigenetic data. Integrative analysis across cell types revealed putative driver TFs in cell lineage-specific differentiation in both mouse and human systems. We have also identified TF combinations that play important roles in specific developmental stages. Furthermore, we validated the functions of predicted novel TFs in murine CD8+ T cell differentiation and showed the importance of Elf1 and Prdm9 in the effector versus memory T cell fate specification and Kdm2b and Tet3 in promoting differentiation of CD8+ tissue resident memory (Trm) cells, validating the approach. Thus, we have developed a bioinformatic approach that provides a global picture of the regulatory mechanisms that govern cellular differentiation in the immune system and aids the discovery of novel mechanisms in cell fate decisions.

sentences: Individual variability of neural computations in the primate retina Variation in the neural code contributes to making each individual unique. We probed neural code variation using [~]100 neural population recordings from major ganglion cell types in the macaque retina, combined with an interpretable computational representation of individual variability using machine learning. This representation captured individual variation and covariation in properties such as nonlinearity, temporal dynamics, and spatial receptive field size, while preserving invariances, such as asymmetries between ON and OFF cells. The covariation of response properties in different cell types was associated with the proximity of lamination of their synaptic inputs. Surprisingly, male retinas exhibited higher firing rates and faster temporal integration than female retinas. Exploiting data from previously recorded macaque retinas enabled efficient characterization of a new macaque retina, and of a human retina. Simulations indicated that combining a vast dataset of healthy macaque recordings with behavioral feedback could be used to identify the neural code and thus improve retinal implants for vision restoration.

sentences: Soil microbial diversity impacts plant microbiomes more than herbivory Interactions between plants and microbiomes play a key role in ecosystem functioning and are of broad interest due to their influence on nutrient cycling and plant protection. However, we do not yet have a complete understanding of how plant microbiomes are assembled. Here, we tested and quantified the effect of different factors driving the diversity and composition of plant-associated microbial communities. We manipulated soil microbial diversity (high or low diversity), plant species (Solanum tuberosum or Solanum vernei), and herbivory (presence or absence of a phloem-feeding insect Macrosiphum euphorbiae), and found that soil microbial diversity influenced the herbivore-associated microbiome composition, but also plant species and herbivory influenced the soil microbiome composition. We quantified the relative strength of these effects and demonstrated that the initial soil microbiome diversity explained the most variation in plant- and herbivore-associated microbial communities. Our findings strongly suggest that soil microbial community diversity is a driver of the composition of multiple associated microbiomes (plant and insect), and this has implications for the importance of management of soil microbiomes in multiple systems.

sentences: Yersinia pseudotuberculosis YopE prevents uptake by M cells and instigates M cell extrusion in human ileal enteroid-derived monolayers. Many pathogens use M cells to access the underlying Peyers patches and spread to systemic sites via the lymph as demonstrated by ligated loop murine intestinal models. However, the study of interactions between M cells and microbial pathogens has stalled due to the lack of cell culture systems. To overcome this obstacle, we use human ileal enteroid-derived monolayers containing five intestinal cell types including M cells to study the interactions between the enteric pathogen, Yersinia pseudotuberculosis (Yptb) and M cells. The Yptb type three secretion system (T3SS) effector Yops inhibit host defenses including phagocytosis and are critical for colonization of the intestine and Peyers patches. Therefore, it is not understood how Yptb traverses through M cells to breach the epithelium. By growing Yptb under two physiological conditions that mimic the early infectious stage (low T3SS-expression) or host-adapted stage (high T3SS-expression), we found that large numbers of Yptb specifically associated with M cells, recapitulating murine studies. Transcytosis through M cells was significantly higher by Yptb expressing low levels of T3SS, because YopE and YopH prevented Yptb uptake. YopE also caused M cells to extrude from the epithelium without inducing cell-death or disrupting monolayer integrity. Sequential infection with early infectious stage Yptb reduced host-adapted Yptb association with M cells. These data underscore the strength of enteroids as a model by discovering that Yops impede M cell function, indicating that early infectious stage Yptb more effectively penetrates M cells while the host may defend against M cell penetration of host-adapted Yptb.

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Do males know? Evidence-driven rainmaking of male bonobos to meet fertile phase of females Female bonobos exhibit prolonged receptivity, one suggested function of which is to lower male mating competition. However, it is questionable whether easier access to receptive females can reduce male-male competition, given the exclusive nature of male reproductive success. We tested whether males could determine the fertile phase of females. We found that ovulation probability predicted male mating effort. High-ranking males copulated with females with higher fertility, and male-male agonistic interactions increased when there were fertile females in the party. When there were multiple females with maximal swelling, males concentrated their mating effort on females with an older infant whose maximal swelling started earlier, and they continued mating efforts until detumescence (rainmaking). These findings suggest that male bonobos distinguish between fertile and non-fertile phases of females and that having more receptive females in the party does not reduce male-male competition for fertile females.

TeaserMales use the rainmakers rule to meet the periovulatory phase of the female bonobo for better reproductive success.

sentences: Untangling Microbiota Diversity and Assembly Patterns in the World's Largest Water Diversion Canal Large water diversion projects are important constructions for reallocation of human-essential water resources. Deciphering microbiota dynamics and assembly mechanisms underlying canal water ecosystem services especially during long-distance diversion is the prerequisite for water quality monitoring, biohazard warning and sustainable management. Using a 1432-km canal of the South-to-North Water Diversion Projects as a model system, we answer three central questions: how bacterial and micro-eukaryotic communities spatio-temporally develop, how much ecological stochasticity contributes to microbiota assembly, and which immigrating populations better survive and navigate across the canal. We applied quantitative ribosomal RNA gene sequence analyses to investigate canal water microbial communities sampled over a year, as well as null model- and neutral model-based approaches to disentangle the microbiota assembly processes. Our results showed clear microbiota dynamics in community composition driven by seasonality more than geographic location, and seasonally dependent influence of environmental parameters. Overall, bacterial community was largely shaped by deterministic processes, whereas stochasticity dominated micro-eukaryotic community assembly. We defined a local growth factor (LGF) and demonstrated its innovative use to quantitatively infer microbial proliferation, unraveling taxonomically dependent population response to local environmental selection across canal sections. Using LGF as a quantitative indicator of immigrating capacities, we also found that most micro-eukaryotic populations (82%) from the source lake water sustained growth in the canal and better acclimated to the hydrodynamical water environment than bacteria (67%). Taxa inferred to largely propagate include Limnohabitans sp. and Cryptophyceae, potentially contributing to water auto-purification. Combined, our work poses first and unique insights into the microbiota assembly patterns and dynamics in the worlds largest water diversion canal, providing important ecological knowledge for long-term sustainable water quality maintenance in such a giant engineered system.

sentences: Interactions between SARS-CoV-2 N-protein and α-synuclein accelerate amyloid formation First cases that point at a correlation between SARS-CoV-2 infections and the development of Parkinsons disease have been reported. Currently it is unclear if there also is a direct causal link between these diseases. To obtain first insights into a possible molecular relation between viral infections and the aggregation of -synuclein protein into amyloid fibrils characteristic for Parkinsons disease, we investigated the effect of the presence of SARS-CoV-2 proteins on -synuclein aggregation. We show, in test tube experiments, that SARS-CoV-2 S-protein has no effect on -synuclein aggregation while SARS-CoV-2 N-protein considerably speeds up the aggregation process. We observe the formation of multi-protein complexes, and eventually amyloid fibrils. Microinjection of N-protein in SHSY-5Y cells disturbed the -synuclein proteostasis and increased cell death. Our results point toward direct interactions between the N-protein of SARS-CoV-2 and -synuclein as molecular basis for the observed coincidence between SARS-CoV-2 infections and Parkinsonism.

sentences: Unique Protein Interaction Networks Define The Chromatin Remodeling Module of The NuRD Complex The combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1, and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodeling module of the Nucleosome Remodeling and Deacetylase (NuRD) complex, a gene repressor complex. Specific paralogues of the MGCC subunits such as MBD2 and CHD4 are amongst the key repressors of adult-stage fetal globin and provide important targets for molecular therapies in beta ({beta})-thalassemia. However, mechanisms by which the MGCC module acquires paralogue-specific function and specificity have not been addressed to date. Understanding the protein-protein interaction (PPI) network of the MGCC subunits is essential in defining underlying mechanisms and developing treatment strategies. Therefore, using pulldown followed by mass spectrometry analysis (PD-MS) we report a proteome-wide interaction network of the MGCC module in a paralogue-specific manner. Our data also demonstrate that the disordered C-terminal region of CHD3/4/5 is a gateway to incorporate remodeling activity into both the ChAHP (CHD4, ADNP, HP1{gamma}) and NuRD complexes in a mutually exclusive manner. We define a short aggregation prone region (APR) within the C-terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the Nuclear Receptor Co-Repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.

sentences: Glucocorticoid receptor activation induces gene-specific transcriptional memory and universally reversible changes in chromatin accessibility Glucocorticoids are stress hormones that elicit cellular responses by binding to the glucocorticoid receptor (GR), a ligand-activated transcription factor. The exposure of cells to this hormone induces wide-spread changes in the chromatin landscape and gene expression. Previous studies have suggested that some of these changes are reversible whereas others persist even when the hormone is no longer around. However, when we examined chromatin accessibility in human airway epithelial cells after hormone washout, we found that the hormone-induced changes were universally reversed after one day. Reversibility of hormone-induced changes are found for GR-occupied opening sites and also for closing sites that typically lack GR occupancy. These closing sites are enriched near repressed genes, suggesting that transcriptional repression by GR does not require nearby GR binding. Mirroring what we say in terms of chromatin accessibility, we found that transcriptional responses to hormone are universally reversable. Moreover, priming of cells by a previous exposure to hormone, in general, did not alter the transcriptional response to a subsequent encounter of the same cue. Interestingly, despite the short-lived nature of hormone-induced changes in the chromatin landscape, we identified a single gene, ZBTB16, that displays transcriptional memory manifesting itself as a more robust transcriptional response upon repeated hormone stimulation. Single-cell analysis revealed that the more robust response is driven by a higher probability of primed cells to activate ZBTB16 and by a subset of cells that express the gene at levels that are higher than the induction levels observed for naive cells. Although our study shows that hormone-induced changes are typically reversable, exposure to hormone can induce gene-specific changes in the response to subsequent exposures which may play a role in habituation to stressors and changes in glucocorticoid sensitivity.

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Trace elements in grey seals from the Gulf of St. Lawrence We measured baseline levels of 19 trace element and mercury speciation for grey seals (Halichoerus grypus) from the Gulf of St. Lawrence (GSL), Quebec, Canada. With interest growing in commercializing grey seal products for human consumption in this region, the goal of this study was to measure essential and non-essential trace elements in grey seals to evaluate health concerns and nutritional benefits. From 2015 to 2019, 120 grey seals were sampled by hunters and researchers at 4 sites in the GSL. Muscle, liver, heart and kidney samples were analyzed for 10 non-essential elements (Sb, As, Be, B, Cd, Pb, Hg, Ni, Tl, Sn) and 9 essential elements (Co, Cr, Cu, Fe, Mg, Mn, Mo, Se, Zn). Both total mercury (THg) and methylmercury (MeHg) were analysed for a subset of samples. Many elements were undetected in liver (Sb, As, Be, B, Cr, Co, Pb, Ni, Tl, Sn) and muscle tissues (same, plus Cd, Mn, Mo). Results showed lower element concentrations in the muscle (Fe, Mg, Se) and livers (Cd, Cr, Hg, Mn, Mo, Se) of young-of-the-year harvested in the winter (< 6 weeks old) compared to older animals feeding at sea. For older seals ([~] 5 months to 29 years), we did not observe progressive age-dependent bioaccumulation. Sex-specific differences were not very pronounced, but a few elements were 30 - 70% higher in the muscle (THg, MeHg) and liver (Mn, Zn) of male seals. Comparison to Canadian dietary reference intakes shows that a weekly portion of liver from young-of-the-year (< 6 weeks old) is a good source of essential elements (Cu, Fe) and that muscle and liver from this age category does not exceed reference values for toxic elements (As, Cd, Pb, MeHg). Ongoing discussions with regional public health professionals will help to develop dietary recommendations for the consumption of older grey seals.

HIGHLIGHTSO_LIWe measured baseline levels of 19 trace elements in grey seals harvested from the Gulf of St. Lawrence.
C_LIO_LIWe evaluated nutritional benefits and health concerns of human consumption of grey seal products.
C_LIO_LIOnce seals began feeding at sea ([~] 5 mo), many element concentrations increased, but did not bioaccumulate progressively with age afterwards.
C_LIO_LISome elements were more concentrated in the muscle (mercury) and livers (manganese, zinc) of male seals.
C_LIO_LIYoung seal (< 6 we) livers are a good dietary sources of copper and iron, while its muscle and liver were below reference values for toxic elements.
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sentences: Automated control of odor dynamics for neurophysiology and behavior Animals use their olfactory systems to avoid predators, forage for food, and identify mates. Olfactory systems detect and distinguish odors by responding to the concentration, temporal dynamics, and identities of odorant molecules. Studying the temporal neural processing of odors carried in air has been difficult because of the inherent challenge in precisely controlling odorized airflows over time. Odorized airflows interact with surfaces and other air currents, leading to a complex transformation from the odorized airflow that is desired to the olfactory stimulus that is delivered. Here, we present a method that achieves precise and automated control of the amplitude, baseline, and temporal structure of olfactory stimuli. We use this technique to analyze the temporal processing of olfactory stimuli in the early olfactory circuits and navigational behavior of larval Drosophila. Precise odor control and calcium measurements in the axon terminal of an Olfactory Receptor Neuron (ORN-Or42b) revealed dynamic adaptation properties: as in vertebrate photoreceptor neurons, Or42b-ORNs display simultaneous gain-suppression and speedup of their neural response. Furthermore, we found that ORN sensitivity to changes in odor concentration decreases with odor background, but the sensitivity to odor contrast is invariant - this causes odor-evoked ORN activity to follow the Weber-Fechner Law. Using precise olfactory stimulus control with freely-moving animals, we uncovered correlations between the temporal dynamics of larval navigation motor programs and the neural response dynamics of second-order olfactory neurons. The correspondence between neural and behavioral dynamics highlights the potential of precise odor temporal dynamics control in dissecting the sensorimotor circuits for olfactory behaviors.

sentences: Human Cardiac Organoids to Model COVID-19 Cytokine Storm Induced Cardiac Injuries Acute cardiac injuries occur in 20-25% of hospitalized COVID-19 patients. Despite urgent needs, there is a lack of 3D organotypic models of COVID-19 hearts for mechanistic studies and drug testing. Herein, we demonstrate that human cardiac organoids (hCOs) are a viable platform to model the cardiac injuries caused by COVID-19 hyperinflammation. As IL-1{beta}is an upstream cytokine and a core COVID-19 signature cytokine, it was used to stimulate hCOs to induce the release of a milieu of proinflammatory cytokines that mirror the profile of COVID-19 cytokine storm. The IL-1 {beta} treated hCOs recapitulated transcriptomic, structural, and functional signatures of COVID-19 hearts. The comparison of IL-1{beta} treated hCOs with cardiac tissue from COVID-19 autopsies illustrated the critical roles of hyper-inflammation in COVID-19 cardiac insults and indicated the cardioprotective effects of endothelium. The IL-1{beta} treated hCOs also provide a viable model to assess the efficacy and potential side effects of immunomodulatory drugs, as well as the reversibility of COVID-19 cardiac injuries at baseline and simulated exercise conditions.

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Peptide-based drug predictions for cancer therapy using deep learning BackgroundTherapeutic drugs used in cancer treatment have ineffectiveness and resistance to drug action problems. Anticancer peptides (ACPs) are selective and toxic to cancer cells and quickly produced. Thus, ACPs can be a satisfactory substitute for therapeutic drugs. We developed AI4ACP, a user-friendly web-server ACP predictor that can predict the anticancer property of query peptides, thus promoting the discovery of peptides with anticancer activity.

ResultOur results revealed that the performance of our ACP predictor trained using the new ACP collection was superior to that of the available high-performance ACP predictors.

ConclusionsAI4ACP is a user-friendly web-server ACP predictor that can be used to determine whether a query sequence is an ACP. This tool can be beneficial for drug development for cancer treatment. AI4ACP is freely accessible at https://axp.iis.sinica.edu.tw/AI4ACP/

sentences: Inter-Subject Correlation during New Music Listening: A Study of Electrophysiological and Behavioral Responses to Steve Reich`s Piano Phase Musical minimalism utilizes the temporal manipulation of restricted collections of rhythmic, melodic, and/or harmonic materials. One example, Steve Reichs Piano Phase, offers listeners readily audible formal structures containing unpredictable events at local levels. Pattern recurrences may generate strong expectations which are violated by small temporal and pitch deviations. A hyper-detailed listening strategy prompted by these minute deviations stands in contrast to the type of listening engagement typically cultivated around functional tonal Western music. Recent research has suggested that the inter-subject correlation (ISC) of electroencephalographic (EEG) responses to natural audio-visual stimuli objectively indexes a state of "engagement", demonstrating the potential of this approach for analyzing music listening. But can ISCs capture engagement with minimal music, which features less obvious expectation formation and has historically received a wide range of reactions? To approach this question, we collected EEG and continuous behavioral (CB) data while 30 adults listened to an excerpt from Steve Reichs Piano Phase, as well as three controlled manipulations and a popular-music remix of the work. Our analyses reveal that EEG and CB ISC are highest for the remix stimulus and lowest for our most repetitive manipulation. In addition, we found no statistical differences in overall EEG ISC between our most musically meaningful manipulations and Reichs original piece. We also found that aesthetic evaluations corresponded well with overall EEG ISC. Finally we highlight co-occurrences between stimulus events and time-resolved EEG and CB ISC. We offer the CB paradigm as a useful analysis measure and note the value of minimalist compositions as a limit case for studying music listening using EEG ISC. We show that ISC is less effective at measuring engagement with this minimalist stimulus than with popular music genres and argue that this may be due to a difference between the type of engagement measured by ISC and the particular engagement patterns associated with minimalism.

sentences: Design of a novel DNA Gyrase B inhibitor with a rhodanine scaffold: in silico and in vitro approaches Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin intermediate-resistant Staphylococcus aureus (VRSA) is one among the WHO high priority pathogens. Among these two, MRSA is the most globally documented pathogen that necessitates the pressing demand for new classes of anti-MRSA drugs. Bacterial gyrase targeted therapeutics are unique strategies to overcome cross-resistance as they are present only in bacteria and absent in higher eukaryotes. The GyrB subunit is essential for the catalytic functions of the bacterial enzyme DNA Gyrase, thereby constituting a promising druggable target. The current study performed a structure-based virtual screening to designing GyrB target-specific candidate molecules. The de novo ligand design of novel hit molecules was performed using a rhodanine scaffold. Through a systematic in silico screening process, the hit molecules were screened for their synthetic accessibility, drug likeliness and pharmacokinetics properties in addition to its target specific interactions. Of the total 374 hit molecules obtained through de novo ligand design, qsl-304 emerged as the most promising ligand. qsl-304 was synthesized through a one-step chemical synthesis procedure, and the in vitro activity was proven, with an IC50 of 31.23 g/mL against the novobiocin resistant clinical isolate of Staphylococcus aureus sa-P2003. Further studies on time-kill kinetics showed the bacteriostatic nature with the diminished recurrence of resistance.

sentences: Outbreak.info: A standardized, searchable platform to discover and explore COVID-19 resources and data To combat the ongoing COVID-19 pandemic, scientists have been conducting research at breakneck speeds, producing over 52,000 peer reviewed articles within the first 12 months. In contrast, a little over 1,000 peer reviewed articles were published within the first 12 months of the SARS-CoV-1 pandemic starting in 2002. In addition to publications, there has also been an upsurge in clinical trials to develop vaccines and treatments, scientific protocols to study SARS-CoV-2, methodology for epidemiological modeling, and datasets spanning molecular studies to social science research. One of the largest challenges has been keeping track of the vast amounts of newly generated disparate data and research that exist in independent repositories. To address this issue, we developed outbreak.info, which provides a standardized, searchable interface of heterogeneous resources on COVID-19 and SARS-CoV-2. Unifying metadata from twelve repositories, we have assembled a collection of over 260,000 publications, clinical trials, datasets, protocols, and other resources as of March 2022. We used a rigorous schema to enforce a consistent format across different data sources and resource types, and linked related resources where possible. This enables users to quickly retrieve information across data repositories, regardless of resource type or repository location. Outbreak.info also combines the combined research library with spatiotemporal genomics data on SARS-CoV-2 variants. The web interface provides interactive visualizations and reports to explore the unified data and generate hypotheses. In addition to providing a web interface, we also publish the data we have assembled and standardized in a high performance public API and an R package. Finally, we discuss the challenges inherent in combining metadata from scattered and heterogeneous resources and provide recommendations to streamline this process to aid scientific research.

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Wide occurrence of putative mobilized colistin resistance genes in the human gut microbiome BackgroundThe high incidence of bacterial genes that confer resistance to last-resort antibiotics, such as colistin caused by MCR genes, poses an unprecedented threat to our civilizations health. To understand the spread, evolution, and distribution of such genes among human populations, with the final goal of diminishing their occurrence in human environments should be a priority. To tackle this problem, we investigated the distribution and prevalence of potential mcr genes in the human gut microbiome we used a set of bioinformatics tools to screen the Unified Human Gastrointestinal Genome (UHGG) collection for the presence, synteny and phylogeny of putative mcr genes, and co-located antibiotic resistance genes.

ResultsA total of 2,079 ARGs were classified as different MCR in 2,046 Metagenome assembled genomes (MAGs), present in 1,596 individuals from 41 countries, of which 215 MCRs were identified in plasmidial contigs. The genera that presented the largest number of MCR-like genes were Suterella and Parasuterella, prevalent human gut bacteria of which Suterella wadsworthensis is associated with autism. Other potential pathogens carrying MCR genes belonged to the genus Vibrio, Escherichia and Campylobacter. Finally, we identified a total of 22,746 ARGs belonging to 21 different classes in the same 2,046 MAGs, suggesting multi-resistance potential in the corresponding bacterial strains, increasing the concern of ARGs impact in the clinical settings.

ConclusionThis study uncovers the diversity of MCR-like genes in the human gut microbiome. We showed the cosmopolitan distribution of these genes in individuals worldwide and the co-presence of other antibiotic resistance genes, including Extended-spectrum beta-lactamases (ESBL). Also, we described mcr-like genes fused to a PAP2-like domain in S. wadsworthensis. Although these novel sequences increase our knowledge about the diversity and evolution of mcr-like genes, their activity and a potential colistin resistance in the corresponding strains has to be experimentally validated.

sentences: Vertebrate whole genome duplications shaped the current 3D genome architecture Topologically associated domains (TADs) are interaction sub-networks of 3D genomes. TAD boundaries frequently coincide with genome breaks while their deletion is under negative selection, suggesting that TADs act as modules facilitating genome rearrangements and metazoan evolution. However, the role of TADs in the evolution of gene regulation and essentiality is not well understood. Here, we show that TADs play a role organising ancestral functions and evolutionary novelty. We discovered that genes co-localise by evolutionary age in the human and mouse genomes, resulting in TADs that have different proportions of younger and older genes. A major transition in the TAD age co-localisation patterns is observed between the genes born as a result of the vertebrate whole genome duplications (WGDs) or before, and those born afterwards. We also found that primate- and rodentspecific genes are more frequently essential when they are located in  aged TADs and connected to genes that have not duplicated since the WGD. Our data suggests that evolutionary success of recent genes may increase when located in functionally relevant TADs with established regulatory networks.

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Nonlinear super-resolution signal processing for subcellular analysis of calcium dynamics Optical (fluorescence) imaging of ionic dynamics has revolutionized neuroscience as it allows the study of neural activity across spatially identified populations. Quantification of fluorescence signals is commonly performed using ratiometric measures, like the {Delta}F/F. Although these measures are robust and easy to implement, they do not take advantage of the temporal information available in time-varying signals. Moreover, since a baseline (reference) period needs to be selected, their application is often limited to the quantification of stimulus-evoked activity. Here, we present a new approach, called ARES, based on the quantification of residuals after linear autoregression. We demonstrate the utility of ARES to quantify the functional dynamics of stimulus representation in cortical networks and show that it improves the spatial and temporal resolution with respect to {Delta}F/F. We further show that ARES can be used to study subcellular calcium dynamics and exemplify its utility to describe the spatiotemporal dynamics of calcium signal localization in compartmental network recordings. ARES offers a novel method for quantitative analysis of optical imaging data

Author summaryTwo-photon calcium imaging is a powerful and non-invasive method to measure and visualize the activity of neural populations in single cell and subcellular resolution. However, the slow dynamics of both the calcium signal itself and its indicators make it difficult to relate the timing and origin of the neural activity to the calcium signal. The most commonly used analysis method, {Delta}F/F, uses a reference baseline to quantify signal ratiometrically. This results in several limitations, especially in accounting for the time varying fluctuations in fluorescence values. Here we present a new method for the analysis of calcium signals, called ARES, in which a sliding window is used to predict the future values of the signal and deviations from this prediction are recorded as the signal. This has the advantage that shifts in baseline do not affect the processing, and that the method has an improved spatial and temporal resolution with respect to {Delta}F/F.

sentences: Genome-wide association mapping of transcriptome variation in Mimulus guttatus indicates differing patterns of selection on cis- versus trans-acting mutations We measured the floral bud transcriptome of 151 fully sequenced lines of Mimulus guttatus from one natural population. Thousands of single nucleotide polymorphisms (SNPs) are implicated as transcription regulators, but there is a striking difference in the Allele Frequency Spectrum (AFS) of cis-acting and trans-acting mutations. Cis-SNPs have intermediate frequencies (consistent with balancing selection) while trans-SNPs exhibit a rare-alleles model (consistent with purifying selection). This pattern only becomes clear when transcript variation is normalized on a gene-to-gene basis. If a global normalization is applied, as is typically in RNAseq experiments, asymmetric transcript distributions combined with "rarity disequilibrium" produce a super-abundance of false positives for trans-acting SNPs. To explore the cause of purifying selection on trans-acting mutations, we identified gene expression modules as sets of co-expressed genes. The extent to which trans-acting mutations influence modules is a strong predictor of allele frequency. Mutations altering expression of genes with high "connectedness" (those that are highly predictive of the representative module expression value) have the lowest allele frequency. The expression modules can also predict whole-plant traits such as flower size. We find that a substantial portion of the genetic (co)variance among traits can be described as an emergent property of genetic effects on expression modules.

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Coupled myovascular expansion directs cardiac growth and regeneration BackgroundHeart regeneration requires multiple cell types to enable cardiomyocyte (CM) proliferation. How these cells interact to create growth niches is unclear. Here we profile proliferation kinetics of cardiac endothelial cells (CECs) and CMs in the neonatal mouse heart and find that they are spatiotemporally coupled.

Methods and ResultsWe show that coupled myovascular expansion during cardiac growth or regeneration is dependent upon VEGF-VEGFR2 signaling, as genetic deletion of Vegfr2 from CECs or inhibition of VEGFA abrogates both CEC and CM proliferation. Repair of cryoinjury displays poor spatial coupling of CEC and CM proliferation. Boosting CEC density after cryoinjury with virus encoding Vegfa enhances regeneration. Using Mendelian randomization, we demonstrate that circulating VEGFA levels are positively linked with human myocardial mass, suggesting that Vegfa can stimulate human cardiac growth.

ConclusionsOur work demonstrates the importance of coupled CEC and CM expansion and reveals a myovascular niche that may be therapeutically targeted for heart regeneration.

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Revisiting colorectal cancer tumorigenesis with spatially-resolved gene expression profiling Early detection and treatment are paramount to the clinical outcome of patients with colorectal cancer (CRC). Deciphering the dynamic interactions that occur between epithelial cells and stromal cells during tumorigenesis requires in-depth analyses of early-stage CRC lesions in spatial context. Here we employed spatially-resolved gene expression profiling to dissect molecular processes that associate with malignant transformation in CRC. We provide the transcriptional landscapes of colorectal cancer tumorigenesis from healthy mucosa, through different degrees of dysplasia, to cancer. The complementary examination of epithelial and stromal fractions allowed us to define whether specific oncogenic processes involved cancer cells, stromal cells, or the tumor microenvironment as a whole. We identified several genes that were consistently deregulated during CRC onset that could serve as clinical biomarkers for early-stage CRC. Furthermore, we uncovered an essential role for the innate immune system during CRC tumorigenesis.

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sentences: Identifying Causal Subsequent Memory Effects Over 40 years of accumulated research has detailed associations between neuroimaging signals measured during a memory encoding task and later memory performance, across a variety of brain regions, measurement tools, statistical approaches and behavioral tasks. But the interpretation of these Subsequent Memory Effects (SMEs) remains unclear: if the identified signals reflect cognitive and neural mechanisms of memory encoding then the underlying neural activity must be causally related to future memory. However, almost all previous SME analyses do not control for potential confounders of this causal interpretation, such as serial position and item effects. We collect a large fMRI dataset and use a novel experimental design and analysis approach that allows us to statistically adjust for all exogenous confounding variables. We find that, using standard approaches without adjustment, we replicate several univariate and multivariate subsequent memory effects and are able to predict memory performance across people. However, we are unable to identify any signal that reliably predicts subsequent memory after adjusting for confounding variables, bringing into doubt the causal status of these effects. We apply the same approach to subjects judgments of learning collected during an encoding period, and show that these behavioral measures of encoding quality do predict memory after adjustments, suggesting that it is possible to measure signals at the time of encoding that reflect causal mechanisms but that existing neuroimaging measures may not have the precision and specificity to do so.

sentences: Are members of the Anopheles fluviatilis complex conspecific? Anopheles fluviatilis sensu lato, a primary malaria vector in India, was identified to be comprised of four cryptic species, provisionally designated as species S, T, U and V. However, Kumar et al. (Mol Ecol Resour, 2013;13:354-61) considered all of the then known three members of this species complex (S, T and U) conspecific. The specific status of species S and T was refuted based on the lack of sufficient barcode gap in mitochondrial-CO1 and the perceived presence of heterozygotes in populations as detected through one of the two species-specific PCR assays employed for the cryptic species identification. The existence of species U was refuted claiming that earlier investigations have already refuted their existence. This conclusion is concerning because of the differential public health implications of members of the Fluviatilis Complex. Here we discuss problems associated with the CO1-based barcode approach for delimitation of cryptic species, the perceived heterozygosity between species S and T based on a species-specific PCR assay, and interpretation of published reports. We demonstrated that fixed differences do exist in the ITS2-rDNA sequence of species S and T with no evidence of heterozygotes in sympatric populations and, that the observed heterozygosity by Kumar et al. in the ITS2-based species diagnostic PCR is due to the high mispriming tendency of the T-specific primer with species S. We infer that mitochondrial DNA-based  barcoding gap, an arbitrary threshold recommended for species delimitation, alone, is inadequate to delimit the members of An. fluviatilis complex.

sentences: In silico analysis and molecular investigation of the nuclear shuttle protein-interacting kinase 1 (NIK1) in six Solanaceous species The first layer of innate immunity in plants is initiated through the perception of microbe-associated molecular patterns (MAMPs) or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs). MAMP/DAMP perception initiates downstream defense responses, a process which ultimately leads to pattern triggered immunity (PTI). In Arabidopsis, the nuclear shuttle protein-interacting kinase 1 (NIK1), among other PRRs, is one of the most important central components of PTI signaling and kinase signaling cascade, since it is involved in the plant antiviral response against geminiviruses. Despite the characterization of the structure and function of the NIK1 receptors made by some groups, studies related to NIK1 importance in the current gene-editing era are missing. By simple in silico analysis, in this study we investigated the NIK1 homologues from six Solanaceous plant species including: tomato (Solanum lycopersicum), potato (Solanum tuberosum), Solanum pennellii, eggplant (Solanum melongena), pepper (Capsicum annum), and Nicotiana benthamiana. The phylogenetic analyses of different NIK1 proteins from Arabidopsis and six Solanaceous plants revealed nine different clades. As expected, we found that these NIK1 orthologs have similar genomic structures suggesting a similar function. We could identify that SotubNIK1, SolyNIK1, SopenNIK1, CANIK1, NibenNIK1, SmeNIK1 have the highest sequence homology with AtNIK1. Additionally, the conserved protein kinase domain (PKD) that is present in NIK1 from Arabidopsis thaliana was bioinformatically analyzed and found in other species. As this highly conserved NIK1 region is present in several crops of economic importance, its potential is highlighted as a possible target site for gene editing, to develop crops tolerant to geminiviral infections.

sentences: Predictable and stable epimutations induced during clonal propagation with embryonic transcription factors Although clonal propagation is frequently used in commercial plant breeding and plant biotechnology programs because it minimizes genetic variation, it is not uncommon to observe clonal plants with stable phenotypic changes, a phenomenon known as somaclonal variation. Several studies have shown that epigenetic modifications induced during regeneration are associated with this newly acquired phenotypic variation. However, the factors that determine the extent of somaclonal variation and the molecular changes associated with it remain poorly understood. To address this gap in our knowledge, we compared clonally propagated Arabidopsis thaliana plants derived from somatic embryogenesis using two different embryonic transcription factors-RWP-RK DOMAIN-CONTAINING 4 (RKD4) or LEAFY COTYLEDON2 (LEC2) and from two epigenetically distinct tissues. We found that both the epi(genetic) status of explant and the regeneration protocol employed play critical roles in shaping the molecular and phenotypic state of clonal plants. Phenotypic variation of regenerated plants can be largely explained by the inheritance of tissue-specific DNA methylation imprints, which are associated with specific transcriptional and metabolic changes in sexual progeny of clonal plants. Moreover, regenerants from roots were particularly affected by the inheritance of epigenetic imprints, which resulted in increased accumulation of salicylic acid in leaves and accelerated plant senescence. Collectively, our data reveal pathways for targeted manipulation of phenotypic variation in clonal plants.

sentences: Fast and reliable sgRNA efficiency testing using HIReff CRISPR/Cas9 is the method of choice for gene editing like the endogenous knock-in of sequences in order to investigate protein function, abundance or intracellular localization. One of the crucial steps in the preparation of CRISPR/Cas9-mediated knock-ins is the design of sgRNAs, which need to be tested carefully in order to minimize off-target binding and reach highest cleavage efficiency. Usually, sgRNA is evaluated via mismatch cleavage assays, like Surveyor or T7 endonuclease 1 assay. We demonstrate that these methods are often highly cost- and time-intensive with a low sensitivity and high fail rate. As an alternative, we present a new HITI-based sgRNA efficiency (HIReff) test to precisely evaluate sgRNA efficiency. HIReff is based on a sophisticated integration vector with on-site generation of a linear donor fragment that allows a comparably easy read-out via fluorescence signal and integrates several internal controls. Next to a quantifiable sgRNA assessment, HIReff provides additional information on the  gene/protein to be studied abundance, subcellular localization and promoter activity and allows derivation of fluorescence protein labeled clonal lines. We highlight benefits of HIReff in comparison to commonly used enzyme-based assays and demonstrate improved practicability and high sensitivity, while being less time-, labor- and costintensive at the same time. Our results suggest HIReff as a fast and easy-to-use alternative for sgRNA efficiency testing.

sentences: Host related factors determine co-occurrence patterns between pathogenic bacteria, protozoa, and helminths in populations of the multimammate mouse, Mastomys natalensis Advances in experimental and theoretical work increasingly suggest that parasite interactions within a single host can affect the spread and severity of wildlife diseases. Yet empirical data to support predicted co-infection patterns are limited due to the practical challenges of gathering convincing data from animal populations and the stochastic nature of parasite transmission. Here, we investigated co-infection patterns between micro- (bacteria and protozoa) and macroparasites (gastrointestinal helminths) in natural populations of the multimammate mouse (Mastomys natalensis). Fieldwork was performed in Morogoro (Tanzania), where we trapped 211 individual M. natalensis and tested their behavior using a modified open-field arena. All animals were checked on the presence of helminths in their gastrointestinal tract, three bacteria (Anaplasma, Bartonella, and Borrelia) and two protozoan genera (Piroplasma and Hepatozoon). Besides the presence of eight different helminth genera (reported earlier), we found that 21% of M. natalensis were positive for Anaplasma, 13% for Bartonella, and 2% for Hepatozoon species. Hierarchical modelling of species communities was used to investigate the effect of the different host-related factors on these parasites infection probability and community structure. Our results show that the infection probability of Anaplasma and Bartonella was higher in adults than juveniles. We also observed that females and less explorative individuals had a higher infection probability with Bartonella. We found limited support for within-host interactions between micro-and macroparasites, as only animals infected with Bartonella were significantly more likely to be infected with Protospirura, Trichuris, and Trichostrongylidae helminths.

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Constitutive expression of JASMONATE RESISTANT 1 elevates content of several jasmonates and primes Arabidopsis thaliana to better withstand drought Jasmonates have a well-documented role in balancing the trade-off between plant growth and defense against biotic stresses. However, the role of jasmonate signaling under abiotic stress is less well studied. Here, we investigated the function of JASMONATE RESISTANT 1 (JAR1) in drought stress in Arabidopsis thaliana. JAR1 converts jasmonic acid (JA) to jasmonyl-L-isoleucine (JA-Ile), the major bioactive form of jasmonates. Comparison of a newly generated over-expression line (JAR1-OE) with jar1-11, a T-DNA insertion line in the JAR1 locus, and Col-0 revealed that constitutively increased JA-Ile production results in stunted growth and a delay in flowering. Upon water limitation, JAR1-OE plants retained more water in their leaves, showed reduced wilting and recovered better from drought stress than the wild type. By contrast, jar1-11 mutant plants were hypersensitive to drought. RNA-seq analysis and hormonal profiling of plants under control and drought stress conditions provided insight into the molecular reprogramming caused by the alteration in JA-Ile content. Especially JAR1-OE plants were affected in many adaptive systems related to drought stress, including stomatal density, stomatal aperture or the formation of reactive oxygen species (ROS). Overall, our data suggest that constitutively increased expression of JAR1 can prime Arabidopsis towards improved drought tolerance.

One sentence summaryConstitutive expression of JAR1primes Arabidopsis thaliana to better withstand drought stress

sentences: The Rise of West Nile Virus in Europe: investigatingthe combined effects of climate, land use and economic changes West Nile Virus (WNV) has recently emerged as a major public health concern in Europe, its recent expansion also coincided with some remarkable socio-economic and environmental changes, including an economic crisis and some of the warmest temperatures on record. Here we empirically investigate the drivers of this phenomenon at a European wide scale by constructing and analyzing a unique spatial-temporal data-set, that includes data on climate, land-use, the economy, and government spending on environmental related sectors. Drivers and risk factors of WNV were identified by building a conceptual framework, and relationships were tested using a Generalized Additive Model (GAM), which could capture complex non-linear relationships and also account for the spatial and temporal auto-correlation. Some of the key risk factors identified in our conceptual framework, such as a higher percentage of wetlands and arable land, climate factors (higher summer rainfall and higher summer temperatures) were positive predictors of WNV infections. Interestingly, winter temperatures of between 2 {degrees}C and 6 {degrees}C were among some of the strongest predictors of annual WNV infections, one possible explanation for this result is that successful overwintering of infected adult mosquitoes (likely Culex pipiens) is key to the intensity of outbreaks for a given year. Furthermore, lower surface water extent over the summer is also associated with more more intense outbreaks, suggesting that drought, which is known to induce positive changes in WNV prevalence in mosquitoes is also contributing to the upward trend in WNV cases in affected regions. Our indicators representing the economic crisis were also strong predictors of WNV infections, suggesting there is an association between austerity and cuts to key sectors, which could have benefited vector species and the virus during this crucial period. These results, taken in the context of recent winter warming due to climate change, and more frequent droughts, may offer an explanation of why the virus has become so prevalent in Europe.

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Measles virus exits human airway epithelia via infectious center sloughing Measles virus (MeV) is the most contagious human virus, but we do not fully understand why. Unlike most respiratory viruses, MeV does not infect the airway epithelium immediately. MeV traverses the epithelium within immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for [~]3-5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may slough off while preserving epithelial function. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In sloughed infectious centers, ciliary beating persisted and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, modeling the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center sloughing. 5-ethynyl-2-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the extruded infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeVs strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.

AUTHOR SUMMARYMeasles virus (MeV) is a respiratory pathogen that infects millions worldwide each year. Although sometimes mischaracterized as an innocuous childhood disease, measles remains a leading cause of death for children under five. MeV is the most contagious human virus and requires vaccination rates above 90% to maintain herd immunity. Global decreases in vaccination rates over the past ten years contributed to recent, widespread MeV outbreaks. We uncover here a novel mechanism by which MeV exits the human airways that may explain why it is much more contagious than other viruses. We document that infected cells containing cell-associated virus slough en masse from the airway epithelial sheet. These expelled infectious centers are metabolically active and can transmit infection to primary human monocyte-derived macrophages more efficiently than cell-free virus particles. Thus, cell-associated MeV can transmit host-to-host, a new paradigm for efficient respiratory virus transmission.

sentences: Kincore: a web resource for structural classification of protein kinases and their inhibitors The active form of kinases is shared across different family members, as are several commonly observed inactive forms. We previously performed a clustering of the conformation of the activation loop of all protein kinase structures in the Protein Data Bank (PDB) into 8 classes based on the dihedral angles that place the Phe side chain of the DFG motif at the N-terminus of the activation loop. Our clusters are strongly associated with the placement of the activation loop, the C-helix, and other structural elements of kinases. We present Kincore, a web resource providing access to our conformational assignments for kinase structures in the PDB. While other available databases provide conformational states or drug type but not both, KinCore includes the conformational state and the inhibitor type (Type 1, 1.5, 2, 3, allosteric) for each kinase chain. The user can query and browse the database using these attributes or determine the conformational labels of a kinase structure using the web server or a standalone program. The database and labeled structure files can be downloaded from the server. Kincore will help in understanding conformational dynamics of these proteins and guide development of inhibitors targeting specific states. Kincore is available at http://dunbrack.fccc.edu/kincore.

sentences: Dengue transmission risk in a changing climate: Bangladesh could experience a longer dengue fever season in the future Our changing climate is already affecting the transmission of vector borne diseases such as dengue fever. This issue presents a significant public health concern for some nations, such as Bangladesh, which already experience regular seasonal outbreaks of dengue fever under present day conditions. To provide guidance for proactive public health planning to potentially mitigate future infections, we explore the impact of climate change on dengue infections by calculating the change in vectorial capacity of Aedes aegypti mosquito at a seasonal level for all regions in Bangladesh under two scenarios for future atmospheric greenhouse gas concentrations. For each of the four climate models used, and for both scenarios, our analysis reveals that the annual vectorial capacity remains at a level that would enable potential dengue epidemic transmission in all regions in Bangladesh. We found a slight decline in vectorial capacity in half of the regions examined during the last two decades of the 21st Century for the lower-concentration scenario, with a pronounced decline in vectorial capacity in all geographic regions beginning in 2060 for the higher-concentration scenario. The likely reason is that in many regions, warming is leading to sub-optimal mosquito breeding temperatures. However, seasonal differences in vectorial capacity dissipates as the climate warms, to the point that there is almost no observable seasonality for the higher-concentration scenario during the last two decades of this century. This finding suggests there is the potential for the dengue season to extend all year, with outbreaks occurring at any time. Consequently, disease surveillance and control activities would need to be geographically and temporally adapted to mitigate dengue epidemic risk in response to climate change.

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Substrate stiffness controls the cell cycle of human mesenchymal stem cells via cellular traction The microenvironment of human mesenchymal stem cells (hMSCs) regulates their self-renewal and differentiation properties. Previously it was shown that hMSCs remained quiescent on soft (0.25 kPa) polyacrylamide (PA) gels but re-entered into cell cycle on a stiff (7.5 kPa) gel. However, how cells behave on intermediate stiffness and what intracellular factors transmit mechanical changes to cell interior thereby regulating cell cycle remained unknown. In this work we demonstrated that PA gels between 1 and 5 kPa act as a mechanical switch in regulating cell cycle of hMSCs. By experiments on cell-cycle exit and re-entry, we found that hMSCs demonstrated a sharp transition from quiescence to proliferation between 1 and 5 kPa. Further studies with ROCK inhibitor Y-27632 revealed that contractile proteins, but not cell spread area, accounts for the sensitivity of hMSCs towards substrate stiffness and hence correlates with their changes in cell cycle. These observations therefore suggest that substrate stiffness regulates hMSC proliferation through contractile forces as generated by cellular contractile proteins in a unique pattern which is distinct from other cell types as studied.

Graphical abstract

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sentences: Gene Expression and Tracer-Based Metabolic Flux Analysis Reveals Tissue-Specific Metabolic Scaling in vitro, ex vivo, and in vivo Metabolic scaling, the inverse correlation of metabolic rates to body mass, has been appreciated for more than 80 years. Studies of metabolic scaling have almost exclusively been restricted to mathematical modeling of oxygen consumption. The possibility that other metabolic processes scale with body size has not been studied. To address this gap in knowledge, we employed a systems approach spanning from transcriptomics to in vitro and in vivo tracer-based flux. Gene expression in livers of five species spanning a 30,000-fold range in mass revealed differential expression of genes related to cytosolic and mitochondrial metabolic processes, in addition to detoxication of oxidative damage. This suggests that transcriptional scaling of damage control mechanisms accommodates increased oxidative metabolism in smaller species. To determine whether flux through key implicated metabolic pathways scaled, we applied stable isotope tracer methodology to study multiple cellular compartments, tissues, and species. Comparing mice and rats, we demonstrate that while scaling of metabolic fluxes is not observed in the cell-autonomous setting, it is present in liver slices and in vivo. Together, these data reveal that metabolic scaling extends beyond oxygen consumption to numerous other metabolic pathways, and is likely regulated at the level of gene expression and substrate supply.

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Caspases switch off m6A RNA modification pathway to reactivate a ubiquitous human tumor virus The methylation of RNA at the N6 position of adenosine (m6A) orchestrates multiple biological processes to control development, differentiation, and cell cycle, as well as various aspects of the virus life cycle. How the m6A RNA modification pathway is regulated to finely tune these processes remains poorly understood. Here, we discovered the m6A reader YTHDF2 as a caspase substrate via proteome-wide prediction, followed by in vitro and in vivo validations. We further demonstrated that cleavage-resistant YTHDF2 blocks, while cleavage-mimicking YTHDF2 fragments promote, the replication of a common human oncogenic virus, Epstein-Barr virus (EBV). Intriguingly, our study revealed a feedback regulation between YTHDF2 and caspase-8 via m6A modification of CASP8 mRNA and YTHDF2 cleavage during EBV replication. Further, we discovered that caspases cleave multiple components within the m6A RNA modification pathway to benefit EBV replication. Together, our study establishes that caspase disarming of the m6A RNA modification machinery fosters EBV reactivation.

Teaser

Cellular m6A RNA modification machinery is cleaved by caspases to foster the reproduction of a common human tumor virus

sentences: SAMHD1 mutations in mantle cell lymphoma are recurrent and confer in vitro resistance to nucleoside analogues The genomic landscape of mantle cell lymphoma (MCL) includes frequent alterations of TP53, ATM, CCND1 and KMT2D. Thus far, the mutational landscape provides little information for treatment stratification. We characterized a cohort of MCL by targeted next generation sequencing and discovered SAMHD1 as a novel recurrently mutated gene (8.5% of investigated cases, 4/47 samples). Furthermore, we provide evidence of in vitro resistance of SAMHD1 mutated patient-derived MCL cells to cytarabine and fludarabine.

sentences: Maternal transmission as a symbiont sieve, and the absence of lactation in male mammals Gut microbiomes of humans carry a complex symbiotic assemblage of microorganisms. As in all mammals, the special mode of feeding newborn infants through milk from the mammary gland enhances the opportunity for vertical transmission of elements of this microbiome. This has potential benefits, but it also brings with it some hazards for the host. We point out here that vertical transmission from both parents would allow host populations to be invaded by elements that are deleterious. In contrast, vertical transmission, when restricted to one parent, acts as a sieve preventing the spread of such elements. Because some transmission from mother to infant is unavoidable in placental mammals, uniparental transmission of symbionts, if it were to be selected, would require some separation of the father from the newborn infant. This paper therefore puts forward the hypothesis that the asymmetry in roles of mother and father, together with the hazards that come with biparental transmission, generate a selection pressure against male lactation in humans, and in mammals in general.

sentences: PIPP: Improving peptide identity propagation using neural networks Peptide identity propagation (PIP) can substantially reduce missing values in label-free mass spectrometry quantification by transferring peptides identified by tandem mass (MS/MS) spectra in one run to experimentally related runs where the peptides are not identified by MS/MS. The existing frameworks for matching identifications between runs perform peak tracing and propagation based on similarity of precursor features using only a limited number of dimensions available in MS1 data. These approaches do not produce accompanying confidence estimates and hence cannot filter probable false positive identity transfers. We introduce an embedding based PIP that uses a higher dimensional representation of MS1 measurements that is optimized to capture peptide identities using deep neural networks. We developed a propagation framework that works entirely on MaxQuant results. Current PIP workflows typically perform propagation mainly using two feature dimensions, and rely on deterministic tolerances for identification transfer. Our framework overcomes both these limitations while additionally assigning probabilities to each transferred identity. The proposed embedding approach enables quantification of the empirical false discovery rate (FDR) for peptide identification, while also increasing depth of coverage through coembedding the runs from the experiment with experimental libraries. In published datasets with technical and biological variability, we demonstrate that our method reduces missing values in MaxQuant results, maintains high quantification precision and accuracy, and low false transfer rate.

sentences: Cellulosic copper nanoparticles and a hydrogen peroxide-based disinfectant protect Vero E6 cells against infection by viral pseudotyped particles expressing SARS-CoV-2, SARS-CoV or MERS-CoV Spike protein. Severe acute respiratory syndrome (SARS) is a viral respiratory infection caused by human coronaviruses (HuCoV) that include SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Although their primary mode of transmission is through contaminated respiratory droplets from infected carriers, the deposition of expelled virus particles onto surface and fomites could contribute to viral transmission. Here, we use replication-deficient murine leukemia virus (MLV) pseudoviral particles expressing SARS-CoV-2, SARS-CoV, or MERS-CoV Spike (S) protein on their surface. These surrogates of native coronavirus counterparts serve as a model to analyze the S-mediated entry into target cells. Carboxymethyl cellulose (CMC) nanofibers that are combined with copper (Cu) exhibit strong antimicrobial properties. S-pseudovirions that are exposed to CMC-Cu nanoparticles (30 s) display a dramatic reduction in their ability to infect target Vero E6 cells, with [~]97% less infectivity as compared to untreated pseudovirions. In contrast, addition of the Cu chelator tetrathiomolybdate protects S- pseudovirions from CMC-Cu-mediated inactivation. When S-pseudovirions were treated with a hydrogen peroxide-based disinfectant (denoted SaberTM) used at 1:16 dilution, their infectivity was dramatically reduced by [~]98%. However, the combined use of SaberTM and CMC-Cu is the most effective approach to restrict infectivity of SARS-CoV-2-S, SARS-CoV-S, and MERS-CoV-S pseudovirions in Vero E6 cell assays. Together, these results show that cellulosic Cu nanoparticles enhance the effectiveness of diluted SaberTM sanitizer, setting up an improved strategy to lower the risk of surface- and fomite-mediated transmission of enveloped respiratory viruses.

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Cell cortex microtubules contribute to division plane positioning during telophase in maize The phragmoplast is a plant-specific microtubule and microfilament structure that forms during telophase to direct new cell wall formation. The phragmoplast expands towards a specific location at the cell cortex called the division site. How the phragmoplast accurately reaches the division site is currently unknown. We show that a previously uncharacterized microtubule arrays accumulated at the cell cortex. These microtubules were organized by transient interactions with division-site localized proteins and were then incorporated into the phragmoplast to guide it towards the division site. A phragmoplast-guidance defective mutant, tangled1, had aberrant cortical-telophase microtubule accumulation that correlated with phragmoplast positioning defects. Division-site localized proteins may promote proper division plane positioning by organizing the cortical-telophase microtubule array to guide the phragmoplast to the division site during plant cell division.

One Sentence SummaryMicrotubules accumulate at the cell cortex and interact with the plant division machinery to direct its movement towards the division site.

sentences: Association between land use, land cover, plant genera, and pollinator abundance in mixed-use landscapes Pollinators are globally threatened by land-use change, but its effect varies depending on the taxa and the intensity of habitat degradation. However, pollinator-landscape studies typically focus on regions of intensive human activities and on a few focal species. Evaluating pollinator responses in landscapes with moderate land-use changes and on multiple pollinator groups would therefore fill an important knowledge gap. This study aims to determine the predictive capacity and effect of habitat characteristics on the relative abundance of multiple pollinator groups in mixed-use landscapes. To do this, we collected field data on the relative abundance of nectivorous birds, bees, beetles, and butterflies across the Tasman Peninsula (Tasmania, Australia). We then applied Random Forests to resolve the effects of land use (protected areas, plantation, and pasture), land cover at different radii (100 m and 2000 m), and plant genera on pollinator abundance. Overall, land cover and plant genera were more important predictors of pollinator abundance than land use. And the effect of land use, land cover, and plant genera varied depending on the pollinating group. Pollinator groups were associated with a range of plant genera, with the native genera Acacia, Leptospermum, Leucopogon, Melaleuca, Pomaderris, and Pultenaea being among the most important predictors. Our results highlight that one size does not fit all--that is pollinator response to different landscape characteristics vary, emphasise the importance of considering multiple habitat factors to manage and support a dynamic pollinator community, and demonstrates how land management can be informed using predictive modelling.

sentences: A burst of transposon expression accompanies the activation of Y chromosome fertility genes during Drosophila spermatogenesis Transposable elements (TEs) must replicate in germline cells to pass novel insertions to offspring. In Drosophila melanogaster ovaries, TEs can exploit specific developmental windows of opportunity to evade host silencing and increase their copy numbers. However, TE activity and host silencing in the distinct cell types of the Drosophila melanogaster testis are not well understood. We reanalyzed publicly available single-cell RNA-seq datasets to quantify TE expression in the distinct cell types of the Drosophila testis. We developed a novel method for identification of TE and host gene expression programs and find that a distinct population of early spermatocytes expresses a large number of TEs at much higher levels than other germline and somatic components of the testes. This burst of TE expression coincides with the activation of Y chromosome fertility factors and spermatocyte-specific transcriptional regulators, as well as downregulation of many components of the piRNA pathway. The TEs expressed by this cell population are enriched on the Y chromosome and depleted on the X chromosome relative to other active TEs. These data suggest that some TEs may achieve high insertional activity in males by exploiting a window of opportunity for mobilization created by the activation of spermatocyte-specific and Y-chromosome-specific transcriptional programs.

sentences: Necroptosis inhibition counteracts axonal degeneration, cognitive decline and key hallmarks of aging, promoting brain rejuvenation. Age is the main risk factor for the development of neurodegenerative diseases. In the aged brain, axonal degeneration is an early pathological event, preceding neuronal dysfunction, and cognitive disabilities. Necroptosis activation mediates degeneration of injured axons, but whether necroptosis triggers neurodegeneration and cognitive impairment along aging is unknown. Here we show necroptosis activation in hippocampal axons during aging. Loss of the necroptotic effector Mlkl was sufficient to delay age-associated axonal degeneration, protecting against decreased synaptic transmission and memory decline in aged mice. Moreover, short-term pharmacologic inhibition of necroptosis in aged mice reverted structural and functional hippocampal impairment. Finally, a quantitative proteomic analysis revealed that necroptosis inhibition leads to an overall improvement of the aged hippocampal proteome, including molecular biofunctions associated with brain rejuvenation. Our results demonstrate that necroptosis contributes to the functional decline of the aged brain, and necroptosis inhibition constitute a potential geroprotective strategy to treat age-related disabilities.

sentences: An ecological perspective on microbial genes of unknown function in soil Genes that remain hypothetical, uncharacterized, and unannotated comprise a substantial portion of metagenomic datasets and are likely to be particularly prevalent in soils where poorly characterized taxa predominate. Documenting the prevalence, distribution, and potential roles of these genes of unknown function is an important first step to understanding their functional contributions in soil communities. We identified genes of unknown function from 50 soil metagenomes and analyzed their environmental distributions and ecological associations. We found that genes of unknown function are prevalent in soils, particularly fine-textured, higher pH soils that harbor greater abundances of Crenarchaeota, Gemmatimonadota, Nitrospirota, and Methylomirabilota. We identified 43 dominant (abundant and ubiquitous) gene clusters of unknown function and determined their associations with soil microbial phyla and other "known" genes. We found that these dominant unknown genes were commonly associated with microbial phyla that are relatively uncharacterized, with the majority of these dominant unknown genes associated with mobile genetic elements. This work demonstrates a strategy for investigating genes of unknown function in soils, emphasizes the biological insights that can be learned by adopting this strategy, and highlights specific hypotheses that warrant further investigation regarding the functional roles of abundant and ubiquitous genes of unknown function in soil metagenomes.

sentences: Deciphering the tissue-specific regulatory role of intronless genes across cancers Intronless genes (IGs) or single-exon genes lacking an intron are found across most Eukaryotes. Notably, IGs display a higher transcriptional fidelity as they are not regulated through alternative splicing, suggesting better predictability biomarkers and easier regulation as targets for therapy. Cancer is a complex disease that relies on progressive uncontrolled cell division linked with multiple dysfunctional biological processes. Tumor heterogeneity remains the most challenging feature in cancer diagnosis and treatment. Given the clinical relevance of IGs, we aim to identify their unique expression profiles and interactome, that may act as functional signatures across eight different cancers. We identified 940 protein-coding IGs in the human genome, of which about 35% were differentially expressed across the analyzed cancer datasets. Specifically, [~]78% of differentially expressed IGs were undergoing transcriptional reprogramming with elevated expression in tumor cells. Remarkably, in all the studied tumors, a highly conserved induction of a group of deacetylase-histones located in a region of chromosome 6 enriched in nucleosome and chromatin condensation processes. This study highlights that differentially expressed human intronless genes across cancer types are prevalent in epigenetic regulatory roles participating in specific PPI networks for ESCA, GBM, and LUAD tumors. We determine that IGs play a key role in the tumor phenotype at transcriptional and post-transcriptional levels, with important mechanisms such as interactomics rewiring.

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A severe form of maternal separation in rat consisting of nineteen-day 6-hour daily separation at unpredictable times minimally affects behavior across the lifespan Maternal separation (MS), a type of early life stress, has been associated with adverse socioemotional and behavioral outcomes throughout the lifespan across multiple species. Comprehensive longitudinal biobehavioral characterization of MS in rats is sparse and conflicting, warranting more studies. We conducted an MS paradigm involving 6-hour daily separation at unpredictable start times from P2 to P21. We hypothesized this severe form of MS would lead to developmentally emerging maladaptive biobehavioral consequences from juvenile through adult periods compared to Controls (C), especially in social behaviors. We tested: (1) own dam odor preference shortly after weaning; (2) juvenile and adult anxiety-like, sociability, and play behaviors using the light-dark test, three-chambered social interaction test, and video-coded juvenile play behavior; and (3) adult coping behaviors and neuroendocrine response using the forced swim test and blood corticosterone responses. Our results show minimal effects on biobehavioral outcomes across the lifespan. Recently weaned MS male rats had a stronger preference for their dams odor. Juvenile MS females spent more time in rough-and-tumble play than C female rats. No differences in sociability were found in the juvenile or adult periods. MS promoted a decrease in anxiety-like behavior that persisted from juvenile to adult periods. Finally, MS led to deficits in coping behavior in male adults, but basal and reactive corticosterone levels were unaltered by MS. More studies are needed to validate our surprising findings and probe the neural mechanisms underlying some of the observed protective effects.

Significance StatementMaternal separation (MS), a type of early life stress, has been shown to lead to short and long-term adverse socioemotional consequences in humans and biobehavioral outcomes in rodents. Available data on MS exposed rats are sparse, conflicting, and often lack longitudinal outcomes. We designed a 6-hour unpredictable daily MS paradigm from postnatal day (P2) to P21 to severely disrupt normal dam-pup interactions. Our results show MS leads to increased preference for own dam odor, persisting reductions in anxiety-like behaviors from juvenile to adulthood, and no effects on sociability. Adverse effects on coping behaviors were uniquely identified in males. The present study warrants reevaluation of the central dogma that MS leads to maladaptive biobehavioral outcomes.

sentences: Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament assembly and organization during terminal ED have revealed essential roles for the pointed-end actin filament capping proteins, tropomodulins (Tmod1 and Tmod3). Additionally, tropomyosin (Tpm) binding to Tmods is a key feature promoting Tmod-mediated actin filament capping. Global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To test a cell autonomous function for Tmod3 and further decipher its biochemical function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability and increased apoptosis. In Mel ds19 cells, both Tpms and actin were preferentially associated with the Triton-X 100 insoluble cytoskeleton during ED, indicating Tpm-coated actin filament assembly during ED. While loss of Tmod3 did not lead to a change in total actin levels, it led to a severe reduction in the proportion of Tpms and actin associated with the Triton-X 100 insoluble cytoskeleton during ED. We conclude that Tmod3-regulation of actin cytoskeleton assembly via Tpms is integral to morphological maturation and cell survival during normal erythroid terminal differentiation.

sentences: Combination of a Sindbis-SARS-CoV-2 spike vaccine and αOX40 antibody elicits protective immunity against SARS-CoV-2 induced disease and potentiates long-term SARS-CoV-2-specific humoral and T-cell immunity The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the worlds population at record speeds. However, there is still demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (OX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus OX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles and metabolic analysis indicate a reprogramming of T-cells in vaccinated mice. Activated T-cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus OX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response that can be used as a new candidate to combat SARS-CoV-2. Given the strong T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as, serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.

sentences: Massive loss of transcription factors and the initial diversification of placental mammals As one of the most successful categories of organisms, mammals occupy a variety of niches on earth as a result of macroevolution. Transcription factors (TFs), the basic regulators of gene expression, may also evolve during mammalian phenotypic diversification and macroevolution. To examine the relationship between TFs and mammalian macroevolution, we analyzed 140,821 de novo-identified TFs and their birth and death histories from 96 mammalian species. Gene tree vs. species tree reconciliation revealed that mammals experienced an upsurge in TF losses around 100 million years ago and also near the K-Pg boundary, thus implying a relationship with the divergence of placental animals. From approximately 100 million years ago to the present, losses dominated TF events without a significant change in TF gains. To quantify the effects of this TF pruning on mammalian macroevolution, we analyzed rates of molecular evolution and expression profiles of regulated target genes. Surprisingly, TF loss decelerated, rather than accelerated, molecular evolutionary rates of their target genes, suggesting increased functional constraints. Furthermore, an association study revealed that massive TF losses are significantly positively correlated with solitary behavior, nocturnality, reproductive-seasonality and insectivory life history traits, possibly through rewiring of regulatory networks.

sentences: Comparative Genome Analysis of 'Candidatus Phytoplasma luffae' Reveals the Influential Roles of Potential Mobile Units in Phytoplasma Evolution Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of  Candidatus Phytoplasma luffae strain NCHU2019 that is associated with witches broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has an exceptionally repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ~25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for neofunctionalization or subfunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

sentences: Enemy or ally: a genomic approach to elucidate the lifestyle of Phyllosticta citrichinaensis Members of the fungal genus Phyllosticta can colonize a variety of plant hosts, including several Citrus species such as Citrus sinensis (orange), Citrus limon (lemon), and Citrus maxima (pomelo). Some Phyllosticta species have the capacity to cause disease, such as Citrus Black Spot, while others have only been observed as endophytes. Thus far, genomic differences underlying lifestyle adaptations of Phyllosticta species have not yet been studied. Furthermore, the lifestyle of Phyllosticta citrichinaensis is ambiguous, as it has been described as a weak pathogen but Kochs postulates may not have been established and the presence of this species was never reported to cause any crop or economic losses. Here, we examined the genomic differences between pathogenic and endophytic Phyllosticta spp. colonizing Citrus and specifically aimed to elucidate the lifestyle of Phyllosticta citrichinaensis. We found several genomic differences between species of different lifestyles, including groups of genes that were only present in pathogens or endophytes. We also observed that species, based on their carbohydrate active enzymes, group independent of their phylogenetic association, and this clustering correlated with trophy prediction. Phyllosticta citrichinaensis shows an intermediate lifestyle, sharing genomic and phenotypic attributes of both pathogens and endophytes. We thus present the first genomic comparison of multiple citrus-colonizing pathogens and endophytes of the genus Phyllosticta, and therefore provide the basis for further comparative studies into the lifestyle adaptations within this genus.

sentences: Optimal evolutionary decision-making to store immune memory The adaptive immune system provides a diverse set of molecules that can mount specific responses against a multitude of pathogens. Memory is a key feature of adaptive immunity, which allows organisms to respond more readily upon re-infections. However, differentiation of memory cells is still one of the least understood cell fate decisions. Here, we introduce a mathematical framework to characterize optimal strategies to store memory to maximize the utility of immune response over an organisms lifetime. We show that memory production should be actively regulated to balance between affinity and cross-reactivity of immune receptors for an effective protection against evolving pathogens. Moreover, we predict that specificity of memory should depend on the organisms lifespan, and shorter-lived organisms with fewer pathogenic encounters should store more cross-reactive memory. Our framework provides a baseline to gauge the efficacy of immune memory in light of an organisms coevolutionary history with pathogens.

sentences: Structure and dynamics of FCHo2 docking on membranes Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an instrumental role in driving CME initiation. The F-BAR domain only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remains elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining bottom-up synthetic approaches on in vitro and cellular membranes. Our results indicate that PI(4,5)P2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like shape protein patches. We show that binding of FCHo2 on cellular membranes promotes PI(4,5)P2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P2-interacting proteins at endocytic sites.

sentences: Mitochondrial Redox Adaptations Enable Aspartate Synthesis in SDH-deficient Cells The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD+ to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss of function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC). Mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports cell proliferation through aspartate synthesis but, unlike other ETC impairments, is not restored by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH impaired cells by concomitant inhibition of ETC complex I (CI). We determine that the benefits of CI inhibition in this context are dependent on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production. We also find that genetic loss or restoration of SDH selects for cells with concordant CI activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. Collectively, these data identify a metabolically beneficial mechanism for CI loss in proliferating cells and reveal that compartmentalized redox changes can impact cellular fitness.

sentences: Aberrant motor contagion of emotions in psychopathy and high-functioning autism Psychopathy and autism are both associated with aberrant social interaction and communication, yet only psychopaths are markedly antisocial and violent. Here we compared the functional neural alterations underlying these two different phenotypes with distinct patterns of socioemotional difficulties. We studied 19 incarcerated male offenders with high psychopathic traits, 20 males with high-functioning autism and 19 age-matched healthy controls. All groups underwent functional magnetic resonance imaging while they viewed dynamic happy, angry and disgust facial expressions or listened to laughter and crying sounds. Psychopathy was associated with reduced somatomotor responses to almost all expressions, while subjects with autism demonstrated less marked and emotion-specific alterations in the somatomotor area. These data suggest that psychopathy and autism involve both common and distinct functional alterations in the brain networks involved in socioemotional processing.

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Large-scale exploration of whole-brain structural connectivity in anorexia nervosa: alterations in the connectivity of frontal and subcortical networks BackgroundAnorexia nervosa (AN) is characterized by disturbances in cognition and behavior surrounding eating and weight. The severity of AN combined with the absence of localized brain abnormalities suggests distributed, systemic underpinnings that may be identified using diffusion-weighted MRI (dMRI) and tractography to reconstruct white matter pathways.

MethodsdMRI data acquired from female patients with AN (n = 147) and female healthy controls (HC; n = 119), aged 12-40 years, were combined across five studies. Probabilistic tractography was completed, and full cortex connectomes describing streamline counts between 84 brain regions generated and harmonized. Graph theory methods were used to describe alterations in network organization in AN. The network-based statistic tested between-group differences in brain subnetwork connectivity. The metrics strength and efficiency indexed the connectivity of brain regions (network nodes), and were compared between groups using multiple linear regression.

ResultsIndividuals with AN, relative to HC, had reduced connectivity in a network comprising subcortical regions and greater connectivity between frontal cortical regions (p < 0.05, FWE corrected). Node-based analyses indicated reduced connectivity of the left hippocampus in patients relative to HC (p < 0.05, permutation corrected). Severity of illness, assessed by BMI, was associated with subcortical connectivity (p < 0.05, uncorrected).

ConclusionsAnalyses identified reduced structural connectivity of subcortical networks and regions, and stronger cortical network connectivity, amongst individuals with AN relative to HC. These findings are consistent with alterations in feeding, emotion and executive control circuits in AN, and may direct hypothesis-driven research into mechanisms of persistent restrictive eating behavior.

sentences: CloneSig: Joint inference of intra-tumor heterogeneity and mutational signatures' activity in tumor bulk sequencing data Systematic DNA sequencing of cancer samples has highlighted the importance of two aspects of cancer genomics: intra-tumor heterogeneity (ITH) and mutational processes. These two aspects may not always be independent, as different mutational processes could be involved in different stages or regions of the tumor, but existing computational approaches to study them largely ignore this potential dependency. Here, we present CloneSig, a computational method to jointly infer ITH and mutational processes in a tumor from bulk-sequencing data. Extensive simulations show that CloneSig outperforms current methods for ITH inference and detection of mutational processes when the distribution of mutational signatures changes between clones. Applied to a large cohort of 8,951 tumors with whole-exome sequencing data from The Cancer Genome Atlas, and on a pan-cancer dataset of 2,632 whole-genome sequencing tumor samples from the Pan-Cancer Analysis of Whole Genomes initiative, CloneSig obtains results overall coherent with previous studies.

sentences: KSTAR: An algorithm to predict patient-specific kinase activities from phosphoproteomic data Kinase inhibitors are one of the largest classes of FDA-approved drugs and are major targets in oncology. Although kinase inhibitors have played an important role in improving cancer outcomes, major challenges still exist, including the development of resistance and failure to respond to treatments. Improvements for tumor profiling of kinase activity would be an important step in improving treatment outcomes and identifying effective kinase targets. Here, we present a graph- and statistics-based algorithm, called KSTAR, which harnesses the phosphoproteomic profiling of human cells and tissues by predicting kinase activity profiles from the observed phosphorylation of kinase substrates. The algorithm is based on the hypothesis that the more active a kinase is, the more of its substrates will be observed in a phosphoproteomic experiment. This method is error- and bias-aware in its approach, overcoming challenges presented by the variability of phosphoproteomic pipelines, limited information about kinase-substrate relationships, and limitations of global kinase-substrate predictions, such as training set bias and high overlap between predicted kinase networks. We demonstrate that the predicted kinase activities: 1) reproduce physiologically-relevant expectations and generates novel hypotheses within cell-specific experiments, 2) improve the ability to compare phosphoproteomic samples on the same tissues from different labs, and 3) identify tissue-specific kinase profiles. Global benchmarking and comparison to other algorithms demonstrates that KSTAR is particularly superior for predicting tyrosine kinase activities and, given its focus on utilizing more of the available phosphoproteomic data, significantly less sensitive to study bias. Finally, we apply the approach to complex human tissue biopsies in breast cancer, where we find that KSTAR activity predictions complement current clinical standards for identifying HER2-status - KSTAR can identify clinical false positives, patients who will fail to respond to inhibitor therapy, and clinically defined HER2-negative patients that might benefit from HER2-targeted therapy. KSTAR will be useful for both basic biological understanding of signaling networks and for improving clinical outcomes through improved clinical trial design, identification of new and/or combination therapies, and for identifying the failure to respond to targeted kinase therapies.

sentences: CLUH controls astrin-1 expression to couple mitochondrial metabolism to cell cycle progression Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.

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Resurrection of a viral internal ribosome entry site from a 700 year old ancient Northwest Territories cripavirus The dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome, named ancient Northwest territories cripavirus (aNCV), was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. Structural prediction of the aNCV IGR sequence generated a secondary structure similar to contemporary IGR IRES structures. There are, however, subtle differences including 105 nucleotides upstream of the IRES of unknown function. Using filter binding assays, we showed that the aNCV IGR IRES could bind to purified salt-washed human ribosomes and compete with a prototypical IGR IRES for ribosomes. Toeprinting analysis using primer extension pinpointed the putative start site of the aNCV IGR at a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR IRES can direct internal ribosome entry in vitro in a manner dependent on the integrity of the PKI domain. Lastly, we generated a chimeric virus clone by swapping the aNCV IRES into the cricket paralysis virus infectious clone. The chimeric infectious clone with an aNCV IGR IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework in the importance of this viral translational mechanism.

IMPORTANCEInternal ribosome entry sites are RNA structures that are used by some positive-sense monopartite RNA viruses to drive viral protein synthesis. The origin of internal ribosome entry sites is not known. Using biochemical approaches, we demonstrate that an RNA structure from an ancient viral genome that was discovered from a 700-year-old caribou feces trapped in subarctic ice is functionally similar to modern internal ribosome entry sites. We resurrect this ancient RNA mechanism by demonstrating that it can support virus infection in a contemporary virus clone, thus providing insights into the origin and evolution of this viral strategy.

sentences: Local Connectivity and Synaptic Dynamics in Mouse and Human Neocortex To elucidate cortical microcircuit structure and synaptic properties we present a unique, extensive, and public synaptic physiology dataset and analysis platform. Through its application, we reveal principles that relate cell type to synapse properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Despite these associations, synaptic properties are heterogeneous in most subclass to subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, while the strength axis accounts for significant heterogeneity within the subclass. In human cortex, excitatory to excitatory synapse dynamics are distinct from those in mouse and short-term plasticity varies with depth across layers 2 and 3. With a novel connectivity analysis that enables fair comparisons between circuit elements, we find that intralaminar connection probability among cell subclasses exhibits a strong layer dependence.These and other findings combined with the analysis platform create new opportunities for the neuroscience community to advance our understanding of cortical microcircuits.

sentences: Estimating genetic correlation jointly using individual-level and summary-level GWAS data With the increasing accessibility of individual-level data from genome wide association studies, it is now common for researchers to have individual-level data of some traits in one specific population. For some traits, we can only access public released summary-level data due to privacy and safety concerns. The current methods to estimate genetic correlation can only be applied when the input data type of the two traits of interest is either both individual-level or both summary-level. When researchers have access to individual-level data for one trait and summary-level data for the other, they have to transform the individual-level data to summary-level data first and then apply summary data-based methods to estimate the genetic correlation. This procedure is computationally and statistically inefficient and introduces information loss. We introduce GENJI (Genetic correlation EstimatioN Jointly using Individual-level and summary data), a method that can estimate within-population or transethnic genetic correlation based on individual-level data for one trait and summary-level data for another trait. Through extensive simulations and analyses of real data on within-population and transethnic genetic correlation estimation, we show that GENJI produces more reliable and efficient estimation than summary data-based methods. Besides, when individual-level data are available for both traits, GENJI can achieve comparable performance than individual-level data-based methods. Downstream applications of genetic correlation can benefit from more accurate estimates. In particular, we show that more accurate genetic correlation estimation facilitates the predictability of cross-population polygenic risk scores.

sentences: Analysis of nucleic acids extracted from rapid diagnostic tests reveals a significant proportion of false positive test results associated with recent malaria treatment Surveillance programs often use malaria rapid diagnostic tests (RDTs) to determine the proportion of the population carrying parasites in their peripheral blood to assess the malaria transmission intensity. Despite an increasing number of reports on false-negative and false-positive RDT results, there is a lack of systematic quality control activities for RDTs deployed in malaria surveillance programs. Our study provides a larger scale comparative evaluation of RDTs used in the 2018 Malaria Indicator Survey (MIS) conducted on Bioko Island, Equatorial Guinea. We conducted a molecular analysis by extraction of nucleic acids from 1,800 negative and 1,065 positive RDTs followed by qPCR analysis. These results were combined with a dataset collected in a comprehensive questionnaire from each MIS participant. Of the 2,865 RDTs that were collected in 2018 on Bioko Island and analysed in our study, 4.7% had a false-negative result. These false-negative RDT results were associated with low parasite density infections. In a substantial proportion of samples, we identified masked pfhrp2 and pfhrp3 gene deletions in which at least one P. falciparum strain carried a gene deletion. Among all positive RDTs analysed, 28.4% were tested negative by qPCR and therefore considered to be false-positive. Analysing the questionnaire data collected from the participants, this high proportion of false-positive RDT results could be explained by PfHRP2 antigen persistence after recent malaria treatment. We conclude that malaria surveillance depending solely on RDTs needs well-integrated quality control procedures assessing the extend and impact of reduced sensitivity and specificity of RDTs on malaria control programs.

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Oxytocinergic modulation of stress-associated amygdala-hippocampus pathways in humans is mediated by serotonergic mechanisms BackgroundThe hypothalamic neuropeptide oxytocin (OXT) may exert anxiolytic and stress-reducing actions via modulatory effects on amygdala circuits. Animal models and initial findings in humans suggest that some of these effects are mediated by interactions with other neurotransmitter systems, in particular the serotonin (5-HT) system. Against this background, the present pharmacological resting state fMRI study aimed at determining whether effects of OXT on stress-associated amygdala intrinsic networks are mediated by 5-HT.

MethodsWe employed a randomized placebo-controlled double-blind parallel-group pharmacological fMRI resting state experiment during which n = 112 healthy male participants underwent a transient decrease in 5-HT signaling via acute tryptophan depletion (ATD) or the corresponding placebo-control protocols (ATDc) before the administration of intranasal OXT or placebo intranasal spray, respectively.

ResultsOXT and 5-HT modulation exerted interactive effects on the coupling of the left amygdala with the ipsilateral hippocampus and adjacent midbrain. OXT increased intrinsic coupling in this pathway, while this effect of OXT was significantly attenuated during transiently decreased central serotonergic signaling induced via ATD. In the absence of OXT or 5-HT modulation this pathway showed a trend for an association with self-reported stress perception in everyday life. No interactive effects were observed for the right amygdala.

ConclusionsTogether, the findings provide first evidence that effects of OXT on stress-associated amygdala-hippocampal-midbrain pathways are critically mediated by the 5-HT system in men.

sentences: Development of a machine learning model to estimate biotic ligand model-based predicted no-effect concentrations for copper in freshwater The copper biotic ligand model (BLM) has been used for environmental risk assessment by taking into account the bioavailability of copper in freshwater. However, the BLM-based environmental risk of copper has been assessed only in Europe and North America, with monitoring datasets containing all of the BLM input variables. For other areas, it is necessary to apply surrogate tools with reduced data requirements to estimate the BLM-based predicted no-effect concentration (PNEC) from commonly available monitoring datasets. To develop an optimized PNEC estimation model based on an available monitoring dataset, an initial model that considers all BLM variables, a second model that requires variables excluding alkalinity, and a third model using electrical conductivity as a surrogate of the major cations and alkalinity have been proposed. Furthermore, deep neural network (DNN) models have been used to predict the nonlinear relationships between the PNEC (outcome variable) and the required input variables (explanatory variables). The predictive capacity of DNN models in this study was compared with the results of other existing PNEC estimation tools using a look-up table and multiple linear and multivariate polynomial regression methods. Three DNN models, using different input variables, provided better predictions of the copper PNECs compared with the existing tools for four test datasets, i.e., Korean, United States, Swedish, and Belgian freshwaters. The adjusted r2 values in all DNN models were higher than 0.95 in the test datasets, except for the Swedish dataset (adjusted r2 > 0.87). Consequently, the most applicable model among the three DNN models could be selected according to the data availability in the collected monitoring database. Because the most simplified DNN model required only three water quality variables (pH, dissolved organic carbon, and electrical conductivity) as input variables, it is expected that the copper BLM-based risk assessment can be applied to monitoring datasets worldwide.

sentences: A metagenomic-based stufy of two sites from the Barbadian reef system We study the microbiome of sea water collected from two locations of the Barbadian coral reefs. The two sites differ in several environmental and ecological variables including their endogenous benthic community in addition to their proximity to urban development and runoffs from inland watersheds. The composition of the microbial community was estimated using whole genome DNA shotgun sequencing. Although both sites exhibit a similar degree of richness, the less urbanized site (Maycocks reef at Hangmans Bay) is strongly concentrated with phototrophs. In comparison, the more urbanized location (Bellairs Research Institute) is enriched for copiotrophs, macroalgal symbionts and marine-related disease-bearing organisms from taxa scattered across the tree of life. Overall, our samples and associated measurements of chemical and environmental qualities of the water are in line with previous marine microbiome profiles of warm ocean surface waters. This suggests our approach captures salient information regarding the state of each coral reef site and sets the stage for larger longitudinal studies of coral reef dynamics in Barbados.

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Hierarchical control of microbial community assembly Metabolic processes that fuel the growth of heterotrophic microbial communities are initiated by specialized biopolymer degraders that decompose complex forms of organic matter. It is unclear, however, to what extent degraders control the downstream assembly of the community that follows polymer breakdown. Investigating a model marine microbial community that degrades chitin, we show that chitinases secreted by different degraders produce oligomers of specific chain lengths that not only select for specialized consumers but also influence the metabolites secreted by these consumers into a shared resource pool. Each species participating in the breakdown cascade exhibits unique hierarchical preferences for substrates, which underlies the sequential colonization of metabolically distinct groups as resource availability changes over time. By identifying the metabolic underpinnings of microbial community assembly, we reveal a hierarchical crossfeeding structure that allows biopolymer degraders to shape the dynamics of community assembly.

One sentence summarySpecialized biopolymer degraders direct the trajectory of microbial community assembly through interconnected modes of nutrient crossfeeding.

sentences: MixEHR-Guided: A guided multi-modal topic modeling approach for large-scale automatic phenotyping using the electronic health record Electronic Health Records (EHRs) contain rich clinical data collected at the point of the care, and their increasing adoption offers exciting opportunities for clinical informatics, disease risk prediction, and personalized treatment recommendation. However, effective use of EHR data for research and clinical decision support is often hampered by a lack of reliable disease labels. To compile gold-standard labels, researchers often rely on clinical experts to develop rule-based phenotyping algorithms from billing codes and other surrogate features. This process is tedious and error-prone due to recall and observer biases in how codes and measures are selected, and some phenotypes are incompletely captured by a handful of surrogate features. To address this challenge, we present a novel automatic phenotyping model called MixEHR-Guided (MixEHR-G), a multimodal hierarchical Bayesian topic model that efficiently models the EHR generative process by identifying latent phenotype structure in the data. Unlike existing topic modeling algorithms wherein the inferred topics are not identifiable, MixEHR-G uses prior information from informative surrogate features to align topics with known phenotypes. We applied MixEHR-G to an openly-available EHR dataset of 38,597 intensive care patients (MIMIC-III) in Boston, USA and to administrative claims data for a population-based cohort (PopHR) of 1.3 million people in Quebec, Canada. Qualitatively, we demonstrate that MixEHR-G learns interpretable phenotypes and yields meaningful insights about phenotype similarities, comorbidities, and epidemiological associations. Quantitatively, MixEHR-G outperforms existing unsupervised phenotyping methods on a phenotype label annotation task, and it can accurately estimate relative phenotype prevalence functions without gold-standard phenotype information. Altogether, MixEHR-G is an important step towards building an interpretable and automated phenotyping system using EHR data.

sentences: Learning to see colours: generating biologically relevant fluorescent labels from bright-field images Fluorescence microscopy, which visualizes cellular components with fluorescent stains, is an invaluable method in image cytometry. From these images various cellular features can be extracted. Together these features form phenotypes that can be used to determine effective drug therapies, such as those based on nanomedicines. Unfortunately, fluorescence microscopy is time-consuming, expensive, labour intensive, and toxic to the cells. Bright-field images lack these downsides but also lack the clear contrast of the cellular components and hence are difficult to use for downstream analysis. Generating the fluorescence images directly from bright-field images would get the best of both worlds, but can be very challenging to do for poorly visible cellular structures in the bright-field images. To tackle this problem deep learning models were explored to learn the mapping between bright-field and fluorescence images to enable virtual staining for adipocyte cell images. The models were tailored for each imaging channel, paying particular attention to the various challenges in each case, and those with the highest fidelity in extracted cell-level features were selected. The solutions included utilizing privileged information for the nuclear channel, and using image gradient information and adversarial training for the lipids channel. The former resulted in better morphological and count features and the latter resulted in more faithfully captured defects in the lipids, which are key features required for downstream analysis of these channels.

sentences: Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy Nanoscale resolution imaging of whole vertebrates is required for a systematic understanding of human diseases, but this has yet to be realized. Expansion microscopy (ExM) is an attractive option for achieving this goal, but the expansion of whole vertebrates has not been demonstrated due to the difficulty of expanding hard body components. Here, we demonstrate whole-body ExM, which enables nanoscale resolution imaging of anatomical structures, proteins, and endogenous fluorescent proteins (FPs) of whole zebrafish larvae and mouse embryos by expanding them fourfold. We first show that post-digestion decalcification and digestion kinetics matching are critical steps in the expansion of whole vertebrates. Then, whole-body ExM is combined with the improved pan-protein labeling approach to demonstrate the three-dimensional super-resolution imaging of antibody- or FP-labeled structures and all major anatomical structures surrounding them. We also show that whole-body ExM enables visualization of the nanoscale details of neuronal structures across the entire body.

sentences: Visual discrimination of optical material properties: a large-scale study Complex visual processing involved in perceiving the object materials can be better elucidated by taking a variety of research approaches. Sharing stimulus and response data is an effective strategy to make the results of different studies directly comparable and can assist researchers with different backgrounds to jump into the field. Here, we constructed a database containing several sets of material images annotated with visual discrimination performance. We created the material images using physically-based computer graphics techniques and conducted psychophysical experiments with them in both laboratory and crowdsourcing settings. The observers task was to discriminate materials on one of six dimensions (gloss contrast, gloss distinctness-of-image, translucent vs. opaque, metal vs. plastic, metal vs. glass, and glossy vs. painted). The illumination consistency and object geometry were also varied. We used a non-verbal procedure (an oddity task) applicable for diverse use-cases such as cross-cultural, cross-species, clinical, or developmental studies. Results showed that the material discrimination depended on the illuminations and geometries and that the ability to discriminate the spatial consistency of specular highlights in glossiness perception showed larger individual differences than in other tasks. In addition, analysis of visual features showed that the parameters of higher-order color texture statistics can partially, but not completely, explain task performance. The results obtained through crowdsourcing were highly correlated with those obtained in the laboratory, suggesting that our database can be used even when the experimental conditions are not strictly controlled in the laboratory. Several projects using our dataset are underway.

sentences: Co-radiation of Leptospira and Tenrecidae (Afrotheria) on Madagascar Leptospirosis is a bacterial zoonosis caused by pathogenic Leptospira that are maintained in the kidney lumen of infected animals acting as reservoirs and contaminating the environment via infected urine. The investigation of leptospirosis through a One Health framework has been stimulated by notable genetic diversity of pathogenic Leptospira together with a high infection prevalence in certain animal reservoirs. Studies of Madagascars native mammal fauna have revealed a diversity of Leptospira with high levels of host-specificity. Native rodents, tenrecids, and bats shelter several distinct lineages and species of Leptospira, some of which have also been detected in acute human cases. Specifically, Leptospira mayottensis, first discovered in humans on Mayotte, an island neighboring Madagascar, was subsequently identified in a few species of Malagasy tenrecids, an endemic family of small mammals. Distinct L. mayottensis lineages were identified in shrew tenrecs (Microgale cowani and Nesogale dobsoni) on Madagascar, and later in spiny tenrecs (Tenrec ecaudatus) on Mayotte. These findings suggest that L. mayottensis (i) has co-radiated with tenrecids on Madagascar, and (ii) has recently emerged in human populations on Mayotte following the introduction of T. ecaudatus from Madagascar. Hitherto, L. mayottensis has not been detected in spiny tenrecs on Madagascar. In the present study, we broaden the investigation of Malagasy tenrecids and describe the presence of L. mayottensis in Malagasy T. ecaudatus and M. thomasi. These results confirm the hypothesis that L. mayottensis was introduced to Mayotte, presumably via T. ecaudatus, and provide additional data on the co-radiation of Leptospira and Tenrecidae.

sentences: Equivalent noise characterization of human lightness constancy A goal of visual perception is to provide stable representations of task-relevant scene properties (e.g. object reflectance) despite variation in task-irrelevant scene properties (e.g. illumination, reflectance of other nearby objects). To study such stability in the context of the perceptual representation of lightness, we introduce a threshold-based psychophysical paradigm. We measure how thresholds for discriminating the achromatic reflectance of a target object (task-relevant property) in rendered naturalistic scenes are impacted by variation in the reflectance functions of background objects (task-irrelevant property), using a two-alternative forced-choice paradigm in which the reflectance of the background objects is randomized across the two intervals of each trial. We control the amount of background reflectance variation by manipulating a statistical model of naturally-occurring surface reflectances. For low background object reflectance variation, discrimination thresholds were nearly constant, indicating that observers internal noise determines threshold in this regime. As background object reflectance variation increases, its effects start to dominate performance. A model based on signal detection theory allows us to express the effects of task-irrelevant variation in terms of the equivalent noise, that is relative to the intrinsic precision of the task-relevant perceptual representation. The results indicate that although naturally-occurring background object reflectance variation does intrude on the perceptual representation of target object lightness, the effect is modest - within a factor of two of the equivalent noise level set by internal noise.

sentences: Evaluating and mitigating clinical samples matrix effects on TX-TL cell-free performance Cell-free biosensors are promising tools for medical diagnostics, yet their performance can be affected by matrix effects arising from the sample itself or from external components. Here we systematically evaluate the performance and robustness of cell-free systems in serum, plasma, urine, and saliva using two reporter systems, sGFP and luciferase. In all cases, clinical samples have a strong inhibitory effect. Of different inhibitors, only the RNase inhibitor mitigated matrix effects. However, we found that the recovery potential of RNase inhibitor was partially muted by interference from glycerol contained in the commercial buffer. We solved this issue by designing a strain producing an RNase inhibitor protein requiring no additional step in extract preparation. Furthermore, our new extract yielded higher reporter levels than previous conditions and tempered interpatient variability associated with matrix effects. This systematic evaluation and improvements of cell-free system robustness unified across many types of clinical samples is a significant step towards developing cell-free diagnostics for a wide range of conditions.

sentences: Population structure and environmental niches of Rimicaris shrimps from the Mid Atlantic Ridge Among the endemic and specialized fauna from hydrothermal vents, Rimicaris shrimp surely constitutes one of the most important and emblematic components of these ecosystems. In the Mid Atlantic Ridge, two species affiliated to this genus co-occur: Rimicaris exoculata and Rimicaris chacei that differ by their morphology, their trophic regime as well as by their abundance. The first forms large and dense aggregations on active vent chimney walls in close proximity to vent fluid emissions, whereas the second is recognized as much less conspicuous, living mostly in scattered groups or solitary further away from the fluids. However, the recent revision of Rimicaris juvenile stages from the Mid Atlantic Ridge shows that R. chacei abundance would be higher than expected at these early life stages. Here, we describe and compare the population structures of R. exoculata and R. chacei at the Snake Pit and TAG vent fields. We show distinct population demographics between the two co-occurring shrimps with a large post settlement collapse in R. chacei population suggesting a large juvenile mortality for this species. We also observe important spatial segregation patterns between the two species and their different life stages. Additionally, our results highlight distinct niches for the earliest juvenile stages of both R. exoculata and R. chacei, compared to all the other life stages. Finally, we discuss the potential factors - predation and competitive interactions among others - that could explain the differences we observe in the population structure of these two species.

sentences: LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the activity of the molecular motor cytoplasmic dynein. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it regulates the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the expression of extracellular matrix (ECM) and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post- transcriptional regulation underlying development and mechanosensitive processes.

sentences: Identification and in silico comparison of transposable elements of Solanum tuberosum subsp. andigena from two localities of Peru The potato is one of the main crops in Peru, being one of the pillars in the countrys economy. It also has a cultural importance, being a basic element of Peruvian gastronomy and an emblematic crop of the Peruvian highlands, where small farmers over time have been able to conserve and cultivate 6048 native varieties. In Peru, around 300,000 hectares of potatoes are planted annually, and in 2019 Peru remained the main potato producer in Latin America, registering an annual production of 5.3 million tons. Therefore, it is necessary to study and characterize the genome of this very important tuber for the country. Although there are many potato genomic studies in the world and in Peru; these have been based on the search for genes of importance, genetic diversity, etc. and little has been deeply studied about the transposable elements (ETs) that are part of its genome. It is known that in many eukaryotes, transposable elements are powerful drivers of genome evolution, and they occupy a considerable part in plant genomes, so with these studies, more could be understood about how these ETs have intervened in the evolution of the potato in Peru; thus, how these mechanisms would help in the future to the survival of the species in scenarios of climate change, among others. This study proposes the identification and comparison of the transposable elements of the only 2 complete genomes of potatoes grown in Peru, found in the NCBI database, one from the department of Puno and the other from Cusco. There are various approaches to search for ETs in genomes, but for a complete and reliable annotation, it has been shown that the best strategy is the adoption of combined approaches, in this study, we will use 2 approaches, one based on homology, with the program RepeatMasker, and the other de novo, with the RepeatModeler2 program.

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Using Neutralization Landscapes to enumerate Antibody Behavior and Decompose Antibody Mixtures Antibodies constitute a key line of defense against the diverse pathogens we encounter in our lives. While the interactions between a single antibody and a single virus are routinely characterized in exquisite detail, the inherent tradeoffs between attributes such as potency and breadth remain unclear. In addition, there is a wide gap between the discrete interactions of single antibodies and how antibody mixtures collectively repel multiple viruses. Here, we develop a new form of antigenic cartography called a Neutralization Landscape that enumerates the full space of antibody-virus interactions for antibodies targeting the influenza hemagglutinin stem. This reference set of antibody behaviors transforms the potency-breadth tradeoff into a readily solvable geometry problem. Using the Neutralization Landscape, we decompose the collective neutralization from multiple antibodies to characterize the composition and functional properties of the stem antibodies within. Looking forward, this framework can leverage the serological assays routinely performed for influenza surveillance to analyze how an individuals antibody repertoire evolves over time in response to vaccination or infection.

One-Sentence SummaryWe describe the full range of behaviors for antibodies targeting the stem of influenza hemagglutinin, enabling us to decompose the collective behavior of antibody mixtures and characterize the individual antibodies within.

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The PMT-driven p-coumaroylation of poplar lignins impacts lignin structure and improves wood saccharification Transgenic poplars (Populus tremula x Populus alba, clone INRA 717-1B4) were produced by introducing the Brachypodium distachyon Bradi2g36910 (BdPMT1) gene driven by the Arabidopsis (Arabidopsis thaliana) Cinnamate 4-Hydroxylase (AtC4H) promoter in the wild-type (WT) line and in a line overexpressing the Arabidopsis Ferulate 5-Hydroxylase (AtF5H). BdPMT1 encodes a transferase which catalyzes the acylation of monolignols by p-coumaric acid (CA). Several BdPMT1- OE/WT and BdPMT1-OE/AtF5H-OE transgenic lines were grown in the greenhouse and BdPMT1 expression in xylem was confirmed by RT-PCR. The analysis of the cell walls (CW) of poplar stems and of corresponding purified dioxan lignins (DL) revealed that the BdPMT1-OE lignins were as p-coumaroylated as the lignins of C3 grass straws. For some transformants, CA levels even reached about 11 mg/g CW and 66 mg/g DL, which by far exceeds those of Brachypodium or wheat samples. This unprecedentedly high p-coumaroylation of poplar lignins affected neither the poplar growth, nor the stem lignin content. By contrast, the transgenic lignins were structurally modified, with an increase of terminal units with free phenolic groups. Relative to controls, this increase argues for a reduced polymerization degree of BdPMT1-OE lignins and makes them more soluble in cold NaOH solution. The p-coumaroylation of poplar samples, up to the levels of C3 grasses, improved the saccharification yield of alkali-pretreated poplar CW. These results establish that the genetically-driven p-coumaroylation of lignins is a promising strategy to make wood lignins more susceptible to the alkaline treatments that can be used during the industrial processing of lignocellulosics.

One-sentence summaryThe expression of a grass p-coumaroyl-CoA:monolignol transferase induces a high p-coumaroylation of poplar lignins and a better saccharification of alkali-pretreated poplar wood without growth penalty

sentences: Systematic comparison of experimental assays and analytical pipelines for identification of active enhancers genome-wide Mounting evidence supports the idea that transcriptional patterns serve as more specific identifiers of active enhancers than histone marks1,2; however, the optimal strategy to identify active enhancers both experimentally and computationally has not been determined. In this study, we compared 13 genome-wide RNA sequencing assays in K562 cells and showed that the nuclear run-on followed by cap-selection assay (namely, GRO/PRO-cap) has significant advantages in eRNA detection and active enhancer identification. We also introduced a new analytical tool, Peak Identifier for Nascent-Transcript Sequencing (PINTS), to identify active promoters and enhancers genome-wide and pinpoint the precise location of the 5 transcription start sites (TSSs) within these regulatory elements. Finally, we compiled a comprehensive enhancer candidate compendium based on the detected eRNA TSSs available in 120 cell and tissue types. To facilitate the exploration and prioritization of these enhancer candidates, we also built a user-friendly web server (https://pints.yulab.org) for the compendium with various additional genomic and epigenomic annotations. With the knowledge of the best available assays and pipelines, this large-scale annotation of candidate enhancers will pave the road for selection and characterization of their functions in a time-, labor-, and cost-effective manner in the future.

sentences: Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions The Sc2.0 project is building a eukaryotic synthetic genome from scratch, incorporating thousands of designer features. A major milestone has been achieved with the assembly of all individual Sc2.0 chromosomes. Here, we describe the consolidation of multiple synthetic chromosomes using endoreduplication intercross to generate a strain with 6.5 synthetic chromosomes. Genome-wide chromosome conformation capture and long-read direct RNA sequencing were performed on this strain to evaluate the effects of designer modifications, such as loxPsym site insertion, tRNA relocation, and intron deletion, on 3D chromosome organization and transcript isoform profiles. To precisely map "bugs", we developed a method, CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS", exploiting directed mitotic recombination in heterozygous diploids. Using this method, we first fine-mapped a synII defect resulting from two loxPsym sites in the 3 UTR of SHM1. This approach was also used to map a combinatorial bug associated with synIII and synX, revealing a highly unexpected genetic interaction that links transcriptional regulation, inositol metabolism and tRNASerCGA abundance. "Starvation" for tRNASerCGA leads to insufficient levels of the key positive inositol biosynthesis regulator, Swi3, which contains tandem UCG codons. Finally, to expedite consolidation, we employed a new method, chromosome swapping, to incorporate the largest chromosome (synIV), thereby consolidating more than half of the Sc2.0 genome in a single strain.

sentences: Overexpression of UCP4 in astrocytic mitochondria prevents multilevel dysfunctions in a mouse model of Alzheimer s disease Alzheimers disease (AD) is becoming increasingly prevalent worldwide. It represents one of the greatest medical challenge as no pharmacologic treatments are available to prevent disease progression. Astrocytes play crucial functions within neuronal circuits by providing metabolic and functional support, regulating interstitial solute composition, and modulating synaptic transmission. In addition to these physiological functions, growing evidence points to an essential role of astrocytes in neurodegenerative diseases like AD. Early stage AD is associated with hypometabolism and oxidative stress. Contrary to neurons that are vulnerable to oxydative stress, astrocytes are particularly resistant to mitochondrial dysfunction and are therefore more resilient cells. In our study, we leveraged astrocytic mitochondrial uncoupling and examined neuronal function in the 3xTg AD mouse model. We overexpressed the mitochondrial uncoupling protein 4 (UCP4), which has been shown to improve neuronal survival in vitro. We found that this treatment efficiently prevented alterations of hippocampal metabolite levels observed in AD mice, along with hippocampal atrophy and reduction of basal dendrite arborization of subicular neurons. This approach also averted aberrant neuronal excitability observed in AD subicular neurons and preserved episodic-like memory in AD mice assessed in a spatial recognition task. These findings show that targeting astrocytes and their mitochondria is an effective strategy to prevent the decline of neurons facing AD-related stress at the early stages of the disease.

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A kinase-dead Csf1r mutation associated with adult-onset leukoencephalopathy has a dominant-negative impact on CSF1R signaling. Amino acid substitutions in the kinase domain of the human CSF1R gene are associated with autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). To model the human disease, we created a disease-associated mutation (Glu631Lys; E631K) in the mouse Csf1r locus. Homozygous mutation (Csf1rE631K/E631K) phenocopied the Csf1r knockout; with prenatal mortality or severe postnatal growth retardation and hydrocephalus. Heterozygous mutation delayed the postnatal expansion of tissue macrophage populations in most organs. Bone marrow cells from Csf1rE631K/+ mice were resistant to CSF1 stimulation in vitro, and Csf1rE631K/+ mice were unresponsive to administration of a CSF1-Fc fusion protein which expands tissue macrophage populations in controls. In the brain, microglial cell numbers and dendritic arborization were reduced in the Csf1rE631K/+ mice as in ALSP patients. The microglial phenotype is the opposite of microgliosis observed in Csf1r+/- mice. However, we found no evidence of brain pathology or impacts on motor function in aged Csf1rE631K/+ mice. We conclude that disease-associated CSF1R mutations encode dominant negative repressors of CSF1R signaling. We speculate that leukoencephalopathy associated with human CSF1R mutations requires an environmental trigger and/or epistatic interaction with common neurodegenerative disease-associated alleles.

Summary StatementThis study describes the effect of a human disease-associated mutation in the mouse CSF1R gene on postnatal development and growth factor responsiveness of cells of the macrophage lineage.

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Investigation on How Dynamic Effective Connectivity Patterns Encode the Fluctuating Pain Intensity in Chronic Migraine Chronic migraine is characterised by persistent headaches for more than 15 days per month; the intensity of the pain is fluctuating over time. Here, we explored the dynamic interplay of connectivity patterns between regions known to be related to pain processing and their relation to the ongoing dynamic pain experience. We recorded EEG from 80 sessions (20 chronic migraine patients in 4 separate sessions of 25 minutes). The patients were asked to continuously rate the intensity of their endogenous headache. On different time-windows, a dynamic causal model (DCM) of cross spectral responses was inverted to estimate connectivity strengths. For each patient and session, the evolving dynamics of effective connectivity were related to pain intensities and to pain intensity changes by using a Bayesian linear model. Hierarchical Bayesian modelling was further used to examine which connectivity-pain relations are consistent across sessions and across patients.

The results reflect the multi-facetted clinical picture of the disease. Across all sessions, each patient with chronic migraine exhibited a distinct pattern of pain intensity-related cortical connectivity. The diversity of the individual findings are accompanied by inconsistent relations between the connectivity parameters and pain intensity or pain intensity changes at group level. This suggests a rejection of the idea of a common neuronal core problem for chronic migraine.

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Complete Protection of Nasal and Lung Airways Against SARS-CoV-2 Challenge by Antibody Plus Th1 Dominant N- and S-Specific T-Cell Responses to Subcutaneous Prime and Thermally-Stable Oral Boost Bivalent hAd5 Vaccination in an NHP Study We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike (S-Fusion) protein and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) with the potential to increase MHC class I/II responses. The adenovirus serotype 5 platform used, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity, can be delivered in an oral formulation that overcomes cold-chain limitations. The hAd5 S-Fusion + N-ETSD vaccine was evaluated in rhesus macaques showing that a subcutaneous prime followed by oral boosts elicited both humoral and Th1 dominant T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.

ONE SENTENCE SUMMARYhAd5 spike + nucleocapsid SC prime/oral boost vaccine elicits humoral and T-cell responses and protects rhesus macaques from SARS-CoV-2 challenge.

sentences: Heterocomplexes between the Atypical Chemokine MIF and the CXC-Motif Chemokine CXCL4L1 Regulate Inflammation and Thrombus Formation To fulfil their orchestrating function in immune cell trafficking in homeostasis and disease, a network of 49 chemokines and 23 receptors capitalizes on features of specificity, redundancy, and functional selectivity such as biased agonism. The discovery of the chemokine interactome, i.e. heteromeric chemokine-chemokine interactions, even across CC- and CXC-class borders, has further expanded the complexity within the network. Moreover, some inflammatory mediators, which are not structurally linked to classical CC-, CXC-, CX3C-, or C-chemokines, can bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified the cytokine macrophage migration inhibitory factor (MIF) as an ACK that binds to the chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF may form heterocomplexes with classical chemokines. We tested this hypothesis, applying an unbiased chemokine protein binding array. The platelet chemokine CXCL4L1, but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12, was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, which also established high-affinity binding (KD{asymp}100-150 nM). The binding interface was predicted by peptide array-based mapping and molecular docking. We next determined whether heterocomplex formation modulates inflammatory and atherogenic activities of MIF. MIF-elicited T-cell chemotaxis as assessed in a 3D-matrix-based live cell-imaging set-up was abrogated, when cells were co-incubated with MIF and CXCL4L1. Heterocomplexation also blocked MIF-triggered migration of Egfp+ microglia in cortical cultures in situ. Of note, CXCL4L1 blocked the binding of Alexa-MIF to a soluble ectodomain mimic of CXCR4 and co-incubation with CXCL4L1 attenuated MIF-triggered dynamic mass redistribution in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. As MIF and CXCL4L1 are abundant platelet products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM- FRET, and proximity ligation assay visualized heterocomplexes in platelet aggregates and clinical human thrombus sections. Moreover, heterocomplex formation inhibited MIF- stimulated thrombus formation under flow and skewed the morphology of adhering platelets from a large to a small lamellipodia phenotype. Together, our study establishes a novel molecular interaction, adding to the complexity of the chemokine interactome and chemokine/receptor network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be promising target structures to modulate inflammation and thrombosis.

sentences: Dynamic Phosphorylation of RGS Provides Spatial Regulation of MAP Kinase And Promotes Completion of Cytokinesis during the Yeast Pheromone Response Yeast use a G-protein coupled receptor (GPCR) signaling pathway to detect mating pheromone, arrest in G1, and direct polarized growth towards the potential mating partner. The primary negative regulator of this pathway is the regulator of G-protein signaling (RGS), Sst2, which induces G GTPase activity and subsequent inactivation of all downstream signaling including a MAPK cascade. The MAPK Fus3 phosphorylates the RGS in response to pheromone, but the role of this modification is unknown. We set out to examine the role of RGS phosphorylation during the pheromone response. We found that RGS phosphorylation peaks early in the pheromone response and diminishes RGS localization to the polarity site and focuses MAPK complexes there. At later time points, RGS is predominantly unphosphorylated, which promotes RGS localization to the polar cap and broadens the distribution of MAPK complexes relative to the Cdc42 polarity machinery. Surprisingly, we found that phosphorylation of the RGS is required for the completion of cytokinesis prior to pheromone induced growth. The completion of cytokinesis in the presence of pheromone is promoted by the formin Bnr1 and the kelch-repeat protein, Kel1, both proteins previously found to interact with the RGS.

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Simultaneous adjustments of major mitochondrial pathways through redox regulation of dihydrolipoamide dehydrogenase (mtLPD1) Thioredoxins (TRX) are pivotal for the redox regulation of enzyme activities to adjust metabolic fluxes towards environmental changes. Previous reports demonstrated TRX o1 and h2 impact on mitochondrial metabolism including photorespiration and the tricarboxylic acid (TCA) cycle. Here, we aimed to unravel potential specificities between regulation modes of both TRXs, especially under conditions with short-term changes in photorespiration. Therefore, short-term metabolite responses of single TRX mutants were analyzed after exposure to altered CO2/O2 ratios during darkness and illumination. This approach was complemented by comprehensive characterization of multiple Arabidopsis mutants lacking either one or both TRX in the wild-type Arabidopsis or the glycine decarboxylase (GDC) T-protein knock down line (gldt1). The results provided evidence for additive effects of combined TRX o1 and h2 deficiency to suppress growth, photosynthesis and mitochondrial metabolism. Quantification of pyrimidine nucleotides in conjunction with metabolite and 13C-labelling approaches revealed a rather uniform impact on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) dependent pathways. Biochemical analysis of recombinant mtLPD1 demonstrated its inhibition by NADH, pointing at an additional measure to fine-tune its in vivo activity. Collectively, we propose that TRX o1 and h2 contribute to the communication of altered subcellular redox-states through direct and indirect regulation of mtLPD1. This regulation module might represent a common intercept for simultaneous adjustments in the operation of photorespiration, the TCA-cycle and the degradation of branched chain amino acids.

One-sentence summaryRedox regulation of mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) simultaneously modulates photorespiration, the tricarboxylic acid (TCA)-cycle and branched chain amino acid (BCAA) degradation in response to rapid environmental changes.

sentences: Identifying evolutionary divergence in gene expression across species and organs: a case-study using Hawaiian Drosophila 1With detailed data on gene expression accessible from an increasingly broad array of species, we can test the extent to which our developmental genetic knowledge from model organisms predicts expression patterns and variation across species. But to know when differences in gene expression across species are significant, we first need to know how much evolutionary variation in gene expression we expect to observe. Here we provide an answer by analyzing RNAseq data across twelve species of Hawaiian Drosophilidae flies, focusing on gene expression differences between the ovary and other tissues. We show that over evolutionary time, there exists a cohort of ovary specific genes that is stable and that largely corresponds to described expression patterns from laboratory model Drosophila species. However, our results provide a demonstration of the prediction that, as phylogenetic distance increases, variation between species overwhelms variation between tissue types. Using ancestral state reconstruction of expression, we describe the distribution of evolutionary changes in tissue-biased expression, and use this to identify gains and losses of ovary expression across these twelve species. We then use this distribution to calculate the evolutionary correlation in expression changes between genes, and demonstrate that genes with known interactions in D. melanogaster are significantly more correlated in their evolution than genes with no or unknown interactions. Finally, we use this correlation matrix to infer new networks of genes that share evolutionary trajectories, and we present these results as a dataset of new testable hypotheses about genetic roles and interactions in the Drosophila ovary.

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SEMplMe: A tool for integrating DNA methylation effects in transcription factor binding affinity predictions MotivationAberrant DNA methylation in transcription factor binding sites has been shown to lead to anomalous gene regulation that is strongly associated with human disease. However, the majority of methylation-sensitive positions within transcription factor binding sites remain unknown. Here we introduce SEMplMe, a computational tool to generate predictions of the effect of methylation on transcription factor binding strength in every position within a transcription factors motif.

ResultsSEMplMe uses ChIP-seq and whole genome bisulfite sequencing to predict effects of methylation within binding sites. SEMplMe validates known methylation sensitive and insensitive positions within a binding motif, identifies cell type specific transcription factor binding driven by methylation, and outperforms SELEX-based predictions for CTCF. These predictions can be used to identify aberrant sites of DNA methylation contributing to human disease.

Availability and ImplementationSEMplMe is available from https://github.com/Boyle-Lab/SEMplMe.

Contactapboyle@umich.edu

Supplementary InformationSupplementary data are available at Bioinformatics online.

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Scoping review of the applications of peptide microarrays on the fight against human infections. IntroductionThis scoping review explores the use of peptide microarrays in the fight against infectious diseases. The research domains explored included the use of peptide microarrays in the mapping of linear B-cell and T cell epitopes, antimicrobial peptide discovery, immunosignature characterisation and disease immunodiagnostics. This review also provides a short overview of peptide microarray synthesis.

MethodsElectronic databases were systematically searched to identify relevant studies. The review was conducted using the Joanna Briggs Institute methodology for scoping reviews and data charting was performed using a predefined form. The results were reported by narrative synthesis in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews guidelines.

ResultsEighty-six articles from 100 studies were included in the final data charting process. The majority (93%) of the articles were published during 2010-2020 and were mostly from Europe (44%) and North America (34 %). The findings were from the investigation of viral (44%), bacterial (30%), parasitic (25%) and fungal (2%) infections. Out of the serological studies, IgG was the most reported antibody type followed by IgM. The largest portion of the studies (78%) were related to mapping B-cell linear epitopes, 10% were on diagnostics, 9% reported on immunosignature characterisation and 6% reported on viral and bacterial cell binding assays. Two studies reported on T-cell epitope profiling.

ConclusionThe most important application of peptide microarrays was found to be B-cell epitope mapping or antibody profiling to identify diagnostic and vaccine targets. Immunosignatures identified by random peptide microarrays were found to be applied in the diagnosis of infections and interrogation of vaccine responses. The analysis of the interactions of random peptide microarrays with bacterial and viral cells using binding assays enabled the identification of antimicrobial peptides. Peptide microarray arrays were also used for T-cell linear epitope mapping which may provide more information for the design of peptide-based vaccines and for the development of diagnostic reagents.

sentences: A retrograde mechanism coordinates memory allocation across brain regions Memories engage ensembles of neurons across different brain regions within a memory system. However, it is unclear whether the allocation of a memory to these ensembles is coordinated across brain regions. To address this question, we used CREB expression to bias memory allocation in one brain region, and rabies retrograde tracing to test memory allocation in connected presynaptic neurons in the other brain regions. We find that biasing allocation of CTA memory in the basolateral amygdala (BLA) also biases memory allocation in presynaptic neurons of the insular cortex (IC). By manipulating the allocation of CTA memory to specific neurons in both BLA and IC, we found that we increased their connectivity and enhanced CTA memory performance. These results - which are corroborated by mathematical simulations, and by studies with auditory fear conditioning - demonstrate that a retrograde mechanism coordinates the allocation of memories across different brain regions.

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Sex dependent glial-specific changes in the chromatin accessibility landscape in late-onset Alzheimer's disease brains In the post-GWAS era, there is an unmet need to decode the underpinning genetic etiologies of late-onset Alzheimers disease (LOAD) and translate the associations to causation. Toward that goal, we conducted ATAC-seq profiling using neuronal nuclear protein (NeuN) sorted-nuclei from 40 frozen brain tissues to determine LOAD-specific changes in chromatin accessibility landscape in a cell-type specific manner. We identified 211 LOAD-specific differential chromatin accessibility sites in neuronal-nuclei, four of which overlapped with LOAD-GWAS regions ({+/-}100kb of SNP). While the non-neuronal nuclei did not show LOAD-specific differences, stratification by sex identified 842 LOAD-specific chromatin accessibility sites in females. Seven of these sex-dependent sites in the non-neuronal samples overlapped LOAD-GWAS regions including APOE. LOAD loci were functionally validated using single-nuclei RNA-seq datasets. In conclusion, using brain sorted-nuclei enabled the identification of sex-dependent cell type-specific LOAD alterations in chromatin structure. These findings enhance the interpretation of LOAD-GWAS discoveries, provide potential pathomechanisms, and suggest novel LOAD-loci. Furthermore, our results convey mechanistic insights into sex differences in LOAD risk and clinicopathology.

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sentences: Large-bodied ornithomimosaurs inhabited Appalachia during the Late Cretaceous of North America Reconstructing the evolution, diversity, and paleobiogeography of North Americas Late Cretaceous dinosaur assemblages requires spatiotemporally contiguous data; however, there remains a spatial and temporal disparity in dinosaur data on the continent. The rarity of vertebrate-bearing sedimentary deposits representing Turonian-Santonian ecosystems, and the relatively sparse record of dinosaurs from the eastern portion of the continent, present persistent challenges for studies of North American dinosaur evolution. Here we describe an assemblage of ornithomimosaurian materials from the Santonian Eutaw Formation of Mississippi. Morphological data coupled with osteohistological growth markers suggest the presence of two taxa of different body sizes, including one of the largest ornithomimosaurians known worldwide. The regression predicts a femoral circumference and a body mass of the Eutaw individuals similar to or greater than that of large-bodied ornithomimosaurs, Beishanlong grandis and Gallimimus bullatus. The paleohistology of MMNS VP-6332 demonstrates that the individual was at least 11 years of age (similar to B. grandis [[~]375 kg, 13-14 years old at death]). Additional pedal elements share some intriguing features with ornithomimosaurs yet suggest a larger-body size closer to Deinocheirus mirificus. The presence of a large-bodied ornithomimosaur in this region during this time is consistent with the relatively recent discoveries of early-diverging, large-bodied ornithomimosaurs from mid-Cretaceous strata of Laurasia (Arkansaurus fridayi and B. grandis). The smaller Eutaw taxon is represented by a tibia preserving seven growth cycles, with osteohistological indicators of decreasing growth, yet belongs to an individual with near reaching somatic maturity of the larger taxon, suggesting the co-existence of medium- and large-bodied ornithomimosaur taxa during the Late Cretaceous Santonian of North America. The Eutaw ornithomimosaur materials provide key information on the diversity and distribution of North American ornithomimosaurs and Appalachian dinosaurs and fit with broader evidence of multiple cohabiting species of ornithomimosaurian dinosaurs in Late Cretaceous ecosystems of Laurasia.

sentences: Moonlighting proteins activate transformation in epigenetically-differentiated phase variants of multidrug-resistant Streptococcus pneumoniae Transformation of Streptococcus pneumoniae GPSC1 has enabled vaccine evasion and the acquisition of antibiotic resistance. Epigenetic phase variants of GPSC1 isolate RMV7, differentiated by rearrangements at the tvr restriction-modification locus, differed [~]100-fold in their transformation efficiency. This variation was recapitulated in knock-in mutants of the relevant tvr alleles. RNA-seq showed the difference was due to blocking of the early competence regulatory cascade. The more transformable variant upregulated expression of manLMN, encoding a carbon source importer. This was shown to be necessary for efficient competence induction, despite being dispensable for growth in rich media. Transformation was promoted by import of N-acetylglucosamine, which activated competence through an orthologue of the gram-negative competence regulator TfoX, and an enzyme likely involved in nucleotide-mediated signalling. A mobile genetic element was more active in the less transformable variant, which limited competence induction through inhibiting CIRCE motif binding by the chaperone regulator HrcA. Correspondingly, both heat shock and decreased Ca2+ concentrations reduced competence by limiting HrcA binding regulatory sequence motifs. Hence both ManLMN and HrcA moonlighted as activators of competence. Such proteins may be important in potentiating horizontal DNA exchange, as selection on their primary function likely constrains them from mutating into alleles that selfishly downregulate transformation.

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Development of lipidoid nanoparticles for siRNA delivery to neural cells Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential utility in either applications in the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX--the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.

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sentences: Interrogating Metabolic Interactions Between Skeletal Muscle and Liver Circadian Clocks In Vivo Expressed throughout the body, the circadian clock system achieves daily metabolic homeostasis at every level of physiology, with clock disruption associated with metabolic disease (1, 2). Molecular clocks present in the brain, liver, adipose, pancreas and skeletal muscle each contribute to glucose homeostasis (3). However, it is unclear; 1) which organ clocks provide the most essential contributions, and 2) if these contributions depend on inter-organ communication. We recently showed that the liver clock alone is insufficient for most aspects of daily liver glucose handling and requires connections with other clocks (4). Considering the pathways that link glucose metabolism between liver and skeletal muscle, we sought to test whether a clock connection along this axis is important. Using our previous published methodology for tissue-specific rescue of Bmal1 in vivo (4, 5), we now show that in the absence of feeding-fasting cycles, liver and muscle clocks are not sufficient for systemic glucose metabolism, nor do they form a functional connection influencing local glucose handling or daily transcriptional rhythms in each tissue. However, the introduction of a daily feeding-fasting rhythm enables a synergistic state between liver and muscle clocks that leads to restoration of systemic glucose tolerance. These findings reveal limited autonomous capabilities of liver and muscle clocks and highlight the need for inter-organ clock communication for glucose homeostasis which involves at least two peripheral metabolic organs.

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Paired heavy and light chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses Understanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2.

HighlightsO_LIA molecular study of IGHV3-53/3-66 public antibody responses reveals critical heavy and light chain features for potent neutralization
C_LIO_LICryo-EM analyses detail the structure of a novel public antibody class member, antibody 910-30, in complex with SARS-CoV-2 spike trimer
C_LIO_LICryo-EM data reveal that 910-30 can both bind assembled trimer and can disassemble the SARS-CoV-2 spike
C_LIO_LISequence-structure-function signatures defined for IGHV3-53/3-66 class antibodies including both heavy and light chains
C_LIO_LIIGHV3-53/3-66 class precursors have a prevalence of 1:44,000 B cells in healthy human antibody repertoires
C_LI

sentences: A bacterial derived plant- mimicking cytokinin hormone regulates social behaviour in a rice pathogen Many plant-associated bacteria produce plant- mimicking hormones which are involved in modulating host physiology. However, their function in modulating bacterial physiology has not been reported. Here we show that the XopQ protein, a type-III effector of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo), is involved in cytokinin biosynthesis. Xoo produces and secretes an active form of cytokinin which enables the bacterium to maintain a planktonic lifestyle and promotes virulence. RNA-seq analysis indicates that the cytokinin produced by Xoo is required for the regulation of several genes which are involved in biofilm formation. We have also identified the Xoo isopentenyl transferase gene, which is involved in the cytokinin biosynthesis pathway and is required for maintaining planktonic behaviour and virulence. Furthermore, mutations in the predicted cytokinin receptor kinase (PcrK) and the downstream response regulator (PcrR) of Xoo phenocopy the cytokinin biosynthetic mutants, but are not complemented by supplementation with exogenous cytokinin. Cytokinin biosynthetic functions are encoded in a number of diverse bacterial genomes suggesting that cytokinin may be a widespread signalling molecule in the bacterial kingdom.

sentences: NetREx - Network-based Rice Expression Analysis Server for Abiotic Stress Conditions Recent focus on transcriptomic studies in food crops like rice, wheat and maize provide new opportunities to address issues related to agriculture and climate change. Re-analysis of such data available in public domain supplemented with annotations across molecular hierarchy can be of immense help to the plant research community, particularly co-expression networks representing transcriptionally coordinated genes that are often part of the same biological process. With this objective we have developed NetREx, a Network based Rice Expression Analysis Server, that hosts ranked co-expression networks of Oryza sativa using publicly available mRNA-seq data across uniform experimental conditions. It provides a range of interactable data viewers and modules for analysing user queried genes across different stress conditions (drought, flood, cold and osmosis) and hormonal treatments (abscisic and jasmonic acid) and tissues (root and shoot). Subnetworks of user-defined genes can be queried in preconstructed tissue-specific networks, allowing users to view the fold-change, module memberships, gene annotations and analysis of their neighborhood genes and associated pathways. The webserver also allows querying of orthologous genes from Arabidopsis, wheat, maize, barley, and sorghum. Here we demonstrate that NetREx can be used to identify novel candidate genes and tissue-specific interactions under stress conditions and can aid in the analysis and understanding of complex phenotypes linked to stress response in rice. Available at: https://bioinf.iiit.ac.in/netrex/index.html

sentences: Plant-associate interactions and diversification across trophic levels Interactions between species are widely understood to have promoted the diversification of life on earth, but how interactions spur the the formation of new species remains unclear. Interacting species often become locally adapted to each other, but they may also be subject to shared dispersal limitations and environmental conditions. Moreover, theory predicts that different kinds of interactions have different effects on diversification. To better understand how species interactions promote diversification, we compiled published genetic data for host plants and intimately associated herbivores, parasites, and mutualists. We first tested whether host and associate population structures were correlated -- an indication of associates locally adapting to hosts -- and tested for confounding correlations with geographic distance or climate variation. We used Bayesian multiple regression to estimate the effect of host plant genetic differentiation on associate genetic differentiation over and above the confounding effects of geography and climate. We found that plant and associate genetic structures are indeed often congruent, but isolation by distance and by climate are also common. Multiple regressions established that the effect of host plants on associates is robust to accounting for geographic distance and climate. Finally, associate genetic structure was significantly explained by plant genetic structure more often in antagonistic interactions than in mutualistic ones. This supports a key prediction of coevolutionary theory, that antagonistic interactions promote diversity through local adaptation of antagonists to hosts, while mutualistic interactions promote diversity via the effect of hosts geographic distribution on mutualists dispersal.

sentences: Atypus karschi Dönitz, 1887 (Araneae: Atypidae): an Asian purse-web spider established in Pennsylvania, USA The Mygalomorph spiders of the family Atypidae are among the most archaic spiders. The genus Atypus Latreille, 1804 occurs in Eurasia and northern Africa, with a single enigmatic species, Atypus snetsingeri Sarno, 1973, restricted to a small area in southeastern Pennsylvania in Eastern USA. This study was undertaken to learn more about genetics of that species, its habitat requirements and natural history. A close relationship to European species could be assumed based on A. snetsingeris occurrence on the eastern coast of the USA, however molecular markers (CO1 sequences) confirmed that A. snetsingeri is identical with Atypus karschi Donitz, 1887 native to East Asia; it is an introduced species. The specific epithet snetsingeri is therefore relegated to a junior synonym of A. karschi. The karyotype of A. karschi has 42 chromosomes in females and 41 in males (X0 sex chromosome system). Chromosomes were metacentric except for one pair, which exhibited submetacentric morphology. In Pennsylvania the above-ground webs are usually vertical and attached to the base of bushes, trees, or walls, although some webs are oriented horizontally near the ground. It was found in a variety of habitats from forests to suburban shrubbery, and over a wide range of soil humidity and physical parameters. Prey include millipedes, snails, woodlice, carabid beetles and earthworms. The number of juveniles in excavated female webs ranged from 70 to 201. Atypus karschi is the first known case of an introduced purse-web spider. It is rarely noticed but well-established within its range in southeastern Pennsylvania.

sentences: Predictive coding of natural images by V1 activity revealed by self-supervised deep neural networks Predictive coding is an important candidate theory of self-supervised learning in the brain. Its central idea is that neural activity results from an integration and comparison of bottom-up inputs with contextual predictions, a process in which firing rates and synchronization may play distinct roles. Here, we quantified stimulus predictability for natural images based on self-supervised, generative neural networks. When the precise pixel structure of a stimulus falling into the V1 receptive field (RF) was predicted by the spatial context, V1 exhibited characteristic{gamma} -synchronization (30-80Hz), despite no detectable modulation of firing rates. In contrast to{gamma} , {beta}-synchronization emerged exclusively for unpredictable stimuli. Natural images with high structural predictability were characterized by high compressibility and low dimensionality. Yet, perceptual similarity was mainly determined by higher-level features of natural stimuli, not by the precise pixel structure. When higher-level features of the stimulus in the receptive field were predicted by the context, neurons showed a strong reduction in firing rates and an increase in surround suppression that was dissociated from synchronization patterns. These findings reveal distinct roles of synchronization and firing rates in the predictive coding of natural images.

sentences:
Evolution and regulation of microbial secondary metabolism Microbes have disproportionate impacts on the macroscopic world. This is in part due to their ability to grow to large groups and cooperatively secrete massive amounts of secondary metabolites that impact their environment. Yet, the conditions enabling secondary metabolism without compromising primary needs remain unclear. Here we investigated the biosynthesis of rhamnolipids, a secondary metabolite that Pseudomonas aeruginosa makes to decrease the surface tension of surrounding liquid. Using a combination of genomics, metabolomics, transcriptomics, and mathematical modeling we show that biosynthesis of rhamnolipids from glycerol varies inconsistently across the phylogenetic tree; instead, non-producer lineages are also those worse at reducing the oxidative stress of primary glycerol metabolism. The link to oxidative stress explains the inconsistent distribution across the P. aeruginosa tree, adding a new layer to the regulation of rhamnolipids--a microbial secondary metabolite important for fitness in natural and clinical settings.

SignificanceThe bacterium Pseudomonas aeruginosa is a major source of hospital-acquired infections. This pathogens knack for virulence relies on its ability to multiply and secrete massive amounts of substances that overwhelm microbial competitors and weaken host defenses. It remains unclear how the bacteria conciliate their need to grow and multiply--a need at the individual-level-- with their ability to secrete products--a need of the population. Here we combined genomics, metabolomics and mathematical modeling to study the biosynthesis of rhamnolipids, a surfactant that P. aeruginosa makes to reduce the surface tension of surrounding liquids. Our study reveals a new link between oxidative stress and rhamnolipid synthesis, which helps explain how this important bacterial product has evolved and how it persists in many lineages of pathogens.

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